Water Quality Parameters

WATER QUALITY PARAMETERS
A symposium cosponsored
by the Canada Centre for
Inland Waters and the
Analytical Chemistry Division
of the Chemical Institute
of Canada
Burlington, Ontario, Canada,
19-21 November 1973
ASTM SPECIAL TECHNICAL PUBLICATION 573

Silvio Barabas, general chairman
List price $29.50

04.573000-16
qSTAMERICAN SOCIETY FOR TESTING AND MATERIALS

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9 BY AMERICAN SOCIETY FOR TESTING AND MATERIALS 1975
L i b r a r y o f Congress C a t a l o g C a r d N u m b e r : 74-28980
NOTE
The Society is not responsible, as a body,
for the statements and opinions
advanced in this publication.
Printed in Baltimore. Md.

June 1975
Second Printing, 1979

Baltimore, Md.
Foreword
The Symposium on Water Quality Parameters--Selection, Measure-
ment, and Monitoring was held on 19-21 November 1973 at the Canada
Centre for Inland Waters in Burlington, Ontario, Canada. The sympo-
sium was cosponsored by the Canada Centre for Inland Waters and the
Analytical Chemistry Division of the Chemical Institute of Canada. Silvio
Barabas, Canada Centre for Inland Waters, presided as the general chair-
man.
This publication was sponsored by the American Society for Testing and
Materials' Committee on Publications.
Related
ASTM Publications
Biological Methods for the Assessment of Water Quality, STP 528 (1973)
$16.25, 04-528000-16
Microorganic Matter in Water, STP 448 (1969) $9.25, 04-448000-16
Manual on Water, STP 442 (1969) $16.50, 04-442000-16

Contents
Introduction
Inorganic Analysis
Asbestos Fibers in Lake Superior--R. w. DUarIAM AND T. PANG 5

Analytical Method 6
Results 7
Discussion 12
Speetrophotometric Determination of Sulfide in Water--
F . A . J. A R M S T R O N G 14
Experimental 15
Recommended Method lS
Results and Discussion 16
Electrochemical Detection of NH4+-NH3 Systems in Water--
J. B A R I C A 20
Determination of Ionized NH4 + with a Univalent Cation
Electrode 21
Determination of Total A m m o n i a with an A m m o n i a Probe 23
Direct Determination of Free Unionized Ammonia 23
Discussion 24
Determination of Total Mercury Levels in Natural Waters--
SUEO NISHI AND YOSHIYUKI HORIMOTO 25
Experimental 26
Analysis of Zn +§ Cd +§ and Pb ++ in Natural Waters by Anodie
Stripping Voltammetry Using a Rotating Pt:Hg Electrode--
PIERRE P I C H E T A N D M O N I Q U E G R A N D M A I S O N 30
Experimental 30
Summary 34
Automated Method for Sulfate Determination in Lake Water--
M. A. SANTIAGO, SAUNDRA FIELEK, AND C. L. SCHELSKE 35
Experimental 36
Conclusion 44
Measurable Inorganic Carbon Parameters in Seawater--
C . S. W O N G , R. D. BELLEGAY, AND A. B. CORNFORD 47
Selection of Parameters 48
Experimental Techniques 49
Discussion 54
Critical Review of Analytical Techniques for the Determination
of Soluble Pollutant Heavy Metals in Seawater--
D. C. BURRELL AND MENG-LEIN LEE 58
Detection Limits and Applicability 59
Seawater Matrix Problems 63
Chemical Speciation of Soluble Fraction 64
Sampling and Pre-Analysis Processing 67
Survey Analyses of Trace Elements in Water by Spark Source
Mass Speetrometry--i. H. CROCKER 71
Experimental 73
Nuclear Activation Determination of Heavy Metals in Great
Lakes Sediments, Soils, and Vegetation--R. E. JERVIS,
AMARES CHATTOPADHYAY, E. CSILLAG, AND B. TIEFENBACH 82
Nuclear Activation Techniques (NAA and PAA) 83
Studies of Lower Great Lake Sediments: Selenium and
Antimony (NAA) 87
Toxic Metal Accumulation in Aquatic Species (NAA) 91
Studies of Heavy Metal Uptake in Marsh Soils: (IPAA) 92
Conclusions 93
Trace Elements in Molluscs in the Kingston Basin--
D. A. LORD, W. G. BRECK, AND R. C. WHEELER 95
Experimental 98
Conclusion 108
Analysis of Total Mercury in Biological and Water Samples by
an Ultrasensitive Kinetic Method--p. J. KE ANn
R . J. T H I B E R T 112
Experimental 114
Procedure l!s
Rapid Determination of Cyanide in Waste Waters--
OM P. BHARGAVA, G. W. DELINE, AND W. G. HINES 122
Experimental 124
Calibrations and Procedure 124
Effect of Variation in NaOH Concentration 124
Effect of Sulfide 125
Effect of Thiocyanate, Thiosulfate, and Ferrocyanide 126
Precision 126
Recovery (Accuracy) 127
Conclusions 127
Rapid Determination of Fluoride in Hydrochloric Acid, Pickle
Liquor, and Gaseous Emissions---oM p. BHARGAVA,
A . A. S C H U L D T , AND W. G. HINES 128
Experimental 130
Reagents 130
Determination of Fluoride in HC1 131
Determination of Fluoride in Pickle Liquor 131
Determination of Fluoride in Gaseous Emissions 131
Interference Study 132
Iron Interference in F Determination in Pickle Liquor 132
Effect of HC1 Concentration 133
Summary 134
Scanning Electron Microscopy and Energy Dispersive X-ray
Mieroanalysis of Nuclear Reactor Corrosion Particles--
A. G . W I K J O R D , G . H . M A Y O R , A N D F . E . D O E R N 136
Experimental 138
Discussion 140
Conclusions 150
Preservation of Wastewater Effluent Samples for Forms of
Nitrogen and Phosphorus--D. F. KRAWCZYK 152
Experimental 153
Results 157
Conclusions 161
Organic Analysis
Guidelines for Quantitative Liquid-Liquid Extraction of Organo-
phosphate Pesticides from Water---i. H. SUFFET, r WU,
AND D. T. L. W O N G 167
Method of Approach 169
Experimental 169
Results 174
Summary 181
Pesticide Residue Screening Methods Utilizing Multldetector
Conflgurations---H. A. M C L E O D A N D D . E . C O F F I N 183
Analysis of Organochlorine Residues in Fish--L. M. REYNOLDS
AND T. COOPER 196
Cleanup of Biological Tissue Extracts 197
Analyses of Fat or Oil Samples for Low Levels of OC Residues 199
PCB Quantitation 200
Some Factors Affecting the Recovery of Polyehlorinated
Biphenyls (PCB's) from Water and Bottom Samplesw
T . A. B E L L A R A N D J. J. L I C H T E N B E R G 206
Experimental 207
Conclusions 218
Liquid Chromatography of Carbamate Pesticides---
A . D . T H R U S T O N , JR 220
Experimental 221
Conclusions 223
Uncoated Teflon as Support and Stationary Phase for Liquid/
Solid Gas Chromatography--K. L. E. KAISER 227
Properties of Teflon as Support 228
Experimental 228
Direct Aqueous Injections 236
Conclusion 240
Applications of Direct Aqueous Injection Gas Chromatography
and Freeze Concentration for the Determination of Organic
Compounds in Water and Waste Waters---M. E. FOX 242
Residual Methanol in Sewage 243
Aircraft Deicer in Sewage 244
Kraft Pulp Mill Effluent Plume 248
Gas Chromatographic Determination of Low Concentrations of
Hydrocarbons in Water by Vapor Phase Extraction--
DONALD M A C K A Y , W. Y. S H I U , A N D A . W . W O L K O F F 251
Theoretical 252
Experimental 254
Polycyelie Aromatic Hydrocarbons in Lake Waters and
Associated Sediments: Analytical Determination by Gas
Chromatography-Mass Spectrometry--M. T. STROSnERAND
G. W. H O D G S O N 259
Experimental 260
Instrumentation 262
Results 262
Summary 269
A Gas Liquid-Gas Solid Chromatographic Method for the
Identification of Sources of Oil Pollution--A. E. GEORGE,
G . T . S M I L E Y , D . S. M O N T G O M E R Y , AND H. SAWATZKY 271
Experimental 273
Discussion 277
Quantitative Analysis of Petroleum Oil Pollutants by Infrared
Speetrophotometry--M i CHAEL G R U E N F E L D 290
Experimental 293
Results and Discussions 295
Biological Analysis
Problems in the Monitoring of Biomass--N. H. F. WATSON,
G . F. C A R P E N T E R , A N D M. M U N A W A R 311
Biomass Estimation 312
Procedure 312
Heavy Metal Toxicity and Algal Bioassays--T. c. HtrrcmNsoN
A N D P. M . S T O K E S 320
Experimental Details 321
Conclusion 342
Algal Assays: Development and Appllcation--T. E. MALONEY
AND W. E. M I L L E R 344
Experimental 346
Conclusion 353
Effect of Body Weight on Uptake of Methyl Mercury by Fish--
A. S. W. D E F R E I T A S A N D J. S. H A R T 356
Experimental 357
Monitoring and Remote Sensing
Experiences in Operating a Continuous Water Quality
Monitoring Network--J. E. H A G A N A N D R. L . E S T E S 367
The System 368
Procedures 373
Cost 373
Automatic Water Quality Monitoring Within the Saint John
River Basin--D. n. CULLEN 375
Monitor Purchase 376
Physical Arrangement and Operation of Monitors 376
Monitor Locations 380
Field Monitor Services 381
Operational Problems and Applied Corrective Measures 384
Monitoring Servicing 388
Data Output 388
Assessment 389
Future Outlook for AWQM's 390
Field Analysis of Dissolved Gases in Lake Waters by Gas
Chromatography--D. A. J. M U R R A Y , D . P O V O L E D O , A N D
R. V. S C H M I D T 391
Experimental 392
Calibration 394
Discussion 395
Underwater Probing with Laser Radar--s. SIZGORIC AND
A. I . C A R S W E L L 398
Lidar Design Considerations 399
The Marine Lidar System 403
Measurements 406
Conclusion 412
Fast Kinetic Spectrometry and Automated Trace Analysis--
C. H. LANGFORD 414
Sensors for Monitoring Water Quality--R. s. INGOLS AND
T . F. C R A F T 418
Design Considerations 418
Sampling 421
Conclusion 423
A Remote Sensing Laser Fiuorometer--R. A. O'NEIL,
A. R. D A V I S , H . G . G R O S S , A N D J. K R U U S 424
Fluorescence 424
Remote Sensing 427
The Laser Fluorosensor 427
Field Trials 429
Conclusion 434
Possible Future Development 434
REX, A Computer Controlled Robot for In Situ Water Quality
Monitoring--g. N . B I R C H 437
The Basic System Configuration 438
The Robot Sensing Head 441
The Measurement of Water Quality Parameters 445
Conclusions 453
Utilization of Data from Continuous Monitoring Networks---
C. G . G U N N E R S O N 456
Data and Analyses 458
Discussions and Conclusions 474
Parameter Selection and Quality Control
Environmental Impact of Experimental Oil Spills in the
Canadian Aretie--w. A. ADAMS, n. F. SCOTT, AND
N. B. SNOW 489
Study Area 491
Methods 492
Experimental Oil Spills 498
Discussion and Conclusions 510
Investigation of the Weathering of a Selected Crude Oil in a
Cold Environment~n. F. ScoTT 514
Study Area Preparation 515
Analytical Procedures 515
Summary 522
Sampling Techniques in Urban Runoff Quality S t u d i e s -
]. M A R S A L E K 526
Composite Sampling Techniques to Determine the Total
Pollutant Yield from a Runoff Event 527
Sampling Techniques to Determine the Pollutant
Concentration Variation 530
Practical Aspects of Sampling Installations in Urban Runoff
Studies 536
Conclusions 541
Stable Carbon Isotope Ratios as Water Quality Indicators--
F. C . T A N A N D G . J. P E A R S O N 543
Carbon Isotope Ratios of Various Carbon Reservoirs 544
Principles of ~C 13 Technique 544
Analytical Techniques 546
Application 547
Selection of Background ~C13 Values 548
Limitations and Applicability of the Method 549
Data Are for Looking At or Quality Control Through
Interpretation--j. M. B E W E R S , I . D . M A C A U L A Y ,
BJ~RN SUNDBY, AND D. E. BUCKLEY 550
Data Interpretation and Corrective Feedback 551
Input Data Inspection 552
Data File Interpretation 554
Sediment Data 563
Conclusions 565
lnterlaboratory Study of the Cold Vapor Technique for Total
Mercury in Water--J. A . W I N T E R A N D H . A . C L E M E N T S 566
Experimental 567
Results 569
Discussion 579
Conclusions 579
STP573-EB/Jun. 1975
Introduction
On 19 to 21 November 1973, the Symposium on Water Quality Para-

meters-Selection, Measurement, and Monitoring was held at the Canada
Centre for Inland Waters (CCIW) in Burlington, Ontario. The symposium
was cosponsored by CCIW and the Analytical Chemistry Division of the
Chemical Institute of Canada.
During the three-day symposium, 127 papers were presented in six
disciplinary sessions bearing on new analytical developments in the fields
of inorganic chemistry, organic chemistry, biology, continuous monitoring
and remote sensing, parameter selection, and laboratory quality control.
Over 600 scientists, coming from all over Canada, the United States, and
representatives from Europe, South America, and Japan, attended the
symposium.
This symposium was organized to provide a suitable forum for the
ever-increasing number of scientists in government, industry, and univer-
sities concerned with the development or application or both of analytical
methods of detecting and measuring water pollutants and to provide
information on their latest findings and practices.
This publication of the proceedings is comprised of 46 selected papers
dealing exclusively with recent methodology on water analysis. All papers
submitted for publication in the proceedings have undergone critical
review by American Society for Testing and Materials appointed reviewers
in accordance with the ASTM standards for publication. Excellent review
papers not dealing with the analytical methodology that were presented at
the symposium have not been included in this volume.
For the convenience of the readers, the papers are grouped in five
sections: Inorganic Analysis, Organic Analysis, Continuous Monitoring
and Remote Sensing, Parameter Selection, and Biological Analysis.
The proceedings will be useful as a reference material in every labora-
tory engaged in water analysis as well as in every laboratory that may be
called upon from time to time to perform water analysis.
The proceedings of the Symposium on Water Quality Parameters--
Selection, Measurement, and Monitoring, the first of its kind in its
comprehensiveness and specific relevance to water, is hopefully only a
precursor to a long series of "proceedings" on water analysis in the years
to come as public recognition of the seriousness of the water quality
problems increases with time.
Copyright9 1975 by ASTM International www.astm.org

2 WATER QUALITY PARAMETERS
Acknowledgment is due to the session chairmen of the symposium for

their efforts in committing outstanding scientists in the field of analytical
water chemistry to present their findings at the symposium: W. J. Traversy
(Inorganic Analysis), A. S. Y. Chau (Organic Analysis), O. Bhargava
(Parameter Selection), W. T. Sayers (Continuous Monitoring and Remote
Sensing), T. G. Bridges (Quality Control), and B. Dutka (Biological
Analysis).
Silvio Barabas,
Head, Analytical Methods Research Section,
Canada Centre for Inland Waters,
Burlington, Ontario, Canada,
general chairman
Inorganic Analysis
R. W. D u r h a m I a n d T. P a n g j
Asbestos Fibers in Lake Superior
REFERENCE: Durham, R. W. and Pang, T., "Asbestos Fibers in Lake Superior,"

Water Quality Parameters, ASTM STP 573, American Society for Testing and Mate-
rials, 1975, pp. 5-13.
ABSTRACT:The discoveryof high concentrations of asbestos fibers in the western arm
of Lake Superior prompted the monitoring of the whole lake for fiber concentrations
in order to determine the extent of transboundary movement. A description of the elec-
tron microscopetechnique used for fiber analysis and identification is given along with
an assessment of the accuracyof the method.
KEY WORDS:water quality, asbestos, environmental tests, fibers, electron microscopes
Asbestos is a commercial product derived from several minerals of the

hydrated silicate type which crystallize in an acicular or needle-like
fashion. When crushed, these crystals have the property of splitting longi-
tudinally into fibers with great tensile strength and flexibility. A con-
siderable fraction of these fibers can be microscopic in size and enter the
atmosphere during the crushing operation as dust. These tiny fibers are
the causitive agent of the disease asbestosis, a scarring of the lungs by
increased fibrous tissue growth, which is an occupational hazard in the
asbestos industry. Unfortunately, the widespread use of asbestos materials
has lead to the spread of this disease to the general public [1],2 but a
more disturbing finding is that exposure to asbestos causes malignant
tumors of the pleura and peritoneum [2]. Such exposure is normally
considered to occur by way of inhalation of asbestos fibers, but there is
now concern that minute fibers may be absorbed from drinking water
through the gut and into the bloodstream. Pontefract and Cunningham
[3] have demonstrated this effect in rats.
The first investigation of asbestos fibers in aqueous suspension was by
Biles and Emerson in 1968 [4]. They concentrated fibers from beer
samples by centrifuging out fibers and sediment, ashing to remove organic
matter, and then resuspending in water. The fibers in this residue were
then scrutinized using a transmission electron microscope. Fibers were
JHead and electron microscopist, respectively, Physical Sciences Labs, Canada Centre for
Inland Waters, Burlington. Ontario L7R 4A6, Canada.
2The italic numbers in brackets refer to the list of references appended to this paper.
Copyright9 1975by ASTMInternational www.astm.org

similar in size and appearance to a standard sample of chrysotile and

electron diffraction patterns of a clump of small fibers confirmed this
observation. These fibers were expected to have come from the chrysotile
asbestos filters used in beer production.
In 1971, Cunningham and Pontefract [5] used similar techniques to
measure the numbers of fibers per liter in beverages and drinking water
from several Canadian cities. They found that Canadian beer and tap
water contained chrysotile asbestos fibers, mostly less than 1 /am in length,
at concentrations ranging from 2 to 10 x 106 fibers per liter with unfiltered
water supplies at the top of the range. Tap water drawn from a lake in
the asbestos mining region of Quebec and unfiltered gave a value of 173 •
106 fibers per liter.
This report apparently caused the Ontario Ministry of the Environment
to assess the water supplies of municipalities in the province drawn from
surface waters. Samples from 22 cities and towns were analyzed by
Ontario Research Foundation and the results have recently been published
[6]. Concentrations ranged from 0.136 x 106 fibers per liter in Ottawa's
tap water to 3.87 x 106 in Sarnia's. The mass concentration was calculated
from the number of fibers, their size distribution and the density of
chrysotile (2.4 g/cm3). For Thunder Bay with 0.83 x 106 fibers per liter,
the most probable particle size was 0.1 to 0.2 ~rn while the estimated
mass concentration was 0.235 nanograms per liter.
The U.S. Environmental Protection Agency is concerned over the effect
on the Duluth water supply of mine tailings containing the asbestiform
mineral cummingtonite [7] being dumped into Lake Superior from mining
and milling operations at Silver Bay in Minnesota. The Department of the
Environment is naturally interested in determining whether there is any
transboundary movement of these fibers, as Lake Superior provides several
Canadian municipalities with their drinking water directly as well as
flowing out into Lake Huron and hence to Erie and Ontario. In order to
evaluate this possibility, we are analyzing for asbestos fibers in water
samples taken from selected stations during the monitor cruises of Lake
Superior undertaken by the Canada Centre for Inland Waters (CCIW) in
1973.
Analytical Method
The concentrations of fibers in Lake Superior waters are too low to
allow direct observation of a drop with an electron microscope so it is
necessary to use a concentration stage in the analysis. The choice is
between filtration with the smallest porosity membrane filter and ultra-
centrifugation. We selected ultracentrifugation after running into difficulty
trying to destroy the membrane filter without losing fibers.
In a typical analysis, 250 ml of lake water were spun at 20 000 rpm
(about 30 000 g) for 2 h to bring down the fibers. These were resuspended
DURHAM AND PANG ON ASBESTOS FIBERS 7
ultrasonically in 1 ml of distilled water without further treatment because

the very low concentration of suspended matter in Lake Superior water
did not interfere with fiber counting. One microliter of this suspension was
transferred to a 3-mm carbon-coated grid and dried under a heat-lamp.
The grid was mounted in a Siemens 101 transmission electron microscope
and scanned at a magnification of 20 000. In order to obtain good
counting statistics, the fibers were counted in all the 230 openings of the
200 mesh grid rather than just a fraction of them. This was because of the
uneven distribution of fibers on the grid and the fact that the drop did not
cover it entirely. The lengths of about 80 fibers on each grid were
measured using a calibrated eye-piece in order to determine the size
distribution of the fibers in each sample. The shortest fiber detectable was
about 0.05 pm in length.
The recovery of fibers in the centrifugation step was checked by centri-
fuging the supernatant for a further 2 h and then counting the fibers and
measuring the size distribution. This was done for two samples resulting
in recoveries of 86 and 85 percent. The size distribution of fibers was
similar to the original so that the less than complete recovery in one
centrifugation was not a fractionation effect. The reproducibility of
counting the fibers in the resuspension was determined by counting dupli-
cate 1 pl aliquots after mounting on grids. The difference was 5 percent
which is compatible with the tolerance of _+1 percent for the micropipette.
Any contamination of the coated grid by ubiquitous asbestos fibers
would be magnified by the concentration factor which was 1000 in this
case. We were not able to eliminate contamination completely in pre-
paring the grids, but by ultrasonic cleaning of the fresh grids and all
glassware, the background contamination averaged 25 fibers per grid.
Each grid was scrutinized in the electron microscope before the sample
was evaporated onto it to determine its fiber background level. This value
was then subtracted from the total number observed after the sample had
been added.
Results
Figure 1 is a map of the Lake Superior shoreline showing the general
location of the sample stations for which results have been obtained.
Table 1 shows the results of the analyses of samples from two cruises in
early summer of 1973. During Cruise 103 two stations only were sampled:
142, between Isle Royale and Thunder Bay, and 203, which is about five
miles off Silver Bay, Minn. The fiber count of the Silver Bay sample was
appreciably higher than that of Isle Royale as it was also for Cruise 104
although the sample station analyzed in that cruise was 138 rather than
142. This station is in Thunder Bay.
Figure 2 is an electron micrograph of an average fiber from Station 142
which has the typical hollow cylinder appearance of a chrysotile fiber [8].
DULUTH
FIG. 1--Lake Superior showing locations of sample stations.
FIG. 2--Chrysotile fibers in water sample from near Isle Royale.
The fibers shown in Fig. 3 are from Station 203 and have a very different
appearance but are typical of the majority of fibers observed from this
sample station. One such fiber shown in Fig. 4 was large enough to
produce the accompanying electron diffraction pattern. This pattern is
identical with that shown in Fig. 5 from a fiber in a water suspension of
tailings from the taconite milling operation at Silver Bay. We have iden-
TABLE 1--Asbestos fiber concentrations in Lake Superior.
Fibers/liter
Cruise Station Depth, m (x 106)
103a 203 ,50 to 15 87.3

103 142 50 to 15 15.5
104b 203 1 9.5
104 138 1 1.4
104 138 85 0.76
104 89 1 2.2
104 2 1 1.7
104 43 1 1.4
aCruise 103, 15-28 June.

bCruise 104, 26 July to 8 August.
FIG. 3---Cummingtonite fibers in water sample near Silver Bay.

FIG. 4---Micrograph (top) and electron-diffraction pattern (bottom) of fiber in water off
Silver Bay,
FIG. 5--Micrograph (top) and electron-diffraction pattern (bottom) of fiber from tailings
of taconite milling operations at Silver Bay.
tiffed this fiber as cummingtonite by comparing its electron diffraction

pattern with that of a standard sample of this mineral.
Figure 6 shows the distribution of the fibers according to length in
Cruise 103 samples from Stations 203 and 142. The distribution of the
fibers in the sample from Station 203 extends over a much wider range
than that of Station 142 with about 20 percent of the fibers being longer
than 1 ~m.
FIG. 6---Fiber distributions in water samples from Silver Bay and Isle Royale.
Discussion
It is quite clear from the results of both cruises that the mining and
milling operation at Silver Bay has a major impact on the asbestos fiber
content of the western arm of the lake. The results of the second cruise,
104, however, show that it has little effect if any on the waters of Thunder
Bay or those of the eastern lake. The CCIW current monitoring program
during 1973 has shown that except for a few early oscillations, there was a
steady counter-clockwise flow around the lake [9]. Such a flow would tend
to move fibers from Silver Bay towards Duluth and then eastwards along
the south shore. These currents are slow, averaging a few centimeters per
second, so that the fibers will gradually settle out or diffuse into the main
body of the lake while the flowing water traverses the immense distances
involved.
Except for Station 203, the values obtained are consistent with those
quoted by Kay [6] for Thunder Bay municipal drinking water which is not
filtered. It is unlikely, however, that a standard water filtration system

would remove an appreciable fraction of fibers with a mean size of 0.3/~a.
The major difference between the results from the two cruises is the
ten-fold higher level found during Cruise 103. This may be due to the
different sampling techniques, as Cruise 103 samples were taken by
bleeding water into the sample bottle during descent from 15 to 50 m
while Cruise 104 samples were taken at discrete depths. However, Station
138 was sampled at 1 and 85 in and the results are not very different. The
lake was not yet stratified during Cruise 103 so collection of a more
concentrated sample through selective layering of fibers at a thermocline
could not have occurred.
The higher values obtained during the first cruise, 103, at Stations 142
and 203 might be related to the prevailing offshore wind direction during
this cruise. The coastline undergoes severe erosion such that offshore
winds could move the material further out into the lake. As the majority
of fibers in the Station 142 sample were chrysotile, its high value could
not he due to a reversal of current flow driving Silver Bay material
northeastwards.
We have too few results at the moment to state categorically that
Canadian waters of Lake Superior have stable, probably historic levels of
asbestos fibers in the 1 to 2 x 106 per liter range. Also, although the
precision of our analytical method is about _+15 percent, we need to
intercompare samples with other laboratories and agree on the shortest
length of fiber to be included before we can claim a particular accuracy
and relate our results directly to the work of others.
References
[1] Langer, A. M., Selikoff, I. J., and Sastre, A., Archives o f Environmental Health, Vol.
22, 1971, p. 348.
[2] Selikoff, I. J. and Hammond, E. C., American Journal o f Public Health, Vol. 59, 1968,
p. 1658.
[3] Pontefract, R. D. and Cunningham, H. M., Nature, Vol. 243, 1973, p. 352.
[4] Biles, B. and Emerson, T. R., Nature, Vol. 219, 1968, p. 93.
[5] Cunningham, H. M. and Pontefract, R. D., Nature, Vol. 232, 1971, p. 332.
[6] Kay, Garnet, Water and Pollution Control, Sept. 1973, pp. 33-35.
[7] Burrell, Stephen, "Amphiboles in Taconite from the Peter Mitchell Mine, Reserve
Mining Company, Babbitt, Minn.," report for the Minnesota Pollution Control Agency
by Dept. of Earth Science, University of Wisconsin, 1973.
[8] Kalousek, G. L. and Muttart, L. E., The American Mineralogist. Vol. 42, 1957, p. 1.
[9] Private communication, E. B. Bennett, Canada Centre for Inland Waters, Burlington.
F. A. J. A r m s t r o n g j
Spectrophotometric Determination
of Sulfide in Water
REFERENCE: Armstrong, F. A. J., "Spectrophotometrie Determination of Sulfide in

Water," Water Quality Parameters, A S T M STP 573, American Society for Testing and
Materials, 1975, pp. 14-19.
ABSTRACT: The hydrosulfide ion HS- has high absorbance in the ultra violet. It pre-
dominates in solution at pH above 8, but is negligible below pH 5. Sulfide up to 5 mg
S2-/liter in water can be determined by the difference between absorbance measure-
ments at 228 nm, first on the sample made alkaline with sodium hydroxide, and then
after acidification. Samples can be stabilized for some hours if made alkaline when col-
lected, and then kept in the dark. Interference may be caused if calcium and mag-
nesium are precipitated by alkali, and difficulty may be found with colored waters with
high ultraviolet absorbance. Standard deviation was ..+__0.1at the 4 mg S2-/liter level.
KEY WORDS. water quality, spectrophotometry, sulfide minerals, environmental tests
Hydrogen sulfide is highly objectionable a n d is toxic to m a n a n d to fish.

It m a y be generated in n a t u r a l waters by bacterial reduction of sulfate,
a n d is frequently f o u n d in the deeper waters of lakes and fjords when
oxygen i n t e r c h a n g e with the a t m o s p h e r e is h a m p e r e d by ice or by stable
density layering.
T h e r e are several m e t h o d s available for d e t e r m i n a t i o n of sulfide. The
most straightforward is the iodimetric d e t e r m i n a t i o n as in one of the
A P H A m e t h o d s [l].Z T h e sulfide electrode [2] appears to be very useful
over a wide r a n g e of concentrations. There are sensitive colorimetric
m e t h o d s which d e p e n d on p r o d u c t i o n of methylene blue from p - a m i n o -
dimethyl aniline [1] or of L a u t h ' s Violet from p - p h e n y l e n e d i a m i n e [3].
O t h e r colorimetric m e t h o d s use the p r o d u c t i o n of colloidal b i s m u t h sulfide
[4] or the reaction with sodium nitroprusside [5]. Generally (except for the
electrode method), these procedures involve a fair a m o u n t of m a n i p u -
lation. T h e range of application of some of these m e t h o d s is shown in
T a b l e 1.
, Head, Analytical Chemistry Group, Freshwater Institute, Winnipeg, Manitoba R3T 2N6,
Canada.
14

ARMSTRONG ON SULFIDE IN WATER 15
TABLE l--Typical concentration rangefor determination of sulfide.

Method Sample Size Range
Iodimetry 200 ml 10 ml 0.01 N (iodine) ~ 8 mg S2-/liter
Sulfide electrode (10-2 to 10-* M) typically 10-4 M or 3.2 mg S~-/liter
Lauth's Violet .~ ml E1 cm600 bn 1.00 ~ 1.2 mg S2-/liter
Bismuth sulfide 10 ml E1 cm350 nm 1.00 ~ 9 mg S2-/liter
Ultraviolet 50 ml E1 cm228 nm 1.00 ~---4.6 mg S2-/liter
nOTE--Limit of detection of absorptiometric methods is about 1/100 of above concentra-
tions.
In the course of some unrelated investigations it was noticed that

alkaline sulfide solutions had high absorbance in the ultraviolet, which
appeared to offer a very rapid and simple method for the determination.
Experimental
A p p a r a t u s - - A Unicam SPS00 spectrophotometer or Cary 14 recording
spectrophotometer was used.
R e a g e n t s - - S o d i u m sulfide approximately 0.1 M. Crystals of the hydrate

Na2S. 9H20 were sparingly washed and dried with filter paper. 24.2 g was
weighed and dissolved in water and made to 1 liter. This solution was
standardized by iodine titration.
Sodium sulfide 10-4M (1.00 ml - 3.20 mg S2-/liter) can be prepared
when required by diluting 1.00 ml of 0.1 M standard to 1 liter.
Recommended Method
The water sample should have been taken and kept with proper care to
avoid loss of hydrogen sulfide, and may contain up to S mg S2-/liter. For
analysis it should be made just alkaline (pH 9 to 10) with sodium
hydroxide, and if this is done "at the time of collection it will help to
stabilize the sample. With waters of low conductivity (<100 /~S/cm)
addition of 1 ml/liter of 0.1 M sodium hydroxide is sufficient. With other
waters the quantity of alkali required should be determined by trial with a
separate portion, using phenolphthalein as indicator.
The absorbance in a 1-cm cuvette, at 228 nm should be measured, and
then without removing the cuvette, the sample should be acidified with
one drop of 0.1 M hydrochloric acid, the contents mixed, and the
absorbance measured again. The difference between the two readings is
proportional to sulfide concentration. Should more than about 1 percent
by volume of alkali have been used in preparation of the sample, the
dilution should be allowed for in the calculation.
A factor for this may be found by carrying out the procedure with a
freshly prepared 10 -4 M sodium sulfide solution.
Results and Discussion

Figure 1 shows the absorption spectrum of a sodium sulfide solution at
pH 9.5. There is a maximum at about 228 nm, and an indication of high
absorption below 200 nm. This has been discussed by Ley and Arends [6]
who point out that the first maximum (which they determined to be at 227
nm) was that of the SH- ion. They found another maximum at 186 nm,
due to OH ion, which of course is also present. Hydrogen sulfide had a
0:7.
O6. I
IxlO-4M SO01UM SULPHIDE
05-.
(32 mcJ Sz-/~ )
o3\
(D
<~ (32-
QI
OO
200 210 220 2:30 240 250 260 270 280 290 3OO
WAVELENGTH (nrn)
FIG. l~Absorption spectrum of sodium aulfide.
much weaker maximum at 189 nm. If the absorption of solutions is

measured at various pH values (Fig. 2), it is seen that it is constant above
pH 8 and zero below pH S. This is in accord with published values for the
dissociation constants of hydrogen sulfide. It can be calculated that HS-
ion exists in negligible quantity at pH less than 5.
Beers Law
The relationship between absorbance and sulfide concentration is linear
up to at least 6 mg S2-/liter. Higher values (up to 9 mg/liter) have been
reported in anoxic sea water, and dilution would be required.
Stability
Changes in concentration of sulfide solutions at the 10 -4 M level (Fig. 3)
were measured over a period of 6 to 7 h, using a recording spectro-
ARMSTRONG O N S U L F I D E IN W A T E R 17
0.7
0.6 _1
7
0.5 84- -
HzS K,: 9.1 x I0 -8 pKa: 7o2/
K2: 1.2xlO -I~ pKa=14.92~
co
04- I
g
t~
L)
Z
113
rr 0 2 - - - - -
o
m
01~
/
O.O-
_Y
2 4 5 6 7 8 9 10 II
pH
FIG. 2--Change o f absorbance o f sulfide solution (10-4 M S 2-) with pH.
07
0.6 A,B
,.,., ~C
0.5
co
0,4
g \
03
w
z
a: 0.2
\
0
m
~D
0.1 84
0
0 I 2 3 4 5 6 7
TIME (hr)
FIG. 3----Change of absorbance o f sulfide solution (I0 -4 M Na2S) with time.

photometer and pumping solutions through a flow cuvette with a peri-

staltic pump. With alkaline solutions in dark bottles there was a decrease
of about 3 percent in 6 h (Curves A, B). This was the same whether the
solution was made by diluting 0.1 M sodium sulfide with oxygenated
water or with water deoxygenated with a stream of helium. There was a
marked decrease if the solution was in a clear bottle and exposed to
fluorescent lighting (Curve C), and a rather sharp decline when the
solution was made acid so that it contained free hydrogen sulfide (Curve
D). (For this experiment the solution was made alkaline with a stream of
NaOH, introduced just ahead of the cuvette.)
Interference
Difficulty with this determination may be expected if much alkali must

be added, or if the background ultraviolet absorbance of the water is too
high. I f much alkali has to be added there is a risk that calcium and
magnesium may be precipitated. This can be minimized by adding the
alkali rather slowly, with gentle mixing, avoiding contact with air.
Figure 4 shows the absorbance of some natural waters from 200 to 400
nm. Curve A is for sea water, and B, C, D, and E are for fresh waters of
color from 8 to 35 platinum units in that order. The absorbance at 228
0.7
E I
06.
05 L
0~..
Z
trl
'~ 0 3 .
0
o3
e,m
02-
OI
B
A
0.0
200 210 220 230 240 250 260 270 280 290 300
WAVELENGTH, nrn
FIG. 4--A bsorption spectra of natural waters.

ARMSTRONG ON SULFIDE IN WATER 19
nm can be appreciable, and of course must not change significantly when

the pH is altered. Measurement of the change showed a decrease of 2
percent of the base absorbance, that is to say, with a strongly colored
water of absorbance 0.5 at 227 nm, it was about 0.01, corresponding to
about 0.04 mg S2-/liter.
Test of Method
Comparisons of results obtained by a Lauth's Violet method and this
one are shown in Table 2.
TABLE 2--Comparison of methods for sulfide in water.
A B
Lauth's Violet Ultraviolet
(Poison and Strickland [3]) Absorbance A-B
mg S2-/liter mg S2-/liter mg S2-/liter
1.37 1.37 0.00

1.48 1.54 --0.06
1.54 1.53 +0.01
2.16 2.13 +0.03
3.53 3.58 --0.05
4.59 4.63 --0.04
6.44 6.35 +0.09
6.51 6.43 +0.08
Reproducibility
There is so little sample manipulation with this method that the error is
practically that of the difference between two absorbance measurements,
which is usually around 1 percent. Here, since absorbance is measured at
a short wavelength, where cleanliness of cuvette surfaces is quite impor-
tant, a somewhat larger error can be expected. Some tests at the 4 mg
S2"/liter level gave a standard deviation _-+-0.1 mg S2-/liter, (n = 8), that
is, relative standard deviation _+2.5 percent.
Acknowledgment
I am grateful for the analytical assistance given by M. Pitze.
References
[l] Standard Methods for the Examination of Water and Waste Water, 13th ed., American
Public Health Association, New York, 1971, p. 551.
[2] Hseu, T. M. and Rechnitz, G. A., Analytical Chemistry, Vol. 40, 1968, p. 1054.
[3] Polson, D. S. C. and Strickland, J. D. H., Analytica Chimica Acta. Voi. 6, 1952, p.
452.
[4] Field, E. and Oldach, C. S., Industrial and Engineering Chemistry, Analytical Edition,
Vol. 18, 1946, p. 665.
[5] Casapieri, P., Scott, R., and Simpson, E. A., Analytica Chimica Acta, Voi. 45, 1969,
p. 547.
[6] Ley, H. and Arends. B., ZeitschriftfilrPhysikalische Chemie, Vol. B15, 1932, p. 311.
J. B a r i c a I
Electrochemical Detection of
NH,+-NH3 Systems in Water
REFERENCE: Barica, J., "Electrochemical Detection of NH,*-NH3 Systems in Water,"

Water Quality Parameters. A S T M STP 573, American Society for Testing and Mate-
rials, 1975, pp. 20-24.
ABSTRACT: Two methods for determining the forms of ammonia nitrogen in water are
described. A univalent cation electrode can be used to detect ionized ammonium if po-
tassium and sodium concentrations of the solution are constant. At pH values less than
7.5 this wilt correspond to the total ammonia concentration in a water sample. Another
approach is to employ a membrane electrode, which is sensitive to unionized ammonia
and subject to fewer interferences. It involves an alkalization of a sample to pH 12 and
subsequent measurement of liberated unionized ammonia (total ammonia). A direct
measurement of free ammonia can be made without alkalization. Suitability of both
methods for determination of high ammonia concentrations (above 0.2 to 0.5 mg/liter
N) in water samples, mainly fish tanks, is discussed. There is no upper limit of de-
tection, and samples therefore do not require any dilution.
KEY WORDS: water quality, nitrogen inorganic compounds, ammonia, environmental

tests
T h e d e t e r m i n a t i o n o f a m m o n i a by c o n v e n t i o n a l c o l o r i m e t r i c m e t h o d s
( n e s s l e r i z a t i o n , p h e n o l h y p o c h l o r i t e r e a c t i o n ) o r by d i s t i l l a t i o n n o r m a l l y
yields t o t a l a m m o n i a c o n c e n t r a t i o n in a w a t e r s a m p l e .
Total a m m o n i a = ionized N H { + unionized N H 3
It is, h o w e v e r , t h e u n i o n i z e d free a m m o n i a in t h e e q u i l i b r i u m e q u a t i o n
[NH4*]" [OH-] = Ki
[NH~]
w h i c h e x e r t s a d v e r s e p h y s i o l o g i c a l or h i s t o p a t h o l o g i c a l effects o n fish. A
c o n c e n t r a t i o n o f 0 . 2 m g / l i t e r N H 3 is g e n e r a l l y a c c e p t e d as t h e t h r e s h o l d
Research scientist, Department of the Environment, Freshwater Institute, Winnipeg R3T

2N6, Canada.
2O
9
Copyright 1975 by ASTM International www.astm.org
BARICA ON NH4+-NH3 SYSTEMS IN WATER 21
LC 50 [113 The concentration of free unionized ammonia depends on the

pH and temperature of the sample, and can be calculated from ionization
constants, Ki, for different temperatures [2].
Two specific ion electrodes, available commercially, make possible the
direct measurement of total, ionized, and unionized ammonia in the
following manner:
1. the ammonium ion (NH4§ using a univalent (monovalent) cation

electrode;
2. unionized free ammonia (NH3) using a membrane-type ammonia
electrode (ammonia probe); and
3. total ammonia (NH4§ + NHa) using the membrane-type ammonia
electrode after converting all NH4§ into NH 3 by alkalization of the
sample to pH about 12.
In this paper a summary of the aforementioned approaches is pre-

sented. Details on reliability, accuracy, standardization, and procedures
are given elsewhere [3, 6].
Determination of Ionized NH4 + with a Univalent Cation Electrode
The univalent cation electrode (Corning monovalent cation electrode

No. 476220 or comparable electrode) is a glass membrane electrode,
sensitive also to other univalent cations, mainly to K § and Na § The final
electrode potential depends on the sum of their activities corrected for the
selectivity constants of the electrode (approximate apparent selectivity at
pH 7 for K§ § = 3 and for K§ § = 8). Although this feature limits
the use of the electrode to low and constant potassium and sodium
background levels, it is possible to use it in controlled systems where
fluctuations in concentration of K § and Na § are negligible and their
concentrations are lower than the concentration of ammonium ion. Such a
situation generally prevails in fish-holding tanks or aquaria as well as in
many hypolimnetic waters of softwater lakes, where the NH4* is the only
univalent cation that fluctuates significantly over a short period of time as
a result of biological processes. This method enables the direct deter-
mination of NH4§ in concentrations above 0.5 mg/liter (as N) without any
dilution or pre-treatment of the sample. The electrode operates in com-
bination with a suitable reference electrode (Ag-AgCI type). A pH meter
with expanded millivolt scale is required for measurements. For lower
concentrations of ammonium (0.5 to 3.0 mg/liter as N), the preparation
of a working calibration curve is required, using 2 to 3 standard additions
of ammonium to an ammonia-free water sample of identical ionic strength
2The italic numbers in brackets refer to the list of referencesappended to this paper.
(aquaria make-up water, tap water, etc.). Semilog paper is used for
plotting the electrode potential on the linear axis and concentration of
NH4* on the logarithmic axis as shown in Fig. 1. The electrode drift
should be checked regularly and the electrode recalibrated before each
series of determinations, at least once a day. For concentrations higher
I00 -
D~-Z--~e_ AMMONIAPROBE
80- l ~ LD~O
60-
40
20-
0
-20 -
~_J- 4 0 -
.J
5: -60-
-80 -
UNIVALENT CATION oo , . ~ o ~
-I00 - EL ECTRODE t e ~ e
-120 -
-140 -
-160-
-180 --f"" I I l 1' I I II I' I I I I I I-II I I
OI 0.5 I 5 I0 20 50
NHa-N mg/I
F I G , 1--Electrode responses to different concentrations of ammonium or total ammonia
nitrogen. I -~ deionized water: 2 = Winnipeg tap water (spec. eond. 900 ~mhos/cm); 3, 4
,fish homing tanks calibration curves. Ammonia added in all cases.
than 3 mg/liter NH4+-N the method of standard known addition can be

used, or the pNH4 can be read directly from the logarithmic scale of the
meter, provided the instrument has been standardized within +3 mg/liter
of the expected NH4+-N concentration [3]. Electrode response to change in
ammonium ion concentrations is very rapid.
BARICA ON NH4+-NH3 SYSTEMS IN WATER 23
A calculation using the ionization constants [2] shows that ammonium

ion concentration at pH = 7.5 or less is practically equal to the total
ammonia concentration.
Determination of Total A m m o n i a with an A m m o n i a Probe

Gas-membrane electrodes, which have recently become available com-
mercially, are sensitive only to unionized ammonia in solution, are more
specific, and subject to less interference than the glass electrode. In
addition, no separate reference electrode is required, as the electrode
system itself represents an electrochemical cell (ORION ammonia elec-
trode Model 95-10 or comparable electrode). Since the probe detects only
free ammonia and is entirely insensitive to ammonium ions, a complete
conversion of ammonium ions to the unionized free ammonia form by pH
adjustment is necessary to obtain a measure of total ammonia nitrogen.
From the ionization constants it follows that at pH higher than 11
practically only free NH 3 is present. Therefore, when pH of the sample is
increased to levels between 12 to 13 by addition of 1 ml of 40 percent
NaOH per 100 ml of the sample, the reading will correspond to total
ammonia nitrogen.
The electrode response to ammonia in alkaline solutions follows the
Nerstian slope only between concentrations 10 -1 to 10-4 M (1.4 g/liter to
14 mg/liter NH3-N). In the normal working range of fish holding tanks or
aquaria (up to 10 mg/liter), the response does not follow the Nerstian
equation. Preparation of working calibration curves in this range is there-
fore essential (Fig. 1). The lowest limit of detection is around 0.1 mg/liter
NH3-N. The method is suitable for sea-water [4] and industrial effluents
[5]. Stabilization time of the electrode is substantially longer since NH 3
must diffuse through the electrode membrane and equilibration times of 2
to 5 min, depending on NH 3 concentration [6], are required.
Direct Determination of Free Unionized A m m o n i a

For direct detection of free NH 3 the same technique can be used as for
the total ammonia, however, millivolt reading is taken without addition of
NaOH. Standardization can be performed similarly to the total ammonia
concentration, but without alkalization. In this case the reading must be
corrected using the Trussell [2] chart (for example, for total ammonia
concentration of 5 and 10 mg/liter N added from standard solution for
construction of a calibration curve without alkalization, the actual con-
centrations of free unionized ammonia at, say, 20~ and pH 8, will be
0.191 and 0.383 mg/liter NH3-N, respectively).
Direct determination of free NH3, using the membrane electrode in a
series of high-ammonia fish tank water samples, several domestic sewage
(lagoon effluent) and oil refinery effluents agreed within 10 percent with
data calculated using the Trussell [2] method.
Discussion
Although the specific ion electrodes cannot yet compete in accuracy
with spectrophotometric determination of ammonia, especially at concen-
trations lower than 1 mg/liter, they are very useful for higher concentra-
tion ranges as they do not require any dilution of sample prior to the
determination, are much faster, and the volume of sample is minimal (20
to 50 ml). At the same time, the electrodes offer the possibility of con-
tinuous monitoring and signaling of dangerous levels of ammonia. The
choice between a glass or a membrane electrode depends on the analyst's
needs and conditions. The glass electrode is about one-tenth of the cost of
an ammonia probe and has much faster response, however, its application
is limited to water with a low and constant background K § and Na § which
does not always prevail. The ammonia probe is more specific and more
versatile.
For theoretical studies on NH4+-NH3 equilibria, both electrodes can be
successfully used simultaneously in the same sample or in a continuous
sample stream, as no chemical pre-treatment is required for ammonium
and free ammonia determination.
References
[I] "Report on Ammonia and Inland Fisheries," European Inland Fisheries Advisory Com-
mission, EIFAC Technical Paper No. II, FAO Rome, 1970.
[2] Trussell, R. P., Journal, Fisheries Research Board of Canada, Vol. 29, 1972, pp. 1505-
1507.
[3] Barica, J., Journal, Fisheries Research Board of Canada, Vol. 28, 1971, pp. 759-764.
[4] Gilbert, R. T. and Clay, A. M., Analytical Chemistry. Vol. 45, 1973, pp. 1757-1759.
[5] LeBlanc, P. J. and Sliwinski, J. F., American Laboratory, July 1973, pp. 51-54.
[6] Barica, J., Journal, Fisheries Research Board of Canada, Vol. 30, 1973, pp. 1389-1392.
Sueo Nishil and Yoshiyuki Horimoto I
Determination of Total Mercury

Levels in Natural Waters
REFERENCE: Nishi, Sueo, and Horimoto, Yoshiyuki, "Determination of Total Mer-

cuff Levels in Natural Waters," Water Quality Parameters, A S T M STP 573, American
Society tbr Testing and Materials, 1975, pp. 25-29.
ABSTRACT: A rapid and simple preconcentration technique for the determination of

trace amounts of mercury in natural waters has been studied. A l-liter water sample
is pretreated by conventional methods using permanganate and sulfuric acid.
Mercury(ll) ion formed is reduced to elemental mercury which is aerated and caught
in a gold trap. The trapped mercury is released by heating the gold trap and driven
into a photometer. A 0.01 ppb level of mercury in natural water is determined success-
fully.
KEY WORDS: water quality, mercury (metal), atomic spectroscopy, environmental
tests, sampling
F l a m e l e s s a t o m i c a b s o r b t i o n s p e c t r o m e t r y c o m b i n e d with t h e reduction-
a e r a t i o n t e c h n i q u e h a s b e e n widely used in the analysis o f trace m e r c u r y
in water. A conventional p h o t o m e t e r used in the analysis o f m e r c u r y often
has t h e sensitivity o f 0.01 /ag or less of m e r c u r y at full scale; however, the
o p t i m a l r a n g e of the d e t e r m i n a t i o n is usually considered to be 0.01 to
0.5 tag.
T h e v o l u m e of t h e s a m p l e w a t e r at t h e r e d u c t i o n - a e r a t i o n stage con-
s i d e r a b l y affects the sensitivity o f the analysis [1],z the l a r g e r the volume
of the s a m p l e , the less t h e sensitivity. T h e m a x i m u m limit of the s a m p l e
volume at the r e d u c t i o n - a e r a t i o n stage is a b o u t 100 ml for a conventional
method.
O m a n g [2] d e t e r m i n e d the c o n c e n t r a t i o n of m e r c u r y to b e less t h a n 0.1
/ag/liter in lake water w i t h o u t any c o n c e n t r a t i o n p r o c e d u r e . However, in
the case of m e r c u r y c o n c e n t r a t i o n s of less t h a n 0.1 /ag/liter, it is prefer-
able to d e t e r m i n e it after the m e r c u r y has been c o n c e n t r a t e d to i m p r o v e
the accuracy. C h a u a n d Saitoh [3] r e c o m m e n d e d the c o n c e n t r a t i o n pro-
~National Chemical Laboratory for Industry, 1 chome, Honmachi, Shibuya-ku, Tokyo,
Japan.
25

26 WATER
QUALITPARAMETERS
Y
cedure of mercury in water, in which extraction with dithizone was
combined with back extraction with hydrochloric acid. Topping and Pirie
[4] proposed a method in which mercury from 4-liter sample water is
aerated into 20 ml of permanganate solution.
This study aims at the improvement of the simple and rapid precon-
centration technique for the determination of trace mercury in water.
Experimental
Figure 1 shows schematically the apparatus used for the experiments.
The photometer used was a Mercury Vapor Analyzer, Toshiba-Beckman
Rotmet
a er
Gold Filter
Gold Trap i Ph~176 l~
Activated
Carbon
Recorder
Ice Trap
Air
F1G. 1--Schematic diagram of preconcentration apparatus.
MV 253, equipped with a 300-mm long, 30-mm internal diameter optical

cell. All reagents were guaranteed reagent grade and used without further
purification except sulfuric acid which was purified by distillation under
reduced pressure.
The reducing solution was prepared by dissolving 100 g of sulfuric acid,
5 g of hydroxylaminesulfate, 2.5 g of sodium chloride, and 50 g of
stannous sulfate in 300 ml of water, then diluting to 500 ml with water,
and separated from the insoluble part by centrifuging.
The standard mercury solution was prepared in the following manner.
Mercuric chloride was dissolved in water to make 100 ppm (as Hg) and
was stored as the stock solution. The working solution was diluted to 1
ppm or 0.1 ppm just prior to use.
The sample was placed in a polyethylene vessel. Immediately after the
sampling, nitric acid was added to make the solution about 0.1 N, and
the sample was analyzed within three days.
NISHI AND HORIMOTO ON MERCURY LEVELS IN NATURAL WATERS 27
Preparation of GoM Trap

Two grams of chloroauric acid (HAuCI4"4H20) of reagent grade are
dissolved in about 20 ml of distilled water and, while stirring, the solution
is added to 15 g of Chromosorb W or Chromosorb P (each 30-60 mesh),
mixed, and dried at 100~ The product is packed in a quartz glass tube
and heated in a stream of air at about 800~ to decompose the chloro-
auric acid, forming a gold layer on the surface of the support. This is
packed in a 5-mm internal diameter quartz tube in a layer about 5-mm
thick. Both ends are stopped with quartz glass wool to produce the gold
trap.
Procedure
One liter of the water sample is combined in a flask with 10 ml of
aqueous sulfuric acid solution (1:1) and 2.5 ml of 5 percent potassium
permanganate solution. The mixture is left overnight at room temper-
ature. If discoloration of the permanganate occurs, an additional amount
of the permanganate solution is added. After the excess permanganate
and the precipitate are decomposed with a 10 percent solution of hydrox-
ylaminesulfate, the flask is connected, as shown in Fig. 1, and 4 ml of the
reducing solution are added. The mixture is aerated carrying the elemen-
tal mercury formed by reduction to the gold trap to be absorbed. Twelve
minutes after aeration begins, the gold trap is heated rapidly to release
the trapped mercury, which is led into the mercury analyzer and
determined.
The same procedure is applied to 1 liter of deionized water containing
no mercury, and the result obtained is taken as the blank value of the
reagents.
Independently, a 100-ml reduction-aeration cell is connected in place of
the sample flask. Each 30 ml of deionized water added with a known
amount (in the range of 0.01 to 0.3/ag) of mercury is reduced by adding 4
ml of the reducing solution. Mercury vapor is trapped in the gold trap
and determined according to the abovementioned procedure. A calibration
curve (the amount of mercury against the peak height) is plotted and
shows a straight line plot.
The amount of mercury in a sample is obtained by subtracting the
blank value of the reagents from the value measured for the sample.

The standard mercury solutions of 0.1 and 0.01/ag were added to 1 liter
of deionized water, and peak heights of the instrument response were
recorded. This procedure was repeated to study the reproducibility, and
the results are given in Table 1. This shows good reproducibility with the
TABLE 1--Reproducibility of mercury recovery from 1 liter of deionized water

spiked with mercury.
Added 0.1 tag Added 0.01 /ag
Mean 0.097 g 0.0095 g

Standard deviation 0.0018 g 0.0003 g
Coefficient of variation, % 2 3
No. of replicates 8 11
coefficient of variation of 3 percent, even at a c o n c e n t r a t i o n of m e r c u r y of

0.01 /ag/liter. Therefore, it is clear that this c o n c e n t r a t i o n method c a n be
applied satisfactorily to q u a n t i t a t i v e analysis.
For analyses of mercury in n a t u r a l waters, it is necessary to convert
mercury, which m a y be present in various chemical forms in the sample,
to bivalent m e r c u r y ion. O m a n g [2] o b t a i n e d good results by t r e a t i n g
samples of n a t u r a l waters a n d effluents with a mixture of sulfuric acid
and permanganate.
I n the present study, n a t u r a l waters were analyzed for total m e r c u r y
using the p r e c o n c e n t r a t i o n t e c h n i q u e after treating with the sulfuric acid
a n d p e r m a n g a n a t e mixture. Results are shown in T a b l e 2. A b o u t 95
percent of the inorganic m e r c u r y at 0.1 p p b was recovered using the
procedure just discussed. Recovery of the m e r c u r y in methyl m e r c u r y was
only 70 percent. W h e n heavily polluted waters or effluents were analyzed
(where mercury is present in complex compounds), overnight p e r m a n -
TABLE 2--Determination of mercury in natural waters.

Hg Hg Net Concen- Recovery
Sample Location of Added Found tration of Mercury
(1 liter) Sampling (tag) ~g) ~g/liter) (%)
Deionized water 0 0.007 (blank value for reagents)

River water Tama River 0 0.029 0.022
River water Tama River 0.1 0.125 0.118 "@
River water Sagami River 0 0.025 0.018 ...

River water Sagami River 0.1 0.121 0.114 96
Lake water Lake Saiko 0 0.017 0.010 ...
Ground water Tokyo city 0 0.121 0.014 ...
Sea water Chigasaki Beach 0 0.311 0.304

Sea water Chigasaki Beach o.1 0.41s 0.408 104
Sea water Chigasaki Beach 0.1 a 0.381 0.374 70
Sea water Chigasaki Beach 0 0.016 0.009 b ...
aHg is added as CH3HgC1.

bDirectly reduced without the oxidation procedure.
NISHI AND HORIMOTO ON MERCURY LEVELS IN NATURAL WATERS 29
ganatc oxidation was not sufficient to convert the mercury completely into
mercury (II) ion.
Results from analyses of sea water indicate a marked increase in analy-
tical value after the permanganate oxidation of samples. This coincides
with the results reported by Hosohara et al [5]. These findings suggest
that more of the mercury in sea water may exist as stable compounds
which cannot be reduced directly by the reduction-aeration method.
Most of the published data [4,6,8] on mercury levels in sea water have
been obtained without the oxidation procedure. Further investigations arc
necessary to establish better methods for the determination of mercury
levels in sea water.
References
[1] Rains, T. C. and Meins, O., Journal of the Association of Official Analytical Chemists,
Vol. 55, 1972, p. 1319.
[2] Omang, S. H., Analytica Chimica Acta, Vol. 53, 1971, p. 415.
[3] Chau, Y. K. and Saitoh, H., Environmental Science and Technology, Vol. 4, 1970, p.
839.
[4] Topping, G. and Pirie, J. M., Anatytica Chimica Acta, Vol. 62, 1972, p. 200.
[5] Hosohara, K., Kozuma, H., Kawasaki, K., and Tsuruta, T., Journal of the Chemical
Society of Japan, Pure Chemistry Section, Vol. 82, 1961, p. 1479.
[6] Burton, J. D. and Leatherland, T. M., Nature, Vol. 231, 1971, p. 1106.
[7] Leatherland, T. M., Burton, J. D., McCartney, M. J., and Culkin, F., Nature, Vol. 232,
1971, p. 112.
[8] Weiss, H. V., Yamamoto, S., Crozier, T. E., and Mathewson, J. H., Environmental
Science and Technology, Vol. 6, 1972, p. 645.
Pierre Pichet I and Monique Grandmaison I
Analysis of Zn § § Cd +§ and Pb § §
in Natural Waters by Anodic
Stripping Voltammetry Using a
Rotating Pt:Hg Electrode
REFERENCE: Pichet, Pierre and Grandmaison, Monique, "Analysis of Zn §247Cd §

and Pb ~+ in Natural Waters by Anodle Stripping Voltammetry Using a Rotating Pt:Hg
Electrode, Water Quality Parameters. A S T M STP 573, American Society for Testing
and Materials, 1975, pp. 30-34.
ABSTRACT: A rotating Pt:Hg electrode used for anodic stripping voltammetry gave
peak heights varying linearly in the concentration range studied (1 to 600 ppb) for Zn §247
Cd **, and Pb §247Good pH buffering appeared essential.
KEY WORDS: water quality, ions, electrodeposition, scanning, environmental tests,

pH, water pollution
In water pollution research there is a need to analyze low levels of metal

ions. One of the methods by which this could be done is anodic stripping
voltammetry (ASV) in which the ions are first concentrated on an elec-
trode by electrodeposition and oxydized afterwards by anodic potential
scanning. The most currently used electrode for ASV is the hanging
mercury drop electrode (HMDE) which gives valuable results but neces-
sitates constant care from the operator. Other electrodes have been
proposed, one of which, the mercury-covered graphite electrode, has
received considerable attention recently [1,2]. 2 The Pt:Hg rotating elec-
trode proposed by Barendrecht [3] attracted our attention in an effort to
develop a semi-automatic ASV method. This paper presents a study of the
various parameters which are implied in a successful utilization of a
rotating Pt:Hg electrode.
Experimental
Apparatus
The Pt:Hg electrode was prepared by welding a 22-gage platinum wire
Professor and research assistant, respectively, Chemistry Department, Universit6 du
Qu6bec a Montr6al, Montrdal, Qu6bec H3C 3P8, Canada.
30
9
PICHET AND GRANDMAISON ON ANODIC STRIPPING VOLTAMMETRY 31
at the end of a 6-mm diameter glass tubing. The electrode end was
ground flat and polished. The platinum wire was etched (approximately
0.1 mm) in aqua regia and subsequently mercury covered [4]. A mercury
drop (0.8-mm diameter) was attached to the platinum electrode. The drop
was obtained from a mercury micro-feeder (Metrohm E 410) which was
also used as an H M D E .
A thermostated (T = 25.0 _ I~ polarographic cell (Metrohm) was
used to perform the experiments. The five-hole stopper was fitted with the
rotating Pt:Hg electrode (loosely fitted), a saturated calomel electrode
(SCE), a platinum counterelectrode, a glass electrode, and a gas bubbler.
The SCE was separated from the analyzed solution by a glass compart-
ment filled with 1 M KNO3.
The Pt:Hg electrode was rotated using a hollow shaft variable speed
stirrer (Caframo RZR1). The current-voltage curves were obtained from a
recorder (Tacussel EPL1) fitted with a polarographic module (Ti Pol)
coupled when necessary to an a-c voltammetric module (Adapal).
Reagents
Deionized, doubly distilled water was used to prepare the stock solu-
tions of metals (1, 10, and 25 p p m by dilution of 1000 p p m stock
solution). HNO3 (Aristar) was a high purity grade reagent and the sodium
acetate buffer was purified with dithizone [5]. N2 and CO2 were of
chromatographic purity.
Procedure
After rinsing the cell with 1 M HNO 3 and distilled water, the solution to
be analyzed was poured into and Nz or Nz q- CO2 mixtures and bubbled
for 15 rain. The Pt:Hg electrode was rotated at approximately 500 rpm.
The electrolysis potential ( - 1 . 2 V versus SCE) was applied for a total of
360 s. In the last 60 s as dur!ng the anodic potential scanning, the
rotation of the electrode was stopped. The current voltage curves were
then recorded at 0.6 V/min. The glass electrode was used to monitor the
pH of the solution which was adjusted with CO2 or an acetate buffer.

As can be seen in Fig. 1, the Pt:Hg electrode has a much more impor-
tant residual current than the H M D E . This residual current is related
either to platinum dissolved in mercury or to incomplete mercury coverage
of the platinum electrode [6]. This is the most serious limitation of the
Pt:Hg electrode versus the H M D E . Examination of data on mercury
covered graphite electrode [2] shows an analogous situation. In practice,
the effect of residual current on the limit of detection can be lessened by
]20hA
4.0
V vs SCE
FIG. 1--Comparison of a hanging mercury drop electrode (HMDE) with a rotating

Pt:Hg eh'ctrode of" similar size (0.8-ram diameter): (a) Pt:Hg. (b) HMDE. (c) Pt:Hg with
current compensation ('~100 hA~V). Solution: 5 ppb Zn § 5 ppb Cd~. 5 ppb Pb *§ 0.05
M KNO,. pH = 5 (acetate-acetic acid bufferO.Ol M). Plate time = 360 s. Anodic scanning
= 0.6 V/mbz.
using a slope compensating device (Fig. 1, Curve c) or by using a-c

voltammetry during the anodic potential scanning (Fig. 2).
One of the drawbacks of the HMDE electrode is that the peak heights
obtained are a function of the solution stirring rates; a variation of 5
percent in the stirring rate gave a 20 percent variation in peak heights [7].
In the case of the Pt:Hg electrode, changing its speed from 500 to 250 or
750 rpm did not vary the peak heights by more than 3 percent. Further-
more, magnetic stirring of the solution was also tried and at about 200
rpm the peak heights were 30 percent greater than without stirring
showing that the results with rotating Pt:Hg electrode are much less
sensitive to solution stirring than with the HMDE.
As can be seen on Fig. 2, the peak heights obtained are greatly pH
dependent, as was found earlier [8, 9], so we tried to control the pH by
using a mixture of COz and N2 as purging gas [8]. However, the repro-
ducibility of peak heights, for successive runs with the same solution, was
poor (pH 5 or 6). The lack of precision could be related to the stirring of
the solution in the vicinity of the Pt:Hg electrode which could lead to
losses of CO2 and corresponding pH variations.
Acetate-acetic acid buffer (10 -2 M) was substituted for CO 2. This
resulted in an improvement in the precision of the method (mean relative
deviation of 2 percent with same solutions).
The variation of the peak heights was studied for various Zn "§ Cd *§
and Pb "§ concentrations using the acetate-acetic acid buffer (typical results
are shown in Table 1). From the results it can be concluded that the peak
heights are proportional to the concentration at least in the concentration
PICHET AND GRANDMAISON ON ANODIC STRIPPING VOLTAMMETRY 33
PT~I ]20nA~~.
VvsSCE
I . . f L l i , i
' ' -10 0 -1.0
FIG. 2--Effect o f p H on peak heights obtained in anodic stripping a-c or d-c voltammetry
with a rotating Pt:Hg electrode. Solution: 10 ppb Zn**, 10 ppb Cd *§ 10 ppb Pb§ 0.05 M
K N O 3, p H adjusted with N2-C02 mixtures. Drop size = 0.8-ram diameter. Plate time =
360 s. Anodic scanning = 0.6 V/rain.
TABLE 1--Variation o f peak heights with concentration (typical results).
Zn*§ Cd+§ pb**
Ca hb h/C h h/C h h/C

(ppb) (nA) (nA/ppb) (nA) (nA/ppb) (nA) (nA/ppb)
3.2 25 7.8 20 6.3 15 4.7

8.0 65 8.1 45 5.6 35 4.4
24 175 7.3 125 5.2 100 4.2
102 750 7.4 550 5.4 430 4.2
260 1950 7.5 1500 5.8 1200 4.6
790 50,00 7.5 4300 5.4 3300 4.2
a c : ion concentration in parts per billion (ppb).

bh : peak heights in nanoamperes (nA).
Solutions: 0.0S M KNO3, pH = 5 (acetate-acetic acid buffer 0.01 M). Drop size =
0.8 mm diameter. Plate time = 360 s. Anodic scanning = 0.6 V/min.
r a n g e s t u d i e d (1 t o 6 0 0 p p b ) . T h i s s t u d y h a s b e e n d o n e i n p u r e w a t e r , a n d
p r e l i m i n a r y r e s u l t s i n d i c a t e t h a t it s e e m s v a l i d i n f i l t e r e d ( 0 . 4 5 / a n filter)
r i v e r w a t e r (Rivi~res d e s P r a i r i e s , Q u 6 . ) .
T h i s s t u d y s h o w s also t h a t we c a n o b t a i n g o o d r e s u l t s in A S V e v e n i f
the mercury surface is used repeatedly (in fact, the drop was changed only
when it fell down accidently). This is somewhat surprising unless we
r e m e m b e r that the graphite surface of a mercury-covered electrode is also
used repeatly with success for days [2].
Summary
A rotating P t : H g electrode used in anodic stripping voltammetry gave
peak heights that were almost independent of the electrode rotation speed
(250 to 750 rpm). Although the residual currents are m u c h higher t h a n
those of equivalent size hanging mercury drop electrodes, measurements
are usable down to the 1 ppb level. The peak heights were shown to be
p H dependent and use of CO2-N2 mixtures for controlling the p H was
found inadequate, but reproductibility of 1 to 2 percent was obtained with
an acetate-acetic acid buffer. The peak heights obtained with the latter
buffer varied linearly with concentration in the 1 to 600 ppb range studied
for Zn §247Pb §247and Cd §
References
[l] Matson, W. R., Roe, D. K., and Carritt, D. E., Analytical Chemistry, Vol. 37, 1965,
p. 1594.
[2] Clem, R. G., Litton, G., and Ornelas, L. D., Analytical Chemistry, Vol. 45, 1973, p.
1306.
[3] Barendrecht, E., Nature, Vol. 181, 1958, p. 764.
[4] Underkofler, W. L. and Shain, I., Analytical Chemistry. Vol. 33, 1961, p. 1966.
[5] Traversy, W. J., Methods for Chemical Analysis of Waters and Wastewaters. Ottawa,
Canada, 1971, p. 54.
[6] Barendretch, E., in Electroanalytical Chemistry, Vol. 2, A. J. Bard, Ed., Marcel
Dekker, New York, 1967, p. 87.
17] Whitnack, G. C. and Sasselli, R., Analytiea Chimica Acta, Vol. 47, 1969, p. 267.
[8] Zirino, A. and Healy, M. L., Environmental Science and Technology, Vol. 6, 1972,
p. 243.
[9] Fukai0 R. and Huynh-Ngoc, L., Radioactivity in the Sea, International Atomic Energy
Agency, Publication 22, 1968.
M . A . Santiago, ~ S a u n d r a Fielek, ~ a n d Co L. S c h e l s k e t
Automated Method for Sulfate

Determination in Lake Water*
REFERENCE: Santiago, M. A., Fielek, Saundra, and Schelske, C. L., "Automated

Method for Sulfate Determination in Lake Water," Water Quality Parameters, ASTM
STP 573. American Society for Testing and Materials, 1975, pp. 35-46.
ABSTRACT: An automated turbidimetric method is presented for the determination of

sulfate in lake water. Turbidity of samples filtered with 0.45 /am HA Millipore filters is
measured at 420 m/am with a Technicon AutoAnalyzer after sulfate is reacted with BaCI2
in HCI reagent. A NH4OH-EDTA rinse prevents coating of the BaSO4 precipitate on the
flowcell walls, glass coils, and plastic tubing of the manifold system, eliminating excessive
baseline drift. There are no interference effects of seven ions most likely to occur in lake
water. Recovery in tests and replicate analyses shows an accuracy better than _+2 percent
and a precision of _+2 percent. The method is used to determine S to 40 ppm sulfate.
Results are compared with those obtained from other automated turhidimetric methods.
KEY WORDS- water quality, sulfates, automatic control equipment, environmental tests,
sampling
The t u r b i d i m e t r i c m e t h o d for m e a s u r i n g sulfate has f o u n d widespread

acceptance in m a n y analytical laboratories [1-13]. z It is preferred over the
classical gravimetric m e t h o d because it offers a faster a n d r e a s o n a b l y
accurate procedure, particularly in the lower c o n c e n t r a t i o n ranges of sul-
fate. T h e t u r b i d i m e t r i c t e c h n i q u e , however, is complicated d u e to the
difficulty in controlling conditions for precipitation of BaSO4 d u r i n g the
m i x i n g a n d transfer to the sample cells. R o s s u m a n d Villarruz [6] listed
15 variables t h a t affect the t u r b i d i m e t r i c procedure for sulfate d e t e r m i n a -
tion. Most of these variables are e l i m i n a t e d [14-17] with a u t o m a t e d pro-
cedures.
A u t o m a t i o n not only permits analysis of a larger n u m b e r of samples b u t
also offers higher precision t h a n m a n u a l methods. I n m a n u a l procedures,
poor reproducibility results from h u m a n errors a n d variations in the time
of reaction, necessitating the need for reactions to go to completion;
*Contribution No. 174 of the Great Lakes Research Division, The University of Michigan.
1Assistant research chemist, research assistant, and research limnologist, respectively, Great
Lakes Research Division, The University of Michigan, Ann Arbor, Mich. 48105.
~The italic numbers in brackets refer to the list of references appended to this paper.
35

however, in automated procedures, addition of reagents, mixing of re-

actants, and time of reaction are highly reproducible. This eliminates the
need for complete reactions and permits a more rapid measurement, thus
making it a satisfactory procedure for routine analysis.
In other automated procedures for sulfate analysis, excessive baseline
drift has resulted from the coating of BaSO4 precipitate on the walls of
the flowcell. Dieu [16] recommended an inter-sample air rinsing. Although
this method is effective in eliminating baseline drift, high sample peaks
are difficult to distinguish from the very high air peak levels. Occasional
"noise" in this method results in a 2.5 percent error, making the peaks
difficult to read. Ferrara et al [15] used a 5 percent ethylenediamine
tetraacetic acid (EDTA) wash followed by a water wash to reduce baseline
drift, but a precipitate still accumulated on the flowcell wall, requiring
careful observation of the baseline to prevent drift problems.
Our method employs an ammoniacal EDTA solution rinse which suc-
cessfully prevents buildup of BaSO4 on the walls of the tubing and
flowcell. This method was adapted for the routine analysis of lake water
samples, taking into account the presence of interfering ions and the
widely varying composition of the lake water at different locations and
times.
Experimental
Apparatus
A Technicon AutoAnalyzer II System, consisting of a sampler, pump
module, two time-delay coils, a colorimeter, and a recorder, was used for
analysis.
Reagents
The BaCl2-HCl-gel reagent was prepared by dissolving 20 g of BaCl2.
2H20 (Reagent Grade) in 500 ml of distilled, deionized water and adding
10 ml of 1 N HCI and 0.5 g of gelatin (USP), then mixing and diluting to
1000 ml in a volumetric flask. The reagent is slightly turbid due to the
traces of sulfate in the gelatin. It was mixed for an hour and then filtered
through a large Millipore filter supported by a screen on a large Bfichner
funnel [18]. Filtration through a fine-fritted glass filter can also be used
but would require a longer filtration time.
The NH4OH-EDTA reagent was prepared by mixing 2.5 ml of con-
centrated NH4OH with 1.86 g of Na2EDTA.2HzO (Reagent Grade) and
2.50 g of NH4Cl (Reagent Grade) in about 900 ml of distilled deionized
water. The pH of the solution was adjusted to 8.0 and the mixture diluted
to 1000 ml in a volumetric flask.
SANTIAGO ET AL ON SULFATE DETERMINATION 37
Standards
A stock solution containing 1000 ppm SO4 was prepared with anhy-
drous NazSO4, previously dried at ll0~ Dilutions were made to give 3,
5, 10, 15, 20, 25, 30, 35, and 40 ppm SO4 as working standards.
Interfering Ions
In separate 100-ml volumetric flasks, mixtures containing a single con-
centration of sulfate and a single concentration of an interfering ion were
prepared. Sulfate concentrations were 5, 10, 20, and 40 ppm. Other ion
concentrations were: 40-100 ppm calcium, 20-100 ppm potassium, 20-100
ppm magnesium, 20-100 ppm sodium, 10 ppm nitrogen (NO3), 100 ppm
PO4, and 10 ppm SiO2.
Lakewater Samples
Water samples were filtered aboard ship through 0.45 /an HA Millipore
filters. The filtrate was stored and frozen in 2-oz polyethylene bottles so
samples could also be used for analyses of different forms of nitrogen and
phosphorus and brought back to the laboratory for analysis. The samples
were thawed out at room temperature prior to analysis.
Procedure
The flow diagram of the automated system is shown in Fig. 1. The
sample is segmented with air, the BaCI2 reagent is introduced, and the
mixture is then diluted with water and allowed to pass through a double
mixing coil and through two time-delay coils for the reaction to take
place. The turbidity formed by the precipitate suspended in the gelatin
solution is measured at 420 m/am. An ammoniacal EDTA rinse is pro-
vided.
Method Development
A series of experiments was conducted in order to optimize the wave-
length of measurement, concentration of reagent, pH of the rinse solution,
volume ratio of sample to reagent, volume ratio of sample to rinse, time of
reaction and mixing, and range of sulfate concentrations for the pro-
cedure.
Wavelength Measurement
Maximum absorbance was obtained at 420 m/am, which is used as the
analytical wavelength.
Sampler Wash I:>
O.IlO NH:~- EDTAWosh
0.045 Air ~er

9ooj I
0.065 Sample
Double Mixing [ [ 0.045 BoCI~ -HCI Reagent
Coil /
0.1 I0 Water
rime
)~Deloy
[
Coils
<~Woste 0.065
I
Waste [
'~ IColorimeter
llSmm
I
I
II I
I
I Tubulo r Cell~ Recorder
1420m," J
FIG. 1--AutoAnalyzer manifbld andflow diagram.
Reagent Concentrations
Concentrations of the components in the BaClz-HCl-gelatin solutions
were varied to give the o p t i m u m s t a n d a r d curve and most reproducible
results. It was f o u n d that the combination of 0.082 M BaC12 -- 0.01 N
HCI - 0.05 percent gelatin gave the best results. Gelatin was a d d e d as a
precipitate-dispersing agent. O t h e r dispersing agents tested were 50 per-
cent ethanol, acetone, and glycerine. Precipitation occurred when the rinse
solution was mixed with 50 percent ethanol (ETOH). Distorted sample
peaks and poor results were observed for acetone and glycerine, re-
spectively.
NH4OH-EDTA Reagent
In aqueous solution, BaSO4 dissociates in the absence of a complexing
agent and at an ionic strength of 0.1 according to the following equation
BaSO4 ~ Ba §247+ S O z 2 K = 10 -9.2 (1)
In the presence of E D T A , Ba** forms a complex
Ba §247-b E D T A -4 ~- B a E D T A -2 K = 10 7"a (2)

It has been shown [19] that the solubility of BaSO4 in the presence of
EDTA can be represented as
S(BaSO,) = 10-9.2 aBa(EDTA) aSO,(H) (3)
where aBaEDTA is the coefficient accounting for the barium concentra-

tion [Ba] reacting with EDTA and can be represented as the ratio of the
complexed [BaEDTA] and uncomplexed [Ba] to the total barium concen-
tration
O~Ba(EDTA) = [Ba] + [BaEDTA] (4)

[Ba]
Using the conditional stability constant of BaEDTA -2, Eq 3 becomes
S(BaSO,) = 10-9"z 1 + KtBaEDTA-0 aSO,tH) (5)

OtEDTA (H)
where C~EDTA(H ) is the coefficient accounting for the effect of pH on the

different forms of EDTA.
Using calculated values [19] for aEDTACH) and aso,cn) and 0.10 M
for EDTA concentration, the conditional solubility product of BaSO4 is a
function of pH (Fig. 2).
Calculations of solubility at the sample-reagent-rinse-sample interfaces
in the continuous flow system of the AutoAnalyzer become complicated
since fluid "slug" volume, "slug" velocity, leakage flow velocity, and
variations in diameters of glass mixing coils [20] have to be considered.
Optimal concentrations of NH4OH, EDTA, and NH4C1 in the rinse solu-
tion were therefore empirically determined by using a series of concentra-
tions of NH4OH-EDTA reagent buffered with NH4C1 at pH 6 to 9.
A solution of pH 8 gave the maximum peak height and high precision
(Fig. 3).
Time of Reaction
The length of time for the reaction to take place was varied with mixing
and time-delay coils. It was found that a time of reaction of at least 20
min was needed to obtain maximum absorbance.
Dilution Line Variation

Deviations from Beer's Law became apparent at sulfate concentrations
greater than 25 ppm (Fig. 4). Increasing the volume of the water
-2-
-I0 I I I 1 I I
2 4 6 8 I0 12
pH
FIG. 2--Conditional solubility product qf BaS04 in the presence of EDTA.
diluent by using a larger diameter manifold tubing eliminated some of the

deviations at high concentrations.
S t a n d a r d Calibration Curve
The optimum working range for this procedure is from 5 to 40 ppm

SO4 where the standard curve is linear (Fig. 5). At concentrations beyond
40 ppm SO4, depression of the peak heights is observed resulting in
negative deviations from the Beer's plot for standards. Sulfate concentra-
tions in Lake Michigan range from 15 to 25 ppm, which is within the
linear portion of the curve.
Data Treatment
A least squares, linear regression is used to calculate the sample con-

centrations. The regression equation is
A = 0.001 + 0.0168 c standard error o f A = 0.0069 (6)

0.8
9 0.012 M NH3-0.0025 M EDTA (pH=6)
0.7 - 9 0.05 M NHs-O.O05 M EDTA (pH=7) ~ ~-lqf
o O.0325MNH3-O.O025 MEDTA (pH=8) J f
0.6- . - . =
ow 0.5
m O.4
o
0.3;
0.2
0.1
I I I I I I I
5 I0 15 20 25 30 35 40
CONCENTRATION (ppm SO4)
FIG. 3 - - S t a n d a r d curves using various concentrations o f rinse solutions.
0.6
0.5--
0.4
000.3
o
0.2
~" 9 A Month-Old BaCIz-Gel Reagent

0.1
9 Filtered Freshly-Made BaCIz-Gel Reagent
o Larger Diameter Water Dilutant Line Used
I I I I I I
5 I0 15 20 25 30 35
FIG. 4---E~'ect qf.filtering BaCI 2 reagent and increasing amount q f dilution.
0.7
0.6
~ 0.4
~
0.5
0.2
i
0.1
0
5 I0 15 20 25 30 35 40 45
FIG. S - - - S t a n d a r d calibration curve.
with sulfate standards between 5 and 40 ppm.

However, a better fit can be obtained with an exponential function
A = m (1 -- e-kd + b (7)
or a hyperbolic function
A=m'( c) (8)
where A = absorbance, c = concentration, b and b' = y-intercepts, k =

extinction coefficient, and m, m', and k ' are constants.
Using a nonlinear least squares regression analysis based on these
models, the absorbance can be expressed as
A = 3.28 (1 -e-o.0oss2c) + 0.28 standard e r r o r A = 0.0066 (9)

or
A = 6.29 + 0.284 s t a n d a r d error A ---- 0.0066 (10)
No significant differences between the linear and the nonlinear models are
observed for concentrations up to 40 p p m SO4. Due to practical con-
SANTIAGO ET AL ON S U L F A T E DETERMINATION 43
sideration and smaller computational requirements, the linear regression

plot was used for routine analysis in our laboratory.
Interference Studies
M a t r i x E f f e c t - - L a k e water is considerably more complex in composition

than the sodium sulfate standards used to develop this method and, as
with all methods based on barium sulfate precipitation, some interferences
may be expected from other major constituents present in lake water.
Possible interferences from certain species in Lake Michigan were tested
with lake water spiked with sulfate. Comparison of the two plots in Fig. 6
shows a non-zero intercept of about 0.192 absorbance units for the upper
curve. This value indicates about 18 p p m SO4, which corresponds to the
concentration obtained for the lake water used.
Assuming 18 p p m SO4 of lake water was added to each of the stan-
dards, the 15 to 40 p p m SO4 region was used for statistical comparison of
the slopes to test for matrix effects.
Using a linear regression analysis on the SOcspiked lakewater data
gives the equation
A = 0.192 + 0.0107 c standard error o f A = + 0.008 (11)
and a linear regression analysis for the absorbance values from 15 to 40

p p m of the standard curve gives
A = 0.02 + 0.0105 c standard error of A --- + 0.02 (12)

o.t5-
0.5
0.4
'~ 0.2
0~ 5 I0 I5 20 25 30 35 40 45
CONCENTRATION (ppm S04)
FIG. 6--Comparison of Beer's plot of $04 standards with and without Lake Michigan water.
4,4 WATER QUALITY PARAMETERS
The difference between the slopes of the plots indicated by Eqs 11 and 12
shows no significant matrix effect in the determination of lakewater
sulfate.
Ion Interference--Many common ions affect the recovery of sulfate
precipitated with BaC12 as the result of coprecipitation with the BaSO4.
Anions coprecipitate causing high results, while cations, usually of lower
atomic weights than barium, coprecipitate to give low results [21].
In order to investigate the interference of major ions present in lake
water, solutions were prepared containing 5, 10, 20, and 40 ppm SO4 with
varying concentrations of calcium, magnesium, sodium, potassium,
phosphate, nitrate, and silicate ions. The ion concentrations chosen
covered the wider ranges than those normally expected in lake water.
Concentration ranges of major ions in Lake Michigan waters, for which
the procedure has been used routinely, are approximately: calcium, 35 to
40 ppm; magnesium, 10 to 15 ppm; sodium, 3 to 6 ppm; potassium, 1 to
3 ppm; SiO2, 0.1 to 2 ppm; NOrN, 0.01 to 0.2 ppln; and PO4, 0.01 to
0.1 ppm.
Except for 5 ppm SO4, good recovery was observed for all concen-
trations of SO4, within the precision of the method (Table 1). Each 5 ppm
TABLE 1.--Recovery of sulfate in the presence of interfering ions.
ppm SO,
Ion (ppm added)a 5 10 20 40
Ca (40-100) 7.22 _-b_0.44 9.44 4- 0.49 18.11 _+_ 0.28 39.64 4- 0.08
K (20-100) 8.55 ..-L0.37 10.36 -F 0.32 19.47 _+_ 0.13 41.01 4- 0.34
Mg (20-100) 9.43 _+_ 0.04 10.93 _+_ 0.32 19.34 _.+_0.54 40.75 -4- 0.23
Na (20-100) 8.41 i 1.83 10.44-t-0.62 19.88_+_0.17 39.91 -t-0.51
N-NO 3 (10) 18.95 _-b_0.22
SiO 2 (10) 19.28 _.+_0.57
PO, (100) 21.50 _+_ 0.20
a Concentrations of ions in parenthesis are: Ca (40, 60, 100 ppm), K (20, 40, 100 ppm),
Mg (20, 40, 100 ppm), and Na (20, 40, 100 ppm). Standard deviation of amount recovered
was estimated for all concentrations of interfering ions.
SO4 mixture was run after a 40 ppm SO4 mixture resulting in absorbance
readings higher than those expected due to contamination from the 40
ppm SO4 mixtures. This type of contamination is normally encountered in
most AutoAnalyzer methods, usually observed as a "shouldering" on the
higher peak. The recovery values obtained for 5 ppm SO4 reflect the
amount of contamination one can expect if samples with large ranges in
concentrations are run successively.
Conclusion
The method presented here provides an automated procedure for the
TABLE 2--Comparison of turbidimetric m e t h o d s f o r s u l f a t e analysis.
Concentration Accuracy
Method Application Range (ppm SO4) Precision (%) (% recovery) Interferences Reference 60
Z
Parallel photometric ... 4-40 1 99 PO4 > 500 ppm [12] 4-4
analysis
(GEMSAEC) 0
BaCIz/"CAD" I'fl
Reagent
Automated BaCl2 urinary 384-960 2 100.6 + 2.2 no interference [16]
Reagent inorganic NaCI (x38) t"
sulfate PO4 (xl0) 0

2-aminopyrimidine atmospheric 1-10 0.7 98 CI, Br > 10 ppm [171 z
chlorohydrate sulfur oxide NO3 > 100 ppm
c
Reagent pollutants F-, fluorosilicate, v-
"13
PO4 (1 ppm)
BaCI2 plant and animal 30-120 1 ... 100 ppm P [71 4..4
[11
materials
BaClz automated sulfer in plant 30-150 ,,o0.5 98 ... [151 o
i'o
materials 4...4
m
BaCI2 fresh water 10-150 '~2 101.3 + 1.1 [61
BaCh automated lake water 5-40 _+__2 98 see T a b l e ' i ' this study
z
7
r o u t i n e analysis of sulfate in lake water samples with a precision of ___2

percent a n d accuracy of ___2percent.
With the use of the AutoAnalyzer, this method ensures that samples
a n d s t a n d a r d s are analyzed u n d e r c o n s t a n t conditions, the m a j o r limita-
tion in c o n v e n t i o n a l t u r b i d i m e t r i c procedures. A c o m p a r i s o n of these
t u r b i d i m e t r i c m e t h o d s is presented in T a b l e 2,
The m e t h o d is sensitive a n d obeys Beer's Law over a range of concen-
trations c o m p a r a b l e to those expected in m a n y lake waters. No serious
interferences have been e n c o u n t e r e d .
Acknowledgments
The authors t h a n k T. B. Ladewski for helpful c o m m e n t s a n d dis-

cussions on the analysis of data.
This work was supported by a contract from the Atomic Energy
C o m m i s s i o n (C00-2003-19), a n d by a gift from the I n d i a n a - M i c h i g a n
Electric C o m p a n y .
References
[1] Sheen, R. T., Kahler, H. L., and Rose, W. H., Industrial and Engineering Chemistry,
Analytical Edition, Vol. 7, 1935, p. 262.
[2] Sperber, I., Journal of Biological Chemistry, Vol. 172, 1948, p. 441.
[3] Nalefski, L. A. and Takano, F., Journal of Laboratory and Clinical Medicine, Vol. 36,
1950, p. 468.
[4] Toennies, G. A. and Bakey, B., Analytical Chemistry, Vol. 25, 1953, p. 1960.
[5] Thomas, J. F. and Cotton, J. E., Water and Sewage Works, Vol. 101, 1954, p. 462.
[6] Rossum, J. R. and Villarruz, P. A., Journal of the American Water Works Association,
Vol. 53, 1961, p. 873.
[7] Blanchar, R. W., Rehm, G., and Caldwell, A. C., Proceedings, Soil Science Society of
America, Vol. 29, 1965, p. 71.
[8] Standard Methods for the Examination of Water and Waste Water, Public Health
Association, New York, 1967, pp. 291-293.
[9] Martin, J. and Stephen, W. J., Analytica Chemica Acta, Vol. 39, 1967, p. 175.
[10] Martin, J. and Stephen, W. J., Analytica Chimica Acta, Vol. 39, 1967, p. 525.
[11] Stefkin, F. S., Uchenye Zapiski, Mordovskii Universitet, No. 81, 1971, p. 32.
[12] Coleman, R. L., Schults, W. D., Kelley, M. T., and Dean, J. A., Analytical Chemistry,
Vol. 44, 1972, p. 1031.
[13] ChemicalAbstracts, Vol. 78, 1973, p. 67801.
[14] "Sulfate Method VIb, via Turbidimetry," Technicon, Ardsley (Chauneey), N.Y., 1959,
p. 10502.
[15] Ferrara, L. W., Floyd, R. S., and Blanchar, R. W. in Technicon Symposia 1965, "Auto-
mation in Analytical Chemistry," L. T. Shiggs, Jr. et al, Eds., Mediad, Inc., New York,
1966, p. 109.
[16] Dieu, J. O., Clinical Chemistry, Vol. 17, t971, p. 1183.
[17] Audouze, B. and Bonometti, G., lnformations Chimie, No. 107, 1972, p. 223.
[18] Shapiro, J., Science, Vol. 133, 1961, p. 1828.
[19] Ringbom, A., Complexation in Analytical Chemistry, Chemical Analysis, Vol. 16. Inter-
science, New York, 1963, pp. 67-70.
[20] Begg, R. D., Analytical Chemistry, Vol. 43, 1971, p. 854.
[21] Fritz, J. and Schenk, G., Quantitative Analytical Chemistry. Allyn and Bacon, Boston,
1966, p. 34.
C. S. W o n g ) R. D. Bellegay, ~ and A. B. Cornford t
Measurable Inorganic Carbon

Parameters in Seawater
REFERENCE: Wong, C. S., Bellegay, R. D., and Cornford, A. B.. "Measurable

Inorganic Carbon Parameters in Seawater," Water Quality Parameters, A S T M STP 573,
American Society for Testing and Materials, 1975, pp. 47-57.
ABSTRACT: Existing techniques in use at the Ocean Chemistry Laboratory for

measureable parameters of inorganic carbon in seawater are discussed. These para-
meters are dissolved inorganic carbon (YCO2), carbonate alkalinity, partial pressure of
CO2 in air and seawater (pCO2), and C13/C12 ratio and Ca4/C12 ratio, measured by
infrared analysis, potentiometric titration, mass spectrometry, and low level radioactive
gas counting, respectively. The significance of the measured variability in atmospheric
CO 2 and exchange with oceanic CO2 is outlined.
KEY WORDS: water quality, carbon, carbon dioxide, environmental tests, carbon
dioxide cycle, monitors, seawater
T h e increase o f c a r b o n dioxide in the a t m o s p h e r e since 1950, resulting

p r i m a r i l y from limestone a n d the b u r n i n g o f fossil fuels, is significantly
altering the b a l a n c e o f the c a r b o n cycle between the a t m o s p h e r e , bio-
sphere, a n d ocean reservoirs. This m a n - i n d u c e d i n p u t shifts t h e n a t u r a l
seasonal c a r b o n d i o x i d e (CO2) e q u i l i b r i a involving biological r e s p i r a t i o n
a n d photosynthesis, a n d p h y s i c a l , a n d c h e m i c a l processes d e p e n d e n t u p o n
such variables as t e m p e r a t u r e , mixing, b u f f e r i n g intensity, a n d c o m p l e x
f o r m a t i o n . A n a s s e s s m e n t o f i n d u s t r i a l CO2 p r o d u c t i o n has b e e n recently
r e p o r t e d by K e e l i n g [1] 2 a n d r e c a l c u l a t e d by Rotty [2] (Table III, p. 145,
R e f 2) for the p e r i o d 1960 to 1971. A s s e s s m e n t of the climatic i m p l i c a -
tions o f b o t h regional a n d global v a r i a b i l i t y in a t m o s p h e r i c CO2 requires a
c a p a b i l i t y for l o n g - t e r m m o n i t o r i n g of the m a n y p a r a m e t e r s o f the c a r b o n
d i o x i d e system. O n l y t h e n will the significance o f a c o n t i n u a l a n n u a l
increase in a t m o s p h e r i c CO2 be d i s c e r n a b l e u p o n the reservoirs a n d t h e i r
environments,
'Head. senior technician, and research scientist, respectively, Ocean Chemistry Division,
Marine Sciences Directorate. Pacific Region, Department of Environment Canada, Victoria,
British Columbia V9A 3S2, Canada.
47

The manning of the Canadian weatherships at ocean Station P at 50~

145~ has offered the Ocean Chemistry Division of Marine Sciences Direc-
torate, Pacific Region, a unique strategic advantage in embarking on a
major effort on the oceanic aspect of a long-term study of the CO, global
environmental problem. Station P, being the only continuously occupied
marine air CO, station, offers the opportunity to answer scientific ques-
tions not resolvable by observations on land-based stations, such as the
Mauna Loa Observatory.
Our main objectives are (1) to monitor the CO z increase in marine air,
(2) to study the factors controlling air-sea COz exchange, (3) to assess the
CO, uptake by the ocean, and (4) to predict quantitatively future air CO z
trends. This paper summarizes the reasoning behind our selection of CO z
parameters, the methods of measuring such parameters, and some results.
Selection of Parameters
The fundamental reactions for the COz system under study involve CO2
exchange between gaseous and aqueous phases and the subsequent hydrol-
ysis of CO2 in the aqueous phase to various ionic forms:
Marine air CO2 (gaseous)

.............................. 1l ..................................................................
Ocean water CO2 (aqueous) d- H20 ~- H2CO3

t~
H § + HCO~-
HCO3- -~ H § + CO~= (aqueous)
Ca*" + CO3=
Sediments Ca*+ q- CO3: ~ CaCO3 (solid)
The bicarbonate-carbonate system in seawater can be described com-

pletely by a set of thermodynamic equations relating the four chemical
parameters of pH (negative logarithm of the hydrogen ion concentration),
pCO, (partial pressure of carbon dioxide in seawater), alkalinity (the
amount of strong acid, in equivalents, required to titrate one kilogram of
sea water to the second end-point, that is, the acid neutralizing capacity
or proton deficiency) and YCO2 (the total inorganic .CO2 species dissolved
in seawater) [3]. The total alkalinity is expressed by ~.A, the carbonate
alkalinity by A, and the solubility of CO2 in seawater by a s.
WONG ET AL ON INORGANIC CARBON PARAMETERS IN SEAWATER 49
A = YCO2" [1 + 2(Kz'/a,H)]/[1 + (all~K, ') + (K2'/aFI)] (1)
= A + K B " Z B / ( K B " + all) (2)
gl'
a H = 2[(ZCO2/as "pCOz) - 1]
x {1 + {1 + 4(K2'IKl")" [~-CO2/as " pCOz) - 1]}l/z} (3)
ZCO2 = A[1 + (K2'/aH) + (oLF~/K, ')]/[1 + 2(K2'/aH)] (4)
pCOz = A(aH/K, ')/as[1 + 2(/(2'/aH)] (5)
a H = (KI'/2)(Y-COz/A){[1 - (A/YCO2)] + {[1 - (A/ZCOz)] z

- 4(Kz'/K1)(A/ZCOz)" [2 -- (A/7.COz)]} l/z} (6)
KI 'K, 'Kg' are the first and second apparent dissociation constants of
the carbonic acid system and first apparent dissociation constant of the
boric acid system, respectively. ZB is the concentration of total boron, and
aH is the hydrogen ion concentration = 10 -pH.
Table 1 summarizes the measurable inorganic carbon parameters in the
seawater system. To study the carbonate chemistry of seawater, measure-
ment of temperature and salinity, as well as of at least two of the
preceding four carbonate parameters is necessary. Both the pCO2 and
XCO2 values may be determined by methods independent of the aqueous
solution chemistry [3]. Therefore, the parameters selected for our air-sea
COz exchange studies are: (1) marine air CO z, (2) pCO2 of seawater, (3)
~CO2, and (4) C14/C z2 ratios and Cz3/C lz ratios for both marine air and
surface seawater. Parameters 1, 2, and 3 are all measured by the infrared
technique because of its high sensitivity for the direct measurement of
COz. However, during the interim period of pCO2 system installation on
the weatherships and infrared intercalibration studies, a time-series XCOz
and total alkalinity program was maintained to characterize the carbonate
chemical system.
The experimental techniques are described in the following section.
Experimental Techniques
Duplicate marine air CO z samples were collected weekly at ocean
weather Station P in evacuated two-liter Pyrex glass bottles fitted with
6-mm-bore, high vacuum stol~cocks with taper joints, greased with
Apiezon-N grease [4]. Extreme precautions were necessary to prevent
contamination of samples [4,5]. Exposures were made on the windward
side away from the ship's exhaust when the wind velocity was greater than
five knots. The analytical system, as shown in Fig. 1, consists of a high
TABLE l--Measurable inorganic carbon parameters in seawater.
Parameter Method
Marine air CO 2 infrared analysis
CO2 (1) vacuum extraction or N2 purging + infrared analysis

(2) gas chromatograph
(3) potentiometric titration
(4) pH-alkalinity
pCO2 (1) equilibration -t- infrared analysis

(2) calculation from 03 pH, alkalinity
or (ii) CO 2. alkalinity
Carbonate alkalinity (1) potentiometric titration

(2) pH-method
(3) calculation from other carbonate chemistry parameters
pH (1) electrode measurements

(2) calculation from other carbonate chemistry parameters
C ~VCI~ mass spectrometer
C'4/C '2 low-level gas proportional counting using (i) CO v

(ii) CH4, or (iii) C2H2
vacuum system, a Toepler pump to transfer air from the sample flask to
the infrared cell, a dry-ice cold trap to remove water vapor, and a URAS-2
nondispersive gas analyzer. This system, an improved version of Keeling,
Harris, and Wilkins [4], has leak-proof stainless steel plumbing and
solenoid control valves for minimum maintenance. More uniform tempera-
ture control is achieved by enclosing the analyzer in a plexiglass box fitted
with an oven and fan. Duplicate analyses agree to within 0.02 percent.
Calibration was made by the technique of Wong [6] using standard
sodium carbonate solutions. Gas reference standards were referred to the
Scripps Institution of Oceanography standard used in the Mauna Loa and
Antarctica air CO2 global monitoring programs.
The relationship between total alkalinity and total CO2 is given in
Eqs 1 and 2. The alkalinity is unaffected by CO2 addition or removal
since this does not affect the charge balance (which inherently defines
alkalinity) of the solution.
The potentiometric titration method of Dyrssen and Sillen [7] as
improved by Edmond [8] was used to determine these two parameters in
seawater samples. The titration chamber consists of a 150-ml flat-
bottomed, four-necked flask, with a glass and standard calomel electrode
pair and 2.5-ml micrometer screw burette glued into three of the necks. A
vertical capillary is fitted into the fourth neck to equalize the pressure in
the titration flask (with the ambient). The sample was stirred with a
CG
O
z
SV9 I ~NOMETER
SV4 m
SV2 VP2
INFRA-RED | >
ANALYZER |
t-
~ FLOWNNTER CG O
z
SV• VS P V ~ VP3
SV3 l | O
-m
RECORDER
SV5 LN2 TRAP O
>
DRY ICE Z
TRAPS
C)
SAMPLE FLASK >
VT O3
O
TPC %rOlLER Z
13
"11
m
--I
P ~ VP1 m
-n
C~
l LN2 TRAP
m
CALIBRATION GASES N 2 GAS
--.t
m
FIG. 1--Marine Air COz Analysis System; with automated calibration (CG) vacuum gages, (SV) solenoid valves, (PV) pump valve, (LNz)
liquid nitrogen, (TPC) Toepler pump valve, (VT) Toepler pump valve, (VS) sample line valve, and (VP) vacuum pump.
teflon-coated stirring bar and titrated with 0.3 N hydrochloric acid. The
electrode potential was measured on an Orion digital pH meter. The
precision has been estimated to be 0.35 percent for alkalinity and 0 .7
percent for ZCOz under laboratory conditions.
pCOz, the partial pressure of COz in seawater, is an important param-
eter for air sea COz exchange studies and can be measured continuously
with the ship underway. Measurements are usually made at a keel depth
of 2 to 3 m below the sea surface. The technique used in the Ocean
Chemistry Division follows that developed by Keeling, Rakestraw, and
Waterman [9]. A URAS-2 nondispersive infrared gas analyzer, shock
mounted to reduce the effects of vibrations on ships, was used with a
Honeywell strip chart recorder to monitor the CO2 in a carrier gas (N2 or
air) equilibrated with a seawater sample at a known temperature and
pressure. The pCO2 measuring system can be separated into three parts:
the air system, the seawater equilibration system, and the calibration
system. These are shown in Fig. 2 and discussed later.
The air system consisted of two air lines of nylon or polyethylene tubing
protected from the ultraviolet component of sunlight (which generates CO2
in the material) by dark-colored garden hoses which were connected to
copper tubing inside the shipboard laboratory. The air sampling intakes
were located on the foremast or at the aft upper helicopter deck, always
upwind from the ships stack exhaust. The intakes were controlled by a
solenoid valve system activated by a change in wind direction. Water
vapor in the air was removed by a refrigeration and defrosting system
capable of maintaining the temperature of a cold trap at - 5 0 ~ The
dried air was heated to 60~ inside the infrared analyzer and then vented.
The equilibration system consisted of a continuous flow of seawater
through a shower head into a closed volume of air, then into a large
volume of seawater in the equilibrator (20 liters). The equilibrated air was
circulated by a diaphragm pump through a --60~ cold trap into the
infrared analyzer and then vented.
Standard gases consisted of five mixtures of COz in N2 carrier gas
ranging in COz concentration between 240 and 340 ppmv (parts per
million volume) stored in 6000-liter gas cylinders. The infrared analyzer
was calibrated by introducing these reference gases into the infrared cell.
The overall accuracy for pCO2 has been estimated to be ___2 percent or
better, and a precision of about 0.3 percent has been attained.
YCOz measurement by the infrared gas analysis method followed the
technique developed by Wong [6]. The dissolved CO2 species were com-
pletely extracted from acidified seawater samples by vacuum. Water vapor
was removed by a dry-ice trap. The pressure and volume of the N2 carrier
gas mixed with the extracted CO2 were determined manometrically, and
the temperature measured. The N2-CO2 mixture was then pumped into an
infrared gas analyzer with a Toepler pump, and the COz concentration
r,
was determined. A precision of 0.15 percent was obtained. The infrared

system was calibrated gravimetrically with standard Na2CO a solutions to
an accuracy of ___0.2percent.
Carbon isotope sampling and analysis techniques for determination of
C14Oz and claoz in air and seawater consisted of the following. A high-
volume fast-pumping system incorporated a Drierite trap for water vapor
removal and sufficient Linde 5A molecular sieves to absorb the CO2 from
approximately 310 m a of marine air. In the shore laboratory, the sieve was
heated to 380~ in vacuum. The evolved COz was passed through a
dry-ice water vapor trap and collected at a liquid nitrogen temperature of
- 1 9 6 ~ for purification. Two hundred liter surface seawater samples
were collected either through the seawater loop or in a modified Gerard-
Ewing sampler [10] and immediately transferred by a closed pumping
system to a poly-lined 45-gal drum on deck. The sample was acidified,
heated with a quartz immersion heater, and recycled within a closed
pumping system containing bubblers in the sample container and a l-liter
solution of COz-free strontium chloride-ammonium hydroxide. The CO2
was precipitated as strontium carbonate, later recovered in the shore
laboratory by acidification with phosphoric acid, dried, and successively
purified using methods similar to Broecker et al [11] and Dyck [12]. The
radiocarbon content was measured by COz gas proportional counting [13]
to an accuracy of one standard deviation. Isotopic fractionation correc-
tions and caa/c 12 ratio in seawater and air samples were determined at
the Ocean Chemistry Laboratories in Victoria using an AEI MS20 Isotope
mass spectrometer and procedures similar to Kroopnick et al [14] to an
accuracy better than 0.2%o.
Discussion
Unpublished data [15] of marine air CO2 concentrations obtained at
ocean weather Station P since May 1969, show seasonal fluctuations
closely in phase with those in the air COz data for the island-based station
at Mauna Loa, Hawaii (19~ [16]. The seasonal amplitude appears to be
latitude dependent, decreasing from north to south: 20 ppmv at 70~
(Alaska, Scandinavia), 15 ppmv at 50~ (Station P), and 10 ppmv at
19~ (Mauna Loa). The annual increase of about 0.93 ppmv/year is 35
percent higher than the average change of 0.68 ppmv/year for the period
1959 to 1968 at Mauna Loa. The man-induced CO2 input rate is relatively
constant in seasonal average due to rapid tropospheric mixing. A higher
rate of increase appears to be the trend for air COz since 1968 as shown in
the Report of the Study of Man's Impact on Climate [17].
This seasonal fluctuation in marine air COz starts its downward trend in
about May, reaching a minimum in August, and starting an upward trend
in September. This can be correlated in an approximate inverse manner
with the air or sea temperature [18] and with the nitrate seasonal changes
in surface seawater [19]. However, a simple explanation of marine air COz
control by oceanic biological production and temperature is complicated
by other possible seasonal oceanographic processes of horizontal advection
of other water masses and vertical diffusion of deep water, which have
different CO2 and nutrient contents. Unfortunately, data required for
these oceanographic processes have not been worked out for this paper.
Figure 3 shows ZCO2 and total alkalinity as determined by the Sillen
and Dyrssen potentiometric titration technique [7]. )-CO2 has an average
of 2.01 mmol/kg. According to the relationship derived by Bolin and
Eriksson [20], a fractional change in pCOz = 12.5 times the fractional
change in ECO 2. A 15 ppmv seasonal change in pCOz should produce a
change in ~-COz of only 0.4 percent which can only be observed by a
technique, with a precision of _+0.15 percent such as the infrared tech-
nique developed by Wong [6]. This change could not be observed by the
potentiometric technique which has a 1 percent precision. Furthermore,
ZCOz determinations are affected more easily by factors such as different
operator techniques and sample handling.
The constant total alkalinity, 2.17 meq/kg (_+0.4 percent), is expected
since over a short period of time, oceanic uptake of COz would not cause
a significant change in carbonate alkalinity (and hence total alkalinity)
which differs by a small borate correction [3,21], by the chemical reaction
COz d- CO 3 ~ d-H20 = 2 HCO3-. However, over a time scale of
centuries, the increasing acidity of the ocean would shift the oversatura-
tion state of the surface water with respect to CaCO 3 to an undersaturated
state, thus causing dissolution of solid CaCO3, and hence an alkalinity
change. It will be interesting to see if the CO3-- ion or alkalinity in the
ocean shifts over periods of 20 to 40 years as a result of this oceanic
uptake of the increased atmospheric CO2.
A sampling program for radiocarbon at Station P was initiated by the
Ocean Chemistry Division in 1971. Earlier radiocarbon data at Station P
collected by the Marine Sciences Directorate illustrated the significance of
such monitoring [22]. Since 1965 the excess of bomb-produced C14Oz in
the troposphere has been decreasing exponentially at a rate dependent
upon the exchange with the stratosphere, the biosphere, and the oceans.
The exchange rate between the atmosphere and mixed layer of the oceans
occurs according to a residence time of 5 to 10 years [23]. The accumu-
lated North Pacific C 14 data [21] is suggestive of a natural C z4 concentra-
tion independent of depth, that is, the radioactive decay of C ~4 is balanced
by a C ~4 influx from the mixed surface layer, most likely attributed to
redissolution of organic carbon and calcium carbonate from the euphotic
zone rather than by physical processes. However, in all areas of the ocean
only a fraction of the total excess bomb-produced C 14which has been trans-
ferred to the ocean remains in the mixed layer and is indicative of circulation
m
0
t'-
t-
.<
"o
m
-i
m
(i)
FIG. 3 - - - C 0 2 and total alkalinity at ocean w e a t h e r Station P.

m i x i n g a n d a d v e c t i o n i n t o t h e d e e p o c e a n . T h e n o n r a d i o a c t i v e fossil fuel C O z
i n p u t to t h e a t m o s p h e r e will h a s t e n a r e d u c t i o n o f C 14 a t m o s p h e r i c c o n -
c e n t r a t i o n s r e s u l t i n g f r o m b o m b p r o d u c t i o n , b u t will as yet h a v e little
i n f l u e n c e on C 14 c o n c e n t r a t i o n s in CO2 d e r i v e d f r o m d i f f u s i v e ( u p w a r d )
fluxes f r o m t h e d e e p o c e a n [21]. W e h o p e t h a t o u r q u a r t e r - y e a r l y r a d i o -
c a r b o n m o n i t o r i n g p r o g r a m at S t a t i o n P for t h e s u r f a c e 500 m will yield
i n f o r m a t i o n on h o w fast b o m b C 14 will b e t r a n s f e r r e d t h r o u g h t h e m a i n
t h e r m o c l i n e into t h e d e e p o c e a n .
References
[1] Keeling, C. D., Tellus, Vol. 25, 1973, p. 174.
[2] Rotty, R. M., Tellus, Vol. 25, 1973, p. 508.
[3] Takahashi, T., Weiss, R. F., Culberson, C. H., Edmond, J. M., Hammond, D. E.,
Wong, C. S., Yuan-Hui Li, and Bainbridge, A. E., Journal of Geophysical Research,
Vol. 75, 1970, p. 7648.
[4] Keeling, C. D., Harris, T. B., and Wilkins, E. M., Journal of Geophysical Research,
Vol. 73, 1968, p. 4511.
[5] Pales, J. C. and Keeling, C. E., Journal of Geophysical Research, Vol. 70, 1965,
p. 6053.
[6] Wong, C. S., Deep-Sea Research, Vol. 17, 1970, p. 9.
[7] Dyrssen, D. and Sillen, L G., Tellus, Vol. 19, 1967, p. 113.
[8] Edmond, J. M., Deep-Sea Research. Vol. 17, 1970, p. 737.
[9] Keeling, C. D., Rakestraw, N. W., and Waterman, L. S., Journal of Geophysical
Research, Vol. 70, 1965, p. 6087.
[10] Gerard, R. and Ewing, M., Deep-Sea Research, Vol. 8, 1961, p. 298.
[11] Broecker, W. S., Tucek, C. S., and Olsen, E. A., International Journal of Applied
Radiation and Isotopes, Vol. 7, 1959, p. 1.
[12] Dyck, W., GSC-DEMR Paper 66-45, 1967.
[13] Corntbrd, A. B. and Wong, C. S., Pacific Marine Science Report No. 74-12, Ocean
Chemistry Radiocarbon Laboratory.
[14] Kroopnick, P., Weiss, R. F., and Craig, H., Earth and Planetary Science Letters, Vol.
16, 1972, p. 103.
[15] Wong, C. S. and Keeling, C. D., unpublished data, 1969 to 1973.
[16] Keeling, C. D., Ekdahl, C. A., Guenther, P. R., Waterman, L. S., and Chin, J. F. S.,
to be published in Tellus.
[17] Report on the Study of Man's Impact on Climate (SMIC), MIT Press, 1971.
[18] Tabata, S., Journal of the Fisheries Research Board of Canada, Vol. 18, 1961, p. 1073.
[19] Anderson, G. C., Parsons, T. R., and Stephens, K., Deep-Sea Research, Vol. 16, 1969,
p. 329.
[20] Bolin, B. and Eriksson, E. in Rossby Memorial Volume, B. Bolin, Ed., Rockefeller
Press, New York, 1959, p. 130.
[21] Edmond, J. M., "The Carbonic Acid System in Sea Water," PhD thesis, University of
California, San Diego, 1970.
[22] Fairhill, A. W., Yound, A. W. and Brandford, P. A., Proceedings, 8th International
Conference on Radiocarbon Dating, Royal Society of New Zealand, 18-25 Oct. 1972,
Vol. 1, 1972, p. 226.
[23] Nydal, R. and Lovseth, K., Journal of Geophysical Research, Vol. 75, 1970, p. 2271.
D. C. BurrelP and Meng-Lein Lee ~
Critical Review of Analytical

Techniques for the Determination
of Soluble Pollutant Heavy Metals
in Seawater*
REFERENCE: Burrell, D. C. and Lee, Meng-Lein, "Critical Review of Analytical

Techniques for the Determination of Soluble Pollutant Heavy Metals in Seawater,"
Water Quality Parameters, A S T M STP 573,American Society for Testing and Mate-
rials, 1975, pp. 58-70.
ABSTRACT. The three most popular procedures for determining soluble trace heavy
metal pollutants in seawater are: atomic absorption spectroscopy, thermal neutron acti-
vation analysis, and anodic stripping voltammetry. The applicability of these techniques
are reviewed in terms of the respective detection limit capabilities, analytical difficulties
imposed by the major ion matrix, chemical speciations, and pre-analysis preparation
requirements.
KEY WORDS: water quality, pollution, seawater, environmental tests, neutron activa-
tion analysis, atomic spectroscopy, electrochemical analysis
The choice of analytical techniques for the determination of the major

potential heavy metal pollutants in seawater--silver, arsenic, c a d m i u m ,
copper, mercury, lead, selenium, z i n c - - i s determined by four major
topics:
1. The detection limit capability and general applicability of the
method for each specific metal. For some individual elements there are
specialized techniques of great sensitivity. However, most laboratories
prefer to develop methods applicable to a range o f metals. T h e heavy
metals are present in seawater in concentrations generally less t h a n 1
gg/liter, which is at, or usually beyond, the direct capability of all the
c o m m o n l y used techniques. Since, therefore, pre-analysis chemistry is the
9 of Marine Science Contribution No. 224, supported in part by U.S. Atomic

Energy Commission Contract No. AT(45-1)-2229.
1Associate professor and graduate assistant, respectively, Institute of Marine Science,
University of Alaska, Fairbanks, Alaska 99701.
58
9
Copyright 1975by ASTMInternational www.astm.org
BURRELL AND LEE ON HEAVY METALS IN SEAWATER 59
rule, it is important to consider how such processing detracts from the

suitability of each technique.
2. The presence of the major constituents. This matrix severely modifies
the analytical schemes developed.
3. Chemical speciation. Metals occur in solution in seawater as a
variety of inorganic and organic species. In times past it was considered a
major achievement to determine an approximation to the total amount of
metal in a sample. However, the demand now is for speciation proportions
and for the delineation of small concentration differences between these
fractions. This is a task which is only now beginning to be addressed;
indeed for most oceanic areas too few data are available for mean total
ranges to be quoted with any degree of confidence.
4. Pre-analysis handling of the sample. This topic has been considered
least, yet is of the highest importance. The introduction of error (particu-
larly nonsystematic) during the sampling, treatment, and storage stages
will clearly negate the benefits of subsequent careful analysis.
It is proposed to briefly consider the three major analytical methods for
seawater--neutron activation, voltammetry, and atomic spectrometry--in
terms of these topics. Although many other analytical techniqes are avail-
able, gas-liquid chromatography [1],2 for example, these have limited
applicability at present.
Detection Limits and Applicability
Neutron Activation
The advantages usually quoted for neutron activation analysis are: low
detection limit capability, universality of application, and a minimization
of processing error, since post-irradiation radiochemical processing losses
are determinable. Of the metals under consideration here, however,
activation analysis is inapplicable to lead and marginally suitable for
cadmium and selenium in natural waters. Only zinc and antimony with
detection limits around 0.2 and 0.005 /~g/liter, respectively, may be
analyzed by instrumental neutron activation without pre- or post-irradia-
tion manipulations using Ge(Li) detection systems and specialized tech-
niques such as the dual coincidence counting method of Cooper and
Perkins [2]. These limitations are imposed because of the presence of,
chiefly, the high concentrations of coexisting sodium and chlorine. Using
pre-irradiation treatments tailored for each specific element to circumvent
these matrix interference problems, Robertson and Carpenter [3] have
suggested detection limits in the range 0.01-0.0001 /~g/liter for silver,
arsenic, cadmium, copper, mercury, and selenium; but the original appeal
of the method has been severely curtailed.
Anodic Stripping Voltammetry

Electroanalytical techniques applicable to seawater are being constantly
improved and Nicholson [4] has observed that theory consistently outruns
practical application. Only the various anodic stripping voltammetric tech-
niques will be considered here. Most pollution orientated laboratories
probably use thin-film mercury electrodes (a glassy-carbon [5] base is
recommended) although only a few elements are determinable by this
system; notably zinc, copper, lead, and cadmium. These metals are,
fortunately, all of considerable current interest. More importantly, with
careful attention to optimization of technique, it is possible not only to
determine these four metals from seawater without pre-analysis concentra-
tion, but also to identify some of the constituent soluble chemical species.
Practical problems with thin-film electrodes are legion. They are diffi-
cult to prepare and to maintain at an optimum performance level. Careful
preparation of the graphite or glassy-carbon substrate is of great impor-
tance, as is plating of the correct thickness and distribution of mercury at
the optimum potential. We consider it preferable to strip off mercury after
each analytical run and subsequently replate a fresh surface just prior to
use. Figure 1 illustrates stripping reproducibility from a "mature" elec-
trode for various standard additions to a seawater sample.
One potential problem which is usually discussed in theoretical ex-
positions [6], but is frequently ignored in practice, is that of the formation
of intermetallic compounds within the mercury layer which decreases the
amount of "free" metal available for stripping. Copper and zinc (that is,
CuaZnb complexes) are the best documented pair of mutual interferents,
but Smith and Redmond [7] have discussed Zn-Cr, Ni-Cr, and Ni-Hg.
For most cell arrangements, the only parameters which can be readily
varied are plating times and scan rates. It is inconvenient, and may lead
to added error, to plate for long periods of time. Decreasing this part of
the cycle, however, necessitates a subsequent rapid scan rate, and the use
of a very rapid response-time recording device such as an oscilloscope
display.
Stripping analysis sensitivity is, to a large extent, dependent upon
separating or diminishing the effect of the capacitance current from the
required peak diffusion (Faradaic) current. Both currents are direct func-
tions of the scan rate [8]. A number of techniques have been employed to
discriminate against the capacitance current:
(a) Dual cells operated in a differential mode [9]. This method would
be ideal were it possible to produce and operate identical cells and
electrodes. Zirino and Healey [10] have applied a modification of this
technique to seawater analysis.
(b) Phase sensitive a-c anodic stripping [11-14]. Rojahn [15] has used
I I I B I I I
r
4
I I I I I I
-700 -600 -500 - 400 - 300 -200
mV
FIG. l--Stripping curves .for standard additions o f lead and copper to a seawater sample,
demonstrating reproducibility. (a through e) O. 1. 2, 5, and 10 x 10 -s M additions o f lead
and copper, respectively, at p H 2.3. Glassy-carbon electrode; 100 m V / s sweep rate.
this method to determine cadmium, copper, lead, and zinc in estuarine

waters.
(c) Differential pulse anodic stripping. This is another method which
makes use of the dissimilar decay characteristics of the two types of
current to produce derivative stripping curves of great clarity.
As noted, anodic stripping voltammetry is limited at present to the
determination of copper, lead, zinc, and cadmium in seawater; but it is
undoubtedly the method of choice for, at least, copper and lead. Figures 2
and 3 illustrate standard addition peaks and the associated analytical
curves for these two metals. It may also be possible to determine mercury
directly on unplated electrodes, and arsenic and possibly selenium can be
analyzed by conventional differential pulse polarography [10]. However,
7 i r i i
r f
-650 -550 -450 -350 -250 -150 -50

mV
FIG. 2--Stripping curves f o r successive 10-8 M standard additions o f lead and copper
rdded to an estuarine sample at p H 2.3. Glassy-carbon electrode," 100 m V / s sweep rate.
/
5 Pb ii
I
I 3 , , , i i
/I
I /
I /Jr
2 Cu
I / J
I j/"
I /
I /
/ J
/
2 / 7
/
/ ~i I I i i
/ 0
i i
-d
- 3 -2 -I 0 1 2
/;
Cone [xlO-BM)
0i /
// i
-I 0 I 2
Cone ( x lO-SM)
FIG. 3--Standard addition analytical curves f r o m data o f Fig. 2.
no applications to seawater or to metals present at seawater concentrations

have been published.
Atomic Spectrometry
Even with completely optimized atomic spectrometric equipment (and at
present this means atomic absorption for all practical purposes), detection
limit capabilities are insufficient for the seawater heavy metal range of
concentrations. This, however, is somewhat immaterial since the presence
of the major dissolved constituents necessitates a separation step which
can conveniently incorporate a concentration of the test metals. Neverthe-
less, to minimize error, only small volume samples should be taken and
pre-arJalysis chemistry should be held to a minimum. With only a modest
concentration it is considered that flameless atomic absorption is eminent-
ly suitable for the determination of silver, cadmium, copper, lead, and
zinc in seawater. Mercury can be determined by the now classic cold-
vapor method; but only after an initial concentration. This method is no
worse than any other for mercury at this low concentration level but is far
from ideal. Atomic absorption methods based upon the dissociation of
hydrides of arsenic and selenium have not been applied to natural sea-
water samples.
Seawater Matrix Problems
Neutron Activation
Irradiation of seawater or sea-salt samples produces large quantities of
24Na, 3sS, and 32p from the sodium and chlorine and also UBr and 42K such
that immediate detection of trace constituents is impossible. After approx-
imately a 40-day cooling period, it is possible to determine zinc and anti-
mony by instrumental neutron activation as noted before. For all other
"pollutant metals" either a pre- or post-irradiation separation, or both, is
mandatory. Treatments have been devised for mercury, arsenic, silver,
selenium and cadmium but those for the latter two metals are unsatis-
factory, seldom used, and not recommended. The disturbing feature of
most of t h e s e methods is the advocation of complex pre-irradiation ma-
nipulations, thus negating to a large extent one of the principal advantages
of neutron activation analysis; namely, the ability to determine loss and
addition processing errors. Co-precipitation and co-crystallization separa-
tions are frequently employed. For example, Weiss and Crozier [18] have
suggested a sulfide precipitation (using a copper carrier) for mercury, and
thionalide co-crystallization is recommended [19,20] for the separation of
arsenic and silver. It is considerably harder to prevent contamination and
loss problems via these solid phases than by using the liquid-liquid ex-
traction common to atomic spectrometric pre-treatments. These few meth-
ods quoted as examples also require post-irradiation radio-chemical treat-

ment which, although yields may be determined, add to the complexity
and analysis time required.

Electroanalysis requires an electrolyte, and, as there are no specific
requirements regarding the strength of the electrolyte for anodic stripping,
the seawater matrix causes no problems. The major anions determine the
specific inorganic speciation of each metal, however, and it may be nec-
essary, for example, to adjust the pH to determine the total soluble
inorganic fraction.
Atomic Spectrometry
We initially researched the possibility of determining trace metals in
seawater by direct differential atomization from a graphite filament reser-
voir and found, as has Segar and Gonzalez [21] using a furnace atomizer,
this to be impossible. For the foreseeable future, atomic spectrometric
analysis of trace metals in this matrix will require a pre-analysis separa-
tion, and chelation and solvent extraction would appear to be the method
of choice. Early application of flame atomic absorption to seawater analy-
sis necessitated the use of solvents which, unless a back extraction step
was included, could be injected directly into the flame; hence, the popu-
larity of MIBK [22]. This solvent (usually coupled with APDC chelation)
supports combustion but has little else to recommend it, and for flameless
atomization solvents such as CHC13 and CCI4 are preferable. For our work
[17], we use almost exclusively a 0.1 percent solution of dithizone in
chloroform to both separate the test metals and to effect a xl0 concentra-
tion, and the organic solution is added directly to the filament. Figure 4
illustrates the resultant x-t.trace for zinc via a series of standard additions
to seawater, and Figs. 5 and 6 show the analysis curves for lead and
copper in estuarine samples.
Chemical Speciation of Soluble Fraction
Neutron Activation
Neutron activation is a method for total analysis, and the only way of
obtaining information on the various chemical forms is by individual
analysis of the chemically separated fractions. This is the case, also, for
atomic spectrometry, but the pre-analysis separation treatments for the
later technique seem to lend themselves to at least a simple fractionation
scheme. Pre-treatments for neutron activation are dominated by a need to
remove all the coexisting sodium an d chlorine.
Zn
West rod
3t~! somple
SE !
I
il
(sw+lOHw/tZn) (sw+SHg//Za) (sw§ 2~/lZn) (SOa water) blo~

(1~ D/T~'~in
CH C/,)
FIG. 4---Atomic absorption x-t recorder trace f o r dithizone/CHCL 3 extracts o f zinc stand-
ard additions to a seawater sampled (xlO concentration factor). (The.first part of each doub-
let is an artifact associated with sample addition and should be disregarded. )
/0 I I i
Pb
3/~,r sample
~n
9... 5
~L
0.4 0.2 0 C.2 0.4 0.6 0.8

ug/9 added
FIG. S--Standard addition analytical curve .tot xlO dithizone/CHCL 3 extracts o f lead
standard additions to a seawater sample.
20 Y
i i i i
3Cu/zsamp/e
)
/5 SE I0 ~
I0
-3 -2
I
-I SW I
I 2
I
,ag/.~ Cu added
F I G . (>--Analytical curve f o r copper: conditions as,fbr Fig. 5,

Electroanalytical data refer specifically to a particular chemical species
and these methods are ideally suited to delineating the various forms of
the metal present in seawater. Indeed, it is frequently difficult to deter-
mine "totals" at all. In fresh water it is possible to distinguish "simple
ionic forms," that is, aquo and hydroxo species, from more complex
organic forms released by acid treatment. In seawater, additional anion
ligands are important, notably yielding chloro complexes. Although zinc
may be determined at close to seawater pH, it is necessary to determine
copper and lead, for example, as illustrated in Figs. 1 and 2, at a pH of
around 2. We are currently working on characterizing the inorganic
speciation of copper and lead in seawater. For example, Fig. 7 illustrates
peaks obtained for copper and lead in 0.7 N KNO 3 at varying pC1 and
pH. Some workers are now beginning to study organo-metallic complexes
in a similar fashion. One major problem which may be hard to resolve
concerns the stability of the complex during the plating cycle. There are,
of course, innumerable practical problems associated with this work, and
it is imperative to optimize and maintain closely circumscribed cell and
electrode conditions, since very small peak potential shifts are being
monitored. Using thin film electrodes, peak potentials are, for example, a
function of the log scan rate. However, anodic stripping voltammetry is a
most powerful tool in attempts to identify the various soluble fractions
present in marine waters.
Atomic Spectometry
The required pre-atomic absorption analysis preparation of seawater
samples can be utilized to effect a crude differentiation between "ionic"
and "total" forms of the metal [23] and, unless deliberate steps are taken,
BURRELL A N D LEE ON HEAVY M E T A L S IN SEAWATER 67
I-- i I I I I I
I ,
+.370 -.020 -.410 =800 § 020 .4/0 800

E / V vs. SCE E / V vs. SCE
FIG. ?--Stripping curves tbr copper and lead in 0.7 M KN03: (left) [C/] = 0-0.1 M,
(right) p H = 3.9, 4.4, 5.5. 6.7, and8.6.
solvent extraction treatment will yield only an "extractable" fraction de-

fined by the system used. Initial acidification of the sample will tend to
release a larger fraction for chelation via hydrogen exchange with natural-
ly occurring organo-metallic complexes. In our own work we have at-
tempted to chelate and extract that fraction available at seawater pH
using a dithizone-CHCl 3 reagent as noted. For certain samples this is
prefaced by an ultraviolet-irradiation oxidation [24] to enable the total
soluble fraction to be extracted and determined.
Sampling and Pre-Analysis Processing

Neutron Activation
Of the three techniques considered here, only neutron activation is not
directly applicable to the analysis of untreated marine water. Irradiation
releases gaseous radiolysis products which can produce dangerously high
pressures in the activation containers. Most usually freeze-dryed sea-salts
are irradiated for subsequent instrumental analysis. For those elements
requiring pre-irradiation separation of matrix constituents, the products of
this treatment are usually solid phases.
Neutron activation requires very careful matching of samples and stand-
ards through the entire analysis procedure. This is a more arduous pro-
cedure than the solution standard additions commonly adopted for polar-
ography and atomic spectrometry. There are, at present, no widely ac-

cepted and available water standards. Sea-salt samples must be matched
exactly with standards with regard to exposure to the neutron flux, loca-
tion within the counting system and chemical composition to duplicate
effects such as self-shielding.

There are no special sampling problems associated with polarographic
analysis. Only an approximately 50 ml or less sample is required. This
may require filtering and acidification. Since subsequent use of thin-film
electrodes will require the addition of mercury, it is convenient to spike
immediately upon collection to additionally "poison" the sample.
Atomic Spectrometry
When flame atomic absorption was predominantly used for heavy met-
als in seawater, large (sometimes very large) samples were required since
there was around a 1000-fold discrepancy between the natural concentra-
tions and detection capability, and pre-analysis concentration was manda-
tory. Inevitably, problems were encountered with regard to the over-the-
side collection devices, initial treatment, and storage. It seemed obvious
to us that absorption losses and contamination additions, due to the large
surface areas contacted and the many processing stages required, tended
to negate the later careful analytical chemistry. In addition, these pro-
cedures were time-consuming and tended to discourage the collection of
replicate data.
For our own work with filament atomization [17], we have devised a
micro-scale, clean sampling, and preparation scheme which incorporates
the minimum number of manipulations and chemical additions. 10-ml
samples are syringe-filtered from small volume PVC sampling bottles
directly into centrifuge tubes. These samples are not acidified but are
subsequently chelated and extracted from the tubes for direct injection
into the carbon filament reservoir. Sorption of metals onto contacting
surfaces at around neutral pH has been well documented [25] and Table 1
summarizes tracer experiments to test retention of the test metals on the
Swinnex syringe filter used (compared with the commonly used Millipore
equipment). At seawater pH, all the metals tested except cadmium tend to
sorb onto the contacting surfaces of the filtering equipment. The flushing
scheme adopted by us for field use was dictated by the behavior of lead,
the most troublesome metal. It is of interest to note that, in the case of
the Millipore filter rig, lead is largely retained, as might be expected,
within the glass frit; this is the area of potential zinc addition contamina-
tion as noted by Burrell and Wood [26]. The delivery tubes and filter
components are also well "aged" in seawater prior to use.
TABLE 1--Tracer studies to determine losses during filtration and extraction efficiencies.
Ag Cd Co Pb Zn
% recoverya 99 98 ... 70 ...

Millepore filter % on filter 0 0 ... 2 ...
%onfrit 0 0 ... 18 ...
Swinnex filter % recoveryb 95 100 90 84 92

required no rinses c 5 1 10 10 S
pH 8.0 7.8 7.8 7.8 7.8
% extraction via routine aqueous e 99 100 98 98 100

procedure in tubes d organicf 99 95 90 82 102
a Percent recovery of tracer after passing 500 ml filtered seawater through standard rig
with 0.45 m membrane.
bpercent recovery after second rinse through filter.
c Approximate number of rinses required to prevent significant sorption losses.
dStandard procedure: dithizone/CHCl3; 1:10 extraction ratio; 10 min shaking time.
e Percent extraction from aqueous phase.
f P e r c e n t recovery in organic phase; counting efficiencies and errors (_+_10 percent) not
comparable with (e).
References
[1] Lee, M-L. and Burrell, D. C., Analytica Chimiea Acta, Vol. 66, 1973, p. 245.
[2] Cooper, J. A. and Perkins, R. W., Nuclear Instruments and Methods. Vol. 99, 1972,
p. 125.
[3] "Marine Pollution Monitoring: Strategies for a National Program," Workshop Report
to NOAA, Oct. 1972.
[4] Nicholson, R. S., Analytical Chemistry, Vol. 44, 1972, p. 478R.
[5] Florence, T. M., Journal of Electroanalytical Chemistry, Vol. 27, 1970, p. 273.
[6] Barendrecht, E., Journal of Electroanalytieal Chemistry, Vol. 2, 1967, p. 53.
[7] Smith, J. D. and Redmond, J. D., Journal of Electroanalytical Chemistry, Vol. 31,
1971, p. 169.
[8] Roe, D. K. and Toni, J. E. A., Analytical Chemistry, Vol. 37, 1965, p. 1503.
[9] Martin, K. J. and Shain, I., Analytical Chemistry, Vol. 30, 1958, p. 1808.
[10] Zirino, A. and Healy, M. L., Environmental Science and Technology, Vol. 6, 1972,
p. 243.
[11] Underkofler, W. L. and Shain, I., Analytical Chemistry, Vol. 37, 1965, p. 218.
[12] Bond, A. M., Analytical Chemistry, Vol. 44, 1972, p. 315.
[13] Bond, A. M. and Canterford, D. R., Analytical Chemistry, Vol. 44, 1972, p. 721.
[14] Velghe, N. and Claeys, A., Journal of Electroanalytical Chemistry. Vol. 35, 1972,
p. 229.
[15] Rojahn, T., Analytica Chimica Aeta, Vol. 62, 1972, p. 438.
[16] Myers, D. J. and Osteryoung, J., Analytical Chemistry, Vol. 45, 1973, p. 207.
[17] Burrell, D. C., Williamson, V. M., and Lee, M-L., 4th International Atomic Spectro-
scopy Congress, Toronto, Nov. 1973.
[18] Weiss, H. V. and Crozier, T. E., Analytica ChimicaActa, Vol. 58, 1972, p. 231.
[19] Portman, J. E. and Riley, J. P., Analytica Chemica Aeta, Vol. 31, 1964, p. 509.
[20] Ray, B. J. and Johnson, D. L., Analytica Chimiea Acta, in press.
[21] Segar, D. A. and Gonzalez, J. G., Analytiea Chimica Acta, Vol. 58, 1972, p. 7.
[22] Burrell, D. C., Analytica Chimica Acta, Vol. 38, 1967, p. 437.
[23] Burrell, D. C., Proceedings, 3rd International Atomic Spectroscopy Congress, Paris,
Sept. 1971, pp. 409-428.
[24] Armstrong, F. A. J., Williams, P. M., and Strickland, J. D. H., Nature, Vol. 211,
1966, p. 481.
[25] Robertson, D. E., Analytica Chimica Acta, Vol. 42, 1968, p. 533.
[26] Burrell, D. C. and Wood, G. G., Analytica Chimica Acta, Vol. 48, 1969, p. 45.
L H. Crocke~
Survey Analyses of Trace Elements

in Water by Spark Source Mass
Spectrometry
REFERENCE: Crocker, I. H., "Survey Analyses of Trace Elements in Water by Spark

Source Mass Spectrometry," Water Quality Parameters, A S T M STP 573, American
Society for Testing and Materials, 1975, pp. 71-81.
ABSTRACT: The potential role of spark source mass spectrometry in water quality sur-
veillance is perhaps not as widely appreciated as it should be. In this paper an attempt
is m a d e to explain what is unique about the technique and how it can a u g m e n t other
more widely used methods of analysis in the study of inorganic constituents of water.
At Chalk River it has been used to examine samples of water from several rivers,
lakes, and streams. Most of the elements present in 100 ml of water in concentrations of
at least one part in 10 H can be determined in a single analysis.
To date most other comprehensive evaluations of trace element concentrations in
water have suffered from a lack either of sensitivity or of broad elemental coverage. It is
probable that spark source mass spectrometry can remedy both of these shortcomings.
Thus, it can fulfill a path-finding role in studies of toxic, dietary significant, or other
especially interesting trace elements.
K E Y W O R D S . water quality, trace elements, mass spectroscopy, environmental tests
A major area of concern in the large spectrum of water quality

problems is that of trace elements, their occurrence in water, and their
short and long-term significance to m a n and the environment. A great
deal has already been discovered about the effects of certain trace ele-
ments on, for example, h u m a n metabolism. Lead, cadmium, and mercury
have well known toxic effects. Iron, zinc, and manganese, conversely, are
beneficial in the correct concentrations. Undoubtedly many, if not most of
the elements in water are useful, or at least not harmful, if specific con-
centrations are not exceeded. A recent report [1] 2 indicated that 43 ele-
' H e a d , Mass Spectrometry and Fuel Analysis Section, General Chemistry Branch, Atomic
Energy of Canada Ltd., Chalk River Nuclear Laboratories, Chalk River, Ontario K0J 1J0,
Canada.
2The italic n u m b e r s in brackets refer to the list of references appended to this paper.
71
9
ments are regularly found in developing human dental enamel and 25

others have been detected. Sixty-eight elements! Who can say which ones
are absolutely essential or nonessential?
Other reports [2] suggest that subtle, complex interactions of trace
elements with other trace elements affect metabolism and physiology to an
extent that we are only beginning to appreciate at present.
To be able to make even a start at understanding immensely complex
elemental interactions, one obviously must have techniques which allow
the detection and determination of the elements present in a given sample,
in the concentration range that is significant in these interactions.
Enough evidence is at hand to demonstrate that parts per million
concentrations of certain elements are significant in many processes. Parts
per billion in the long term will probably also prove to be significant in
many other processes.
It is necessary to examine our arsenal of sensitive multi-element ana-
lytical techniques to see if we are adequately prepared to attack this
problem. At present there are no perfect comprehensive trace surveillance
techniques. Most techniques are incapable of extensive simultaneous
multi-element determinations, although their sensitivity for individual ele-
ments may be exceptional. An obvious example is neutron activation
analysis (NAA). Others have broad elemental coverage but often are not
sufficiently sensitive to be effective for the application under discussion.
An example would be emission spectroscopy.
There is a technique that fulfills both basic requirements--sensitivity
plus comprehensive elemental coverage--and that technique is spark
source mass spectrometry (SSMS).
In this discussion I wish to avoid at all costs the hazard of appearing to
be backing a single analytical technique to the exclusion of others. On the
contrary, I am an advocate of the cooperative multi-technique approach to
analysis problems.
Water quality surveillance is not my major line of work. My group
devotes most of its energies to the analysis of nuclear fuel and related
materials, and when we analyze water it is most likely to be reactor
cooling water or feedwater to one of the heavy water plants.
However, because of our trace analysis requirements, we have been
fortunate in assembling at one place many of the modern techniques that
are relevant to various areas of study, including that of water analysis.
One of these techniques is SSMS, a method that is used at only one
other laboratory in C a n a d a - - a t NRC, Ottawa. Having had this instrument
for about five years now, I am somewhat surprised that it is not more
widely used. My intention is to describe the role of SSMS in water analysis
and to justify its consideration for this role, not neglecting to estimate the
economics of its use.
CROCKER ON SPARK SOURCE MASS SPECTROMETRY 73
Experimental
Figure 1 is a simple schematic of the mass spectrometer showing its es-
sential features. In common with all mass spectrometers it has a source
where ions of the sample are formed, accelerated, and shaped into a
beam; an analyzer where the ion beam is sorted according to mass; a
detector to record the mass analyzed ions; and a high-vacuum system
which allows these manipulations to take place.
Na.I. PLATE) ION ACCELERATION.

(No.3. PLATE) BEAM DEFINING
/ 'EAR'TH SLI'I~ p ~ - E ' -
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.
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- / \~/., pJ SECONDARYEMISSION
~ R PLATE.
I ELECTROSTATIC / "~,/~JPHOTOGRAPHIC PLATE. j
(N~ I ~___~o,Mo. , ~
EART. I .... I
MAGNETIC ANALYSER.
FIG. 1--Schematic o f mass spectrometer (702).
[on Source
The source is the unique feature of this instrument which defines the
advantages and disadvantages of SSMS. A vacuum spark is produced in
which a high voltage radio frequency discharge occurs between two over-
lapping electrodes of the material to be analyzed. The ions produced are
those of all elements comprising the electrodes plus any others in the
spark gap as vapor or gas. The point energy input is large; the tempera-
ture is estimated to be about 50 000 K. Thus, reasonably similar ioniza-
tion efficiencies are observed for most elements, and a preponderance of
singly charged ions is produced along with a smaller number of multiply-
charged ions. In general, the multiply-charged ions are useful in identifi-
cation of elements and provide a means of circumventing interferences at
some mass numbers.
A disadvantage of this efficient ionization process is that the ions are
formed with an energy spread of several kilovolts. Thus double focusing is
necessary. Mattauch-Herzog geometry is used which simultaneously

focuses all masses along a plane, permitting simultaneous photoplate
recording of all elements, and integration over a selected interval of time
for high sensitivity.
Ion Detection: Photoplate

A graded series of exposures of a photoplate are usually recorded, as in
Fig. 2. This is an example of a photoplate produced during the analysis of
a lake water sample. Multiple mass spectra are recorded here, represent-
ing a range of total ion charges collected from 10 -13 coulombs up to 10 -7
coulombs. In the longest exposure all of the elements present in the elec-
trode from lithium to uranium were recorded if their concentration in the
lake water was about 10 p g / m l or more (10 parts in 1012). In the shortest
exposures, only ions of the matrix element are recorded. In this case the
matrix is graphite, so that lzC* and lac* ions, only, appear.
The second photoplate shown in the slide was obtained by sparking
" p u r e " graphite that is used as a sample support in these analyses. Here
the multiply-charged carbon ions are visible, along with a few contami-
nants such as Z3Na, 14N2§, and 1602§
Photoplate interpretation, calibration, and standardization are done
much as in emission spectrography, although the spectra are simpler to
interpret. Several workers in other countries have automated the evalua-
tion of photoplates [3,4]. A very sophisticated system is required.
Electrical Detection
As an alternative method of ion detection our instrument is equipped
with an electron multiplier, which in conjunction with a total ion current
monitor enables us to record mass spectra electronically. This is a quicker
and usually more accurate method than photoplate detection. The elec-
trical detection schematic is shown in Fig. 3. In this procedure the
separated ion beams are swept consecutively across the electron multiplier
dynode by magnetic scanning. Values of electron currents from each ion
species are automatically ratioed with the instantaneous total ion current,
and the logarithmic sum is displayed on an ultra-violet-sensitive strip
chart recorder.
A portion of a chart from a typical water analysis is shown in Fig. 4.
The portion carries the mass spectrum from mass number 54 to mass
number 70, in the concentration range 6.5 x 10 -s mg/liter to 6.5 x 10 -z
mg/liter. A mass scan from zasu to 6Li is normally completed in 10 min.
This would usually be repeated at three different amplifications to cover
the entire concentration range. Because the response is linear, a single
calibration is suitable for a range of concentrations up to a factor of 10 s.
Precision of results is about _+30 percent, and with extensive calibration
0
0
(3
7~
m
73
0
2s
-13
713
7g
0
c
:IJ
t')
m
6")
"13
I'n
0-d
:7J
0
i"11
:13
-<
FIG. 2 - - P h o t o p l a t e spectra f r o m the s p a r k source mass spectrometer: (top) sample o f lake water residue ashed at low temperature and ",4
m i x e d with p u r e graphite, a n d (bottom) s a m p l e o f p u r e graphite used in m i x above, ol
J U.V. ~ 1 SUHRING J I DIGITAL ]

PK.SWITCHING I I RECORDER~-IAMPL,F,ERI VlV0LTMETERI
~OTENT)O"ETERSI IAMPLIFIERI IAMPL FIER
i
CYCL,CSCAN MON,TOR l I STORAOE I I PEAK I

MOTORDRIVEN NTEGRATOR I IOSCILLOSCOPEI ~NTEGRATORI
POTENTOMETER )
I ~ MONITOR I I | PEAK [
I ......./ AMPLIFIERI L __~AMPLIFIERr ........
I
[AUTOMAT,CI --o~v ::':=~] il
SPARK ~I ACCELERATING [! !I
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/'~,~X
- ,PIMONITOR MULTIPLIER
........ / ~ / COLLECTOR
ELECtrOStaTIC /~,/'-'IPHOTOGRAPHICPLAT[
A N A L Y Z E r ' ' - - - - - HIGH MASS
MAGNETICANALYZER
FIG. 3---Spark source mass spectrometer with electrical and photographic detection o f
mass separated ions.
j~ IOOO
I ]-I ~ --
II II [ I II -- . ~ - - I I
IIA II II
nA ,o
eo"ll~11'"~
II II I I II + I1 II II IW ~,.+ a=,, c..~
.F'e ,,~1+ )Fe'§ IF;. 4- l+ .Ni § Ni§ Ni* i ~- Zn+Cu§ Zn§ Zn,,,. Zn-i. Ga+
II II I i l I I II = II /I II = H IlJJ
54 55 56 57 58 59 60 61 62 63 64 65 66 67 68| 6911 70
FIG. 4--Portion of" ultraviolet chart.from log ratio scan o f lake water sample.
the accuracy can be made to approximate the precision. Without calibra-

tion some elements would be determined with only an order of magnitude
accuracy, which is still quite useful.
The electronic mode of ion detection and measurement lends itself
readily to automated data handling and several designs of appropriate
equipment are now available [5], including complete commercial installa-
tions [6].
Provision has also been made for electrostatic peak switching whereby
several mass numbers of interest may be monitored consecutively after
selection from an oscilloscope display. Enhanced precision and accuracy
are possible using this mode because more time is spent recording each
mass. This technique is ideally suited to stable isotope dilution analysis of
from one to three elements at a time [7]. Much enhanced accuracy results
from this technique.
Sampling and Electrode Preparation

Water samples can be collected by any of the approved methods,
bearing in mind always that maintaining the sample free of additional
trace contamination from the sampling apparatus and during the subse-
quent storage of the sample may not be a simple task. Bottles molded
from high pressure, noncatalyzed process polyethylene contaminate water
less than any other common material and are relatively inexpensive. An
extra precaution that can be taken, if necessary, is to freeze the water for
long-term storage in polyethylene.
Airborne contamination must also be avoided during sample handling.
This is best accomplished by the use of clean hoods serviced with a
positive laminar flow of filtered air. We often use a similar technique to
preconcentrate the water samples before analysis. That is, the water is
evaporated from a polyethylene sheet under a flow of filtered air until only
a solid residue remains. We have also used freeze-drying for the same
purpose, but prefer the first method. If much organic material is in the
residue it is destroyed by low temperature radio-frequency electrodeless
ashing [8], because it may interfere in the subsequent SSMS analysis.
There are several ways of preparing electrodes for spark source analysis.
The ash or dryed residue can be mixed with an equal amount of 1 jma-
size ultra-purity graphite powder, with or without a known amount of a
standard element, and the mix homogenized on a Wig-L-Bug shaker. The
mixture is then molded into a pair of solid electrodes in a polyethylene
mold inside a steel die using a hydraulic lab press.
Alternatively, the water sample can be deposited on a gold disc with or
without other preconcentration and the dried residue sparked with a gold
counter electrode. Of course, the gold plus all its impurities appear in the
subsequent mass spectrum and the minimum detection limit is not as low
as with the first procedure.
Another variation is to freeze a drop of water onto a suitable cupped
electrode using liquid nitrogen coolant and spark with a gold probe
electrode directly to the ice [9,10]. The sensitivity in this case, without
preconcentration, is similar to that obtained in most solid matrices; that is
about one part in 10 s.

In the course of our work at Chalk River we have had occasion to
analyze water samples from a number of lakes, rivers, and streams. An
average analysis usually results in the determination of 30 or more ele-
ments ranging in concentration from p p m (mg/liter) to 10 -s p p m (10
ng/liter). An example of such an analysis is seen in Table 1.
This is a fairly typical analysis of 100 ml of Ottawa River water taken
near Chalk River. About three dozen elements were determined by SSMS;
19 by neutron activation analysis (NAA). The actual instrument time for
the mass spectrometer analysis was less than an hour, during which three
complete scans of the entire mass range were done. Several more hours
were involved in sample preparation and computation of the results. By
way of contrast, the activation analyses involved two separate irradiations
in the high flux NRU reactor, followed by counting with a 70 cm 3 Ge(Li)
detector after various decay periods of up to two weeks following irradia-
tion. Reduction of the counting data was computer-assisted. Other work-
ers have reported allowing as much as 30 days decay time for a complete
activation analysis in which up to three dozen elements were measured in
lake water [I1]. It is fair to say that trace element survey results can be
obtained much more quickly and with less effort by using SSMS than by
using neutron activation analysis.
A criticism frequently applied to SSMS is that it does not provide
accurate results. I f only one or two elements were determined in each
analysis I would consider that a significant comment. Obviously, such is
not the case. Furthermore, the degree of accuracy varies directly with the
amount of calibration done and the precautions taken in preparing the
sample, obtaining the mass spectrum, and reducing the data. How many
neutron activation results would be accurate without meticulous calibra-
tion? A number of the determinations made by both methods and shown
in Table 1 are in reasonable agreement. Others are significantly different.
No one should be surprised. Nor can anyone say offhand which results are
the more accurate.
In Table 2 a set of lake water analyses are reported, some of which were
also done by neutron activation. Results are similar to the preceding ones
TABLE 1--Trace analysis, Ottawa River (in rag/liter).
Element SSMS NAA
1 Be 0.08 ...
2 B 0.02 ...
3 F 0.2
4 Na 5.9 1.8
5 Mg 6.5 4.8
6 AI 1 ...
7 Si 14 ...
8 P 0.05 ...
9 S 9
10 C1 13 1.1
11 K 3.5 0.94
12 Ca 18
13 Sc 0.0008 0.00011
14 Ti 0.06 ...
15 V 0.002
16 Cr 0.006 0.0001
17 Mn 0.03 0.22
18 Fe 0.78 0.78
19 Co 0.015 0.0038
20 Ni 0.013
21 Cu 0.014 0.28
22 Zn 0.036 0.041
23 As 0.015
24 Br 0.048 0.044
25 Rb 0.007 0.0021
26 Sr 0.06 ...
27 Y 0.0005 ...
28 I 0.004 ...
29 Cs 0.00005
30 Ba 0.03 0.01
31 La 0.0007 0.0005
32 Ce 0.0016 0.00063
33 Pr 0.00025
34 Ag 0.00015 0.0~2
35 Cd 0.0001 0.00001
36 Pb 0.01
37 Hg ND a 0.0~95
aND = not detectable.
in scope, sensitivity, and the degree of agreement between both methods

of analysis.
T h e p o i n t is t h a t we h a v e h e r e a r e a d i l y o b t a i n e d q u a l i t a t i y e a n d s e m i -
quantitative analysis of a water sample. Should additional information be
r e q u i r e d a b o u t s p e c i f i c e l e m e n t s , i n c l u d i n g b e t t e r a c c u r a c y , t h e n we a r e i n
a favorable position to make an intelligent selection of other analytical
methods, specific to particular elements and possibly, more accurate, to
apply to the problem.
The other significant criticisms that I have frequently heard are that
TABLE 2 - - T r a c e analysis, M a s k i n o n g e L a k e (in mg/liter).
Element SSMS NAA
1 Be 0.002 . ..
2 B 0.O06 ...
3 F 0.06
4 Na 2.1 2.2
5 Mg 1.2 2.3
6 AI 0.025 99
7 Si 9 ...
8 P 0.02 99
9 S 3 o J
10 CI 4.8 1.2"
11 K 1.7 1.2
12 Ca 6
13 Sc 0.0007 0.000009
14 Ti 0.016 ...
15 v 0.0005
16 Cr 0.009 0.00008
17 Mn 0.04 0.25
18 Fe 0.53 0.53
19 Co 0.016 0.0052
20 Ni 0.012 I
21 Cu 0.01 0.46"--
22 Zn 0.008 0.051
23 As 0.003
24 Br 0.029 0.059"--
25 Rb 0.002 0.0012
26 Sr 0.03 9
27 Y 0.0002 ...
28 I 0.003
29 Ba 0.015 0.0046
30 La ND a 0.00003
31 Ce ND 0.00006
32 Pb 0.004 ...
aND -- not detectable.
SSMS is m u c h too expensive in capital cost a n d requires highly t r a i n e d

personnel for its operation. Let me a t t e m p t to establish a perspective on
these points.
O n e can purchase a spark source mass spectrometer complete with
a u t o m a t e d d a t a h a n d l i n g for $180 000 to $200 000. O n the basis of eight
samples a day a n d 36 elements d e t e r m i n e d per sample, the i n s t r u m e n t
capital cost per e l e m e n t d e t e r m i n e d , over a five year period would be fifty
cents. T h a t represents 374 000 elemental d e t e r m i n a t i o n s . As for highly
t r a i n e d personnel, let me say t h a t I a m all in favor of t h e m . Unless
c i r c u m s t a n c e s change radically, however, we will c o n t i n u e to t r a i n o u r
technical institute graduates on the job. Two such people, one of whom
should have some electronics t r a i n i n g , can h a n d l e the work load I have
just outlined. T h e highly t r a i n e d scientist will certainly be required, b u t
his role will be to evaluate the m o u n t a i n s of analytical results p r o d u c e d
both by SSMS a n d by whatever other analytical techniques he is able to

call u p o n to assist him in his studies.
Acknowledgments
T h e cooperation of W. F. Merritt in sample p r e c o n c e n t r a t i o n a n d
ashing and in n e u t r o n activation analysis, and of C. H. K n i g h t in mass
spectrometric analysis is gratefully acknowledged.
References
[1] Losee, Fred, 7th Annual Conference on Trace Substances in Environmental Health,
1973.
[2] Maugh, T. H., Science, Vol. 181, 1973, p. 253.
[3] Frisch, M. A. and Reuter, Wilhad, Analytical Chemistry, Vol. 45, 1973, p. 1889.
[4] Degreve, F. and de Ribes, D. C., International Journal of Mo~s Spectrometry and
Ion Physics, Vol. 4, 1970, p. 125.
[5] Conzemius, R. J., Rhinehart, W. A., and Svec, H. J., Talanta, Vol. 19, 1972, p. 1147.
[6] Bingham, R. A. and Elliott, R. M., Analytical Chemistry, Vol. 43, 1971, p. 43.
[7] Paulsen, P. J., Aivarez, Robert, and Kelleher, D. E., Spectrochimica Acta, Voi. 24B,
1969, p. 535.
[8] Gleit, C. E. and Holland, W. D., Analytical Chemistry, Vol. 34, 1962, p. 1454.
[9] Chupakhin, M. S., Kryuchkova, O. I., and Semenov, A. D., Zhurnal Analiticheskoi
Khimii, Vol. 27, 1972, p. 1895.
[10] Owens, E. B., Analytical Letters, Vol. 3, 1970, p. 223.
[11] Copeland, R. A. and Ayers, J. C., Environmental Research Group, Special Report No.
1, University of Michigan, 1972.
R. E. Jervis2 Amares Chattopadhyay,2 E. Csillag, 3 and
B. Tiefenbach'
Nuclear Activation Determination of

Heavy Metals in Great Lakes
Sediments, Soils, and Vegetation
REFERENCE: Jervis, R. E., Chattopadhyay, Amares, Csillag, E., and Tiefenbach, B.,
"Nuclear Activation Determination of Heavy Metals in Great Lakes, Sediments, Soils,
and Vegetation," Water Quality Parameters, A S T M STP 573, American Society for
Testing and Materials, 1975, pp. 82-94.
ABSTRACT: A combination of neutron and photon activation techniques have been

developed and applied for detecting traces of heavy metals from water pollution sources.
Air-dried portions of sediments from several regions of the lower Great Lakes, organic
cultivated soils, and selected edible vegetation from a heavily-irrigated and fertilized
marsh crop-growing area were subjected either to neutron irradiation for 25 to 50 h or
to 15 to 44 MeV bremsstrahlung from the University of Toronto electron linear accel-
erator for 1 to 4 h. Many of the neutron activation determinations at low concentrations
required post-activation chemical separations, whereas an advantage of the photonuclear
technique for use with such environmental samples was that multielement deter-
minations could be done completely by an instrumental method.
Elements of environmental interest determined in the activated samples included:
arsenic, antimony, cadmium, chromium, lead, mercury, molybdenum, selenium, thal-
lium, zinc, and several others. The concentration ranges involved were from 0.1 to 500
ppm, and the typical analytical accuracy achieved varied from 5 to 15 percent as verified
by comparison with other techniques and with standard reference materials. These
studies have helped to identify some heavy metal water pollution sources and to eluci-
date the fate of such metals in sediment, soil-water-vegetation ecosystems.
KEY WORDS: water quality, sediments, soils, vegetation, environmental tests, heavy
metals, neutron activation analysis
Recently, there has been an increasing awareness of heavy metal pollu-

tion of the environment, especially that caused by mercury and arsenic in
i n l a n d w a t e r s , c a d m i u m i n soil, a n d l e a d i n t h e u r b a n a t m o s p h e r e .
~Professor, Nuclear and Radiochemstry, Department of Chemical Engineering and

Applied Chemistry, Institute for Environmental Studies, University of Toronto, Toronto,
Ontario MSS 1A4, Canada.
2Assistant professor, Trace Analysis Research Centre, Dalhousie University, Halifax. Nova
Scotia, Canada.
3Radiochemist, Canada Centre for Inland Waters, Environment Canada, Burlington,
Ontario, Canada.
4Research Associate, Department of Chemical Engineering and Applied Chemistry, Uni-
versity of Toronto, Toronto, Ontario M5S 1A4, Canada.
82
9
Copyright 1975by ASTM International www.astm.org
JERVIS ET AL ON HEAVY METALS IN THE GREAT LAKES 83
However, the determination of heavy metals in complex environmental

materials such as lake-bottom sediments, soil, airborne particulate, vege-
tation, and biological tissues is often difficult at the low concentrations in-
volved. Among the techniques capable of being developed for reliable
determination of submicrogram quantities of a number of elements are
nuclear radioactivation, spark source mass spectrometry, and atomic
absorption. The first two methods have excellent scope for multielement
analysis at the trace level, both qualitative and quantitative, whereas the
latter is best when applied for determination of single elements, or se-
quentially for a small number of them. Several excellent reviews of the
salient features of the activation method have been published [1-3]. s
Simultaneous multielement analyses are useful not only because of the
importance of a large number of metals in environmental hazard assess-
ment, but also because of the need to consider synergistic effects, either
the protective effect of one agent against the toxicity of another such as
selenium-mercury or selenium-cadmium protection [4] or a potentiating
effect of one agent in augmenting the toxicity of another.
In this laboratory nuclear radioactivation, techniques have been de-
veloped and applied for high sensitivity multielement analysis of sedi-
ments, soils, vegetation, and other foodstuffs. Both neutron activation
(NAA) and photon activation (PAA) analyses are reported in this paper,
some assisted by post-activation radiochemical separation techniques and
other determinations completed by instrumental or nondestructive activa-
tion analysis. Instrumental analysis, when feasible, can eliminate tedious
chemical procedures and permit a more comprehensive multielement sur-
vey. New photon activation techniques developed in this study have greatly
increased the scope of instrumental analysis so that up to 30 elements
have been determined in single 1-g samples. Atomic absorption aided by
chemical separation has also been used for cadmium and lead as an
accuracy check of the activation techniques. Several standard reference
materials (SRM) obtained from the U.S. National Bureau of Standards
(NBS) have been analyzed by the same techniques, also for accuracy com-
parison (see Tables 1 and 2).
Nuclear Activation Techniques (NAA and PAA)
Sample Activation
Dry samples of 0.05 to 1-g weight were irradiated either with reactor
neutrons er photons from the University of Toronto Linac electron ac-
celerator for periods ranging from 1 to 50 h, depending on the sensitivity
required. The comparator method of activation analysis has been used in
which all the parameters of activation, growth, and decay of respective
sThe italic numbers in brackets refer to the list of references appended to this paper.
TABLE 1--Accuracy of activation analyses (NBS/EPA SRM: fly ash).
Concentration (ppm)
Trace Element NAA 1PAA NBS Value
As 58.2 + 4 . 3 60.7 + 2.6 61 .:k. 3

Cd 1.06 4- 0.16 1.52 4- 0.07 1.45 + 0.06
Cr 131 + 6.1 132 + 5
Hg 0.12":~0.01 0.135 4- 0.01 0.15
Pb ... 70.7 -t- 2.6 70 + 2
Sb _.. 7.14 -4- 0.56
Se 9.85+0.1 9.48 -F 0.8 9.3 "-+- 1.0
TI _. 3.64 + 0.34 4
Zn 78 (?)' 214 + 16 210 :l:. 20
(incomplete
dissolution)
TABLE 2--Accuracy of activation analyses (NBS SRM: orchard leaves).
Concentration (ppm)
NAA NBS Value (based on

Trace Element (based on as-is weight) IPAA dry sample weight)
As 11.4 + 1.6 10.2 -t- 1.0 11 ..-17_2a

Cd 0.11 -t-0.013 0 . 1 1 + 0.01 0.11 -1-0.02
CI 685 4- 32 (700)
Hg 0.136".~ 0.003 0.14 ..+_ 0.01 0.155 ..+_0.015
Pb ... 44.2 _+_ 2.1 45 +3
Sb 3.15 _+_ 0.26 b
Se <6.09 .. 0.08 +0.01
Sr ... 36.2 "-4- 2.0 (37)
Zn 22.7 _+_0.4 24.2 4- 1.5 25 _.+_3
a NBS value recently revised; private communication from P. LaFleur.

bNBS estimate is 2 to 4 ppm.
r a d i o n u c l i d e s a n d d e t e c t o r efficiency are c a l i b r a t e d b y i r r a d i a t i n g k n o w n
m i c r o g r a m w e i g h t s o f e a c h e l e m e n t to b e d e t e r m i n e d a l o n g w i t h t h e
unknown samples.
N e u t r o n a c t i v a t i o n was c a r r i e d o u t b o t h at t h e U n i v e r s i t y o f T o r o n t o
SLOWPOKE r e a c t o r at 1011 to 1012 n e u t r o n s / c m 2 s f l u x a n d at t h e
M c M a s t e r r e a c t o r at 2 x 1013 flux. P h o t o n a c t i v a t i o n was a c c o m p l i s h e d by
e x p o s i n g s a m p l e s on a s l o w l y - r o t a t i n g s a m p l e a s s e m b l y b e h i n d a 3 - m m -
t h i c k t u n g s t e n p l a t e in w h i c h a p r i m a r y e l e c t r o n b e a m u p to 44 M e V in
e n e r g y was c o n v e r t e d to a b r e m s s t r a h l u n g p h o t o n s p e c t r u m . S p a t i a l
p h o t o n f l u x v a r i a t i o n was m i n i m i z e d by t h e r o t a t i n g s a m p l e p l a t f o r m o n
which samples and comparator standards were passed through the beam
u n i f o r m l y . T h e e l e c t r o n e n e r g y was v a r i e d f r o m 15 to 44 M e V to o p t i m i z e
the determination of selected groups of elements, namely, 15 MeV for

antimony and cadmium; 20 MeV for arsenic, scandium, and tellurium; 22
MeV for titanium and zirconium; 35 MeV for chromium, lead, mercury,
molybdenum, selenium, and zinc; 44 MeV for bismuth and vanadium. A
mean electron beam current of about 100-200 gamp was used, corre-
sponding to a photon flux at the sample position of 2 x 1013 photons/s.
Details of the technique have been submitted for publication [5].
Activity Determination
Depending on the activation efficiency of particular trace elements of
interest and their concentration levels, detection and quantitative determi-
nation may often be completed by instrumental resolution of multi-com-
ponent radiation spectra or may require radiochemical separation of the
radioactive nuclides before radiation assay. The chemical steps used in the
latter situation for arsenic, cadmium, mercury, antimony, selenium, and
zinc are discussed in the following paragraphs.
Activated specimens for instrumental radiation analysis were first trans-
ferred to clean unirradiated containers and then accurately positioned
close to a 60-cm 3 high-resolution Ge(Li) gamma detector coupled to a
Canberra Model 8100 spectrometer. Gamma-ray spectra were measured at
various intervals after the end of the sample irradiation period in order to
detect additional components after the short-lived nuclides decayed. De-
termination of particular nuclides was based on gamma rays that were
well-resolved from other gammas. Details of the choice of radionuclides
and their gamma rays found optimal for determination of about 30
elements after neutron or photon activation are beyond the scope of this
paper but are, in part, published separately [5]. The nuclides chosen for
elemental determination are shown on a periodic chart in Fig. 1.
Analytical Accuracy
The results obtained for a variety of inorganic and biological samples
studied by these methods, which were in the concentration range approxi-
mately from 10 ppb to a few ppm, were generally precise to about _+10
percent (as indicated in Tables 1 to 6). The accuracy can be inferred (1)
by the agreement obtained with different, independent activation techniques
and with atomic absorption as shown in Tables 6 and 7; and (2) by com-
parison of the results obtained for NBS SRM's with their certified or
"information" values. In Tables 1 and 2, analytical results obtained for
NBS/EPA fly ash and for orchard leaves both by NAA and instrumental
photon activation analysis (IPAA) are compared with NBS values. It can
be seen that analytical precision for both techniques ranged from 2 to 15
percent at the ppm level, and accuracy can be inferred to be from 2 to 25
percent at worst.
NUCLIDE STUDIED
HALF-LIFE
GAMMA-RAY ENERGY IN KeY
22Na z ~ Na s ~mC:
2.6y 15h 3~a
1274 1368 ~128

~s K
22h
~SC
3.9h
~ISc ~*v
1.8d [6d
*'Cr'*Snli'"l''eoL""i
23h 303d 12.'h 71.3d 36h
~SZn
!45d
?WAs Itmse
17.9d 57m
373 1156 1039 983 310 835 865 811 378 L154 596 103
i smSl aIzr I~MO lo6mAgtllmcdllSm~117m$~122Sb 129T(
70~ 78.4h 67h 8.4d Lgm 100m 69m
]47 393 14d |12.8d
232 909 140 158 564 459
a t97mHg2O2Tl 20~mp~20SBi
6.5d
668
28.7~
268
.h112 6
134 12' 899
::
FIG. l--Nuclides analyzed by instrumental photon activation ana~sis.
TABLE 3---Antimony and selenium in lower Great Lakes sediments.
Location and Station Core Section (cm) Selenium (ppm) Antimony (ppm)
Lake Huron
C (south end)
0-5 (top)
5-10
,}
1.3
1}
1.3
10-15 1.0 +50% 1.1 --9%
15-20 1.2 1.2
Lake Huron 0-5 (top) 0.96 0.28
A (Bruce Point) 15-20 0.86 +11% 0.30 --7%
Lake Huron 0-5 (top) 0,16 0.28
B (midwest) 15-20 0.13 +23% 0.20 +40%
St. Clair (Corunna) (grab sample) 0.43 0.67
St. Clair (Courtwright) (grab sample) 0.43 0.92
St. Clair (Sombra) (grab sample) 0.36 0.34
St. Clair (lake delta) (grab sample) 0.31 0.44
Lake Erie
M (midwest)
0-5 (top)
5-10
10-15
161
0.8
0.6 +100%
7I
1.0
0.8
+70%
15-20 0.9 1.0

Lake Erie 0-5 (top) 0.24 0.46
+78% +100%
N (central) 15-20 0.13 0.23
Lake Ontario
P (Niagara basin)
0-5 (top)
5-10
10-15
1t
1.0
0.8 +60%
1,I
0.6
0.7 +100%
15-20 0.7 0.7
Lake Ontario 0-5 (top) 1.5 1.8
O (Hamilton) 15-20 0.17 +780~176 0.37 +390%
Lake Ontario 0-5 (top) 0.36 0.44
Q (Toronto) 15-20 0.03 +980070 0.23 + 8 7 %
NOTE--All concentrations are expressed in ppm, dry weight basis.

JERVlS ET AL ON HEAVY METALS IN THE GREAT LAKES 87
TABLE 4---Toxic heavy metals in aquatic species.
Species and Location As Cd Hg Se Zn
Nova Scotia oyster ... 0.80 0.073 0.39 750

Nova Scotia oyster ... 0,45 0.098 0.30 1320
Lake St. Clair Walleye 0,068 0.007 3.6 0.21 2.5
Lake St. Clair N. Pike 0.036 0.007 2.2 0.44 3.0
Lake St. Clair Y. pickerel 0.071 0.008 1.23 0.23 2.9
Lake Erie perch 0.017 0.014 0.70 0,29 8.2
Lake Ontario W. perch 0.095 0.004 0.62 0.46 2.6
Lake Ontario w. perch 0.060 0.010 0.36 0.37 2.5
Lake Ontario whitefish 0.015 0.16 0.28 2.0
Lake Ontario sheepshead 010i5 0.006 0.20 0.19 2.9
Lake Ontario S,M. bass 0.058 0.004 0,096 0.16 3.3
Lake Simcoe whitefish 0.012 0.006 0.036 0.37 0.86
Lake Simcoe whitefish 0.038 0.008 0.064 0.31 3.8
Miscellaneous foodstuffs 0.01-3.0 0,002-0.29 0.01-0.25 0.03-1.6 5-250
NOTE--All concentrations in ppm, fresh weight basis, determined by NAA with radio-
chemical separation.
Studies of Lower Great Lake Sediments: Selenium and Antimony (NAA)

Sediment cores and several "grab" samples were obtained, with the
assistance of Canada Centre for Inland Waters, Ontario Ministry of the
Environment, and the Great Lakes Research Centre, Institute of Environ-
mental Science and Engineering, from several locations in Lake Huron, St.
Clair River, Lake Erie, and Lake Ontario. These locations are shown ap-
proximately in the map of the lower Great Lakes region (Fig. 2).
Cores were separated into 5-cm sections which were homogenized and
air-dried. Replicate analyses of 1-g portions showed them to be homo-
geneous to about +_6 to 8 percent in most trace components.
Among the trace elements detected in sediment samples by NAA were
mercury, arsenic, antimony, selenium, zinc, cadmium, silver, sodium, and
scandium. Selenium and antimony were selected for special study: sele-
nium, because of its recently reported [4] protective effect against mercury
and cadmium toxicity; antimony, because it has been little studied in the
environment, although it is used rather widely in industry, and is asso-
ciated often with lead in its use, thus might be related to lead in industrial
waste and in sediment uptake. It has been reported that mercury contents
of bottom sediment over the lower lakes region ranges from 0.1 to 10
ppm.
After about 50-h neutron activation, traces of U4Sb and 755e nuclides were
radiochemically separated by carrier dilution and repeated precipitation of
SbzS s and elemental selenium, respectively. Final assay was done by gamma-
spectrometry to eliminate traces of other metals and corrections applied for
TABLE S--Concentration of elements in soil by depth (ppm, dry has/s), tr
Soil Depth (cm)
Element Surface 1 Surface 2 0 to 7.5 7.5 to 15.0 15.0 to 22.5 22.5 to 30.0 30.0 to 37.5 37,5 to 45.0 -4
m
-n
Antimony 2.11 1.85 1.73 0.66 0.58 0.93 0.75 0.70
D
Arsenic 1.72 1.61 1.85 0.96 0.78 0.85 0.50 0.42 C
Barium 285 270 252 293 300 328 295 330 3~
i"
Bismuth 1.52 1.46 1.33 N.D. a "01 N.D. N.D. 1.21
Cadmium 1.45 1.52 1.71 0.97 0.85 1.15 1.32 0.67 -<
Calcium 20 000 20 000 14 000 11 500 12 300 10 550 11 400 12 200 -u
Cesium 4.82 4.91 2.22 1.95 1.83 2.65 1.78 2.26
Chlorine 72.2 68.5 45.1 32.6 26.0 38.7 11.6 8.42
Chromium 14.3 12.1 18.5 30.2 35.4 45.8 28.6 26.7 rrl
Cobalt 8.52 8.65 8.24 7.66 5.48 7.32 4.37 5.28 --4
Indium 2.60 2.12 2.95 1.56 1.12 1.86 0.93 0.56 tll
Iron 28 000 28 350 21 200 18 000 19 750 22 400 24 000 27 750 fD
Lead 32.2 30.0 20.1 18.5 17.8 15.2 12.4 11.6
Magnesium 780 765 640 420 400 525 546 583
Manganese 1350 1275 840 625 715 1120 1200 1430
Mercury 1.45 2.50 1.18 1.62 1.80 1.20 1.30 N.D.
Molybdenum 2.12 2.00 1.32 1.05 0.84 1.18 1.25 1.47
Nickel 8.11 7.85 6.21 6.64 5.23 7.55 4.81 3.57
Scandium 3.22 3.41 3.65 3.06 4.31 5.26 5.52 5.88
Selenium 1.10 1.43 1.22 0.81 0.62 1.05 0.91 0.53
Silver 0.92 0.85 0.68 0.52 "00.40 N.D. N.D. N.D.
Sodium 6000 5950 5000 4320 4170 3860 3700 3610
Strontium 80.5 82.8 65.6 52.3 41.4 56.7 75.0 89.3
Tellurium 6.25 6.10 4.33 2.17 ,ol.2 N.D. N.D. N.D.
Thallium 0.22 0.20 0.17 N.D. N.D. 0.18 N.D. N.D.
Tin 14.0 13.5 12.2 12.5 10.1 12.3 13.1 14.2
Titanium 101 105 122 145 160 168 173 185
Vanadium 10.5 11.6 15.2 21.4 26.1 30.0 31.4 32.3
Zinc 165 152 148 128 94 65 48 42
Zirconium 200 200 278 323 405 467 500 676
aN.D. = not detected.

GEORGIAN ST. LAWRENCERIVER f
+ FULL CORE ANALYZED

OHIO . TOP AND BOTTOM ANALYZED
FIG. 2--Map of lower Great Lakes region.
TABLE 6--lntercomparison of elemental concentrations by different analytical techniques.
Content (ppm, dry basis)
Method Sample No. As Cd Cr Pb Hg Zn
Instrumental PAA 1 1.72 1.45 14.3 32.2 1.45 165

2 1.61 1.52 12.1 30.0 2.50 152
Destructive PAA 1 ......... 31.3 . . . . . .
2 ......... 31.0 . . . . . .
Instrumental NAA 1 ...... 13.5 . . . . . . 170
2 ...... 12.5 . . . . . . 160
Destructive NAA 1 1.65 1 . 4 2 ...... 1.41 161
2 1.62 1 . 5 7 ...... 2.51 150
Atomic Absorptiona 1 ... 1.45 ... 32.8 . . . . . .
2 ... 1.55 . . . 31.3 . . . . . .
a After chemical separation.
carrier recovery. After dissolution of irradiated sediment in h o t mixed

m i n e r a l a c i d s , s o m e i n s o l u b l e silica r e s i d u e r e m a i n e d , b u t it w a s f o u n d t o
c o n t a i n n e g l i g i b l e , if a n y , s e l e n i u m a n d a n t i m o n y , a n d p r e s u m a b l y o n l y
t r a c e i n c o r p o r a t e d in t h e g e o l o g i c a l p a s t o f t h e s e d i m e n t m a t e r i a l .
In Table 3 are listed the selenium and a n t i m o n y c o n t e n t of G r e a t Lakes
s e d i m e n t s , d i s t r i b u t i o n w i t h d e p t h in a t y p i c a l c o r e a n a l y z e d f r o m e a c h
l a k e , a n d t h e d e g r e e o f e n r i c h m e n t in r e c e n t s e d i m e n t l a y e r s c o m p a r e d t o
TABLE 7--Lead content of soil by IPAA, RPAA, and A A S methods.
Concentration (ppm, dry-weight basis)
Soil Depth (cm) IPAA RPAA AAS
Surface 1 32.2 31.3 32.8

Surface 2 30,0 31.0 31.3
0.0 to 7.5 20.1 20.5 21.2
7.5 to 15.0 18.5 17.8 19.0
15.0 to 22.5 17.8 18.1 18.6
22.5 to 30.0 15.2 14.8 16.2
30.0 to 37.5 12.4 13.0 14.1
37.5 to 45.0 11.6 12.0 12.8
underlying layers deposited more than 80 years ago. It is evident that,

apart from an area adjacent to Port Huron in Lake Huron, the lowest con-
centrations occur in Lake Huron and progressively higher values through
to Lake Ontario. However, although these trends are slight, an analysis of
the enrichment in upper sediment layers corresponding to the last 20 to 30
years shows an even greater trend down through the system with large
increases in both antimony and selenium in Lake Ontario near Hamilton
and of selenium near Toronto. Depth profiles for selenium and antimony
and the approximate deposition dates of the successive 5-cm layers are
shown in Figs. 3 and 4, respectively. Mean sedimentation rates measured
in Lake Ontario near Rochester were used to estimate the time of deposi-
TOP TOP TOP

0 1972
1950
x
S
',- 10 1930 '<O
11 j 1.9o
w
0 ,,,8
is! t910 o
20
0.5 2.5 0.5 2.5 0.5 2.5
CONCENTRATION Se (PPM) CONCENTRATION Se (PPM) CONCENTRATION Se (PPM)
LAKE HURON LAKE ERIE LAKE ONTARIO
POINT C POINT M POINT P
FIG. 3---Vertical distribution of selenium at Stations C, M, and P.

JERVIS ET AL ON H E A V Y M E T A L S IN THE G R E A T L A K E S 91
TOP TOP TOP

0 1972
zgso
10
15 191o g
20 1890
0.5 2.5 0.5 2.5 0.5 2.5
CONCENTRATIONSb (PPM) CONCENTRATIONSb (PPM) CONCENTRATIONSb (PPM)
LAKE HURON LAKE ERIE LAKE ONTARIO
POINT C POINT M POINT P
FIG. 4---Vertical distribution o f antimony at Stations C, M, and P.
tion of successive layers for all locations, although it is recognized that

sedimentation rates undoubtably vary through the lakes.
Toxic Metal Accumulation in Aquatic Species (NAA)

Since Great Lakes sediments have been shown in this and other studies
to contain lead, arsenic, mercury, cadmium, selenium, antimony, and
zinc, some determinations have been made by sequential NAA with radio-
chemical separation of arsenic, cadmium, mercury, selenium, and zinc in
Great Lakes fish taken from various locations. The NAA tissues were
dissolved slowly overnight at low temperature, and after elimination of
nitric fumes, hydrochloric acid (HC1) was added and the mixture applied
to anion exchanger. The metals of interest were eluted sequentially at
lower acidity, with the aid of specific complexing elution agents.
The concentrations of these potentially toxic heavy metals accumulated
in various species of freshwater fish, representative of different levels in
the aquatic food chain, are summarized in Table 4. These concentrations
are compared also with Nova Scotia oyster, with fish from the Trent
waterway near Lake Simcoe, and with residue levels in a variety of
Canadian foodstuffs obtained from a national sampling representative of
the bulk of the Canadian diet.
Little variation is evident among the regions except for mercury, nor
apart from that element are the concentrations in fish arising from water
pollution sources much greater than accumulations in other foodstuffs
from agricultural and market-gardening activities. Although safe levels for

the residues of these metals in foodstuffs other than fish have not been
officiaUy set in Canada, relatively few items appear to be in excess of the
guidelines temporarily set up of 0.1 to 0.5 ppm for several metals [6]. The
levels in oyster are appreciably higher for zinc and cadmium, and also for
arsenic according to other such measurements.
A comparison of NAA and PAA techniques for the determination of
ppb (10 9) of cadmium in such biological materials is detailed in a recent
manuscript [7]. The accuracy of these techniques is demonstrated in
Tables 1 and 2 for standard materials.
Studies of Heavy Metal Uptake in Marsh Soils: (IPAA)

Since the Holland Marsh vegetable-growing area was formed by par-
tially draining a marsh, it was of interest to study the accumulation of
metals from drainage water onto the organic " m u c k " soil, accumulation
from irrigation, fertilizers, herbicides, and pesticides, and their mobiliza-
tion during tillage and vegetable cultivation. " M u c k " soil samples from
several locations in Holland Marsh, vegetation growing in it, water, and
fertilizers were taken for trace metal analysis. The nature of the various
biological specimens made them more suitable for IPAA.
Up to 30 trace elements have been determined by IPAA in typical
Holland Marsh organic soils as a function of depth [5], and a listing of
results for one location is presented in Table 5. While a number of
elements which were probably derived during the formation of the soil are
at concentrations relatively constant with depth, others that may be added
to the top layers from agricultural chemical or atmospheric fallout, such
as lead, arsenic, cadmium, calcium, and zinc, are found in increased
surface concentrations. Distribution of cadmium and zinc between soil at
different depths and a vegetable plant growing in the same soil is depicted
in Fig. 5. It can be seen from this example that appreciable cadmium and
zinc are added to upper layers by fertilizer but that the vegetable shows a
lower concentration than the adjacent soil [5].
The accuracy of these techniques is demonstrated both in Table 3 by
the agreement among different techniques for the same sample (to _+5
percent in most cases) and by comparison of results obtained for SRM's
(Tables 1 and 2) with NBS certified or "information" values. The SRM
orchard leaves serve as a representative vegetation material, while the
NBS/EPA fly ash is of similar bulk composition to soils or sediments.
The PAA for trace lead determination in soil [8-11], vegetation, and in
human tissues such as hair is of special importance because of recent
interest in defining hazards from environmental lead pollution. A sensi-
tivity of several nanograms has been achieved by IPAA, and its accuracy
can be inferred from Table 7 where the IPAA technique (based on 67-min
FIG. 5----Zinc and cadmium content o f soils in a carrot patch, ppm, dry basis.
2~ is compared with a method involving radiochemical separation

(RPAA) (52 h 2a~Pb) and with AAS using a Jarrell-Ash Mark II spectro-
photometer after chemical separation.
Coneluslons
A review of these several studies of trace heavy metals accumulated
from water sources into sediments, aquatic species, and marsh soil and its
vegetation indicate the useful scope of nuclear radioactivation techniques
for sensitive and accurate multielement analyses.
Relocation and d i s t r i b u t i o n of sediments b i n d i n g various heavy metals

t h r o u g h the lower G r e a t lakes can be inferred from analysis of s e d i m e n t
cores a n d " g r a b " samples, a n d f u r t h e r studies with the same sediments
are proposed for other heavy metals.
Studies of the b i n d i n g of heavy metals on organic soils, sediments, a n d
sewage sludges are of potential significance to future land disposal of
sediments a n d sewage sludge as alternatives to dispersal in i n l a n d water
bodies.
References
[1] Lyon, W. S., Analytical Chemistry, Vol. 45, 1973, p. 386A.
[2] Meinke, W. W., "The Ultimate Contribution of Nuclear Activation to Analysis,"
International Conference on Modern Trends in Activation Analysis, Saelay, France,
1972, Plenary Lecture.
[3] Hislop, I. S. in Proceedings, International Conference of Photonuclear Reactions and
Applications, B. L. Berman, Ed., Vol. II, 1973, p. 1159.
[4] Parizek, J. in Proceedings, IAEA Symposium on Nuclear Activation Techniques in the
Life Sciences, Bled, Yugoslavia, International Atomic Energy Agency, 1972, p. 177.
[5] Chattopadhyay, A. and Jervis, R. E., Analytical Chemistry, Vol. 46, 1974, p. 1630.
[6] Department of National Health and Welfare Guidelines on Metal Residues in Foods,
Ottawa, Canada, 1972.
[7] Jervis, R. E., Tiefenbach, B., and Chattopadhyay, A., Canadian Journal of Chemistry,
Vol. 52, 1974, p. 3008.
[8] Chattopadhyay, A. and Jervis, R. E., Radiochemical and Radioanalytical Letters, Vol.
11, 1972, p. 331.
[9] "Abnormally High Environmental Lead Distributions and Effects on the Local Com-
munities," Report No. 1, Institute of Environmental Science and Engineering, Univer-
sity of Toronto, Toronto, Ontario, Canada, Nov. 1973.
[10] "Abnormal Lead Distributions and Effects on the Human Population," Report No. 2,
Institute of Environmental Science and Engineering, University of Toronto, Toronto,
Ontario, Canada, Nov. 1973.
[11] Roberts, T, M. et al, Science, Vol. 186, 1974, p. 1120.
D. A . Lord, 1 W. G. B r e c k , 2 a n d R. C. W h e e l e r 2
Trace Elements in Molluscs in the

Kingston Basin
REFERENCE: Lord, D. A., Breck, W. G., and Wheeler, R. C., "Trace Elements in
Molluscs in the Kingston Basin," Water Quality Parameters, A S T M STP 573, American
ABSTRACT: Freshwater molluscs (UNIONACAE) have been sampled extensively in the

Kingston Basin area of the Lake Ontario/St. Lawrence River system. Soft tissue of the
animals were homogenized and freezedried, and then analyzed for specific trace ele-
ments.
An analytical procedure was developed whereby the elements cobalt, chromium, cop-
per, lead, and zinc were determined directly on the freezedried solid material by atomic
absorption spectroscopy, using a heated graphite furnace. Prior to analysis, samples
were "diluted," using naphthalene as an inert solid diluent. In the species Lampsilis
radiata, cobalt, chromium, copper, lead, and zinc levels of approximately 1.1, 19, 36,
4.7, and 225 ppm (on dry weight), respectively, were found. In addition, samples of
seston material were separated from water samples collected in the Kingston Basin, by
dialysis and freezedrying, and subsequently also directly analyzed for some of these trace
elements. Chromium and copper levels of 41 and 594 ppm (on dry weight) were found.
Due to the high trace element levels found in the seston material, it is postulated that
trace element enrichment in bivalve molluscs takes place by an essentially two-step
mechanism, the first being the prior preconcentration (complexing, inclusion, precipi-
tation) of trace elements into the particulate material in the water column, followed by
ingestion of this particulate material by the organism. The use of such sedentary parti-
culate feeding organisms, such as fresh water mussels (which are easily collected), is
recommended as a suitable method for long-term in situ monitoring of water quality
with respect to specific chemical species present in the aquatic environment.
KEY WORDS: water quality, trace elements, aquatic animals, mollusca, mussels,
environmental tests
W i t h i n t h e w a t e r s h e d o f t h e G r e a t L a k e s s y s t e m , live m o r e t h a n f o r t y
m i l l i o n p e o p l e , w h o use t h e s e w a t e r s for b o t h t h e i r l i v e l i h o o d a n d r e c r e a -
tion. Man's endeavors have understandably imposed upon these water
r e s o u r c e s , b r i n g i n g i n t o f o c u s t h e n e e d for t e m p e r a n c e in a c t i v i t y p l u s a
r e q u i r e m e n t for c o n s t a n t m o n i t o r i n g o f t h e a q u a t i c e n v i r o n m e n t .
B a s e d on it's m o r p h o m e t r y a n d s u r f i c i a l s e d i m e n t d i s t r i b u t i o n , L a k e
O n t a r i o m a y b e c o n v e n i e n t l y d i v i d e d i n t o f o u r b a s i n s , s e p a r a t e d by sills
1Project biologist, Environment Canada, Halifax, Nova Scotia, Canada.
2Department of Chemistry, Queen's University, Kingston, Ontario, Canada.
95
Copyright 9 1975 by ASTM International www.astm.org

[1], 2 Fig. 1. Three of these basins, the Niagara, Mississauga, and Roch-
ester Basins, are deep (deeper than 100 m on average), while the fourth,
the Kingston Basin, is far shallower (less than 20 m on average). Lake
Ontario obtains about 80 percent of its water from the Niagara River
(representing a mix of the waters of the first four large Great Lakes), the
FIG. l--Lake Ontario, basin nomenclature.
remainder being supplied by the Lake Ontario drainage basin. Due to the
large volume of the lake as compared to the outflow, a direct throughflow
of water is not obtained, and upon imposition of geographical stresses
such as wind and temperature, specific circulation patterns within the lake
are established [2]. The exact circulation at any time will depend on
prevailing conditions, but neglecting the shallower Kingston Basin, an
average circulatory pattern of a counterclockwise gyre can be considered
to be present in Lake Ontario, both on the surface and in deeper waters,
which serves to assist the mixing of the waters within the lake. The lake is
generally considered to be well mixed over its entire volume, as is evi-
denced by the uniform horizontal and vertical distribution of conservative
elements within the waters [3], and because Lake Ontario is the last in the
series of the Great Lakes, the mixed waters of Lake Ontario can be
considered as representing at least a mix of the waters of the five Great
Lakes.
No such patterns of circulation and consequent mixing occur within the
Kingston Basin. The volume of this basin is sufficiently small, that simply
to maintain the required outflow into the St. Lawrence River system, the
LORD ET AL ON TRACE ELEMENTS IN MOLLUSCS 97
water mostly flows through the basin, in laminar fashion [2]. The inflow
into the Kingston Basin consists mainly of epilimnetic waters from Lake
Ontario, so that the waters of the Kingston Basin are representative of the
well-mixed surface waters of Lake Ontario; these then pass through a
geographical funnel into the St. Lawrence River system. The Kingston
Basin therefore, represents a unique location for a study of Great Lakes
waters.
Of increasing concern in the aquatic environment are the levels of
"heavy metals," especially from industrial discharge, and the need for
monitoring levels of these elements is recognized. Living organisms can
serve as excellent qualitative as well as quantitative indices of their en-
vironment, since plants and animals that are constantly exposed act as
long-term monitors to reflect the integrated state of their environmental
experience [4]. They can show pathways and points of accumulation of
pollutants and toxicants in biological systems, and frequently, can simplify
the complex task of relating physical and chemical measurements to
biological effects. For example, Beeton [5] has shown how the simple
concept of a change in species population in Lake Erie can readily indicate
the changes in the environment to which the species was exposed.
It is well documented that filter-feeder organisms, such as bivalve
molluscs, are able to concentrate a variety of compounds from their
aquatic environment, including the trace elements cobalt, chromium, cop-
per, lead, and zinc [6-11]. North America contains the richest and most
varied fauna of freshwater molluscs in the world, and bivalve molluscs
are represented by two families, the UNIONACAE, or freshwater mussels,
and the smaller and less conspicuous fingernail clams, SPHERIIDAE
[12]. Both families abound in the Kingston Basin. These organisms are
sedentary, are well distributed, have some degree of longevity, and can
serve as excellent organisms for monitoring the water on a regular basis.
The present study was undertaken to establish levels of the trace ele-
ments cobalt, chromium, copper, lead, and zinc in the waters flowing
through the Kingston Basin, using freshwater mussels of the family
UNIONACAE as the indicator organism. By similar measurements on the
same species of mussels collected at different times or places, a relative
comparison of water quality with respect to specific elements may be
obtained.
The analysis of biological materials for low levels of heavy metals has
always been an exacting task, with particular problems being contamina-
tion and efficient calibration. At present, the most popular method of
analysis of biological materials is by atomic absorption spectroscopy (AAS)
analyzing a liquid solution prepared from the material. With the advent of
commercial flameless atomizers, the technique of AAS has been expanded
to enable absolute sensitivities in the order of 10-11 to 10 -12 g to be
obtained for many heavy metals, though normal operation still includes
dissolving the biological material and analyzing the resultant solution (for
example, see the review by Willis [13]).
Direct atomization of solid materials in a graphite furnace represents a
promising alternative method of sample handling, eliminating the lengthy
decomposition step and possible sample contamination. So far, the direct
atomization technique has been successfully used mainly for the deter-
mination of trace elements in metals [14,15], but has also been used for the
determination of trace elements in biological materials [16] and in rocks
[17]. Apparently no laboratory is using this method on a routine basis.
In this work, a method was developed for determining the heavy metals
cobalt, chromium, copper, lead, and zinc in dried biological material
(freeze-dried mussels) directly on the solid material, by flameless AAS.
Experimental
The program was initiated by sampling freshwater mussels in the
Kingston Basin. During 1972, approximately 50 separate locations were
sampled, representing all the major water areas in the basin (Fig. 2).
Throughout the sampling, three species of freshwater mussels were found.
They are Elliptio complanata, Anodonta grandis, and Lampsilis radiata.
All can be readily classified by shell shape [12]. Lampsilis radiata were
found wherever there were mussels, while Elliptio complanata were
present at most locations. Anodonta grandis were far more sparsely dis-
tributed, but when they did occur, seemed to prefer sandy, firm condi-
tions, rather than the soft, silty conditions for which their shells appear
designed [12].
All sampling was done by scuba. The mussels were readily seen in the
sediment or in cracks between the rocks, frequently actively filtering. They
were well distributed, proliferating in shallow and shoaly areas, and were
absent only in very weedy areas, on flat rock, or in depths over 75 ft. At a
given sampling area, about 30 to 50 live mussels were randomly selected
from the substrate. The samples were brought to the surface, the animals
classified and counted, and six of each species retained as a representative
sample for that location. Animals were returned to the laboratory, where
they were shucked, the six in each sample pooled and homogenized
together, and the resultant homogenate freezedried, yielding an even-
textured, light brown powder. All samples were stored in polyethylene
bottles.
Sample analyses were conducted by flameless AAS, directly on the solid
sample. The spectrometer used is a single beam instrument, mounted on
an optical bench. The major components consist of; a Jarrell-Ash Model
82-000 series 0.5 m Ebert scanning sl~ectrophotometer with a grating
blazed for maximum energy at 2500 A, a P.A.R. Model 122 lock-in
amplifier coupled with a P.A.R. Model 125 mechanical light chopper
ONTARIO
9 _./F"~S,~ o~
$" f1":-..~.- )
9 5"4
"46 / l " ' ,[ ,.,/ - f r--
O
~65" 79 i -n
.~"~ ~ ,7. l STATE E~
~3 I 43 m
I
6"4 6 I ~ "
I--
I
34 O
~- I :5 Z
I 42 <
I
t I
' " C:Pd -n
, D O
m
"r m
r-
m
r ~ ~ ~ t . . . . 2,1 /;
m
z
6o
J
KINGSTON
0
kilLES I0 r-
r-
I--
A = Kingston Harbour D ----- K i n g s t o n ] ~ a s i n - - - e a s t 0

(o
B = North Channel E = Kingston Basin--west
C = Adolphus Reach P = Seston Sampling Point. North Channel
F I G . 2--Kingston Basin sampling points. 1972: Positions 1 to 54; and 1973: Positions 60 to 69. $
(chopping frequency used was 333 Hz), and a Simpson Model 2742
high-speed recorder. Direct atomization of the solid samples was achieved
in a Perkin-Elmer HGA-2000 graphite furnace, and a D2-1amp was used
to measure background absorption.
Initial experiments with copper indicated that the increased sensitivity
obtained with the graphite furnace resulted in embarrasingly large ab-
sorbances for the mussel samples. To avoid the use of smaller samples it
was necessary to "dilute" the solid analyte with an inert solid carrier.
Naphthalene (Fisher N-134) was chosen as the diluent material, as it is
easily obtained relatively pure, and can be totally volatilized before the
commencement of the atomizing stage. All samples were diluted about ten
fold, by grinding together the appropriate quantities of naphthalene and
sample in an agate mortar with pestle, giving a fine powder. For ease of
handling, samples were pelletized before being weighed into tantalum
boats. Samples were delivered into the graphite furnace using the appro-
priate sampling spoon, and sample sizes of 1 to 5 mg were conveniently
handled by this procedure.

As has been shown by others, care must be taken when using a graphite
furnace for analytical work, not to lose any analyte during the preatomiza-
tion stage [18]. In this work, a procedure was established for determining
preatomization losses from the solid samples, thereby enabling a suitable
ashing temperature to be selected. Basically, the ashing temperature used
should be low enough so as not to lose any analyte during ashing, but
should be high enough to remove sufficient matrix material which would
otherwise give excessively high background absorption during atomization.
A similar process has been recommended for the determination of lead in
milk [19].
The procedure is self explanatory by reference to Fig. 3, from which it
may be deduced that a "safe" ashing temperature of 900~ for the
analysis of chromium may be used. Similarly, standard experimental
conditions may be gaged for other elements, and conditions used in this
work are given in Table 1. Analyses were not conducted at the most
sensitive resonance lines for the elements chromium, copper, and zinc in
order to obtain sample absorbances of the desired levels (absorbance
values of 0.1 to 0.4 considered desirable).
A typical analytical trace with three peaks (naphthalene, smoke, metal)
for a copper analysis is shown in Fig. 4, and all absorbances calculated
from these traces are based on peak height measurements.
Calibration presents a significant problem with solid samples, and the
only reliable procedure to calibrate is by using standards of similar com-
position and size to those of the samples being analyzed. For this purpose,
0"2I,q
0 0.020
I 0.015
I
I
.E I
I
I 0.010
o.10 I
I
I
0.05 0.005
()
, I , , , , I , , , I , ,'~ I
500 10oo 1500 2000
Ashing Temperature,~C
FIG. 3--Determination o f ashing temperature, chromium.
t,.
o=
C3.
0
I I I I
0 20 70 95
Time (s)
FIG. 4--Typical analytical trace f o r analytical conditions f o r copper at 3274 ~: dry = 20 s

at 150~ ash = 50 s at 700~ and atomize = 25 s at 2200~
TABLE 1--Standard analytical conditions.

Co Cr Cu Pb Zn
Wavelength (.~) 2407 3605 3274 2833 3074
Ashing temperature. ~ 1000 900 1000 500 5130
Atomizing temperature, ~ 2600 2500 2200 2000 2000
bulk standard mussel material was prepared and analyzed, and then used
as a calibration standard. Throughout this work, "two separate lots of
standard mussel material were used, identified as B1L and B2L (suffix
" L " refers to the species Lampsilis radiata), and these standards were
initially analyzed by standard addition procedures to the solids.
Samples of standard mussel material were spiked with solutions con-
taining appropriate amounts of the elements of interest, and the resulting
mixtures freezedried. Similar techniques for the preparation of spiked
solid samples have been previously described [20]. The spiked solid sam-
ples were analyzed directly, and the concentration of the unspiked ma-
terial calculated by extrapolation. Calibration curves were then prepared
from the standard mussel material, and all subsequent results were
obtained by comparison with these calibration curves. All copper and
chromium results were obtained by comparison with standard B1L ma-
terial while all cobalt, zinc, and lead results were obtained by comparisons
with standard B2L material.
To test the precision of this method, a sample of Orchard Leaves
(National Bureau of Standards, Standard Reference Material 1571) was
analyzed by the same technique. The results obtained on analysis of the
standard mussel samples and on the Orchard Leaves are shown in Table 2.
TABLE 2--Analysis of mussel standards and orchard leaves.

Orchard Leaves
B1L B2L Found CertifiedContent
Co 1.7 -4- 0.4 0.5 + 0.2 (0.2)
Cr 10 ~_'1.0 15 4- 1.3 3.1 -I- 0.5 (2.3)
Cu 37 4- 4 35 -t- 4 15 ..-k2.5 12 -4- 1
Pb ... 3.6 4- 0.6 45 _-k 3 45 _+_3
Zn ... 342 ._+__60 25 _-k.4 25 _-k_3
r~OrE--numbers in parenthesis indicate concentrations not certified.
Background correction was used for all analyses, and for cobalt, very
high background levels were obtained, as it was shown that only about 55
percent of the sample absorbance signal was due to atomic cobalt. For
lead, background levels were approximately 1S percent of sample absorb-
ance, while for copper, chromium, and zinc, background absorbance

levels were 9, 2, and 4 percent, respectively.
Due to the small sample sizes used (1 to 5 mg), sample homogeneity
was investigated to determine the minimum number of replicate analyses
required to perform an acceptable analysis, where acceptability was
defined as the determined result being within _10 percent of the "true"
value, with 95 percent confidence. On this basis it was found that four repli-
cate analyses gave acceptable results, so any single measurement reported is
the mean of four separate determinations; and all concentrations quoted
refer to analysis of the homogenate of the entire animal (excluding shell)
and are expressed on a dry weight basis.
For the purpose of comparing different bodies of water, the study area
was divided into six regions, as shown in Fig. 2. This division attempts to
differentiate between what will be called Great Lakes Waters, which is
really the epilimnetic outflow from Lake Ontario, and near-shore, or
mixed waters. For example, Region C represents waters just from the
Adolphus Reach, while Region B represents Adolphus Reach waters
mixed with Kingston Basin waters entering the North Channel via the
Upper Gap. Region F represents open lake water flowing over the Duck-
Galloo Sill, while Regions D and E represent open Kingston Basin waters,
east and west, respectively. Region A represents a mix of all these waters,
except perhaps those from Region D. In this division, no provision is
made for the variety of substrates in which the animals occur. Mussels are
essentially filter feeders, feeding n,a,~.,~ on the detritus in the water
column [21,22]; and although they may prefer, or will be found in greater
abundance in certain areas, the nature of the substrate will only influence
the distribution of the organisms, but not their feeding habits.
Tables 3 and 4 indicate the distribution of trace elements with region in
the species Lampsilis radiata and Elliptio complanata. Insufficient sam-
ples of Anodonta grandis were collected to make a similar comparison
worthwhile. The three "offshore" Regions, D, E, and F show comparable
levels of trace element inclusion for all elements in both species. This is
not too surprising as these represent samples from locally unaffected
well-mixed waters. Therefore, results from these three regions were
combined, and the median values calculated and called the "Kingston
Basin Mussel Standard." Any subsequent comparisons could then be
made relative to these "standard" values.
From Tables 3 and 4 it can be seen that only the element lead shows
significant differences in concentration with region, as mussels from the
Adolphus Reach contain appreciably lower levels of lead than those from
the Kingston Basin, this difference being more pronounced for the species
Lampsilis radiata than it is for Elliptio complanta. It is tempting to infer
similar trends for some of the other elements, however, although the mean
values indicate trends (for instance, in both species mean cobalt and zinc
TABLE 3--Distribution o f trace elements with region: Lampsilis radiata.
No. of
Region Samples Co Cr Cu Pb Zn
Kingston Harbour (A) 7 1.1 20 32 4.2 253

0.6-1.0 12-27 14-76 3.1-4.9 99-333
North Channel (B) 11 0.9 15 38 1.6 198
0.6-1.9 11-21 14-49 0.7-2.8 121-343
Adolphus Beach (C) 4 1.4 14 32 1.1 273
1.0-1.8 12-17 25-43 0.5-1.7 220-376
Kingston Basin-- 8 1.1 16 34 4.5 205
east (D) 0.8-1.5 12-21 22-56 2.9-6.4 166-246
Kingston Basin-- 12 1.2 19 39 4.9 239
west (E) 0.9-1.6 13-24 20-70 2.4-6.4 156-246
Duck-Galloo Sill (F) 6 1.1 21 34 4.4 223
0.6-1.4 15-28 22-56 2.9-6.7 170-254
Kingston Basin 26 1.1 19 36 4.7 225
Mussel Standard 0.6-1.6 12-28 18-70 2.4-6.7 153-346
NOTE~AII results are expressed as ppm and are given as means and range, except for the
Kingston Basin Mussel Standard, which is given as median and range.
TABLE 4--Distribution of trace elements with region: Elliptio complanata.
No. of
Kingston Harbour (A) 6 1.2 32 33 2.9 138

0.6-1.8 16-88 21-45 1.9-3.1 88-310
North Channel (B) 11 1.5 34 37 2.3 181
0.3-2.8 12-49 20-78 1.0-3.9 96-306
Adolphus Beach (C) 3 1.6 37 35 1.6 216
1.5-1.7 29-61 18-47 1.3-2.1 140-333
Kingston Basin-- 7 0,8 43 36 3.3 110
east (D) 0.6-1.2 24-82 26-46 1.8-4.2 63-150
Kingston Basin-- 11 1.0 47 44 3.2 117
west (E) 0.3-3.1 32-75 29-56 1.8-5.9 71-245
Duck-Gallo Sill (F) 6 0.7 32 43 3.2 117
0.2-1.3 18-45 30-70 1.4-5.3 74-199
Kingston Basin 24 0.9 41 42 3.2 119
Mussel Standard 0.2-3.1 18-82 26-70 1.8-5.9 71-245
NOTE--All results expressed as ppm on dry weight and are given as mean and range, except
for the Kingston Basin Mussel Standard, which is given as median and range.
contents are higher in the Adolphus Reach than in the Kingston Basin),
concentration ranges in all locations are great and overlap to a large
extent. Only a fairly intensive sampling program and statistical evaluation
of results would confirm any such small trends, and a program of this
kind appears to have no immediate merit.
From the values for lead, the effects of the inflow of Kingston Basin
waters into the Upper and Lower Gaps are clearly seen. Mussel samples
from the Adolphus Reach (C), the North Channel (B), and the Kingston
Harbour (A) show progressively higher lead concentrations, with the
concentration in Kingston Harbour mussels closely approaching that of
the Kingston Basin Standard. Thus, the level of any contaminant (or lack
thereof) in Adolphus Reach waters is rapidly diminished by dilution with
Kingston Basin waters, so that no significant c h e m i c a l effect on the St.
Lawrence river by the Adolphus Reach would be anticipated, provided the
level of any input is not overwhelmingly excessive.
During 1973, a limited mussel sampling program was undertaken,
because analysis of the previous year's collection was still underway. Nine
separate locations were sampled and are shown in Fig. 2 as sampling
points 60 through 69. As can be seen from the locations, samples were
collected from only three of the six regions, and at sampling locations,
only the species L a m p s i l i s radiata was analyzed. However, results are
interesting and worthy of comparison, and are given in Table 5. Once
again, the same variation in lead content with location was found to
occur, with mussels from the Kingston Basin having significantly higher
lead levels than mussels from inshore regions. With only four results
available from which to calculate means, levels of the other four elements
are considered comparable to 1972 results, although cobalt levels do
appear to be consistently higher in all samples collected during 1973.
No previous measurements of trace elements in these two species are
available with which these results can be compared, and so no sound
comment can be made as to whether these values represent pristine, or
TABLE S--Trace elements in Lampsilis radiata, 1973.

No. of
Kingston Harbour (A) 3 2.0 8.3 51 3.9 303
1.2-3.0 7.0-9.6 47-55 3.1-4.4 228-345
Kingston Basin-- 4 1.5 15 40 4.7 269
west (E) 1.0-1.9 11-20 22-57 3.4-5.8 214-304
North Channel (B) 2 2.1 14 36 1.3 286
1.8-2.3 9-19 25-47 1.1-1.5 235-337
NOTE--All results expressed as means and range.
natural levels, or whether they evidence significant levels of contamina-

tion. Only future measurements and comparisons will be able to clarify
this point.
It is common to express results of trace element measurements such as
these, as a concentration factor of the organism relative to the water in
which it lived. However, such water analyses usually express concentra-
tions of a filtered sample and on a volume basis, and it is considered that
the inferences drawn from the high concentration factors that are obtained
when using these numbers, especially when considering filter feeders, can
be misleading.
Studies of the feeding habits of UNIONACAE have shown that these
organisms feed primarily on the particulate matter in the water [21,22], a
well-accepted fact. Hence, any trace element variations in the molluscs
directly reflect trace element differences especially in the particulate
material of a water body; and for adequate assessment of water quality,
these differences must then be related to the total concentration of any
given element in the water.
For instance, it has been shown by Stiff [23] that in fresh waters,
copper may be found associated with suspended solids or in different
soluble chemical states, and that in waters other than those that are oligo-
trophic, particulate copper (material retained by a 0.45 /gn filter) con-
tributes 60 to 90 percent of the total copper in a sample. Riley has shown
a similar distribution of copper to exist in some lakes in Connecticut [24].
Any colloidal material (which may be defined as having a size range from
1 nm to 1 Wn) present in water would be a natural scavenger for
adsorption and inclusion of metals ions, particularly those, like copper,
which form stable complexes, implying that the copper associated with
particulate and colloidal material would represent an even greater fraction
of the total copper present than only that which does not pass through a
0.45/am filter.
The amount of particulate matter in most waters is small, and estimates
for levels of particulate material in Lake Ontario vary from 0 to 8/ag/liter for
organic seston, and a median of 2 to 3 mg/liter for nonfiltrable
residue [2]. So if much of the copper in a water sample is associated with
this particulate material, it would be expected that the copper concentra-
tion of the particulate material itself would be significantly large.
Some results obtained in studies of the seston content of the water, and
in particular, its phytoplankton content, conducted in these laboratories
and reported elsewhere [25], lend support to this view.
During 1973, water samples were collected in the North Channel off
Collins Bay, Position P as shown in Fig. 2, at 5-m and 20-m depths,
and seston material isolated from these samples. To a 3-liter volume
of each water sample was added Lugol's solution to stop any fur-
ther growth of organisms; the solution was then dialyzed to remove ions
L O R D ET AL ON T R A C E E L E M E N T S IN M O L L U S C S 107
and molecules in solution; then flash evaporated to reduce sample volume

to about 100 ml; and finally freezedried to obtain a dry "seston" sample.
Copper and chromium contents of these samples were measured by
flameless AAS, using the solid technique described, and results for the
period May to October are shown in Fig. 5, concentrations being
expressed on a volume basis.
O Cr content of sample collected at 5 m

Q Cr content of sample collected at 20m
3
._
r I
,~ 80
60
MAY JUNE JULY AUG. SEPT. OCT. 1973

Sampling Dole
FIG. 5--Trace elements at Seston, North Channel 1973. Samples collected at Position P
[see Fig. 2].
For both copper and chromium, it can be seen that there is quite a
pronounced fluctuation in trace element levels, in what appears to be a
pulse-like form. Also, deeper samples generally contained slightly higher
levels of copper than did the 5-m samples, while the reverse occurred for
chromium. But more pertinent to this discussion are the absolute levels of
trace elements that were found, with concentrations being expressed as a
function of the mass of the particulate material separated, rather than as
a function of the volume of water containing this particulate material. For
instance, for the samples collected on 25 July, 0.156 g of solid material
was isolated from a 3-liter volume of the sample taken at a depth of 5 m.

Analysis for copper and chromium indicated levels of 594 and 41 ppm,
respectively. This then represents the composition of the food that mussels
ingest. So in this respect, the mussels have no choice but to accept any
high concentrations of trace elements that are present in particulate
matter; and certainly for the elements copper and chromium, it would
appear that a major contribution to the concentrations of these trace
elements in mussels is made through the high level of occurrence of these
elements in the particulate material on which the mussels feed.
This is not a new concept. It has been suggested by Goldberg [26] that
the three main routes by which trace elements are included in organisms
are by (a) preconcentration in the food chain, (b) ingestion of particulate
material, and (c) complexing of metals by coordination into a physiologi-
cally active system, while Brooks et al [I0] in a study on trace element
levels in marine bivalves, concluded that particulate ingestion of absorp-
tion constituted a significant factor to the enrichment of trace elements
within the molluscs studied.
However, both authors, whose works are worthily Considered "classic"
in the field of trace element uptake by organisms, believe that direct
complexation and incorporation into the organism constitutes the prime
mechanism of trace metal enrichment in bivalve molluscs, though neither
of these authors, nor anyone subsequently, has investigated the chemical
composition of the principal food of these organisms.
Based on the results of this study, in particular, those of chromium and
copper levels in seston, it is felt that these original ideas on enrichment
should be expanded, such that it be considered that trace element
enrichment in bivalve molluscs takes place by a two-step mechanism: the
first being the prior preconcentration of these elements in the particulate
material (that is, seston) in the water column, followed by ingestion of this
particulate material by the organism with subsequent incorporation into
its body. Hence chemical properties of an element, in particular, those of
transition elements such as tendency to form stable complexes and
insoluble hydroxides, will favor preeoncentration into the particulate
material, with subsequent high concentrations being observed in organ-
isms feeding on this material. Additional studies on trace element levels
in seston being conducted by Queen's University, will help to further
clarify some of these ideas.
Conclusion
In selecting a living organism (that is, species) to monitor any given
chemical system in the water, consideration should be given to three
factors: the natural selectivity of the organism for that chemical system.
the distribution of the species, and the lifestyle of the organism (for
example, sedentary or mobile). Two species of freshwater mussels,
namely, Lampsilis radiata and Elliptio complanta appear well suited to
meet the requirements of a long-term monitor for the trace elements
cobalt, chromium, copper, lead, and zinc, these organisms being in
abundance over most of the Kingston Basin. The concentration of trace
elements into these mussels appears to be achieved by these organisms
acting as both a physical and chemical system, first filtering the water,
and then incorporating chemical species contained in its food into its own
body.
The use of such organisms for monitoring purposes seems sensible, and
worthwhile conclusions may be obtained provided information generated is
viewed as pertaining to the total ecosystem, and not just the organism
being monitored. For instance, to obtain a more complete picture on the
cycling of trace elements in an aquatic environment and to relate changes
in trace element levels in organisms to the organism's environment, a
knowledge of the distribution of trace elements between solid and liquid
phases, and the speciation (especially in the liquid phase) is needed. The
element copper has been well investigated in this regard [23,24], where
studies have shown that in most lucastine environments, copper is
primarily associated with particulate material. The element cadmium is
receiving increased attention, and studies on this element [27,28] have
shown that most of the cadmium in fresh waters is soluble, albeit
complexed, therefore, totally dissimilar in its distribution compared to
copper.
The levels of lead in Great Lakes waters are significantly higher than
lead levels in the nearshore Adolphus Reach waters. These high levels are
probably due to industrial activity. Analysis of dissected mussels has
shown that these higher levels of lead are found throughout the bodies of
mussels, indicating long-term exposure. Levels of cobalt, chromium,
copper, and zinc were comparable between Kingston Basin waters and
inshore waters; and for all elements, no significant variation between
levels measured in 1972 and those measured in 1973 mussels were found.
The direct analysis for trace elements in solid organic material was
found to be a viable technique for the elements cobalt, chromium, copper,
lead, and zinc. Problems were encountered when attempting to determine
cadmium concentrations by this method, as cadmium losses were oc-
curring before complete ashing of solid sample~ had been achieved.
Cadmium analyses are presently being conducted after prior solution of
the samples. This technique should be viewed as no more than an
additional method of conducting flameless atomic absorption analyses, the
major advantages being that the use of solid samples requires little sample
handling and preparation, and contamination possibilities are minimized.
Acknowledgments
The authors would like to thank A. H. Clarke of the National Museum,
Ottawa, for assisting us with sample identification, and D. Brisbin, who
spent a summer helping with sample collection and preparation of the
mussels. The authors also acknowledge with sincere appreciation the help
afforded them at the Canada Centre for Inland Waters by M. Munawar
and N. Watson as well as the valuable assistance of Alexa Morrison and
Carol Wallingford who spent vacation time learning taxonomy for comple-
tion of the work on the system.
Work was supported by provision of a Queen's University Graduate Fel-
lowship, and a National Research Council research grant.
References
[1] Thomas, R. L., Kemp, A. L. W., and Lewis, C. F. M., Journal of Sedimentary
Petrology, Vol. 42, 1972, p. 66.
[2] "Report to the I. J. C. on Pollution of Lake Erie, Lake Ontario, and the International
Section of the St. Lawrence River," 1969, pp. 27-177.
[3] Weiler, R. R. and Chawla, V. K., Proceedings, 12th Conference on Great Lakes
Research, 1969, p. 801.
[4] Mans Impact on the Global Environment (Report of SCEP), MIT Press, 1971.
[5] Beeton, A. M., Limnology and Oceanography, Vol. 10, 1965, p. 240.
[6] Vinogradov, A. P., "The Elementary Chemical Composition of Marine Organisms,"
Memoirs, Sears Foundation for Marine Research, No. II, New Haven, Conn, 1953.
[7] Bryan, G, W., Journal of the Marine Biological Association of the United Kingdom,
Vol, 53, 1973, p. 145.
[8] Mathis, B. J. and Cummings, T. F., Journal of the Water Pollution Contral Federation,
Vol. 45, 1973, p. 1573.
[9] Bertine, K. K. and Goldberg, E. D., Limnology and Oceanography, Vol. 17, 1972,
p. 877.
[10] Brooks, R. R. and Rumsby, M. G., Limnology and Oceanography, Vol. 10, 1965,
p. 521.
[11] Kopfler, F. C. and Mayer, J., Proceedings, National Shellfisheries Association, Vol. 63,
1973, p. 27.
[12] Charke, A. H., Jr. and Berg, C. O., Memorandum 367, Cornell University Agricultural
Experiment Station, Ithaca, N.Y., 1950.
[13] Willis, L P., Endeavour, Vol. 32, 1973, p. 106.
[14] L'Vov, B. V., Pure and Applied Chemistry, Vol. 23, 1970, p. 19.
[15] Headridge, J. B. and Smith, D. R., Talanta, Vol. 19, 1972, p. 833.
[16] Talmi, Y. and Morrison, G. H., Analytical Chemistry, Vol. 44, 1972, p. 1455.
[17] Langmyhr, F. ]. and Thomassen, Y., Zeitschrifl Fuer Analytische Chemie, Vol. 264,
1973, p. 122.
[18l Findlay, W. I., "Preatomization Losses in Flameless Atomic Absorption," presented at
the 4th International Conference on Atomic Spectroscopy, Toronto, 1973.
[19] Manning, D. C., Atomic Absorption Newsletter, Vol. 5, 1973, p. 37.
[20] Jaworski, J. F., Burdo, R. A., and Morrison, G. H., Analytical Chemistry, Vol. 46,
1974, p. 805.
[21] Allen, W. R., Biological Bulletin, Vol. 27, 1914, p. 127.
[221 Allen, W. R., BiologicalBulletin, Vol. 40, 1921, p, 210.
[23] Stiff, M. J., Water Research, Vol. 5, 1971, p. 585.
[24] Riley, G. A., Ecological Monographs, Vol. 9, 1939, p. 56.
[25] Breck, W. G., Lord, D. A., and van Loon, G., "Related Chemical and Biological
Studies of Seston in the Kingston Basin," presented at CIC/CCIW Symposium on
Water Quality Parameters, Burlington, 1973.
[26] Goldberg, E. D. in Chemical Oceanography, ]. P. Riley and G. Skirrow, Eds., Aca-
demic Press, London, 1965, p. VI, Chapter 5.
[27] Gardiner, J., Water Research, Vol. 8, 1974, p. 23.
[28] Gardiner, J., Water Research, Vol. 8, 1974, p. 147.
P. J. K e t a n d R. J. T h i b e r t 2
Analysis of Total Mercury in

Biological and Water Samples by
an Ultrasensitive Kinetic Method*t
REFERENCE: Ke, P. J. and Thibert, R. J., "Analysis of Total Mercury in Biological

and Water Samples by an Ultr~ensitive Kinetic Method," Water Quality Parameters,
A S T M STP 573, American Society for Testing and Materials, 1975, pp. 112-121.
ABSTRACT: A wet digestion has been developed to prepare biological and water
samples for a kinetic determination of total mercury using an iodide-catalyzed reaction
between cerium (IV) and arsenite (III). A mercury-free control, prepared using ion-
exchange with a selective chelating resin, was used by adding mercury standards to
make a calibration curve. Both inorganic and organic mercury can be determined by the
method described either in biological or water samples containing mercury in the range
of 0.05 to 2.0/~g per ml. The procedure can be used satisfactorily down to the 0.05-ppm
level for urine and fresh water with an overall error of less than 5 percent. The method
can also be employed for the determination of mercury in blood serum or seawater with
an error of 10 percent or less and gives results which compare favorably with other
procedures.
KEY WORDS: water quality, mercury (metal), mercury poisoning, kinetics, environ-
mental tests, ion exchanging
M e r c u r y p o i s o n i n g in h u m a n s h a s b e e n k n o w n e v e r since t h e d i s c o v e r y
of metallic mercury. Although organic mercury compounds may be
p r e s c r i b e d as d i u r e t i c s a n d i n o r g a n i c c o m p o u n d s a r e u s e d to t r e a t a
v a r i e t y o f s k i n a i l m e n t s , it is well k n o w n t h a t e x p o s u r e to c e r t a i n o t h e r
f o r m s o f m e r c u r y c a n be f a t a l . T h u s , t h e g r e a t c o n c e r n in t h e m i n d s o f
m a n y p e o p l e w h e n it was d i s c o v e r e d t h a t fish f r o m m e r c u r y - c o n t a m i n a t e d
w a t e r s c o n t a i n e d m e r c u r y in t h e toxic m e t h y l f o r m [1].a M e r c u r y in m a n y
*This work was supported by a grant from the National Research Council of Canada and
1~resented in Mikrochimica Aeta, 1973.
t This paper is taken in part from a doctorate thesis of P. J. Ke, submitted to the
University of Windsor.
~Research Scientist, Department of the Environment, Fisheries and Marine Service, Hali-
fax Laboratory, Halifax, Nova Scotia B3J 2R3, Canada.
2Professor, Head of Clinical Biochemistry Division, Department of Chemistry, University
of Windsor, Windsor, Ontario, Canada.
3The italic numbers in brackets refer to the list of references appended to this papeL
112
9
KE AND THIBERT ON MERCURY 113
forms is known to occur in trace amounts through the environment

[2-4]. The recent findings of high levels of mercury in biological samples
and natural waters have emphasized the importance of the pollution
problems with this heavy metal [1,5]. The clinical implications of mer-
cury toxicity due to the consumption of mercury in food is a well-
documented but poorly understood phenomenon [5-7]. It is still not
known what normal concentrations of mercury might be expected in
our internal and external environment, since there are a lack of gen-
erally accepted and highly sensitive procedures for the determination of
mercury.
Research on the methodology of mercury analysis has been extensively
studied during the past few years. Most of the methods were developed by
utilizing various instrumental techniques. In applying these procedures to
a variety of specimens containing mercury at micro levels, various
difficulties have been experienced. However, it has become possible to
employ kinetic methods in trace analyses in addition to the normal
equilibrium techniques [8-9]. Kinetic-based techniques have several in-
herent features such as an extremely high sensitivity and rapid and con-
venient operation, which would make them useful in the microanalysis of
mercury.
The oxidation of arsenious acid with a ceric salt catalyzed by iodide ion,
had been first studied for the microdetermination of iodine [10] which has
been generally adopted and modified by clinical laboratories for the
determination of protein-bound iodine [11-13]. The interference of mer-
cury in this reaction had been observed by Sandell and Kolthoff [10]. The
first application of the iodide-catalyzed ceric-arsenite reaction for the
analysis of mercury by paper chromatography was reported in 1957 [14].
A highly sensitive kinetic method for the determination of mercury at the
nanogram (rig) level has been developed [15].
The digestion of samples was performed with a mixture of sulfuric and
hydrochloric acids in order to avoid contamination of the final reaction
mixture by other strong oxidants and reductants. Separation of mercury
either by extraction with dithizone [16], diethyldithiocarbamate [17], or
alkylphosphorothioic triamides [18], or by ion-exchange methods using a
weakly basic cellulose [19], or a selective chelating resin [20], cannot be
employed for this study due to the reaction between the thio-containing
reagents from the extraction or the elution, and the reactants in the final
reaction mixture. Thus, a digested sample was simply diluted to eliminate
the interfering ions to non-interfering levels without pre-separation; this
sample can be directly and quantitatively analyzed for mercury by the
kinetic method.
In the present study, a method for the determination of total mercury in
biological and water samples by a kinetic procedure is described.
Experimental
Reagents
Digestion Mixture---The digestion mixture (26 N H2SO4 and 2 N HCI)
was made by mixing 730 ml of concentrated sulfuric acid with 170 ml of
concentrated hydrochloric acid.
Selective Chelating Resin--A mercury selective chelating resin, Srafion
NMRR, was donated by Ayalon Water Conditioning Co. Ltd., Haifa,
Israel. Before use, this chelating resin was soaked for 4 h in 0.5 N HC1.
Mercury Standards
A. Stock Inorganic Hg-Solution IA (200 /ag per ml) was prepared by
dissolving 0.3462 g of mercuric nitrate---Hg(NO3)2. H20, British Drug
House--in deionized water with 10 ml of 3 N nitric acid (HNOa), fol-
lowed by dilution to a volume of 1 liter. This Hg-Solution IA was stand-
ardized by titration with potassium chloride using diphenylcarbazone as
indicator. Stock Inorganic Hg-Standard IIA (2 /ag per ml) and Working
Inorganic Hg-Standard IliA (20 ng per ml) were prepared by dilution as
previously reported [15].
B. Dimethyl mercury, prepared according to Dunlop et al [21], was used
to make the organic Hg-standards. Stock Organic Hg-Solution IB (200 /ag
per ml) was made by dissolving 0.2306 g of dimethyl mercury in a solution
containing 4 ml of N,N-dimethyl formamide, 10 ml of 3 N HNO3 and 10
ml deionized water. Gentle stirring was required until the dimethyl
mercury was completely dissolved. This solution was then diluted to a
volume of 1 liter with deionized water. Stock Organic Hg-Standard liB (2
/ag per ml) and Working Organic Hg-Standard IIIB (20 ng per ml) were
prepared as before. Since mercury salts are slowly adsorbed onto glass,
dilute mercury reagents should be prepared daily.
Other Reagents--Working Ce (IV) Solution (0.005 M), Working As
(III) Solution (0.0005 M) and Working Iodide Solution III (40 ng per ml)
were prepared as previously reported [15].
Apparatus
Spectral absorbance measurements were made with a Beckman Model
DU Spectrophotometer equipped with a Gilford Model 220 Absorbance
Indicator and a Gilford Model 2000 Multiple Sample Absorbance Re-
corder. The cuvette chamber was equipped with a heating jacket and was
maintained at a temperature of 37 _ 0.05~ with a Haake thermostated
circulating water pump.
Sample Preparation
Sample A (river water), Sample B (lake water), and Sample C (city
water) were taken from the Detroit River, Lake Erie, and the City of
Windsor in Ontario, respectively. Samples E and F were seaWater. Sample
E (from Inugua, Bahamas) was obtained from the Department of Biology,
the University of Windsor; Sample F was taken from the seawater supply
inlet of Halifax Laboratory of the Fisheries Research Board of Canada.
All water samples were filtered through W h a t m a n No. 50 filter paper with
suction and stored in polyethylene bottles.
Samples P and Q were pooled human blood serum, obtained from local
hospitals. Samples R and S were urine samples from a local hospital, and
HC1 was used as a preservative. Samples P, Q, R, and S were used
directly.
Procedure
Acidic Digestion
One milliliter of sample and 2 ml of digestion mixture were placed in a
single-neck (T14/20) distillation flask (10 ml). Three acid-washed glass
beads were put in the flask and then a reflux column with condenser was
connected to the flask. The digestion was carried out by heating with a
temperature-regulated hot plate and the mixture was refluxed for 2 h.
After the digested sample solution had been cooled to room temperature,
a few milliliters of deionized water were used to rinse the condenser and
reflux column, and were added directly to the flask. The digestion flask
was then disconnected and the sample solution was transferred to a 25-ml
volumetric flask. Two more rinses of the reflux apparatus were combined
with the sample solution in the volumetric flask. The digested sample
solution was diluted to 25 ml with deionized water.
Preparation of Hg-Free Control--A 2S-ml buret packed with 5 g of a
selective chelating resin (Srafion NMRR) was used to remove the mercuric
ion from the digested sample solution. Some acid-washed glass wool was
placed into the buret to support the chelating resin. The height of the
packed column was approximately 10 cm. The packed resin was washed
with 100-ml deionized water before used.
A blank (1 ml of deionized water) was digested in the same way as the
samples. The first portion (15 to 20 ml) of the digested blank solution (25
ml) was used to prepare the Hg-Free Blank Control by ion-exchange as
previously mentioned. The second portion of the digested blank solution
was directly used for the kinetic determination with the Hg-Free Blank
Control. The difference of absorbances for the blank solution and the
Hg-Free Blank Control was a constant of 0.050 absorbance units with less
than 2 percent error for eight independent determinations. This constant
is a function of the composition of the column and the anions that are
eluted from the column.
The used chelating resin can be regenerated by elution with a 5 percent
aqueous solution of thiourea to which S ml of HC1 have been added per

liter of solution. After elution, the excess thiourea must be completely
washed out with a large quantity of deionized water, usually about S00
ml. However, new chelating resin gives better results for the preparation
of the Hg-Free Controls.
Kinetic Determination--To two sets of eight numbered test tubes (150
by 50 mm) were added, 1 ml of blank (No. 1), 1 ml of blank control (No.
2), 1 ml of sample control (No. 3), 1 ml of sample solution (No. 4), and 1
ml of sample control in each of the next four tubes (Nos. S to 8)
containing added Hg-standard of 0 (No. S), 10 (No. 6), 40 (No. 7), and 80
ng (No. 8). Deionized water may be required to adjust the final volume to
10 ml. Working As(Ill) Solution, Iodide Solution III, and Working
Ce(IV) Solution were used as previously described [15]. The kinetic
measurement was made spectrophotometrically at 275 nm as reported
earlier [15].
Zero time was taken as the time when 1 ml of Working CAS-Solution
was added to the first tube. The reaction mixture (10 ml) was homog-
enized by a Vortex-Genie mixer for a few seconds. A cuvette of 10-mm
light path was rinsed three times, then filled with the reaction mixture,
and placed in the cuvette chamber. The whole operation must be
completed within 50 s. The reactions in the next three test tubes should be
initiated at 1 rain intervals in the same manner. Until the fourth cuvette
was placed in position, the cuvette chamber was covered and the
absorbance changes were automatically recorded at 275 nm for a time
interval of 15 s for each sample. The recorder was stopped at 25 min after
the first reaction was started. The averege of the absorbances for the
samples and controls at 20 rain of reaction time were used to calculate the
differential absorbance. 4
The mercury content of the sample (Table 1) was determined using the
calibration curve as shown in Fig. 1.

The iodide-catalyzed reaction between cerium(IV) and arsenite(III) is a
two-step reaction [22]:
2Ce(IV)) ~2I-~ (As(V)
2Ce(III) I2 As(Ill)
4Differential Absorbance (AA) is the difference in absorbance under a particular set of
conditions [14]. This can be expressed by the following equation:
AA = A s - A s c - 0 . 0 S
where As and Asc denote absorbance for a sample and a sample control, respectively.
TABLE 1--Determination of mercury in a sample of water by a kinetic method.
Sample Hg Added (ng) A AA a
X-I (Blank) 0 0.211

X-II (Hg-Free Blank Control) o o.161 olosob
A-II (Hg-Free Sample Control) 0 O.160 0.000
A-II (Hg-Free Sample Control) 10 0.175 0.015
A-II (Hg-Free Sample Control') 20 O.188 0.028
A-II (Hg-Free Sample Control) 40 0.216 0.056
A-II (Hg-Free Sample Control) 80 0.272 O.112
A-I (Sample Solution) 0 0.231 0.021
a AA (Sample A) = As(A-I) - - Asc(A-II) - - 0.050. All data presented are the average of
triplicate determinations.
OFor seven determinations, this constant is a function of composition of the column and
the anions that are eluted from the column to be 0.050 absorbance units with less than 2
percent error.
ofA
0"15 O 9j O~
<
,q 0.10
0"05
o 26 ' 6b ' I 0'0

Hg ( ng}
FIG. l--Change of differential absorbance (AA) with the mercury content in a synthetic
sample. Curve A = inorganic Hg-sample without the digestion. Curve B = digested
Hg-sample with inorganic (e) or of organic mercury (& ).
Ce(IV) reacts with iodide to form Ce(III) and elemental iodine; elemental
iodine then reacts with As(III) to form iodide and As(V). The iodide ion
thus regenerated can react again with ceric ion. The reduction of the
yellow-colored ceric ion (IV) to the colorless cerous ion (III) is used to
measure the rate of reaction which is dependent on the concentration of
iodide present. The situation becomes more complicated when mercuric
ion is present in the reaction mixture [15,25].
A number of studies of the iodide-catalyzed As(III)/Ce(IV) reaction
have been published [23,24]. The following factors have been of primary
concern with respect to conditions governing the reaction rate: acidity,
temperature, relative concentrations of As (III) and Ce(IV), the effect of
iodide concentration, the selection of the wave-length, and the interference
of diverse ions. Optimum reaction conditions were investigated and
reported previously .[15] to obtain a greater sensitivity in the determination
of mercury.
The kinetic method by the iodide-catalyzed As (III)/Ce(IV) reaction has
been successfully used for the determination of inorganic mercury [15].
Even using the non-catalytic As(III)/Ce(IV) reaction, an inorganic mer-
cury sample can be satisfactorily determined with less sensitivity [25].
Since organic mercury is more important in pollution and clinical investi-
gations, a synthetic organic mercury sample, dimethyl mercury, was used
to test the kinetic method described in this study. Table 2 shows that
inorganic mercury can be determined as well as organic mercury. From
the results listed in Table 2, it should not be surprising that organic
TABLE 2--Kinetic determination of inorganic or organic mercury in a synthetic sample

containing 40 ng of rig (11") after digestion.
Hg(II) Found, ng
Treatment Organic Hg Inorganic Hg
No digestion 9 40.3
Digestion at room temperature 39.8
Digestion with reflux for i h 4blzi a 40.5
Digestion with reflux for 2 h 39.8 a 40.1
a The data presented are the average of duplicate determinations.
mercury before decomposition had no affect on the reaction rate of the

iodide-catalyzed reaction between arsenite(III) and serium(IV). However, a
2-h acidic digestion can completely decompose the organic mercury
compounds to form this inorganic mercuric which can be determined by
this method (see Fig. 2).
An extraction with dithizone and its derivatives [I6-18], has been used
for spectrophotometric estimation of the mercury content of various
samples. Hopefully, ion-exchange by a selective chelating resin at low pH
[20] may be employed to modify some instrumental analyses of mercury in
various materials. However, those separation techniques cannot be applied
for the present kinetic determination of mercury due to the reduction of
ceric ion (IV) in the reaction mixture by the thio-containing reagents
which should be used as an extractant or elutant. In fact, there was no
separation operation used in the proposed method for the determination
of mercury in natural waters and biological samples. A simple dilution
can eliminate interferences to a working level at which mercury can be
[
i.SI/o~O--O v ~ B
E l'Ot~,~ -~ -" " " -" A
~ 0.5
0
DIGESTION HOUR
FIG. 2--The completion of acidic digestion for water and serum samples with added
organic and inorganic mercury. Curve A = A sample contains 1 ml of river water, 0.5 lag o f
btorganic Hg and 0.5 tag o f organic Hg. Curve B = A sample contains 1 ml of serum, 0.5 lag
of inorganic Fig and 1.0 tag of organic Fig.
determined by the kinetic method in the range from 0.05 to 2.0/ag per ml
of sample. In this determination, a Hg-free control prepared by means of
ion-exchange with a selective chelating resin [20] was used to make the
standard curve. The results of the determination of mercury in a natural
water are presented in Table 1.
Recovery investigations were made for organic and inorganic mercury
added to the water or biological samples, and the results are shown in
Table 3. It is believed that the described procedure can be satisfactorily
used for the determination of both inorganic and organic mercury in the
water and biological samples. The incomplete recovery of the added
mercury in seawater as well as in serum may be due to some unexpected
interfering substances which are present in the sample.
By using the proposed procedure, a n u m b e r of samples of biological
and water materials have been analyzed for mercury content. The
accuracy and reproducibility of the kinetic method are listed in Table 4.
Errors less than 5 percent for fresh water and urine, and 10 percent for
TABLE 3~Recovery o f mercury added to the water and biological sample using a kinetic
determination with acidic digestion.
Hg Added (~g)
Sample Inorganic Organic % Recovery

A (river water) 0.50 ... 103.2
C (city water) 1.00 0.50 95.8
F (seawater) 1.00 0,50 91.7
P (serum) 0.50 0.50 90.6
R (urine) 0.50 101.7
S (urine) 1.00 0.~2 95.1
TABLE 4--Kinetic determination of total mercury in biological and water samples.
Sample No. of Determinations Hg-Content,/ag/ml R.S.D.%
A (river water) 7 0.350 4.6

B (lake water) 7 0.306 5.2
C (city water) 6 0.090 5.8
E (seawater) 5 0.081 7.6
F (seawater) 5 0.192 9.6
P (serum) 7 0.095 10.5
Q (serum) 5 0.116 8.4
R (urine) 6 0.091 6.2
S (urine) 2 0.052 ...
seawater and serum, can be expected for a sample containing 0.05 to 2.0
/ag of mercury per milliliter. The proposed method can be successfully
employed in the determination of total mercury in urine and fresh water;
the level of mercury poisoning is accepted to be 0.5 /ag per ml for water
and 0.2 /ag per ml for urine [26]. Even for blood serum or seawater, the
kinetic determination of mercury can be recommended for a quantitative
analysis.
References
[1] Bligh, E. G., Canadian Institute of Food Technology Journal, Vol. 5, 1972, p. A6.
[2] Klein, R. and Herman, S., Science, Vol. 172, 1971, p. 872.
[3] Bache, C. A., Gutemann, W. H., and Lisk, D. J., Science, Vol. 172, 1971, p. 951.
[4] Curley, A., Sedlack, V. A., Girling, E. F., Hawk, R. E., and Barthel, W. F.,
Science, Vol. 172, 1971, p. 65.
[5] Hammond, A. L., Science, Vol. 171, 1971, p. 788.
[6] Goldwater, L. J., Scientific American, Vol. 224, 1971, p. 15.
[7] Kevorkian, J., Cento, D. P., Uthe, J. F., and Hagstrom, R. A., American Journal of
Public Health, Vol. 63, 1973, p. 931.
[8] Yatsimirskii, K. B., Kinetic Method of Analysis, Pergamon Press Ltd., New York,
1966.
[9] Mark, N. B., Jr. and Rechnitz, G. A., "Kinetics in Analytical Chemistry," Chemical
Analysis, Vol. 24, 1968.
[10] SandeU, E. B. and Kalthoff, 1. M., Journal of the American Chemical Society, Vo[. 56,
1934, p. 1426.
[11] Zak, B., Willard, H. H., Myers, G. B., and Boyle, A. J., Analytical Chemistry,
Vol. 24, 1952, p. 1345.
[12] Foss, O. P., Hankers, L. V., and van Slyke, D. D., Clinica Chimica Acta, Vol. 5,
1960, p. 301.
[13] Ke, P. J. and Thibert, R. J., Mikrochimica Acta, 1973, p. 569.
[14] Braun, T. E., Mikrochimica Acta, 1957, p. 128.
[15] Ke, P. J. and Thibert, R. J., Mikrochimica Acta, 1972, p. 768.
[16] Official Methods of Analysis. 10th ed., Association of Official Agricultural Chemists,
Washington, D.C., 1965, p. 375.
[17] l-lakkila, E. A. and Waterbury, G. R., Analytical Chemistry, Vol. 32, 1960, p. 1340.
[18] Handley, T. H., Analytical Chemistry. Vol. 36, 1964, p. 2467.
[19] Kuroda, R., Kiriyama, T., and Ishda, K., Analytica Chimica Acta, Vol. 40, 1968,
p. 305.
[20] Law, S. L., Science, Vol. 174, 1971, p. 285.
[21] Dunlop, A. N., Kominar, R. J., and Price, S. J., Canadian Journal of Chemistry,
Vol. 48, 1970, p. 1269.
[22] Pileggi, V. J. and Kessler, G., Clinical Chemistry, Vol. 14, 1968, p. 339.
[23] Chaney, A. L., Advances in Clinical Chemistry, Vol. 1, 1958, p. 81.
[24] Meyer, K. R., Dickenman, R. C., White, E. C., and Zak, B., American Journal of
Clinical Pathology, Vol. 25, 1955, p. 1160.
[25] Ke, P. J. and Thibert, R. J., Mikrochimica Acre, 1973, p. 15.
[26] Shandar, A. and Simson, R. E., Medical Journal of Australia, Vol. 2, 1971, p. 1005.
Om P. Bhargava,' G. W. Deline,' and W. G, Hines'
Rapid Determination of Cyanide in

Waste Waters
REFERENCE: Bhargava, Om P., Deline, G. W., and Hines, W. G., "Rapid Deter-
minatlon of Cyanide in Waste W a t c h , " Water Quality Parameters, ASTM STP 573,
ABSTRACT: A simple, direct, and rapid method is presented for the determination of
cyanide in waste waters employing a selective ion electrode. The method can tolerate up
to 100 ppm of sulfide in the sample and incidentally detects sulfide down to 0.05 ppm.
The range of the method is 0.03 to 100 ppm cyanide and a single determination can be
completed in about 3 min including the filtration of sulfide, if present, in the sample.
The method has been in routine operation for about three years.
KEY WORDS: water quality, cyanides, waste water, environmental tests, electrodes
The determination of cyanide in effluent waters (blast furnace thickener

overflow, open cut sewage, coke oven operations, etc.) in our plant, was
formerly carried out by a distillation plus titration technique. The
tolerance level of cyanide in these samples is 0.1 ppm. At this level of
cyanide the titrimetric method is not very accurate, as it is at the limit of
its detectability. Also, this method is very time-consuming, taking 3 to 4 h
per sample.
The reagent p-phenylenediamine z is used for the absorptiometric deter-
mination of cyanide in water. The initial treatment involves acidification
to liberate hydrocyanic acid followed by addition of bromine to form the
active species (cyanogen bromide) which forms a color complex with
p-phenylenediamine. The method is quite sensitive---down to 0.005 ppm.
However, thiocyanate ion (present in some effluents) also reacts under the
conditions of the procedure, and thus it is not suited for routine applica-
tion. Some four years ago the availability of a cyanide ion selective
electrode, coupled with the simplicity of the approach, attracted our
attention.
'Supervisor, Analytical Chemistry, chemical technologist, and general supervising metal-
lurgist, respectively, Metallurgical and Chemical Laboratories, The Steel Company of
Canada, Ltd., Hamilton, Ontario LSN 3T1, Canada.
2Bark, L. S. and Higson, H. G., Talanta, Vol. 11, 1964, p. 621.
1 22

BHARGAVA ET AL ON CYANIDE IN WASTE WATERS 123
Investigations were carried out with an Orion ion-selective electrode

which is reported to detect cyanide down to 0.03 ppm. The sensing
element of the Orion cyanide ion-selective electrode is a membrane layer
which is composed of a mixture of silver sulfide and silver iodide. The
electro-chemistry of the reaction at the electrode involves a precipitate ion
exchange reaction
2 CN- + AgI --" Ag(CN)2- + I-
at the boundary phases of the membrane layer and responds to cyanide

activity according to the Nernst equation
RT
E = E 0 - 2.3 - - ~ log aCN-
where the slope - 2 . 3 ( R T / F ) at 25~ is about 59 mV for a ten-fold

increase in cyanide level, acN- represents the activity of the ion in solu-
tion. The theory, material, and design have been discussed and reviewed,
among others by Durst,3 Riseman 4 and Toth and Pungor.S
At high concentrations of cyanide there is an excessively rapid corrosion
of the electrode sensing element, thus there is a limited range in which the
electrode can be used. The suggested range is stated as 10 -2 M (260 ppm)
down to 10 -6 M (0.026 ppm). Depending on the cyanide concentration of
the samples the electrode should last for 200 to 1000 h; at levels below 10
ppm it should last as long as 1000 h. To ensure minimum corrosion of the
sensing element, the range used in our investigations was 100 ppm cyanide
down to 0.03 ppm cyanide.
The selective ion electrode does not measure concentration of cyanide
directly; rather it determines the activity 4 of the ion in solution, that is,
ion in the unbound form CN-. The behavior of the cyanide ion is affected
by the presence of other ions, even if they do not complex the cyanide ion
or interfere with the response of the electrode. The activity is a function of
the total ionic strength of the solution. Concentration measurements can
be made on samples that vary widely in the total ionic strength by
preparing both samples and standards with a solution containing a high
level of noninterfering ion. This solution, called an ionic strength adjustor,
swamps out variations in the original total ionic strength of the samples.
Cyanide is subject to hydrogen complexation below pH 11, so the ionic
strength adjustor used must also free cyanide bound to hydrogen for
concentration measurement. At a pH of 12 virtually all the cyanide is in
the unbound form CN-. Therefore, all standards were prepared in 1 M
~Durst, R. A., NBS Special Publication 314, 1969.
4Riseman, J. M., American Laboratory, 1969, p. 133.
SToth, K. and Pungor, E., Ana!ytica Chimica Acta, Vol. 51, 1970, p. 221.
sodium hydroxide to raise the pH of the sample as well as to act as an

ionic strength adjustor.
Experimental
The investigations were made by using an Orion cyanide ion electrode
94-06 in conjunction with an silver-silver chloride (Ag-AgCI) reference
electrode, on a Sargent Digital pH meter. A Fisher Scientific magnetic
stirrer with micro polyethylene-clad bar magnet was used for stirring.
Potassium cyanide standard main Solution A containing 1000 ppm of
CN- was prepared by dissolving 2.505 g KCN and 40.0 g NaOH in water
and diluting the contents to 1 liter. This solution is stable for one month
and is stored in polyethylene bottles. A working standard Solution B
containing 10 ppm CN- was prepared by dissolving 40 g NaOH in water,
adding 10.0 ml of the main standard Solution A and diluting to 1 liter
with water. This was prepared fresh each day and stored in polyethylene
bottles.
Calibrations and Procedure

To a series of 100-ml volumetric flasks were pipetted 10 ml and 1 ml of
standard Solution A and 10, 1, 0.7, 0.5, and 0.2 ml aliquots of Solution B
providing 100, 10, 1, 0.1, 0.07, 0.0S, and 0.02 ppm cyanide solutions,
respectively. After adding 1 ml of S percent lead nitrate solution (for
reasons outlined later) to each of the volumetric flasks, the volumes were
made up to the mark with 1 M sodium hydroxide solution and transferred
to 150 ml polyethylene beakers.
While stirring the solution with the magnetic stirrer, the potential
measurements in millivolt were made with the cyanide ion-selective
electrode and Ag-AgCl electrode as reference, on the Sargent Digital pH
meter.
A calibration curve was plotted on semilog paper: concentration
(cyanide ppm) on the logarithmic axis and the potential (millivolts) on the
linear axis. The resultant relationship is linear.
Test samples were made 1 N in sodium hydroxide by dissolving 4 g of
NaOH pellets in 100 ml of sample. After adding 1 ml of S percent lead
nitrate solution, to precipitate sulfides if any, a portion of the test solution
was filtered. The electrode response in millivolts was then measured like
the calibration standards and concentration of cyanide read from the
graph.
Effect of Variation in N a O H Concentration
The sodium hydroxide concentration used in the method developed is

1 M. A study was carried out to determine how critical the NaOH concen-
tration was. The samples used contained 1.35 and 4.4 ppm cyanide, re-
spectively. The results in Table 1 show that a change of 0.1 M concentra-
tion in NaOH results in a potential change of I mV which is equivalent to
a 2 to 5 percent change in cyanide concentration. It can therefore be seen
that a 0.1 M change in NaOH concentration is significant. For precise
TABLE l--Effect o f NaOH concentration on cyanide determination.
NaOH Sample 1 Sample 2

M mV ppm mV ppm
0.8 825 1.40 853 4.8

0.9 825 1.40 852 4.6
1.0 824 1.35 851 4.4
1.1 823 1.30 850 4.3
1.2 822 1.25 849 4.2
work it is important to control NaOH concentration within 0.05 M. In our

most recent investigations it was found that 0.1 N NaOH concentration is
quite adequate to act as an ionic strength adjuster as well as to release the
cyanide ion, provided both the standards and samples are 0.1 N in sodium
hydroxide.
Effect of Sulfide
Sulfides in the sample solution will poison the cyanide electrode. To
overcome this problem lead nitrate was added to precipitate any sulfide
present. The sulfide precipitate initially was not filtered before the
potential measurement. It was observed that the sulfide, although it
appeared to be completely precipitated, still interfered with the cyanide
determination yielding extremely high results. It was found that filtration
to remove the sulfide precipitate prior to the potential measurement was
effective in eliminating the problem.
The extent of the effect of sulfide concentration in the solution from 1
to 100 ppm on the determination of cyanide in the range 0.1 to 100 ppm
was investigated. After adding the sulfide to the test solution, it was then
precipitated with lead nitrate and filtered before potential measurement,
as specified in the procedure. Table 2 shows that the treatment was
effective in overcoming the disturbing effect of any sulfide originally in a
sample.
Since lead nitrate is added to both standards and samples to precipitate
sulfide, the possibility of interference from rather excessive amounts of
lead nitrate was studied. It was found that there is no appreciable change
in potential of the test solutions up to 2500 ppm of lead nitrate.
TABLE 2--Effectiveness of lead nitrate in offsetting sulfide interference.
CN- Found (ppm)

Sulfide Added (ppm)
(followed by lead nitrate treatment)
CN- Present (ppm) 1 10 50 100
0.10 0.10 0.10 0.13 0.10

1.0 1.0 1.0 1.0 1.0
10.0 10.1 10.1 10.0 10.5
Effect of Thiocyanate, Thiosulfate, and Ferrocyanide

Effluent waters f r o m coke oven c o n t a i n traces o f t h i o c y a n a t e s a n d
thiosulfates. T h e r e was a r e m o t e possibility of some traces of ferro-
cyanides. T h e effects of these ions both individually a n d collectively were
investigated. T h e results are shown in T a b l e s 3 a n d 4.
TABLE 3---Effect of thiocyanate.
CN- Found (ppm)

Thiocyanate Added (ppm)
CN- Present (ppm) 11.6 41.5 70.5 99.5
0.026 0.026 0.027 0.028 0,029

0.26 0.26 0.26 0.26 0,26
2.6 2.6 2.4 2.4 2.4
Z6.0 26.0 26.0 25.0 25,0
TABLE 4---Effect of thiosulfate.

CN- Found (ppm)
Thiosulfate Added (ppm)
CN- Present (ppm) 12.3 36.9 73.8 98.4
0.26 0.26 0.26

2.6 2.4 2.4
26.0 26.0 26.0 25.0 25.0
It is evident f r o m the results t h a t u p to 100 p p m each of t h i o c y a n a t e

a n d thiosulfate do not interfere in the investigated r a n g e of cyanide
concentration.
T h e test solutions (Table 3 a n d 4) were t r e a t e d with up to 106 p p m of
b o t h ferro and f e r r i c y a n i d e ion. No interference was f o u n d .
Precision
A precision study was c a r r i e d o u t with several aliquots from t h e s a m e
parent solution. Table 5 shows that the electrode gives reproducible results
with a precision of _+5 percent of the cyanide concentration.
TABLE S---Precision tests.

Sample mV CN- (ppm)
Standard 1 ppm 820 1.1
Thickener 856 5.4
Thickener 856 5.4
Thickener 855 5.3
Thickener 854 5.2
Open Cut 839 2.7
Open Cut 839 2.7
Open Cut 839 2.7
Open Cut 840 2.7
Recovery (Accuracy)
Several effluent water samples were prepared, and a standard addition
(3 ppm) of cyanide was made to each. The potentials of these solutions
were measured before and after the addition. Table 6 demonstrates that
the cyanide recovery is satisfactory ranging from 90 to 100 percent.
TABLE 6--Recovery tests.

CN- Present CN- Added CN- Found
Sample (ppm) (ppm) (ppm) % Recovery
1 1.3 3.0 4.0

2 4.0 3.0 7.0 1~
3 0.4 3.0 3.3 97
4 3.8 3.0 6.7 97
Conclusions
A simple direct and rapid method has been devised for the determina-
tion of cyanide in effluent waters employing a cyanide selective electrode
in conjunction with a pH meter. The method can toley.ate up to 100 ppm
of sulfide in the sample and incidentally detects sulfide concentration
down to 0.05 ppm. Thiocyanates, thiosulfates, ferrocyanides, and ferri-
cyanides up to 100 ppm do not interfere. The range of the method is from
0.03 to 100 ppm cyanide, and a single determination can be completed in
about 3 min including the filtration of sulfide precipitated from the
sample. The method has been in routine operation for over three years.
Om P. Bhargava, 1 A. A. Schuldt, 1 and W. G. Hines ~
Rapid Determination of Fluoride in

Hydrochloric Acid, Pickle Liquor,
and Gaseous Emissions
REFERENCE: Bhargava, Om. P., Schuldt, A. A., and Hines, W. G., "R~id Deter-
mination of Fluoride in Hydrochloric Acid, Pickle Liquor, and Gaseous Emissions,"
Water Quality Parameters, ASTM STP 573, American Society for Testing and Mate-
rials, 1975, pp. 128-135.
ABSTRACT- Rapid methods for the determination of fluoride in hydrochloric acid,
pickle liquor for hydrochloric acid regeneration systems, and gaseous emissions from
steelmaking slags, using a fluoride ion-selective electrode, are presented. A single deter-
mination down to 1 ppm including standardization can be completed in hydrochloric
acid in about S rain permitting easy monitoring of incoming shipments. Fluoride con-
tents in the gaseous emissions down to 0.01 ppm are readily determined after absorption
in sodium hydroxide solution.
KEY WORDS-"water quality, fluorides, hydrochloric acid, chemical cleaning, environ-
mental tests, slagging
The traditional sulfuric acid pickling of steel results in spent acid as

well as ferrous sulfate, both posing disposal problems and contributing
to pollution. The switch to hydrochloric acid (HCI) pickling which permits
subsequent regeneration has resulted in overcoming this pollution prob-
lem. (The by-product, ferric oxide, is obtained in a high state of purity,
suitable for use as a pigment, or increasingly in the m a n u f a c t u r e of
magnetic tapes.) However, if contaminated with fluoride, HCI is detri-
mental to various fittings and components used in the HCI regeneration
plant. Concentration of fluoride must be limited to less than 5 p p m if
catastrophic corrosion is not to occur. As a safeguard the m a x i m u m limit
to fluoride level in the incoming fresh commercial HC1 shipments has been
set at 2 ppm. The HCI shipment in the t a n k car, etc., has to wait for the
fluoride analysis before it can be unloaded. This, therefore necessitates a
rapid method for fluoride determination in fresh HC1.
Besides this problem, there is our ever present concern for environ-
' Supervisor, Analytical Chemistry, senior utilities engineer, and general supervising metal-
lurgist, respectively, The Steel Company of Canada, Ltd., Metallurgical & Chemical Labs.,
Hamilton, Ontario LSN 3TI, Canada.
128

BHARGAVA ET AL ON RAPID DETERMINATION OF FLUORIDE 129
mental pollution as a result of the use of fluorspar as a fluxing constituent

in slags produced during steelmaking. While we have taken significant
steps to reduce the problem, the need for analytical monitoring of the
local atmosphere for fluoride also exists.
A direct colorimetric method using zirconium alizarol cyanine chelate
was investigated prior to the availability of a fluoride ion-selective elec-
trode. The time required for the color development was 1 h. In addition,
the method suffered from lack of sensitivity. The sensitivity was improved
by taking a large sample, fixing the fluoride by treatment with aluminum
and preconcentration by taking to dryness. The fluoride was then sepa-
rated as fluosilicic acid by a Willard-Winter ~ steam distillation. Treat-
ment of an aliquot of the condensate with zirco~yl-alizarin chelate 3
followed, and the absorbance was measured after 2 h. The sensitivity and
precision of the method were both acceptable. However, the method was
quite time-consuming (taking at least 4 h) and hence was impractical for
routine control purposes.
Some five years ago the availability of an Orion fluoride ion-selective
electrode and the minimal manipulation and its applicability to our
problem led us to investigate and to devise a rapid method for the
determination of fluoride in HCI.
The use of a fluoride ion-selective electrode is quite wide-spread in
determining fluoride in various materials. However, the determination of
this element in HCI or pickle liquor has not been reported to our
knowledge, and there was a need, therefore, to confirm the promised
efficacy of this method on the actual materials to be monitored, especially
where high concentration of iron could occur as in the pickle liquor.
The sensing element of the Orion fluoride ion-selective electrode is a
lanthanum fluoride single crystal membrane which separates an internal
filling solution from the sample solution. The lanthanum fluoride single
crystal responds selectively to the fluoride ion activity according to the
Nernst equation
RT
E ---- E o - 2.3 - - ~ log A~-
where the slope --2.3 ( R T / F ) at 25~ is about 59 mV for a ten-fold

increase in fluoride level.
The activity is a function of the total ionic strength of the solution and
of the formation of complexes. The normal variation in ionic strength
from sample to sample can be overcome by adding an excess of a buffer
which adjusts the ionic strength. This serves the dual purpose of releasing
2Willard, H. H. and Winter, O. B., Industrial and Engineering Chemistry, Analytical
Edition. Vol. 5, 1933, p. 7.
3Ashley, R. P., Analytical Chemistry, Vol. 32, 1960, p. 834.
the fluoride from its hydrogen complex at pH levels of 5 or above,

preferably near 7. Interference from ferric and aluminum ions can be
obviated by complexing them with tartrate or citrate.
Sometimes it is not possible to analyze the sample immediately. The
sample has to be stored, for example, the determination of fluoride in
gaseous emissions. The gaseous stream is passed through a sodium
hydroxide solution both to absorb and to stabilize fluoride. However, the
hydroxyl ion interferes with the fluoride determination since the electrode
responds also to hydroxyl ion at pH 9 or above. At pH 7 and below,
however, the hydroxyl ion concentration is 10-7 M or less and no
detectable interference occurs. Anions such as carbonates and phosphates
do not directly interfere with the electrode response to fluoride. They do,
however, make the solution basic, increasing the hydroxide concentration.
Adjusting the pH to 5 eliminates this interference. At this pH, anions
commonly found with fluoride, such as CI-, Br-, I-, SO4=, NO3-, PO4-,
acetate, and citrate, even when present in excess of a thousand-fold or
more, can be tolerated.
Experimental
The investigations were carried out using an Orion fluoride ion-selective
electrode 94-09 in conjunction with a silver/silver chloride (Ag-AgCl)
reference electrode, on a Sargent Digital pH meter.
A magnetic stirrer with a micro polyethylene clad bar magnet was used
for stirring.
Reagents
All reagents were of analytically pure grade. Distilled water was used
throughout and reagents stored in polyethylene bottles.
1. Hydrochloric Acid: sp gr 1.19 (free from fluoride).
2. Sodium Hydroxide Solution 1 N: dissolve 40 g NaOH in water and
dilute to a liter.
3. Sodium Acetate Buffer: dissolve 500 g of CH3COONa" 3H20 and
dilute to a liter with water.
4. Potassium Acetate Buffer: make 4 M acetic acid solution by diluting
283 ml of glacial acetic acid to 1200 ml with water. While cooling
and stirring add 50% w/v KOH solution to adjust the pH to 5.0.
(Approximately 350 ml of KOH solution are required.)
5. Ammonium Citrate Buffer: dissolve 11.3 g of (NH4)2HC6I-IgO7 in
water and dilute to a liter with water.
6. Ammonium Hydroxide: sp gr 0.88.
7. Iron Solution: dissolve 10.0 g of pure iron in HCI. The solution is
taken to dryness and residue dissolved in about 75 ml of 5 N HC1
(15% v/v) and diluted to 100 ml with the same acid in a volumetric
flask. This solution provided 100 mg iron per ml, 5 N in HC1.
8. Standard Fluoride Solution: sodium fluoride (0.221 g) is dissolved in
water in a polyethylene beaker and diluted to a liter with water in a
volumetric flask. This Solution A provides 100 ~g F- per ml. Ten ml
of Solution A diluted to 100 ml provide Solution B 10/ag F- per ml.
Solution B is stable for at least two weeks.
Determination of Fluoride in HCI

To a series of 150-ml beakers add 50 ml of sodium acetate solution and
5.0 ml of fluorine-free HC1 (Reagent 1). With a pipette add, 0, 5,
10, and 20 /ag of F- to provide 0, 1, 2, and 4 ppm F-, respectively. For
routine analysis, 0 and 2 ppm F- standards are sufficient to analyze a
batch of samples. Set the pH meter to read 1400 mV on the millivolt
scale. Place the fluoride ion and reference electrodes into the solution. Stir
for 2 to 3 min or until a stable response is obtained. Plot a calibration
curve on semilog paper; concentration (fluoride ppm) on the logarithmic
axis and the potential (mV) on the linear axis. The resultant relationship
is linear.
Treat the unknown HCl sample exactly as just described, but without
the fluoride addition. Interpolate ppm F- from the graph.
Determination of Fluoride in Pickle Liquor

To a series of 150-ml beakers pipette 5 ml of 5 N (15% v/v) HCI fol-
lowed by the addition of 50 ml of ammonium citrate solution (Reagent 5).
Adjust pH to 5 on the pH meter by adding diluted (1 q- 1) ammonium
hydroxide (about 5 ml are required). With a pipette add 0, 5, 10, and 20
/ag of F-, respectively, into the beakers. Dilute the contents to 65 ml with
water. Immerse the fluoride ion-selective electrode and reference electrode
into the solution. Stir for 2 to 3 min or until the potential is stable.
Measure the potential as described. Treat the unknown pickle liquor
sample likewise.
The concentration of HC1 in the pickle liquor, received for regeneration,
is in the range of 3 to 4 N (10 to 12% v/v) while that of iron is below 6
percent. The method just described is quite suitable in the stated range,
as will be amplified later.
Determination of Fluoride in Gaseous Emissions

To a series of 150-ml polyethylene beakers transfer 20 ml of potassium
acetate buffer (Reagent 4) and S ml of 1 N NaOH. Add 0.1, 0.5, 1, 2,
and 5 /ag of fluoride using appropriate standard fluoride solution. Dilute
to 30 ml with water. Immerse the fluoride ion-selective electrode and
reference electrode into the solution. Stir for 2 to 5 rain or until a stable
potential response is obtained. Measure the potential as described earlier.
A metered volume (for example, 10 ft 3 at 0.3 ft 3 per rain) of the gaseous
emissions at known temperature and pressure is drawn through a filter (to
isolate the particulate matter) into a series of three gas impingers
(preferably of plastic) each containing 200 ml of 1 N NaOH.
The absorbed fluoride in each of the impingers is determined by
pipetting 5-ml aliquots of the solutions. After addition of 20 ml of
potassium acetate buffer and diluting to 30 ml with water, the potential is
measured with the fluoride ion-selective electrode, exactly as described
before. The concentration of fluoride in the solution is read from the
calibration graph and the fluoride level computed in the gaseous
emissions.
Interference Study
The commercial HCI shipments did not present any problem with
regard to interference in the determination of fluoride. The hydrochloric
acid content did not vary apppreciably. There is no possible contamina-
tion by fluoride in our plant pickle liquors used for regeneration. How-
ever, such may not be the case with pickle liquor received for regeneration
from-outside sources. Thus, it is important to monitor fluoride in such
pickle liquors. The concentrations of both iron and HC1 in pickle liquor
vary from 2 to 8 percent and from 10 (3.3 N) to 12 percent (4 N),
respectively. The effects of these two variables were investigated down to 2
ppm F-.
Iron Interference in F-Determination in Pickle Liquor

Tests were made using iron solution providing 1-ml equivalent to
100-rag Fe in 5 N HCI. (The concentration of HCI was kept constant at
5 N.) The amount of iron in the tests was varied. Total volume was made
up to 5 ml with 5 N HCI. After addition of 50 ml of a m m o n i u m citrate,
the pH was adjusted to 5 on the pH meter and finally the solution was
diluted to 65 ml with water. Potential measurements were then made with
the fluoride ion-selective electrode. The results are shown in Table 1.
It is evident from Table 1 that at 2 p p m F-, there is no appreciable
effect of iron up to 10 percent Fe. Eight percent iron does not interfere
significantly at 4 p p m F- and can be tolerated in determining up to 10
p p m F-. Normally, the pickle liquor for regeneration from outside sources
containing F- in excess of 2 p p m is not accepted. There had been isolated
and rare instances when a pickle liquor (from outside) up to 5 p p m F ~
had been processed. Fluoride accumulates in the regeneration system, but
this is never allowed to exceed 5 ppm. However, it could be concluded
TABLE 1--Effect o f iron concentration on F - determination in pickle liquor.

F- Found (ppm)
Percent Iron Present
F- Added (ppm) 0 2 6 8 10
2.0 2.0 2.1 2.1 2.0 1.9
4.0 4.0 4.0 3.8 3.7 3.2
10.0 10.0 10.0 9.3 9.3 8.2
20.0 ~.0 ~.0 18.6 17.4 16.2
that up to 8 percent iron has no significant interference in determining

fluoride up to 5 ppm.
In instances where the concentration of iron in pickle liquor is
abnormally high, the fluoride determination can be carried out by
doctoring the standards with equivalent amounts of iron with satisfactory
results. Confirmation tests were made on pickle liquor samples using the
Willard and Winter 2 steam distillation., The agreement was quite satis-
factory: 5.0 ppm F- by direct fluoride ion-selective electrode versus 5.2
ppm F- through the steam distillation procedure.
Effect of HCI Concentration

Normally the free HCI concentration in the pickle liquor received for re-
generation is in the range I0 to 12 percent, that is, 3.3 to 4 N in HCI. It
has already been shown that iron in the range 2 to 8 percent does not
interfere in the F- determination. In the following investigation, iron con-
centration was kept at 6 percent and the I-ICI concentration was varied
from 9 to 15 percent, that is, 3 to 5 N and the total volume (iron plus
acid) held at 5 ml. After the addition of 50 ml of ammonium citrate, the
pH was adjusted to 5.0 with diluted (I + I) ammonia. The volume was
finally made up to 65 ml with water. The potential measurements were
then made as before, with the fluoride ion-selective electrode.
Table 2 shows there is no significant effect of free HCI concentration on
fluoride determination within the range of 9 to 15 percent HCI.
TABLE 2--Effect o f HCl concentration on F - determination on pickle liquor.

F- Found (ppm)
Percent Free HCI Present
F- Added (ppm) 9 12 15
2 2.0 2.2 2.4
4 3.7 4.0 4.0
10 10.0 10.0 10.0
20 20.0 20.0 20.0
The determination of fluoride in gaseous emissions absorbed in 1 N

N a O H is quite straight-forward. In the method described, 5-ml aliquot is
taken for determining fluoride down to 0.2 /~g F-. However, in our
investigations it was also established that aliquots ranging from 2 to 6 ml
(in 1 N NaOH) could be processed to determine fluoride exactly as
described in the method without any adjustment. This is illustrated in
Table 3.
TABLE 3---Effect o f N a O H concentration on F - determination in gaseous emissions.

Add 1 N Add Water
Buffer (ml) Add F- (g) NaOH(ml) (ml) Found pH mV
20 0.1 2 8 5.00 --245
20 0.1 4 6 5.07 --245
20 0.1 6 4 5.13 --258
20 1.0 2 7 5.03 --234
20 1.0 4 5 5.10 --234
20 1.0 6 3 5.15 --228
20 10.0 2 7 5.04 --185
20 10.0 4 5 5.09 --185
20 10.0 6 3 5.16 --185
With the present sampling technique based on absorbing fluoride from

10 ft 3 of the gaseous emissions at 0.3 ft 3 per min in 200 ml of 1 N N a O H
and assuming 0.2 ~g F- (easily determinable) in the recommended 5-ml
aliquot, the lower limit is 0.04 p p m F-. However, this could be lowered
quite considerably (0.01 p p m or lower) by taking larger volumes of the
gaseous emission through the absorption train. Determination of higher
concentration (several orders of magnitude) does not pose any problem.
We have also used the fluoride ion-selective electrode for determining
fluoride in particulate matter (containing large amounts of iron oxide), by
fusing the latter with N a O H pellets in a nickle crucible at 600~ and
extracting the melt in a citrate buffer. Likewise, this technique was found
suitable for determining fluoride in "hot top covering reagents" in
steelmaking practice.
Summary
Rapid methods for the determination of fluoride in HCI, pickle liquor
for HCI regeneration system, and gaseous emissions from steelmaking
using a fluoride ion-selective electrode are presented. This technique has
also been applied for determining fluoride in slags, particulate matter,
and "hot top covering reagents" in steelmaking operations. A single
determination down to 1 ppm, including standardization, can be corn-
pleted in HC1 in about 5 min permitting easy monitoring of incoming

shipments. Fluoride contents in the gaseous emissions down to 0.01 ppm
are readily determined after absorption in NaOH solution.
Acknowledgment
The assistance of J. F. Donovan and G. Overholt is gratefully acknowl-
edged.
A. G. Wikjord, 1 G. H. M a y o r , ! a n d F. E. D o e r n I
Scanning Electron Microscopy

and Energy Dispersive X-ray
Microanalysis of Nuclear
Reactor Corrosion Particles
REFERENCE: Wikjord, A. G., Mayor, G. H., and Doern, F. E., "Scanning Electron
Microscopy and Energy Dispersive X-ray Mlcroanalysis of Nuclear Reactor Corrosion
Pm'tieles," Water Quality Parameters, ASTM STP 573, American Society for Testing
ABSTRACT: The nature and origin of particulates in the heat transport system of
CANDU water-cooled nuclear reactors are relevant to the operation of these stations.
The analysis of corrosion particles is difficult because of their small size and low concen-
tration. A scanning electron microscope equipped with an X-ray analyzer has been used
successfully for the simultaneous characterization of the morphology, size, and elemental
composition of these particles.
The good resolution and the large depth of field of the scanning electron microscope
are suited for the examination of micron-sized particles. Also, the solid state Si (Li)
detection system is readily amenable to automated data manipulation and thus permits
a rapid qualitative assay of a representative number of particles from any given sample.
In this study, individual particles having masses as low as 10-Is g have been analyzed
and X-rays arising from electron capture decay of SSFe have been observed in some of
the samples. The techniques discussed are potentially applicable to a variety of problems
related to the quality of environmental waters.
KEY WORDS: water quality, nuclear reactors, electron microscopy, X-ray analysis,
environmental tests
C o r r o s i o n p r o d u c t s p r e s e n t as p a r t i c u l a t e m a t t e r in v e r y low c o n c e n t r a -
t i o n s in t h e p r i m a r y h e a t t r a n s p o r t c i r c u i t o f p r e s s u r i z e d w a t e r - c o o l e d
n u c l e a r r e a c t o r s s u c h as t h e C A N D U - P H W 2 p r e s e n t a p r o b l e m w h i c h is
n o t e n c o u n t e r e d in f o s s i l - f u e l e d e l e c t r i c a l p l a n t s . T h r o u g h a p r o c e s s o f
i n - c o r e n e u t r o n a c t i v a t i o n f o l l o w e d by o u t - c o r e d e p o s i t i o n , c o r r o s i o n
p a r t i c l e s c a n p l a y a m a j o r role in t h e m i g r a t i o n o f r a d i o a c t i v i t y t h r o u g h o u t
t h e p r i m a r y c o o l a n t c i r c u i t . I n o r d e r to u n d e r s t a n d a n d c o n t r o l a c t i v i t y
'Research chemist and chemical technologists, respectively, Analytical Science Branch,
Atomic Energy of Canada Limited, Whiteshell Nuclear Research Establishment, Pinawa,
Manitoba ROE 1L0, Canada.
~Canada Deuterium Uranium-Pressurized Heavy Water Reactor.
136
9
WIKJORD ET AL ON NUCLEAR REACTOR CORROSION PARTICLES 137
transport, it is necessary to elucidate the mechanisms of corrosion and

particle transport. Enquiries into these mechanisms in the laboratories of
Atomic Energy of Canada Limited have inevitably raised questions about
the composition, structure, and size distribution of corrosion particles.
A variety of techniques has been employed to characterize reactor
corrosion products, the more prominent of which are listed in Table 1.
TABLE 1--Methods for characterization and analysis of corrosion particles.

Polarized Light Microscopy(Petrography)a
Electron Microscopy
Electron Probe Microanalysis
X-ray Diffractiona
X-ray Fluorescence
Emission Spectrography
Atomic Absorption Spectrophotometry
),-Spectrometry
Electron Spectrometry(ESCA)a
Zeta Potential/Surface Charge Measurements
Ultrafiltration
a Structure sensitivetechniques.
Most of the methods of analyzing and characterizing corrosion particles,

whether an elemental assay by emission spectrography, or a structural
analysis by X-ray diffraction, can only provide information averaged over
large numbers of particles. There are only a few methods for studying
individual microparticles. These microanalytical techniques--not to be
equated with trace methods of analysis--involve some type of microscopic
observation of the particle under study. Notable examples include the
electron microprobe which consists essentially of an optical microscope
and an X-ray spectrometer in an electron column instrument and the zeta
meter which utilizes an optical microscope to follow the motions of
particles in an electric field.
This paper deals with a microanalytical technique known simply as
analytical electron microscopy [1-4],3 a term referring in this instance to
energy dispersive X-ray analysis in conjunction with scanning electron
microscopy. The method takes advantage of the secondary electrons and
characteristic X-rays generated during electron bombardment to examine
simultaneously the microtopography of individual particles and to de-
termine their composition.
The intent of this paper is to describe the use of analytical electron
microscopy in characterizing individual reactor corrosion particles which
originate in the primary coolant circuits. The relevance of the findings to
the nuclear industry will not be discussed.
Experimental
Sampling Procedure
In the course of this investigation, corrosion particles were sampled

from a variety of Canadian nuclear installations, including in-reactor and
out-reactor experimental loops as well as the primary heat transport
system of CANDU-PHW power reactors. Since filterable particles are
normally present in these systems at levels of 10 to 100 /ag of particulates
per kilogram of coolant, it was necessary to concentrate the particles on
filters. Filtration assemblies of design similar to that shown in Fig. 1, with
filters supported on the sintered stainless steel discs, were valved into the
purification circuits of the various reactor systems where conditions were
nominally 10 MPa (15130 psi), 40~ and pH 10. It was desirable to ensure
a low concentration of particles (104 to l0 s per cm 2) on the filter in order
to avoid conglomeration and difficulty in interpretation. Since the density
FIG. l--Schematic diagram of the in-line filtration assembly for sampling corrosion
particles from reactor circuits. The filters are supported on the sintered disc. The material of
construction is stainless steel.
of particles found on the filters depended on factors such as flow rate,

filtration time, particle concentration in the coolant, and filter pore size,
preliminary filtrations were usually necessary to determine the optimum
sampling period for a given system. In this work, filtration times between
5 rain and 1 h were chosen. Three types of filters were used: silver
membranes (Selas Flotronics), cellulose esters (Millipore Corporation),
and polycarbonate membranes (Nuclepore Corporation). In a few in-
stances, when hot filtrations were conducted at maximum process tem-
perature (~300~ silver membrane filters were used in a modified filter
holder. For electron microscopic examination, filters were mounted di-
rectly on specimen studs with an electrically conductive adhesive. When
dealing with radioactive particulates, the specimens were allowed to stand
to let the radioisotopes with shorter half-lives decay. Small portions of the
filters were then mounted on studs in a fume hood and transferred to the
specimen chamber of the microscope using rubber gloves.
Scanning Electron Microscopy
A Cambridge Mark 2A "Stereoscan" was operated with an electron gun
potential of 20 kV; the resolution capability was about 20 nm. During
viewing, specimens were tilted at an angle of 45 ~ from the column axis.
Photographs were taken of the video display using a Polaroid camera. The
nonconducting Millipore and Nuclepore filters were coated with a thin
layer (~10 nm) of gold to avoid electrostatic charging and image
distortion.
X-ray FluorescenceAnalysis
An EDAX Model 707 energy dispersive X-ray analyzer comprising a
Si(Li) solid state detector was attached to the Stereoscan. The system
resolution was 160 eV (full-width half-maximum 4) for the 5.9 keV MnK
X-ray. An 8 /am thick beryllium window isolated the detector from the
microscope column and precluded detection of elements lighter than
fluorine. The absorption of soft X-rays of the light elements by the gold
coating on some specimens was not considered serious since there was
little interest in elements with atomic numbers below that of aluminum.
The X-ray spectra were processed with an EDIT system [5] which enabled
such manipulations as smoothing statistical scatter, background subtrac-
tion, and peak integration. The spectra were recorded either by photo-
graphing the video display or by printing on teletype.
Radiochemical Analysis
y-Spectrometric analysis of radioactive " c r u d " was performed using an
'The resolution is defined as the full width, in electron volts, of the X-ray peak measured
at one-half the maximum intensity.
80-cm 3 closed end coaxial Ge(Li) detector (Nuclear Diodes) coupled to a

Nuclear Data 2200 multichannel analyzer with a 2048 channel memory.
The spectral information was recorded on magntic tape and evaluated by
computer [6].
Particle Size Analysis

Particle sizing was performed directly from electron micrographs using
a x7 eyepiece fitted with a "globe and circle" graticule following the
procedure described by McCrone et al [4], The size parameter was
expressed as the diameter of a circle with a cross-sectional area equivalent
to the projected area of the particle. For sizing studies by electron
microscopy, only a few hundred micrograms of particulates were collected.
In some instances, when milligram quantities of corrosion particles were
collected and backwashed from the filter, a Coulter Electronics Model TA
particle counter was employed.
Discussion
Principle of Operation
Figure 2 shows the analytical functions of the scanning electron
microscope and the energy dispersive X-ray analyzer. When electrons
emitted from the heated tungsten filament are accelerated to high energies
and focused onto the specimen, two phenomena occur which provide the
basis of the technique. Firstly, low energy secondary and back scattered
electrons are emitted; these are picked up on a collector to enable electron
INCIDENT
ELECTRON
BEAM
Si (Li) ELECTRON
- X-RA, I % EcT~
COLLECTOR
LUORESCE CE t,, -1"~ OPTICAL I
IMAGE I
\
\
/
I
PARTICLE- ~ ~ EXC|TATION VOLUME
FIG. 2--Functional aspects of" the analytical electron microscope technique.

optical imaging of the specimen surface. Secondly, electronic rearrange-

ments within the constituent atoms generate characteristic X-rays which
are analyzed by the Si(Li) semiconductor detector and a multichannel
analyzer. Descriptions of these techniques can be found in greater detail
elsewhere (see, for example, Refs 7, 8, and 9).
Choice of Filter
Each of the three types of filters employed for collecting particles of-
fered unique advantages and disadvantages which were important in
different stages in the operation. Thus, factors such as wet strength,
thermal and chemical stability under process conditions, minimum pore
size, and flow properties were important in sampling; surface texture and
electrical conductivity affected the ease of making microscopic examina-
tion; the composition of the filter had to be considered for possible X-ray
interferences. No filter was ideal in all aspects.
Selas Flotronics silver membrane filters proved to be the most practical
since they were stable under the most severe process conditions and they
did not become electrostatically charged during electron bombardment.
The strong interference of the AgL~ X-ray at 2.98 keV was not a serious
limitation because X-rays of other corrosion metals did not appear in this
region. Traces of chlorine produced a weak K~ peak at 2.62 keV.
However, these filters were unsuitable for collecting the smallest particles
since the minimum pore size available was only 0.2 /an. In addition, the
granular texture of the surface did not lend itself readily to particle sizing
studies.
Millipore filters, of cellulosic composition and available with average
pore sizes as low as 25 nm, have obvious advantages in collecting the very
fine particles. Since they are nonconductive, they require a thin coating of
gold, platinum, or carbon before viewing in the microscope. Spurious
peaks due to contamination with Si and A1 were found to be troublesome.
Nuclepore polycarbonate filters were the best suited for particle sizing
because of their smooth surface. They are noted for their uniform pore
structure [10] and show no interferences in X-ray analysis. They are
nonconducting and are commercially available with pore sizes as low as 30
rim.
Particle Morphology and Composition

The particles appeared for the most part as discrete entities irregular in
shape; crystals of regular geometry were observed only infrequently. In
some cases, regular crystal faces were seen protruding from an otherwise
irregular structure. The great majority of particles analyzed contained iron
as the principal constituent, presumably in an oxide (or mixed oxide)
form; other metals such as chromium, nickel, aluminum, and silicon
were frequently observed, although in smaller concentrations; elements

such as manganese, copper, zirconium, sodium, calcium, magnesium,
zinc, and sulfur were detected only infrequently.
The capability of the technique to reveal simultaneously the micro-
morphology and elemental composition of the particles is illustrated in
Figs. 3 to 7. The X-ray spectra were generally recorded with a stationary
electron beam (spot analysis), although the very large particles (>20 jan)
were analyzed by scanning the beam over the entire particle (area
analysis).
Figure 3 shows a globular-shaped corrosion particle containing iron as
the only detectable constituent. It is possible that the particle is composed
of magnetite (Fe304) or hematite (FezOa) since these structures have been
identified in corrosion products by X-ray diffraction methods [11]. Note
the internal cavity opening to the surface of the particle.
In Fig. 4, a particle is seen lodged in the pore of a silver membrane
filter. The principal constituents are zirconium and silicon--the AgL a
peak is due to interference from the substrate. The small pits appearing
on the surface of the filter were thought to be formed during manufacture.
A "flake-like" particle containing appreciable quantities of iron,
chromium, and nickel is shown in Fig. 5. It is possible that it has spinel
structure, for example, a solid solution of nickel and chromium in
magnetite. Other work has shown that spinels of the type AB204 (where A
= Ni or Fe and B ---- Cr or Fe) play an important role in the transport of
radionuclides in water reactor circuits [11].
Figure 6 shows a submicron-sized particle straddling a pore of the filter
(average pore size 0.45 /an). Iron is again the major constituent; alumi-
num a minor one.
Figure 7 shows a polycrystalline structure consisting of iron, nickel, and
aluminum. Note the protruding crystal faces. Since the density of particles
on the filter was small, it is not likely that this is an aggregation formed
during filtration. The crystals appear to be cemented together and the
assembly probably existed as a single entity in the reactor system. Since
the excitation volume of the analysis spot was larger than the individual
crystaUites, it was not possible to discern differences in their composition.
By the analytical electron microscopic technique, it is possible to
qualitatively analyze particles as small as 10 -is of a gram. The inferior
resolution capabilities of the semiconductor detector as opposed to a
crystal spectrometer was not found to be a serious handicap, since
interelement interferences due to overlapping X-ray lines were usually
clearly delineated. However, there was difficulty in discerning the K ~ peak
of a given metal from the K ~ of its lighter neighbor in the period classifi-
cation. Thus the MnK ~ peak was obscured by the K a of chromium.
There was no attempt to quantify the results by converting the X-ray
intensity data to concentrations. This was because of a lack of suitable
7~
m
-I
i-
0
7.
z
c
c)
i-"
m
>
]3
m
o
0
]3
C)
0
]3
0
rj )
0
z
.11
-I
FIG. 3---Corrosion particle .from an out-reactor e x p e r i m e n t a l loop (RDL5, W N R E ) on a 0.45 tam silver m e m b r a n e .
m
ffl
m
:13
0
c
u-
-<
"13
:D
m
-i
m
~J
o)
FIG. 4---Radioactive corrosion particle f r o m an in-reactor e x p e r i m e n t a l loop (IL2, W R - I , W N R E ) on a 1.2 ~ m

silver m e m b r a n e .
o
o
m
I--
0
z
z
c
0
l=-
m
-m
-11
m
0
0
0
0
11
0
z
"IO
FIG. 5 l R a d i o a c t i v e corrosion .panicle f r o m an in-reactor experimental loop (1L2, WR-1, W N R E ) on a 1.2 ~m

silver membrane. r-
m
..k
m
r
t-
[-
-<
-0
:13
m
-.I
m
(1)
FIG. 6---Radioactive corrosion particle f r o m the primary heat transport system o f a C A N D U - P H W reactor
(NPD, Rolphton) on a 0.2 ~m silver membrane. A weak M n K a (5.9 ke V) X-ray is visible in the X-ray spectrum
using a lower vertical scale attenuation.
r
0
33
I'n
[.-
0
z
z
c
C)
r-
Ill
m
0
0
33
0
0
~J
0
o9
0
z
-8
FIG. 7--Polycrystalline particle from an out-reactor experimental loop (RD-5, W N R E ) on O.45 "/am silver membrane.
F
m
..&
4~
standards for the diversity of particles encountered and the uncertainty in

absorption corrections owing to surface irregularities of many of the
particles.
Particle Size
One of the main objectives of this work was to obtain information on
the size distribution of particulates in reactor circuits. Delineation of size
parameters has important implications in such matters as: (1) choice of
filters for the purification system; (2) the dynamics of crud movement,
deposition, and release; and (3) the interpretation of scattered light signals
in turbidimetric measurements.
The size of the particles collected on filters generally fell within the
range 0.1 to 50 / a n - - a range well suited to the use of the scanning
electron microscope. The lower size observed paralleled the pore size of
the filter employed. There were variations between samples taken at
different times or from different systems. Figure 8 shows a comparison of
size distribution plots obtained by microscopy and by Coulter counter for
similar sample populations. Since a much larger sample was necessary for
the Coulter counter analysis, a longer sampling period was also required.
Thus, while the comparison shown in Fig. 8 does not refer strictly to the
same population, the two samples were taken from the same reactor
system under the same operating conditions. It can be seen that the
number frequency distribution obtained by microscopy gives greater
import to the smaller particles than does the volume measurement of the
Coulter counter. The agreement between the two methods is good,
considering the differences in the sizing and sampling techniques.
FIG. 8--Cumulative and d(fferential size distribution plots o f typical reactor particulates:
Coulter counter versus microscopy.
X-rays from Radioactive Corrison Products

Corrosion particles taken from in-reactor circuits are radioactive due to
the presence of neutron activation products and fission products. Figure 9
shows a typical ),-ray spectrum of particulate matter collected on a filter;
the principal isotopes resulting from neutron activation corrosion products
iOs I I (:3 I r I I I I I
__ ,,,_ z
104 -
Z ~ (-) Z-- ~~' Z -- , -~ - ~I - -0 ~ - Z~- ~ r-, --,
~I-- (~; l--
Ch I~" I0" I ' , -
~ Z ~ --
E-
z
103 :;OE (/) Z ~ NN
SAMPLE SIZE = 9.74 xlO'4g
10 2 DECAY TIME ; 2,18h

C O U N T I N G DURATION = 6 0 0 s
I I I I I I I I I
I0 0
150 300 450 600 750
105 I I i I
0
0
I0 4
(/) NO'~ ,^
(/) (~0~ zOdtO i t It.
<2
I--
z 103
L" ___~==
o
L)
I0 2
I0 I I I I I I I I
675 825 975 1125 1275 1425
ENERGY (keV)
FIG. 9--Typical y-ray spectrum of nuclear reactor corrosion particles.
are SgFe (45d), SlCr (27.8d), SaCo (71.4d), 6~ (5.8y), and S4Mn (313d).
The question arises as to what extent X-rays attributable to radionuclides
may interfere with electron-induced X-ray fluorescence analysis in the
microscope.
To assess this possibility, all that is required is to measure "back-
ground" X-rays with the Si (Li) detector while the specimen is mounted in
the microscope but with the electron gun turned off. Figure 10 shows a 5
min background count taken from a small portion of a lightly loaded
filter. The weak MnK~ peak at 5.9 keV arises from electron capture decay
of SSFe, a neutron activation product of S4Fe.
EC
|~Fe (n, 7) |~Fe ~ ~VlnK~
FIG. lO---Spectrum showing MnKe~ X-ray arising .from SSFe decay in radioactive parl6ele~
with high iron content.
The magnitude of the MnK a peak in counts/second was invariant during

subsequent X-ray fluorescence analysis by electron excitation of the
specimen. The unsuspecting analyst might have assumed that there were
detectable amounts of manganese in the specimen! The appearance of the
MnK~ background peak for this particular specimen (which, incidentally,
consisted mostly of iron) was not surprising since it also showed strong
y-rays attributable to sgFe at 1099 and 1292 keV, respectively. Although,
in this instance, K-capture of SSFe interfered with the determination of
traces of manganese in the specimen, generally speaking, X-rays origi-
nating from nuclear decay processes have not posed serious limitations to
the X-ray fluorescence technique.
While X-rays from other activation products are theoretically possible
by electron capture or internal conversion processes [12], none have been
detected to date.
Conclusions
The scanning electron microscope is ideally suited to characterizing
individual reactor corrosion particles in terms of their size, shape, and
morphology. The analytical versatility of the semiconductor X-ray detector
lends itself to rapid qualitative assay of large numbers of particles. The
technique is insensitive to elements with atomic numbers below fluorine,
and interelement interferences due to overlapping peaks presented minor
difficulties. As a microanalysis technique, analytical electron microscopy is
seen as a valuable a d j u n c t to methods which provide i n f o r m a t i o n on a

macroscopic scale. The technique can provide u n i q u e i n f o r m a t i o n in
problems c o n c e r n i n g particulate c o n t a m i n a t i o n in water systems.
Acknowledgments
The authors are grateful to m e m b e r s of the Chemical Technology
B r a n c h , Whiteshell Nuclear Research E s t a b l i s h m e n t , for providing the
filter specimens from a variety of water reactor installations. They also
wish to t h a n k J. D. Chen and m e m b e r s of the Radiochemistry Laboratory
for p e r f o r m i n g the V-spectrometric analyses.
References
[I] Chandler, J. A., Micron, Vol. 3, 1972, p. 85.
[2] Beaman, D. R. and Isasi, J. A., Materials Research and Standards, Vol. 11, No. 11,
1971, p. 8.
[3] Hotz, H. P., Proceedings, 7th International Conference on Electron Probe Analysis,
1972.
[4] McCrone, W. C. and Delly, J. G., The Particle Atlas--Principles and Techniques, 2nd
ed., Vol. 1, Ann Arbor Science Publishers, Ann Arbor, 1973.
[5] EDAX International Inc., Prairie View, I11.
[6] Felawka, L. T., Molnar, J. G., Chen, J. D., and Boase, D. G., Atomic Energy of
Canada Limited, Report AECL-4217, Pinawa, Man., 1973.
[7] Hearle, J. W. S., Sparrow, J. T., and Cross, P. M., The Use of the Scanning Electron
Microscope, Pergamon Press, 1972.
[8] Russ, J. C., "Elemental X-ray Analysis of Materials," EDAX International, Prairie
View, I11., 1972.
[9] Woldseth, Rolf, "X-ray Energy Spectrometry," Kevex Corporation, Burlingame, Calif.,
1973.
[10] Fleischer, R. L., Viertl, J. R. M., and Price, P. B., Review of Scientific Instruments,
Vol. 43, No. 11, 1972, p. 1708.
[11] Rummery, T. E., Atomic Energy of Canada Limited, private communication.
[12] Pillay, K. K. S. and Miller, W. W., Journal of Radioanalytical Chemistry, Vol. 2,
1969, p. 97.
D. F. K r a w c z y k ~
Preservation of Wastewater Effluent

Samples for Forms of Nitrogen and
Phosphorus
REFERENCE: Krawczyk, D. F., "Preservation of Wastewater Effluent Samples for

Forms of Nitrogen and Phosphorus," Water Quality Parameters, ASTM STP 573,
ABSTRACT: Samples of water containing concentrations of total organic carbon at

levels greater than 20 rag/liter when preserved with 40 mg/liter of mercuric chloride did
not provide a complete inhibition of microbiological growth. A complete inhibition was
noted at a preservation level of 400 rag/liter of mercuric chloride.
A study was conducted on chemical changes of forms of nitrogen and phosphorus in
wastewater effluents preserved with 400 rag/liter of mercuric chloride.
A variety of wastewater treatment plant effluent samples was combined into one
composite. The composite was then divided into ten equal samples. Each sample was
analyzed separately for Kjeldahl nitrogen, ammonia nitrogen, nitrite nitrogen, nitrate
nitrogen, orthophosphate phosphorus, and total phosphate phosphorus after 10, 30, 60,
80, and 100 days. Due to a logistical handling problem, the samples were usually pro-
cessed in order in batches. An analysis for total organic carbon, total inorganic carbon,
and dissolved inorganic mercury was conducted after 100 days.
Small but measurable differences were observed for all constituents from one period
to another. The ammonia, nitrite, nitrate nitrogen system was most susceptible to
change. The Kjeldahl nitrogen analysis showed the greatest variation among replicates
and sets.
The study indicated that wastewater samples can be stored at room temperature after
preservation with 400 mg/liter of mercuric chloride for periods of up to 100 days with
only minimal changes in the form of nitrogen and phosphorus.
KEY WORDS: water quality, waste water, environmental tests, phosphorus, nitrogen
In conducting reruns (reanalysis) on samples preserved for analysis with

40 mg/liter of mercuric chloride there were indications that the 40
rag/liter level was not always adequate to inhibit biological activity. The
preliminary evidence indicated that when the level of organic material was
at a 20 mg/liter organic carbon concentration, neither sulfuric acid (0.2
ml concentrated/liter) nor mercuric chloride (40 mg HgCl2/liter) would
inhibit microbial growth. A study was conducted using samples from a
local wastewater treatment plant, a nearby creek, and an effluent from a
1Chief, Laboratory Services Branch, U.S. Environmental Protection Agency, Pacific
Northwest Environmental Research Laboratory, Corvallis, Ore. 97330.
152

KRAWCZYK ON NITROGEN AND PHOSPHORUS IN WASTE WATER 153
fish hatchery. The study indicated that microbial growth (total plate count
growth at 37 and 25~ in a tryptone-glucose-yeast agar) would occur when
0.2 ml of concentrated sulfuric acid per liter of sample or 40 rag/liter of
mercuric chloride was used as a preservative. No growth was observed for
31 days (length of study) when 400 mg/liter of mercuric chloride was used
as shown in Table 1. Samples collected during the first phase as shown in
Table 1 were kept at room temperature during the period of this study. A
considerable amount of fibrous material was present in the "Creek" and
"Weak Sewage" samples. In examining the carbon data in Table 1 some
inconsistencies appear indicating an apparent rise in total organic carbon.
The inconsistency is in all likelihood the result of the nature of the
samples due to variability in the amount of fibrous material. There is no
intent to imply that a rise occurred in the total organic carbon level in the
samples. The only purpose of presenting Table 1 is to indicate that in an
enriched growth media (tryptone-glucose-yeast agar) growth was observed
in samples that were unpreserved, preserved with 0.2 ml/liter of H2SO4,
and preserved with 40 mg/liter of HgCl2, when the total organic carbon of
the water sample approached 20 mg/liter.
Hellwig reported that 60 to 80 mg/liter of HgCl2 preserved polluted
river water for 18 days [1].z In a later study on preservation of wastewater
samples Hellwig used 890 mg/liter of mercuric chloride to preserve the
samples for 43 days [2].
Jenkins reported that in an estuarine environment 40 mg/liter and
storage at 4~ for a maximum of eight days was a suitable preservation
technique for forms of nitrogen [3]. However, for forms of phosphorus,
Jenkins recommended use of 40 mg/liter mercuric chloride and storage at
-10~ with no observable changes in 31 days, again in an estuarine
environment [4]. Brezonik and Lee, and Howe and Holley reported on the
preservation of forms of nitrogen using mercuric chloride [5,6]. The
studies by the latter investigators pointed out problems that one encoun-
ters with use of sulfuric acid as a preserving agent.
When it became apparent that if the total organic carbon content
approached 20 mg/liter, a 40 mg/liter concentration of HgClz would not
inhibit microbial growth, a ten-fold increase in the mercuric chloride level
was chosen as the next increment to be used in determining the efficiency
of preservation.
Experimental
When wastewater treatment plant samples which have been preserved
with 400 mg/liter of mercuric chloride arrive in the laboratory they are
assigned a laboratory number designating the week of collection and are
computer scheduled for analysis. Each sample is then mixed in a blender
TABLE 1--Changes in total plate count growth as a function of time.
Plate Count Prate Count,

TOC TOC Unpreserved 0.2 m l / l concen- Plate Count, Plate Count, -I
r'rl
Sample Col- Days TOC H2SO 4 HgCI, Sample, trated H2SO 4 40 m g / l HgCI 2 400 m g / l HgCI 2
lected from after Unpreserved, Preserved, Preserved, Colonies/100 Colonies/100 Colonies/100 Colonies/100
D
Location Composite r ag/ lit e r 40 m g / l i t e r 40 m g / l i t e r ml ml ml ml c
i---
Creek 0 18.8 19 19.6 >600 x 103 59.4 x 103 72.7 x 103 <0.1 x 103
1 18.6 16 19.4 6600 x 10J 13.5 x 103 44.5 x 103 <0.1 x 103 -<
2 17.4 18 18 8650 • 101 8.3 x 103 18.0 x 103 <0.1 x 103 'o
3 16.2 16.4 18.0 1.2 • 107 5.2 x 103 2.4 x 103 <0.1 x 103 xl
6 18.4 17.4 17.6 1.4 x 107 2.2 x 103 1.0 x lO3 <0.1 x 103
7 19.2 19.8 19.6 2.5 x 107 3.7 x 103 3.5 x 103 -~0.1 x 103
m
14 19.6 17 21.6 4.0 x 106 1000 x 103 0.8 x 103 <0.1 x 103 .-4
32 14 24 22.4 2.17 x 106 0.05 x 103 <0.1 x 103 <0.1 x lO3 m
20
60
Weak sewage 0 18.6 26.8 I7.6 1.25 x 10e 7.5 x l0 s 5.3 x 104 <0.1 x 103
1 18.6 28.0 18.6 2.3 x 108 2.2 x l0 s 7.6 x 104 <0.1 x 103
2 14.2 26.2 16.8 2.4 x 108 6.6 x 104 7.9 x 104 <0.1 x 103
3 16.6 24.0 16.2 S.0 x 107 8.0 x 104 S.O x 104 <0.1 x 103
6 13.8 28 16.6 6.0 x 107 3.4 x 10~ missing <0.1 x 103
7 15.8 3S.8 19.0 1.8 x 107 6.2 x 10s 4.4 x l ~ <0.1 x 103
14 6.0 34 20 4.75 x lOs 2.8 x lOs 1.5 x 103 <0.1 x 103
32 8.0 34.8 22 4.35 x lOs 4.6 x 104 < 0 . 1 x 103 <0.1 x 103
Fish hatchery 0 2.7 2.2 2.5 1.4 • l0s 0.5 x 103 <0.5 x 103 <0.1 x lO3
effluent 1 2 2.1 2.5 3.9 x l0s <0.1 x 103 <0.1 x 103 <0.1 x 103
2 1.9 2.0 2.6 3.8 • l0s <0.1 x 103 <0.1 x 103 <0.1 x 103
3 2 2.2 2.5 3.0 x l0 s <0.1 x 103 <0.1 x 103 <O.l x 103
6 < 1 1 2.7' 1.9 x 10 r <0.1 x 103 <0.I x 103 <O.l x IO3
7 1 2.2 2.7 1.9 x 107 <0.1 x 103 <0.1 x 103 <0. l x 103
14 2 1 2.7 8.0 x IOs <0.1 x 103 <0.1 x 103 <0.1 x 103
32 < 1 1 2.9 6.0 xlO s <0.1 x103 <0.1 x 103 <0.1 x 103
to provide uniform particle size for analysis of Kjeldahl nitrogen and total
phosphate. After blending, the samples are passed through a glass fiber
filter (free from organic binder) and the filtrate is stored in borosilicate-
stoppered tubes (stopper contains a teflon coated cap) at room tempera-
ture. Ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and orthophos-
phate phosphorus analyses are performed on the filtrate using automated
techniques. The ammonia analysis is performed using the indophenol blue
reaction [7]. Nitrate is reduced to nitrite in a cadmium reduction column.
This nitrite and the nitrite in the original sample are determined using the
classical "Griess" reaction [8]. The single reagent phosphomolybdate
complex reduced by ascorbic acid and catalyzed by antimony salt with
elimination of arsenic interference is the procedure used for orthophos-
phate analysis [9,10].
Total phosphate phosphorus and Kjeldahl nitrogen analyses were per-
formed on the unfiltered sample stored at room temperature. Samples for
total phosphate were diluted 10 to 1 using a programmed dilutor and were
dispensed into 11.5 ml calibrated glass test tubes. Sulfuric acid containing
ammonium persulfate is added to the diluted sample. The test tube is
capped snugly with a Teflon lined closure. The samples are autoclaved at
132~ for 30 min. After cooling, the autoclaved samples are placed into
the sampling cycle and analyzed using the orthophosphate phosphorus
procedure just noted [9,10]. For the Kjeldahl analysis a S-ml sample is
digested with sulfuric acid, potassium sulfate, and mercuric oxide as
catalysts. After digestion the final volume is adjusted to 50 ml and the
sample is analyzed for ammonia nitrogen using the indophenol blue
reaction [7].
The question was asked concerning the length of time the forms of
nitrogen and phosphorus would remain unchanged after preservation with
400 mg/liter of mercuric chloride and laboratory handling procedures.
Samples of wastewater treatment plant effluents which had been
preserved with 400 mg/liter of mercuric chloride at time of collection for
this study were obtained from the Eutrophication Survey Branch and were
mixed together. The information on the samples is shown in Table 2. The
mixed samples were then distributed into ten containers and were
provided with ten different laboratory numbers for Run 1. For each
subsequent run a different set of lab numbers was used. The samples were
treated as if they had been received as legitimate and individual samples.
The treatment of samples was so designated to remove any bias in
handling samples. Thus for each period, ten individual samples were
cataloged and analyzed as ten separate entities. Although the samples all
originated from one composite, the variation in the number of replicate
analyses was a function of scheduled replication as part of the Analytical
Quality Control Program.
At the end of the last group of analyses the samples were analyzed for
-I
m
TABLE 2--Characteristics of samples compositedfor preservation study. ~u
O
Number of Total Ortho- t--
Type of Type of Days From Kjeldahl Ammonia Nitrite Nitrate Phosphate phosphate I""
Treatment Composite Collection Nitrogen, Nitrogen, Nitrogen, Nitrogen, Phosphorus, Phosphorus, -<
Plant (period) to Compositing mg/liter rag/liter mg/liter mg/liter rag/liter mg/liter
"o
Low-Rate trickling filter 8h 55 12.6 7.7 0.82 0,02 4.7 3,8
Activated sludge 24 h 52 18.0 3.4 0.06 0.58 5.7 4.2
Trickling + sand filter 11 h 78 11.0 0.6 0.15 5.0 3.4 2.6 m
Trickling filter 8h 54 7.4 0.6 0.15 1.5 3.0 2.1 -i
n'/
Trickling filter (high rate) 2h 18 25.0 5.7 0.11 1.3 3.2
o)
Activated sludge 2h 18 12.6 1.7 1.4 2.2 12.6
Primary 5h 17 6.5 0.1 0.01 0.6 2.3 1.1
Trickling filter 8h 19 17.0 10.0 0.30 7.5 6.3
Activated sludge 7h 17 1S.0 4.7 1.2 0.22 0.4
Trickling filter (high rate) 2h 17 ... 13.0 0.12 0.37 6.2
Trickling filter (high rate) +
effluent stabilization pond 24 h 17 ... 21.0 0.01 0.03 7.6
Activated sludge 2h 18 ... 2.9 0.02 0.05 4.2
Trickling filter + activated
sludge 24 h 17 12.0 0.2 0.5 25.2 6.6
Secondary treatment 8h 16 2.0 0.06 0,79 4.3
Activated sludge 9h 16 14.0 4.1 0.12 0,72 4.9
s u s p e n d e d solids, i n o r g a n i c c a r b o n , o r g a n i c c a r b o n , a n d dissolved mer-

cury. T h e results o f t h e analyses for these constituents are i n d i c a t e d in
T a b l e 3. T h e level o f s u s p e n d e d solids is not typical for w a s t e w a t e r
effluents. T h e c o n c e n t r a t i o n o f dissolved m e r c u r y in the s a m p l e is an
i n d i c a t i o n o f the source of the s u s p e n d e d solids a s s u m e d to be m e r c u r o u s
c h l o r i d e a n d o t h e r p r e c i p i t a t e d m e r c u r i a l s which p r e c i p i t a t e d either on
initial m i x i n g or t h r o u g h r e a c t i o n as a f u n c t i o n o f storage time.
TABLE 3--Characteristics of sample after compositing for preservation study.
Relative
Mean Standard Standard Number
Value, Deviation, Deviation, Range, of
mg/liter mg/liter xl00 rag/liter Replicates
Suspended solids 401.0 22.0 5.5 51.0 10

Total inorganic carbon 18.8 1.02 5.4 3.3 10
Total organic carbon 76.0 16.0 21.0 55.0 10
Dissolved mercury 2.8 0.19 6.8 0.5 10
Results
T h e d a t a for the i n d i v i d u a l analyses a r e p r e s e n t e d in T a b l e s 4 t h r u 9.
In T a b l e 10 a s u m m a r y o f the S t u d e n t t-test is p r e s e n t e d [12]. E a c h
possible c o m b i n a t i o n was e x a m i n e d ,
In t h e K j e l d a h l d e t e r m i n a t i o n at the 95 p e r c e n t c o n f i d e n c e level,
differences were observed between all runs e x c e p t the first and last (Table
10). T h e differences could be d u e to changes within the samples or errors
in measurement. In the j u d g m e n t o f the writer m o r e c r e d e n c e is assigned
to the errors in m e a s u r e m e n t t h e o r y since the relative s t a n d a r d deviation
for the all runs category is 0.089 (Table 4). T h e value for relative s t a n d a r d
d e v i a t i o n a n d r a n g e c o m p a r e s favorably with d a t a r e p o r t e d by W i n t e r a n d
TABLE 4--Summary of data for Kjeldahl nitrogen measurement.

Analysis Relative
Performed Mean Standard Standard Number
Days after Run Value, Deviation, Deviation, Range, of
Compositing Number mg/liter rag/liter xl00 mg/liter Replicates
4-10 1 17.77 0.583 3.3 1.9 11

27-33 2 21.26 1.23 5.6 3.3 11
51-58 3 19.52 0.905 4.6 3.0 12
81 4 18.54 0.885 4.8 2.2 10
103 S 17.43 1.03 5.7 2.9 10
All Runs 18.95 1.67 8.9 6.9 54
Data taken from Ref 11 4.14 1.06 25.5 3.97 31
TABLE S--Summary of data for ammonia nitrogen measurement.
Analysis Relative
Compositing Number mg/liter mg/liter • mg/liter Replicates
4-10 1 3.68 0.075 2.2 0.2 11

27-33 2 3.15 0.113 3.5 0.4 11
51-58 3 2.96 0.178 6.0 0.6 14
76 4 3.08 0.181 5.8 0.6 10
101 5 3.13 0.206 6.8 0.7 10
All Runs 3.19 0.297 9.4 1.2 56
Data from Ref 11 1.75 0.24 14.0 1.01 24
Data from Ref 11 1.96 0.28 14.0 1.17 24
TABLE 6---Summary of data for nitrite nitrogen measurement.
Analysis Relative
Compositing Number mg/|iter mg/liter xl00 mg/liter Replicates
4-10 1 0.751 0.0030 0.4 0.01 11

27-33 2 0.74S 0.0069 0.9 0.02 11
51-58 3 0.666 0.0063 0.9 0.02 15
76 4 0.678 0.0092 1.3 0.03 10
101 S 0.698 0.0140 2.0 0.04 11
All Runs 0.705 0.0362 5.1 0.11 58
TABLE 7--Summary of data for nitrate nitrogen measurement.
Analysis Relative
Compositing Number mg/liter rag/liter • rag/liter Replicates
4-10 1 2.59 0.024 0.8 0.1 11

27-33 2 2.71 0.030 1.1 0.1 11
51-58 3 2.71 0.070 2.6 0.2 11
76 4 3.13 0.095 2.9 0.3 10
101 S 2.71 0.095 3.7 0.3 10
All Runs 2.77 0.195 7.1 0.8 53
TABLE 8--Summary of data for orthophosphate phosphorus measurement.
Analysis Relative
Compositing Number mg/liter mg/liter x 100 mg/liter Replicates
4-10 1 4.60 0.045 0.9 0.2 11

27-33 2 4.67 0.047 1.1 0.1 11
51-58 3 4.56 0.051 1.1 0.1 14
81 4 4.65 0.053 1.1 0.1 10
102 5 4.79 0.070 1.5 0.2 11
All Runs 4.65 0.097 2.2 0.4 57
TABLE 9---Total phosphate phosphorus.
Analysis Relative
Days after Run Value Deviation Deviation Range of
Compositing Number mg/liter mg/liter xl00 mg/liter Replicates
4-10 1 6.09 0.105 1.8 0.3 9a

27- 33 2 6.58 0.125 2.0 0.4 11
51-58 3 6.97 0.179 2.6 0.5 11
81 4 6.92 0.114 1.6 0.3 10
115 5 6.98 0.147 2.1 0.4 11
All Runs 6.73 0.321 5.4 1.3 52
Data taken from R e f l l 0.81 0.13 16.0 0.78 33
aTwo outliers were not considered. The "Dixon" outlier test used by Natrella was the
basis for rejection.
TABLE l O--T-test [12]~significant difference between samples at 95 percent confidence.
Run K-N NH3-N NO2-N NO3-N Ortho P Total P
1 versus 2 yes yes yes a yes yes yes

1 versus 3 yes yes yes yes yes a yes
1 versus 4 yes a yes yes yes yes a yes
1 versus 5 no yes yes yes yes yes
2 versus 3 yes yes yes no yes yes
2 versus 4 yes no yes yes no yes
2 versus 5 yes no yes no yes yes
3 versus 4 yes a no yes yes yes no
3 versus 5 yes no yes no yes no
4 versus 5 yes a no yes yes yes no
aNo difference at 99.5 percent confidence level.

Midgett, for Kjeldahl nitrogen at 4.1 and 4.61 rag/liter levels [11]. Thus,
even though the t-test indicates differences, application of judgment on
complexities of the test will permit the assessment that there has been no
significant change in the samples from the time of first analysis to the
time of last analysis. Furthermore, our normal procedure for quality
control chart production requires the use of duplicate analysis which
would provide for a greater latitude in differences. The use of ten to
eleven replicates provides an excellent means to identify errors in measure-
ment due to sampling techniques of analysis as well as chemical changes
in samples. If chemical changes due to storage were the cause, then a
pattern would be observed in Fig. 1 showing changes in Kjeldahl similar
to that reported by Hellwig [1]. Since such changes were not observed, the
cause of difference seen in the t-test is attributed to sampling and
measurement error.
The data for ammonia analysis in Table 5 again require application of
judgment since the initial run data are significantly different from any of
the other runs. However, with the exception of Run 2 versus Run 3, there
are no significant differences between the other runs. An assumption is
made that errors in measurement caused the discrepancy in comparing
Run 1 with all others and Run 2 with Run 3. In Table 5 when comparing
0 Kjeldahl Nitrogen
Mean Kjeldahl Nitrogen
Z4 Ammonia Nitrogen
o Nitrate Nitrogen
22
jo-.____~._ Q Nitrite Nitrogen
/ / ~ O
18
16
g
14
12
~E 10
4
-~- _ _ ~ - - - @ ~ ~
0 - - 0 0-
2
I I o I I o ~ I -0 [ I i~ |
10 20 30 40 50 60 70 80 90 100 llO
DAYS
FIG. 1--Formsofnitrogenlevelsas afunctionoftime.
data from this study with data reported by Winter and Midgett, using
relative standard deviation, the judgment is made that there was no
change in the sample during the time period of analysis.
The nitrite nitrogen analyses have changed over time as indicated in
Table 6, but the changes have been small although statistically significant.
The pattern of change does not appear to bear relationship to other forms
of nitrogen either in magnitude or direction. The fact that even after 101
days the change in concentration was only from 0.75 to 0.70 mg/liter
indicates that nothing very drastic occurred in oxidation, biological
utilization, or biological conversion. Here again, Hellwig reported sig-
nificant changes where biological activity took place. Unpublished studies
of the Lake Huron Program Office also confirmed the scope of change
where biological activity proceeds unimpeded [13].
Examination of Table 7 points out that the nitrate nitrogen analyses
have been variable. In some cases (2 versus 3, 2 versus 5, and 3 versus 5
in Table 10) the t-test indicates that no significant differences are
observed in the data. If judgment is applied, the decision could be made
to assign the difference as a function of the sample handling employed
rather than to a true change in the sample. The same comments apply
here in examining the magnitude of change as were made in the Kjeldahl
and nitrite section.
The precision of the orthophosphate analysis is worthy of note as shown
in Table 8. Although the precision is the best of all the analyses
conducted, only in the case of Run 2 versus Run 4 did the t-test indicate
that no significant difference was observable. The relative standard
deviation appears to be much better in a difficult matrix than that
reported by Winter and Midgett [11]. The reliance on judgment would
lead to the conclusion that an error in measurement caused the differences
between runs and no significant changes in sample composition were
evidenced.
With the exception of the ammonia analysis the total phosphate analysis
compared most favorably in run contrasts using the t-test. Thus, Runs 3
versus 4, 3 versus 5, and 4 versus 5 as shown in Table 10 when the t-test
was applied to the data indicated that there was no significant difference
in the samples. Inspection of Table 9 points out a problem in Run 1. This
run provided the only two outliers (using the Dixon criteria in Ref 14) in
seven sets of data.
Conclusions
Samples of wastewater effluents can be preserved with 400 mg/liter of

mercuric chloride for periods of up to 100 days when stored at room
temperature. Although the statistical t-test indicated observable differen-
ces in the majority of the comparisons, judgment in examining the total
picture provides basis that there was no major change in the samples. An
illustrative example is provided in Fig. 1 where, graphically, the forms of
nitrogen are presented. The mean values are plotted as a function of time.
The magnitude of the differences in the Kjeldahl analysis cannot be
explained by the differences observed in the other forms. In fact, the
Kjeldahl differences wipe out any meaning to the nitrite determination.
However, the ammonia, nitrite, and nitrate data provide credence to the
fact that nothing really changed in the sample over the hundred-day
period.
The phosphate determinations, both orthophosphate and total phos-
phate, are more precise than the nitrogen analyses. The relative standard
deviation expressed as a percentage for the orthophosphate analysis at the
4.6 mg/liter level was 2.2 percent. Using the criteria of relative standard
deviation the orthophosphate analysis had the best precision on a com-
parative basis of any of the six analyses performed. The total phosphate
which is the orthophosphate determination preceded by a digestion step
was superior to all nitrogen forms except the nitrite test, again using the
relative standard deviation as the criteria.
The potential for growth of organisms was present in the plastic bottles.
The storage of the bottles in the laboratory at room temperature, mixing-
aerating, and the availability of nutrients for growth would have provided
changes in orthophosphate and forms of nitrogen unless the samples had
been adequately preserved. Inspection of Table 1 would indicate that the
seeding of organisms from the variety of wastewater effluents was a
distinct possibility especially since aseptic techniques were not used in
sample handling. Despite all the favorable factors for biological changes
no major changes in nutrient chemistry of the samples was observed. The
only conclusion one can draw from the lack of change is the effectiveness
of the preservation technique over the hundred-day period.
Why does Table 10 appear to indicate that there are differences
between runs? The t-test measures random variables. Thus, sampling
errors, errors in measurement that are truly random, and errors due to
sample deterioration must be considered random. If a sample is deterio-
rating (chemically changing through oxidation or reduction), then these
changes could be plotted as a function of time. In examining Fig. 1, no
indication is provided that chemical changes in samples were the source of
the random variable. Then where did the random variable come from to
indicate that mean and variance were not normally distributed? My
conclusion is that the random variables were those of sampling and
measurement (instrumental measurement). In Table 11 the wobble of the
method is presented in the form of what is acceptable from round robin
studies and what was observed in the present study. In my judgment
assessment can be made with the acceptable wobble. In all cases the
TABLE 11--Estimated differences in replicates.
Acceptable Relative Observed Relative

Standard Deviation, Standard Deviation,
Measurement xl00 Xl00
Kjeldahl nitrogen 25 a 8.9

Ammonia nitrogen 14 a 9.4
Nitrite nitrogen 10 b 5.1
Nitrate nitrogen 10 b 7.1
Orthophosphate phosphorus 6a 2.2
Total phosphate phosphorus 15 a 5.4
a Ref II.
bEstimate from Analytical Quality Control Charts.
o b s e r v e d w o b b l e was s i g n i f i c a n t l y b e t t e r t h a n w h a t was a c c e p t a b l e w h i c h
is a n o t h e r i n d i c a t i o n t h a t t h e c h a n g e s w e r e n o t s i g n i f i c a n t f r o m a
standpoint of practicality.
References
[1] Hellwig, D. H. R., Air and Water Pollution Int. Journal, Vol. 8, 1964, pp. 215-228.
[2] Hellwig, D. H. R., Water Research, Vol. 1, 1967, pp. 79-91.
[3] Jenkins, D., "A study of methods suitable for the analysis and preservation of nitrogen
forms in an estuarine environment," report to the USPHS, Region IX, WSPC Division
SERL No. 65-13, College of Engineering and School of Public Health, University of
California, Aug. 1965.
[4] Jenkins, D., "A study of methods suitable for the analysis and preservation of
phosphorus forms in an estuarine environment," report to the USPHS, Region IX,
WSPC Division SERL No. 65-18, College of Engineering and School of Public Health,
University of California, Nov. 1965.
[5] Brezonik, P. L. and Lee, G. F., Air and Water Pollution International Journal, Vol.
10, 1966, pp. 549-553.
[6] Howe, L. H. and Holley, C. W., Environmental Science and Technology, Vol. 3, 1969,
pp. 478-481.
[7] Solorzano, L., Limnology and Oceanography. Vol. 1, 1969, pp. 799-801.
[8] "Methods for Chemical Analysis of Water and Wastes," Environmental Protection
Agency AQCL, Cincinnati, Ohio, 1971, pp 175-183, 195-197.
[9] Murphy, J. and Riley, J., Analytica Chimica Acta, Vol. 27, 1962, pp. 31-36.
[10] Johnson. D. L., Environmental Science and Technology, Vol. 5, 1971, pp. 411-414.
[11] Winter, J. A. and Midgett, M. R., "Method Study 2, Nutrient Analyses, Manual
Methods," Environmental Protection Agency, AQCL, Cincinnati, Ohio, 1970.
[12] Natrella, M. G., Experimental Statistics, National Bureau of Standards Handbook 91,
Chapter 21. U.S. Government Printing Office, Washington, D.C., 1963, pp. 21-1 to
21-6.
[13] Buckley, R. M., private communication, Program to Compute Long Term Oxygen
Demand and Nitrogen Balance from Laboratory Data Sheets and Data Runs on a
Variety of Stations from Lake Huron Program Study, 1966.
[14] Natrella, M. G., Experimental Statistics, National Bureau of Standards Handbook 91,
Chapter 17, U.S. Government Printing Office, Washington, D.C., 1963, pp. 17-1 to
17-6.
0
CQ
m l
n l
I. H. Suffet? C. Wu,' and D. T. L. Wong t
Guidelines for Quantitative

Liquid-Liquid Extraction of
Organophosphate Pesticides
from Water
REFERENCE: Suffet, I. H., Wu, C., and Wong, D. T. L., "Guidelines for Quantita-
tive Liquld-Liquid Extraction of Organophosphate Pesticides from Water," Water Qual-
ity Parameters, A S T M STP 573, American Society for Testing and Materials, 1975,
pp. 167-182.
ABSTRACT: A systematic approach for the development of quantitative analysis of

organophosphate pesticides from water is presented. Information is presented on the
different steps of aqueous pesticide residue analysis including liquid-liquid extraction,
water and solvent phase separation, solvent drying, and a two-step evaporation of the
extract which permits solvent volume reduction to 0.S ml for recovery at low levels.
The p-value method of approach is used as a guide to optimize the liquid-liquid
extraction step. The p-value determined ia ~,..... a water is shown to be applicable to
other types of water including river water, sea water, and secondary sewage effluent.
Recovery studies are presented at nanogram/liter concentrations where the solvent evap-
oration procedure is shown to be critical.
KEY WORDS: water quality, pesticides, environmental tests, organophosphates, recov-

ery, extraction
T h e analysis of a representative e n v i r o n m e n t a l sample for a specific

a q u e o u s pesticide residue should provide actual a q u e o u s c o n c e n t r a t i o n
d a t a for i n t e r p r e t i n g ecological p r o b l e m s such as toxicity, persistence, a n d
t r a n s p o r t in a q u a t i c e n v i r o n m e n t s . T h e procedure should be r e p r o d u c i b l e
a n d q u a n t i t a t i v e . At c o n c e n t r a t i o n levels of n a n o g r a m s / l i t e r , the efficiency
of any analytical step d e t e r m i n e s the lower limit of detectability a n d the
precision for the overall method. Some small q u a n t i t y of a c o m p o u n d is
n o r m a l l y lost d u r i n g the different analytical steps of extraction, enrich-
m e n t , a n d c l e a n u p . T h e analytical errors for each step of the procedure
1Associate professor of Chemistry and Environmental Science; research assistant, Environ-
mental Engineering and Science; and research assistant, Department of Chemistry; re-
spectively, Drexel University, Philadelphia, Pa. 19104. Mr. Wu is presently a project en-
vironmental engineer with McFarland-Johnson-Gibbons Engineers, Inc., Woodbury, N.J.
08096.
167
9
should be quantitatively understood to optimize quantitative methodology

and minimize losses. The effects of many variables acting simultaneously
or successively warrant observation.
A systematic approach is needed for the development of quantitative
analysis of pesticides dissolved in a wide spectrum of natural water
samples. Fundamental information is necessary to provide an analyst with
the flexibility of choosing the best method to fit his type of sample. The
aim of this paper is to provide some guidelines for this purpose.
Organophosphate pesticides were chosen as an illustration in this study in
an attempt to characterize the efficiency of different steps of quantitative
analysis in natural waters.
The first step of aqueous organic residue analysis involves liquid-liquid
extraction (LLE) to isolate the organic compound from the aqueous phase,
that is, to transfer materials of interest to the solvent or remove unwanted
materials from the sample. Maximum recovery of a compound from the
aqueous phase depends upon the polarity of the compound, water quality
characteristics (for example, ionic strength and pH), polarity of the
solvent, and the type of LLE process employed [1]. 2
The state-of-the-art for prediction of the extraction capabilities of a
binary solvent system has been presented by Peppard [2]. He states that
the behavior of a given solute in a given extraction system cannot be
predicted in any but the roughest manner; however once determined it can
be relied upon in any future experiments. Faust and Suffet [3] have
described seven general criteria for quantitative extraction of organic
pesticides from aqueous systems. The analyst must select an a q u e o u s
volume, pH, ionic strength, solvent, water to solvent ratio, number of
extractions, and method of extraction. A solvent needing minimal cleanup
should be utilized. Natural waters should be pretreated to constant
conditions of pH and ionic strength to control and promote consistent
results from any analysis.
The p-value method of approach [4] is utilized as a guide to optimize
the selection of parameters for the LLE step of analysis [5-7]. In this
paper the relationship of p-value measurement in distilled water to those
determined in natural waters is investigated for some organophosphate
pesticides. The relationship of the E-value to the level of recovery at 10, 1,
and 0.1 percent of the concentrations used for the p-value test is also
studied with a vortex stirring method [8-10]. This method consists of
vortex stirring, water solvent phase separation, solvent drying, and a
two-step evaporation of the extract initially to less than 10 ml followed by
microcondensation to 0.5 to 5 ml for final recovery. Cleanup was not
found necessary in this study. This paper describes quantitative investiga-
tions of the two-step evaporation process. The relationship between
recovery at different pesticide residue levels will be shown.
SUFFET ET AL ON ORGANOPHOSPHATE PESTICIDES 169
Method of Approach
The distribution coefficient K is the basis of LLE. It can be described
in terms of an E-value (that is, fractional amount of solute partitioned
into the solvent from the aqueous phases under specific water to solvent
ratio, and water quality characteristics).
EIVn E
K - = (1)
( i - E)/Vp ,~(l - E)
v~
a = Vp (2)
a is the ratio of Vn to Vp,, the volumes of the solvent and water phases,
respectively, after an extraction step. The fraction extracted under any
specific condition, E, is related to the fraction extracted under equi-
librated equivolume conditions, p [4].
E
(3)
P ~- E(~- I)
p-Values are presented on a fractional basis and yield direct recovery

data of a 1:1 water to solvent ratio with equilibrated solvents.
Figure 1 shows the relationship between an experimental E and a
p-value calculated from the same data. The E-values were obtained by
Suffet for 2,4-D in pH 2, 0.2 M orthophosphate buffer, extracted with
benzene at a = 0.10 [7]. The figure shows the experimental mean and the
calculated 3 standard deviation (SD) interval. An apparent contraction of
the standard deviation of the p-value relative to that of the E-value occurs
when a p-value >0.90 is calculated from E-values determined at low a.
This is due to the asymptotic nature of the relationship [1].
Experimental
p- Value Determination
All p-values are determined at 25 + 0.5~ at pH 4.2 in 0.2 M
orthophosphate buffer. This fixes the aqueous solutions at the pH of
minimal hydrolysis and maintains a consistent ionic strength. The prepa-
ration of samples, reagents, and the p-value method utilized have pre-
viously been described [5] except that the aliquot of solvent (1 ml of
acetone) which was transferred to the volumetric flask (1000 ml) for
spiking an aqueous sample was not evaporated under a stream of
nitrogen. Thus, the aqueous phase contained 0.1 percent acetone.
0.2
I
T~ II 09 I
FIG. 1--The relationship between experimental E- and p-values and a 3 standard
deviation interval calculated from t h e m e a n E- and p-values. L I E o f 2,4-D [2,4-dichloro-
phenoxy acetic acid) from 0.2 M pH 2.0 orthophosphate buffer at 25~ by benzene at a
water to solvent ratio o f 10:1 for eleven determinations,
In brief, the p-value determination is made by shaking 20 ml of

aqueous buffer containing pesticides with 2 to 4 ml of solvent in a glass
stoppered graduated cylinder. After the phases have separated, the solvent
phase is dried over Na2SO4 and analyzed by gas-liquid chromatography
(GLC). The solvent volume is corrected for losses during drying [I1]. Six
solvents representing a wide range of polarity were utilized for partition;
benzene, hexane, ether, ethyl acetate, chloroform, and dichloromethane.
Since the highest water to solvent ratio will give the most accurate p-value
in the 0.90 to 1.00 range (Fig. 1), determinations were made in the
highest practical water to solvent ratio. This is of prime interest for
complete recovery systems and is the usual ratio for residue samples.
The purity of the organophosphate pesticides studied were checked by
GLC with both flame photometric and electron capture detector responses,
infrared, and thin layer chromatography. Table 1 shows the organo-
phosphate pesticides studies, and the aqueous concentrations used for
p-value analysis and recovery studies.
The effect of natural water quality characteristics upov the p-value
method was investigated. Table 2 shows the natural water characteristics
of the river water, synthetic sea water, and organic water which is
secondary sewage effluent containing organic matter utilized in this study.
All natural waters were filtered through a 0.45 ban Millipore filter.
Extracted samples were quantitated for all p-value determinations by
TABLE 1--Experimental conditions for the p-value determination and for recovery studies
of a mixture of organophosphate pesticides.
Experimental Concentration (/ag/l)
One-Step
Compounda ChemicalName Quoted ~ Purity p-Value RecoveryStudyb
1. Parathion O,O diethyl O-p- 99.7 283 124
nitrophenyl ester
of phosphorothioic
acid
2. DEF Butyl phosphoro- 95.2 178 and 226 282
trithioate
3. Ethion Ethyl methylene 132 and 181 204
phosphorothioate
4. Trithion S-[(p-chloro- 95.0 199 and 298 402
phenyt) thiometh-
yl]-O,O-diethyl
ester of phosphor-
odithioic acid
5. EPN Phenyl-O-ethyl O- 162 252
p-nitrophenyl ester
of phosphorothioic
acid
a Analytical grade of manufacturer. All compounds~iresoluble at these concentrations.
b100% value for direct analysis, 10, 1, and 0.1% of these amounts were also studied.
comparison to standard curves. Samples were run between standards to

assure GLC quantitative accuracy. All organophosphate mixture prepara-
tions, standard solutions, solvent extractions, and GLC injections for these
studies were completed in the same day to minimize errors resulting from
solvent evaporation, pesticide decomposition, and instrument variation.
R e c o v e r y Studies
The vortex stirring method [8-10] of liquid-liquid extraction was used

for recovery studies. Organophosphate pesticides were serially extracted
under p-value conditions (pH 4.2, 0.2 M orthophosphate buffer) from
distilled water, river water, and organic water. Synthetic sea water was
treated by adjusting to pH 4.2 with phosphoric acid.
Two-liter widemouthed glass jars covered by a cap lined with aluminum
foil and a magnetic stirrer were utilized at laboratory temperatures (23 to
25~ in the vortex stirring method. Benzene was utilized as the extracting
solvent at a ratio of 1000 ml aqueous solution to 100 ml benzene.
The time required to develop partition equilibrium of the organo-
phosphate pesticides with the vortex stirring method for a 10:1, water to
solvent ratio was investigated between different types of natural waters.
The concentration of the pesticides utilized for p-value determination was
chosen for this work to enable successive samples of solvent phase to be
..k
,,,4
Ix3
TABLE 2---Water quality characteristics o f Schuylkill River water, outfall o f a secondary sewage treatment plant, and synthetic sea water.
Secondary Sewage Effluent

Schuylkill River Water --4
(Organic Water) Synthetic Sea Water m
I II I II Ia II b Percent D
r--
Alkalinity (rag/liter 75 48 pH 7.5 7.7 NaCI 58.4q i'--

as CaCO3) MgClz 26.46 -<
Hardness (rag/liter 165 97 Settleable Solids 0 0 Ca "z (rag/liter) 400 Na2SO 4 9.750
as CaCO3~ (rag~liter) 6H20 "17
Color (units) 20 40 Suspended Solids 66 75 Mg *z (rag/liter) 1 272 CaCLz 2.765
(mg/liter) KCI 1.645
Specific rn
Conductance (pmho) 437 246 % Volatile Solids 91 85 Na" (rag/liter) I0 550 NaHCO 3 0.477 m
C1- (rag/liter) 30 9 Total Carbon 97 90 HCOf (rag/liter) 138 KBr 0.238 ,(/)
(mg/liter)
Inorganic Carbon 2 3 SO~-2 (rag/liter) 2 649 SrCI 2. 6H~O 0.095
(rag/liter)
COD (rag/liter) 17.2 16.0 Total Organic 95 87 CI- (rag~liter) 18 980 NaF 0.007
Carbon (rag/liter) F- (mg/liter) 1.3 H3BO 3 0.071
SO4-2 (rag/liter) 100 48 Ionic 0.72
Strength M
pH 7.9 7.8 pH 7.8 pH 8.2
Dissolved Solids 306 188
(rag/liter)
Total Solids 324 270
(rag/liter)
a See Ref 16.

b See Ref 17.
analyzed directly. The vortex extractions were allowed to settle for 2 min
before taking an aliquot of extracting solvent. A correction factor for
solvent removal was used to calculate the equilibrium process on a
common E-value basis [11].
Recovery studies utilized 90 rain of mixing by the vortex method to
assure complete equilibrium for successive extractions. Extraction was
followed by successive steps of separation of the water from the solvent
phase and enrichment of the extract in a Kuderna-Danish (K-D) evapo-
rator in two steps: first to under 10 ml on a stream bath and then to a
final volume in a micro K-D evaporator heating block [11] (Kontes
Glassware, Vineland, N.J.).
Recovery studies were performed at 10, 1, and 0.1 percent of the initial
pesticide concentration level shown in Table 1, with a final evaporation
volume to 10, 1, and 0.5 ml, respectively. In one run, final evaporation
was completed with a carborundum chip ebullator, 3-ball Synder column
K569001-03, and a concentrator tube K-570050. In a second run, final
evaporation was completed with nitrogen ebullition [12], a 3-ring column
K-569251-0314, and concentration tube K-570050. The nitrogen ebullator
was approximately S0 bubbles per second, which was ten times faster than
that used by Beroza and Bowman [12]. Glassware was obtained from
Kontes Glass Co., Vineland, N.J.
Analytical
Quantitative analysis was made utilizing GLC. A Tracor model MT-220
equipped with two independent Melpar Flame Photometric Detectors
(FPD) was used. Venting valves were installed in the heated zone between
GLC outlet and FPD to prevent "flame out," which decreased FPD
detector stability [13]. Solvent tailing was controlled by venting up to 1
rain under the GLC conditions used. Chromatograms were recorded on
1-mV Beckman and 1-mV Perkin Elmer recorders with chart speed
maintained at 0.5 in./min and 10 mm/min, respectively. GLC attenuation
was varied for p-value analysis and recovery studies. The amperes full
scale used was 1.6 to 12.9 x 10-8. Nitrogen carrier gas, hydrogen, and
oxygen were passed through a filter drier before entering the GLC. The
optimun signal to noise ratio of the detector was set with flow rates of
hydrogen at 150 ml/min, air at 35 ml/min, and oxygen at 20 ml/min [13].
The GLC columns used were packed with 10 percent DC-200 on 80/100
mesh Gas Chrom Q [I4]. A nitrogen flow rate of 120 ml/min was used.
The inlet temperature was set at 240~ The relative retention times
related to Parathion were DEF 1.74, Ethion 2.20, Trithion 2.56, and EPN
3.60.
Results
p- Value Determination
Table 3 shows the E- and p-values for five organophosphate pesticides
studied in six different solvents. Phase equilibrium was reached within 20
min [1I]. The average p-value is greater than 0.96 for the extraction of
these organophosphate pesticides from 0.2 M pH 4.2 orthophosphate
buffer. Therefore, in this case the average E-value under a consistent
water to solvent ratio is a better judge of solvent choice. Inconsistency in
the precision of the data is noted for the chlorinated solvents dichloro-
methane and chloroform as reflected by the standard deviations of the E-
and p-values. Figure 2 shows the flame photometric detection gas chroma-
(CH3)2CO STD. CH2CI = STD
l 1 I
2
I
3
|
4
i
5
I
6
TIME
I
7
II
~MINUTES )
I i i
1 2 3 4 5 6
n l l
FIG. 2--A comparison of gas-liquid chromatograms of orthophosphate pesticides in

acetone and dichloromethane showing the taifing effect of the dichloromethane solvent with
venting valve installed.
TABLE 3~Liquid-liquid extraction o f organophosphate pesticide from 0.2 M pH 4.2 orthophosphate buffer at 25 ~ -+ 0.5~
Benzene Hexane Ether

Go
Compound # E p # E p # E p c
m
Parathion 13 0.97 -+ 0.04 1.00 _+ 0.01 14 0.98 +_ 0.02 1.00 -+ 0.00 2 0.82 -+ 0.01 0.98 _+ 0.00 --I
12 0.90_+0.0 7 a 0.99_+0.01 a m
DEF 14 0.90 _+ 0.06 0.99 _+ 0.01 15 0.87 -+ 0.05 0.99 -+ 0.01 13 0.79 _+ 0 . 0 6 a 0.97_+0.01 a
Ethion 13 0.88 -+ 0.07 0,99 -+ 0.01 14 0.84 -+ 0,08 0.98 _+ 0.01 13 0.74 _+ 0.06 a 0.96 _+ 0.01 a ),
I"
Trithion 13 0.87 -+ 0.06 0,99 -+ 0.01 14 0.82 _+ 0,07 0.98 _+ 0.01 13 0.73 -+0.05 a 0.96 _+ 0.01 a
EPN 8 0.96 +_ 0,05 1.00 -+ 0.01 12 0.95 _+ 0.06 1.00 -+ 0.01 12 0.84 _+ 0.04 a 0.98 -+ 0.01 a O
z
O
2o
Ethyl Acetate Dichloromethane Chloroform
z
O
Compound # E p # E p # E p "0
"1"
O
Parathion 2 0.92 -+ 0.01 0.99 _+ 0.00 6 1.10 _+ 0.09 1.01 _+ 0.01 7 0.96 _+ 0.05 1.00 _+ 0.01 60
12 0.90 +_ 0.06 a 0.99 _+ 0.0 1a -'1-
DEF IS 0.84+-0.08 a 0.98+-0.01 a 9 0.97-+0.06 1.130-+0.01 7 0.83 _+ 0.02 0.98 _+ 0.00
Ethion --4
14 0.84 _+ 0.07 a 0.98 _+ 0.01 a 6 0.94 -+ 0.09 0.99 _+ 0.01 8 0,89 _+ 0.10 0.99 ___O.01 m
Trithion 14 0.82 -+ 0 . 0 7 a 0.97 _+ 0.01 a 7 1.03 + 0.27 1.00 _+ 0.02 8 0.89 _+ 0.10 0.99 _+ 0.01 "D
EPN 12 0.90 +_ 0.10 a 0.99 -+ 0.02 a 7 1.12 ___0.16 1.01 -+ 0.01 7 1.02 _+ 0.10 1.00 + 0.01 m
o'~
.-I
NOTE--Number of runs : #, and E and p-values _+_ standard deviation.
a All water to solvent ratios are 10:1 except those m a r k e d a where the ratio is 5:1.
b Data exceeding 3 s tan da rd deviations of the average of "acceptable" da t a are discarded. m
60
t2n
tograms of standard solutions of the organophosphate pesticide mixture

(Table 1) in acetone and dichloromethane. The gas chromatograms of the
organophosphate pesticide mixture in dichloromethane show a tailing
effect which limits precise analysis by the standard curve technique.
Chloroform behaves similarly to dichloromethane under the gas chromato-
graphic conditions. Chromatograms of the organophosphate mixture in
other solvents behave as in acetone.
Suffet and Faust [5] have previously determined the p-value of para-
thion to be 0.89 in hexane, 0.89 in benzene, 0.84 in ethyl acetate, and
0.93 in ether at pH 3.1 in 0.2 M orthophosphate buffer at 25~ These
p-values are much lower than the p-values at pH 4.2 determined in the
present study. The concentration of parathion to be extracted from the
water phase was 50 times higher (mg/liter range) in the previous study [5]
than those in this study (/ag/liter range).
In the previous study [5], the aliquot of solvent (10 to 15 ml of acetone)
which was transferred to a volumetric flask (100 ml) for spiking an
aqueous sample was evaporated under a stream of nitrogen. Only 1 ml of
solvent was added to a 1000 ml volumetric flask and diluted to volume
(0.1 percent acetone) in the present study. Therefore, no evaporation was
necessary.
In order to investigate the experimental differences of pH and acetone
evaporation, a p-value study of parathion at high concentrations (3.09
mg/liter) was made in duplicate. First, parathion was extracted from
water as in the low concentration studies completed here. Secondly, the
parathion was extracted into water from a solvent phase, p-Values
calculated by both methods are within 0.01 of the values reported in Table
3. An experiment to test the evaporation effect was completed by
evaporating 10 ml of acetone containing 0.039 mg of parathion to less
than 0.S ml under a nitrogen stream in a 100 ml volumetric flask.
Variable parathion losses were observed under different rates of nitrogen
flow and the height of the nitrogen stream above the liquid. Apparently,
the evaporation of a spiked solvent was a procedural problem in the initial
parathion study.
Benzene was chosen for recovery studies because of a predominance of
high E- and p-values determined for many organophosphate pesticides at
pH 4.2 in 0.2 M orthophosphate buffer [11]. Analysis of benzene extracts
were precise and benzene lacks aqueous solubility. Hexane could have
been used in this case, but for other organophosphates benzene was
considered the better solvent [11].
The Relationship Between p-Values Determined in Distilled Water

and Other Types of Water
Table 4 shows the relationship between p-values of DEF and ethion as
TABLE 4--The effect of different natural waters on the p-value determination of

organophosphate pesticides from 0.2 M, pH 4.2 orthophosphate buffer at 25 4- 0.5~
DEF Ethion
Number p-Value ..-E Number p-Value 4-

of Standard of Standard
Solvent Water Runs Deviation Runs Deviation
Distilled 14 0.99 + 0.01 13 0.99 -I- 0.01

Benzene River 3 0.98 dr_ 0.01 5 0.97 4- 0.02
Sea 3 0.99 ._-E0.00 5 0.98 + 0.00
Orgamc 3 1.00 ..+__0.02 3 0.98 4- 0.02
Distilled 15 0.99 4- 0.01 14 0.98 + 0.01
Hexane River 3 0.98 4- 0.01 5 0.98 + 0.01
Sea 3 0.99 -4- 0.00 5 0.98 4- 0.00
Organic 3 1.01 -I- 0.02 3 1.02 4- 0.03
Distilled 13 0.97 + 0.01 13 0.96 + 0.01
Ether River 3 0.97 + 0.01 S 0.94 4- 0.03
Sea 3 0.96 + 0.01 5 0.92 4- 0.04
Orgamc 3 0.96 + 0.01 3 0.95 4- 0.01
Distilled 15 0.98 + 0.01 14 0.98 4- 0.01
Ethyl River 3 0.97 4- 0.01 5 0.96 4- 0.01
Acetate Sea 3 0.97 4- 0.01 5 0.96 4- 0.01
Orgamc 3 0.98 4 - 0.00 3 0.98 4- 0.00
Distilled 9 1.00 4- 0.01 6 0.99 4- 0.01
Dichloro- River 3 1.00 4- 0.03 5 0.98 4- 0.02
methane Sea 3 1.01 4- 0.01 5 0.98 4- 0.02
Orgamc 3 0.97 4- 0.06 3 0.95 ..+__0.08
Distilled 7 0.98 4- 0.00 8 0.99 4- 0.01
Chloroform River 3 0.97 + 0.04 5 0.96 4- 0.04
Sea 3 0.99 4- 0.01 5 0.98 4- 0.01
Orgamc 3 0.95 4- 0.05 3 0.96 + 0.04
NOTE--All water to solvent ratios are 10:1 except some 5:1 for ether and ethyl acetate.
Apparent p-values are determined in different natural waters.
d e t e r m i n e d in distilled w a t e r , a n d a p p a r e n t p - v a l u e s d e t e r m i n e d in f i l t e r e d
river w a t e r , o r g a n i c w a t e r , a n d s y n t h e t i c sea w a t e r . T h e f i l t e r e d r i v e r a n d
o r g a n i c w a t e r s w e r e a d j u s t e d to t h o s e u t i l i z e d for t h e p - v a l u e c o n d i t i o n in
d i s t i l l e d w a t e r o f p H 4.2, 0 . 2 M o r t h o p h o s p h a t e b u f f e r . T h i s e n a b l e s a
q u a n t i t a t i v e c o m p a r i s o n b e t w e e n p - v a l u e s d e t e r m i n e d with a c o n s i s t e n t set
o f w a t e r q u a l i t ~ c h a r a c t e r i s t i c s . O n l y t h e p H was a d j u s t e d for sea w a t e r .
T h e s t u d e n t t-test was used to d e t e r m i n e w h e t h e r t h e d i f f e r e n c e o f
a p p a r e n t p - v a l u e s u s i n g e a c h w a t e r a n d p - v a l u e s u s i n g distilled w a t e r are
d u e to r a n d o m e r r o r . T a b l e 4 shows t h a t t h e p - v a l u e s o b t a i n e d are
c o n s i s t e n t w i t h i n r a n d o m e r r o r at t h e 2S c o n f i d e n c e interval. T h e a d j u s t -
m e n t o f w a t e r q u a l i t y c h a r a c t e r i s t i c s to p - v a l u e c o n d i t i o n s o f p H a n d ionic
s t r e n g t h a r e i m p o r t a n t p a r a m e t e r s for c o n t r o l o f L L E e f f i c i e n c y a l o n g w i t h
t h e c h o i c e o f solvent.
Recovery Studies of a LLE Step at High Concentration

The time required to develop partition equilibrium was studied for the
vortex stirring method. The fractional amount Ova) of organophosphate
pesticides extracted by 100-ml of benzene from 1 liter of 0.2 M ortho-
phosphate buffer at pH 4.2 was determined. The 100 percent pesticide
concentration level was chosen for this work to enable successive samples
of solvent phase to be analyzed directly. Four different aqueous types were
studied: distilled, river, organic, and synthetic sea water (Table 2).
Figure 3 shows plots of the equilibrium experiments for DEF and
parathion. The trend indicated in Fig. 3 was typical for other organo-
phosphates studied. The type of water extracted and the compound
FIG. 3---The time to develop an extraction equilibrium o f D E F and parathion between 1

liter o f different types o f water adjusted to p H 4.2 with 0.2 M orthophosphate buffer at room
temperature and 100 ml benzene by the vortex stirring method. F I is the fraction extracted.
It is equivalent to E,
extracted did not appear to have any effect upon the equilibrium time for
the vortex stirring method.
Table 5 shows a set of first step vortex recovery data at the 100 percent
concentration level compared to the E-value data shown on Table 3. The
fraction recovered (F a) for a one-step vortex recovery study is equal to an
apparent E-value, since direct recovery data (1000-ml buffer to 100-ml
benzene) is a p-value analysis (20-ml buffer to 2-ml benzene) run with
large water and solvent volumes at the same 10:1 water to solvent ratio.
The vortex data confirms the E-values of Table 3. The apparent E-values
from direct recovery data (1000 ml of aqueous solution) are all higher
than E-values from p-value experiments with 20 ml of aqueous solution.
TABLE 5--A comparison of E-values and apparent E-values from recovery studies.
Number of Table 3 Recovery a

Compound Runs E-Value #a E-Value
Parathion 13 0.97 + 0.04 4 1.01 4- 0.02

DEF 14 0.90 ._+_0.06 11 1.00 -t- 0.05
Ethion 13 0.88 -4- 0.07 7 0.96 4- 0.04
Trithion 13 0.87 -4- 0.06 8 0.95 4- 0.05
EPN 8 0.96 -4- 0.05 4 0.98 4- 0.03
a Recovery E-value equals the fraction recovered (F~) for a one-step vortex recovery study
(direct GLC analysis) at a water to solvent ratio of 10:1 (1000 ml water to 100 ml benzene).
Recovery Studies of a LLE Step at Low Concentration

Figure 4 shows the average replicate results of one-step vortex recovery
studies performed at the four different pesticide fortification levels (10, 1,
and 0.1 percent of the initial level, Table 1) for parathion, DEF, and
ethion. The 0.1 percent level is in the 100 to 400 rig/liter concentration
DEF TRITHION ETHION

10C
0.1
//
,I, II ,.,' ,.,, II II ,,., ,'.o II
F,
FIG. 4--A comparison o f the fraction extracted (F~) versus the change o f absolute
concentrations of three organophosphate pesticides. Table 1 lists the 100 percent concen-
tration.
range. Data is pooled from the extraction of different waters and two
different runs for final evaporation steps to observe trends.
Figure 4 shows that as the pesticide fortification level is lowered, the
fraction recovered (Fa) is also decreased. Evaporation losses appear to be
the prime cause of less recovery at lower levels. Table 6 shows the percent
TABLE 6--Percent recovery q- standard deviation o f nanogram amount of pesticides

from evaporation o f lO0 to 1 ml o f benzene under different experimental conditions.
Percent Recovery ..+..Standard Deviation
Experimental Carborundum Chips Nitrogen Stream

Concentration with 3-ball Snyder with 3-ring Distilling
Pesticides (ng/100 ml) Micro-Column a Column b
Parathion 12.3 80.12 -I- 4.65 90.82 ..~ 2.64

DEF 28.4 81.90-t- 3.84 90.47 _.+_0.74
Ethion 20.0 83.75 -I- 2.91 91.52 _-E 1.38
Trithion 42.0 84.32 _+__4.68 93.22 _:E 1.57
EPN 35.2 84.57 -t- 1..50 94.55 _.-E4.69
Average Recovery = 82.93 -t- 1.88 92.11 -t- 1.72
a 3-ball Snyder micro-column K-569001-03 (19/22) and concentrator tube K-570050 (19/22).
b3-ring distilling column K-569251-0314 (19/22) and concentrator tube K-570050 (19/22).
recovery of nanogram amounts of the pesticides for benzene used in this

study, when evaporated from 100 to 1 ml in the two-step evaporation
procedure. The average recovery was 10 percent lower for the carborun-
dum chip with the 3-ball Snyder micro-column, as compared to the
nitrogen stream with a 3-ring distilling column.
Three successive blank GLC analyses were run before each recovery
study to determine if any contamination was present (Fig. 5). The solvent
blank (evap.) shows solvent impurities and glassware contamination from
the two-step K-D evaporation of 100 ml of solvent. The complete solvent
blank shows the solvent impurities and glassware contamination from all
the glassware involved in the LLE, separation, and drying procedures. The
water blanks are aqueous blanks of 1 liter of 0.45 /am Millipore filtered,
unfortified sample, under p-value condition with final evaporatio n to 0.5
ml. A 0.5-ml limit for evaporation is used as large errors appear due to
smaller volume measurements. All fortified samples were run in their
respective glassware after these blanks.
Flame photometric detection (P-mode) has been described as highly
specific and sensitive to P-containing compounds [15]. For example, 90
percent Of the food extracts chromatographed showed very few inter-
ferences in the P-mode [15]. Only in high fat samples were significant
interfering peaks found. Figure 5 shows the results of one set of blanks.
Certain GLC peaks appear to be specific for a type of water. For example
DISTILLED
TIME (MINUTES)
FIG. 5---A comparison o f the GLC response characteristics o f blank analysis for solvent,
solvent evaporation, and different types o f water adjusted to pH 4.2 with 0.2 M ortho-
phosphate buffer at room temperature.
the 3-min peak is specific for synthetic sea water. Other peaks appearing
in all six chromatograms seem to be in the solvent or on the evaporation
glassware (that is, those between 0 to 1 min). The peak at 1 to 2 min
seems to be due to glassware contaminants from other than the evapo-
ration step. Organic water has a stronger GLC peak in this range. The
peaks of the organophosphate pesticides studied here did not interfere
with any of the GLC peaks observed as impurities.
Summary
This study provides insight into the problems of aqueous pesticide
residue analysis by isolation of the LLE step and other steps of analysis at
nanogram to microgram/liter concentration levels. The effects of natural
water characteristics on extraction procedures appear minimal when pH
and ionic strength are adjusted to consistent conditions. The vortex
stirring method was found suitable for extracting trace concentrations of

pesticides from distilled water, river water, synthetic sea water, and
secondary sewage effluent.
Significant pesticide recovery errors occurred at the lowest fortification
level (125 to 400 ng/liter, 0.1 percent) and were attributable to evapo-
ration losses. Evaporation procedures must be carefully chosen and
checked to assure good recovery at nanogram per liter levels. In the
present study, flame photometric detection (P-mode) did not require
aqueous extract cleanup.
Acknowledgment
This research was supported by Research Grant No. 801179 (formerly
16020FYT), Office of Water Research, Environmental Protection Agency,
Washington, D.C.
References
[1] Suffet, I. H. and Faust, S. D., Advances in Chemistry Series, Vol. 111, 1972, p. 11.
[2] Peppard, D. F., Advances in Inorganic Chemistry and Radiochemistry, Vol. 9, 1966,
p. 1.
[3] Faust, S. D. and Suffet, I. H., Water and Water Pollution Handbook, Vol. III,
L. Ciaccio, Ed., Marcel-Dekker, New York, 1972, Chapter 23, p. 1249.
[4] Beroza, M., Inscoe, M. N., and Bowman, M. C., Residue Reviews, Vol. 30, 1969, p. 1.
[5] Suffet, I. H. and Faust, S. D., Journal of Agricultural and Food Chemistry, Vol. 20,
1972, p. 52.
[6] Suffet, I. H., Journal of Agricuhural and Food Chemistry, Vol. 21, 1973, p. 288.
[7] Suffet, I. H., Journal of Agricultural and Food Chemistry, Vol. 21, 1973, p. 591.
[8] Standard Methods for the Examination of Water and Wastewater, 13th ed., American
Public Health Association, Washington, D.C., 1971.
[9] Kawahara, F. K., Eichelberger, J. W., Reid, B. H., Stierli, H., Journal of the Water
Pollution Control Federation, Vol. 39, 1967, p. 572.
[10] Schafer, M. L., Peeler, J. T., Gardner, W. S., and Campbell, J. E., Environmental
Science and Technology, Vol. 3, 1969, p. 1261.
[11] Suffet, I. H., unpublished data, 1974.
[12] Beroza, M., Bowman, M. C., and Bierl, B. A., Analytical Chemistry, Vol. 44, 1972,
p. 2411.
[13] Sowinski, E. J. and Suffet, I. H., Journal of Chromatogri~ptiic Science, Vol. 9, 1971,
p. 632.
[14] Watts, R. R. and Storherr, R. W., Journal of the Association of Official Agricultural
Chemists, Vol. 52, 1969, p. 513.
[15] Bowman, M. C., Beroza, M., and Hill, K. R., Journal of the Association of Official
Agricultural Chemists, Vol. 54, 1971, p. 346.
[16] Sverdrup, H. U., Johnson, M. W., and Fleming, R. H., The Oceans, Prentice Hall,
Englewood Cliffs, N.J. 1959.
[17] Specification for Substitute Ocean Water, ASTM D 1141-52, Annual Book of ASTM
Standards, Part 31, 1974, p. 47.
H. A . M c L e o d j a n d D. E. C o f f i n I
Pesticide Residue Screening

Methods Utilizing Multidetector
Configurations
REFERENCE: McLeod, H. A. and Coffin, D. E., "Pesticide Residue Screening

Methods Utilizing Multideteetor Configurations," Water Quality Parameters, A S T M
STP 573, American Society for Testing and Materials, 1975, pp. 183-195.
ABSTRACT. Multiple detection systems are reviewed as an approach to improve selec-

tivity, increase the number of compounds detected, aid in confirming identity, and
reduce the time of analysis. Various configurations of separators, detectors, and
operating parameters are outlined. The advantages, disadvantages, scope, and practical
application to screening biological samples for organic residues are illustrated by refer-
ence to several published methods for pesticide residues and drugs.
KEY WORDS" water quality, pesticides, residues, environmental tests, sampling
Approximately 1000 chemical identities are said [1]~ to be used as

pesticides on a global basis. In Canada, some 650 are registered for use
under the PCP Act [2]. Following their application many pesticides are
metabolized into one or more toxic metabolites. These facts underscore
the necessity for analytical methods that are capable of extracting residues
from many different types of samples; separating them from the sample
matrix and determining their identity and concentration.
The status of methodology as reviewed by Beroza and Bowman [3],
McCully and McLeod [4], and Frehse [5] indicates emphasis in develop-
ment has been on cleanup of the sample extract, evaluation of different
gas-liquid chromatography (GLC) columns, and improvement in various
detectors. It is now possible to isolate many different pesticides into a
solution suitable for determinative procedures such as GLC or TLC. For
example, Table 1 lists the polar and nonpolar pesticides and their
metabolites that are known to be recovered from a number of different
plant and animal tissues by a method [6] used in our laboratory.
~Research scientist and division chief, respectively, Food Research Laboratories, Health
Protection Branch, Health and Welfare Canada, Ottawa, Ontario K1A 0L2, Canada.
183
9
TABLE 1--Pesticides and some metabolites quantitatively recovered (80 percent) from
fortified (0.05 mg/kg) samples of plant and animal tissues.
Aldrin p,p'-DDT Heptachlor
Arociors 1254, 1260 p,p'-DDD Heptachlor epoxide
Atrazine 2,4-d acid Malathion
Azinphosmethyl 2,4-d esters Malaoxon
a-BHC Diazinon Methoxychior
/1-BHC Dicofol Parathion
y-BHC (Lindane) Dieldrin Paraoxon
Captan Dinotrophenylanisole Phosaione
cis-Chlordane Dursban Phosphamidon
trans-Chlordane Endosulfan Simazine
Coumphos Ethion Sumithion
Dasanit Fenitrothion 2,4,5-t acid
p,p'-DDE Fenitroxon 2,4,5-t esters
o,p'-DDT HCB Trithion
GLC column packings have been developed and operating parameters

determined for separating many of the pesticides known to be recovered
by the various multiresidue methods [3-6]. Morley and McCully [7]
compiled from the literature listings of relative (parathion) retention times
of pesticides and some metabolites for column packings ranging from the
nonpolar (for example, OV-1) to the very polar (for example, DEGS)
columns. Their data indicate some compounds are discretely separated
but that total or partial overlap occurs for many others. The number of
compounds listed for each packing ranged from 145 to 172. Multi-
detectors, operating in their characteristic response modes, can detect and
distinguish among many of these compounds including some with over-
lapping retention volumes.
The analyst has at his disposal a number of detectors, suitable for
single, dual, or multiple configurations, for monitoring the G L C effluent
[8]. Those most frequently used today, and some that are novel, are listed
in Table 2 in order of the number of response modes. For example, the
EDC [9] has one mode only, that of electron absorption. Similarly for the
FID [10]....The A F I D [11] operates in one mode at a time but selection of
appropriate operating parameters favors detection of phosphorous, nitro-
gen, or halogens. Similarly for the MCD [12] and CECD [13].
The most versatile and rewarding detectors are those capable of dual or
multiple roles simultaneously. The SFTD [11] is available commercially
and is so constructed that it may operate as an FID and AFID simul-
taneously. A novel device, recently reported, is the PFTD [14]. It has two
burner nozzles forming the arms of a Y, and a single input channel, the
tail of the Y, for burner and carrier gases. This arrangement reduces the
dead air space found with the SFTD and enables the analyst to have the
F I D / A F I D or A F I D / A F I D combination. The SPED [15] is a flame
emission detector capable of monitoring phosphorus and sulfur simul-
MCLEOD AND COFFIN ON PESTICIDE RESIDUE SCREENING 185
TABLE 2--A classification of GLC detectors according to their number of

different response modes.
Single:
1. Electron Capture (ECD), detects electron absorbing compounds.
2. Flame Ionization (FID), detects various organic compounds.
Single but Selective; Depending an Operating Parameters:
1. Alkali Flame Ionization (AFID), detects phosphorus or nitrogen.
2. Microcoulometric (MCD), for phosphorus, sulfur, or chlorine.
3. Coulson Electrolytic Conductivity (CECD), for nitrogen, sulfur, or chlorine.
Dual:
1. Stacked Flame Thermionic (SFTD), for FID and phosphorus or nitrogen.
2. Parallel Flame Thermionic (PFTD), for FID and phosphorus, FID and nitrogen, or
phosphorus and nitrogen.
3. Sulfur Phosphorus Emission (SPED), for sulfur and phosphorus.
4. Dual Mode Indium Flame Detector (DMIFD) determines halogen as indium complex
emission, for example, INC1 at 360 nm.
Tri or Multiple:
1. Flame Photometric (FPD), for phosphorus, sulfur, and FID.
2. Dual Flame Photometric (DFPD), for phosphorus, sulfur, chlorine, and FID.
3. Mass Spectrometer (MS), for most pesticide molecular and fragment ion species.
taneously. It uses fiber optics to isolate the photomultiplier tubes from the
high temperature of the burner. It has appeared only recently and unlike
its predecessor, the F P D [16], does not have an ion collector probe for
F I D response mode. A n o t h e r dual detector of recent origin is the D M I F D
[17], and it operates in the F I D / h a l o g e n (360 nm) emission modes.
The F P D is capable of operating in three modes simultaneously;
phosphorus emission at 526 nm, sulfur at 396 nm, and as a flame
ionization detector. Response ratios for c o m p o u n d s containing phos-
phorus, sulfur and, in some cases, for the F I D response, have been
proposed [16,18] as characteristics useful in identifying unknowns.
A n o t h e r novel detector based on flame emission is the D F P D [19]. It is
a stacked flame with optical arrangements to select in the lower flame
phosphorus at 526 nm, and sulfur at 394 n m and in the u p p e r flame,
halogens, for example, chlorine as InCl at 360 nm. This detector m a y
prove to be a significant step forward in multiresidue screening because,
unlike the ECD, it can detect organochlorine c o m p o u n d s in sample
extracts with minimal cleanup. A practical illustration of this was given
using extracts from wild birds (starlings) and from carrots. E C D interfering
c o m p o u n d s obliterated lindane response from the starling extract and
endrin, ronnel, and lindane response from the carrots; the D F P D
responded to the pesticides only.
The mass spectrometer as a G L C detector is unique. It can be
considered as a multiple mode detector of the highest order, with the
potential o f determining all pesticides and their metabolites [20-23]. M a n y
o f the available cleanup procedures give extracts suitable for analysis.
Sensitivity was lacking in older models but recent technical advances

involving computer acquisition, processing, and interpretation of data; the
ability to integrate ion currents of selected mass over energy (m/e) values
over extended time periods; chemical ionization reactions that give intense
quasi-molecular ions and less complex spectra; make it possible to detect
hanD and picogram quantities. Equipment cost is the main deterrent to its
universal application. There remains a need for other detector systems in
multiresidue analysis as well.
The gas splitter (separator) ratios selected for multidetector configu-
rations are dependent on the relative sensitivities of the detectors for the
compounds to be determined. They are usually 1:10 for ECD/FID or 1:1
for ECD/AFID combinations [24]. Figure 1 illustrates several types of
column, splitter, and detector combinations subjected to collaborative
assay for the analysis of organochlorine and organophosphorus pesticides.
They are taken from the associate referees' report to the AOAC by Wessel
[25] in October 1967 and have been incorporated into their Methods of
Analysis [26].
Camoni et al [27] have described in detail a multiresidue method that
used a 1:1 ECD/AFID combination in parallel for organochlorine/organo-
phosphorus analysis. Their report was first presented to the 6th Congresso
Internazionale sulla Protezione delle Piante held in Vienna, September
1967. Thirty-nine dual chromatograms were used to illustrate the
ECD/AFID response to 14 different pesticides in 18 different types of
[, IN-SERIES DUAL DETECTION S~STEM;
N,120M1/MIN . .
COLUMN ECD p KCITD
2. IN-SERIES SPLIT DUAL DETECTION SYSTEM;
N, 120M1/MIN N,60MI/MIN
COLUMN. m ECD---4~I:I SPLITTER i KCITD
1
ATMOSPHERE
3. PARALLEL DUAL DETECTION SYSTEM;
N,60M1/MIN
PURGE
N,60MI/MIN
ECD
N,120MI/MIN [
COLUMN' i:I
SPLITTER
I N,60M1/MIN
KCITD
~CD = ELECTRON CAPTURE DETECTOR

KCZTD = POTASSIUM CHLORIDE THERMIONIC DETECTOR
FIG. l--Multidetector configurations used in a coUaborat~e study reported by Wessel [2S].

vegetables before and after spiking. These data made it possible to resolve
overlapping of organochlorine and organophosphorus GLC peaks, for
example, aldrin and disyston, kelthane, heptachlor epoxide, and methyl
parathion.
A comprehensive paper by Brandenberger [28] describes the application
of two separate dual channel systems in parallel for toxicological analysis
in forensic medicine. Relative retention times and two detector response
ratios, F I D / E C D (coded REY for relative electron capture yields) and
A F I D / E C D were characteristic of individual hypnotics, organophosphate,
and organohalogen (CI, Br) compounds that were used in their identifi-
cation and quantitation. For example, if during the initial screening
analysis REY values between 100 and 500 were observed, they were
indicative of phosphoric acid esters; that is, parathion = 400, mevinphos
: 100, while an organochlorine such as aldrin = 1000. The corres-
ponding A F I D / E C D quotients were 1, 20, and 0, respectively. These
characteristics were used by the author to diagnose a fatal case of
mevinphos poisoning originally believed to be from parathion.
Except for the references [24-28] cited previously there are few reports
on the practical application of multidetector systems using combinations
of two or more detectors. Our laboratory has used the E C D / F P D
combination to obtain simultaneous response of ECD, P526, $394, and
FID. A description of this system was given at the joint CIC-ACS meeting
in Toronto, May 1970 [29], as part of an overall multiresidue method.
Recently, we have been studying the operating characteristics of a GLC
system consisting of a temperature programmed column, three-way
effluent splitter, and five detectors. Our objective was to devise a system
to detect and estimate the concentration of pesticides and their metabo-
lites by the following characteristics:
1. electron capture,
2. phosphorus,
3. sulfur,
4. nitrogen, and
5. flame ionization.
Three detectors, ECD, Melpar FPD, and CECD were selected. They
were connected in parallel using a three-way stainless steel effluent
splitter.
The GLC column effluent distribution pattern, purge, and burner gases
are shown in Fig. 2. The gas flow rates and ratios for the FID burner
gases are shown as variable because it may be necessary to adjust
individual burners for the best combination of sulfur, phosphorus, and
FID responses. An illustration of multiple response under a given set of
conditions is shown in Fig. 3 for a mixture of parathion and piperonyl
butoxide. The sensitivity of detectors relative to one another may be
estimated from this figure as they are in the same scale. Detector
GLC Column Effluent
He-107
He-8 He-59
He-40
N-89~ H-Vart ~H-31
EC FID $394 P526 Coulson

NH 3
FIG. 2--Direction and flow (ml/min) o f various gases through three-way splitter and
associated detector systems.
electrometer settings, except for the ECD which is attenuated to keep the
response on the 1' mV scale, are set for maximum response commensurate
with minimum noise level.
To circumvent the flameout that occurs with the Melpar FPD at each
injection, we reversed the manufacturer's hydrogen and oxygen-air gas
inputs to the burner. Normally, the oxygen-air mixture is mixed with the
carrier gas before the hydrogen. The total mixture then enters the burner
jet. If this pattern of gas mixing is reversed, that is, the carrier gas is
mixed with the hydrogen and then the oxygen-air mixture, flameout does
not occur. This modification has two distinct advantages:
1. it enables the Melpar FPD to fit into an automated system by
eliminating the need for complex relighting equipment, and
2. the first half minute of detector response is retained and not lost
through bypassing.
There is probably a third advantage involving detector response, which
has not been assessed as yet. Because gas flow is not interrupted by
switching, and the flame remains lit through successive injections, repro-
ducibility of response could be more consistent.
Adapting the CECD in the nitrogen mode to a three detector system
meant helium would be required as a carrier gas. It is not usual to pass
helium through an electron capture detector and the effect was not
known. In point of fact, only a small fraction (5 to 10 ml/min) is diverted
for ECD monitoring and when this is supplemented with 80 to 90 ml/min
COULSON N I ~
23 n8 PARA'fl41C~
ng PARA'R~I (~N
80 ng
PIPLRONYL
BLITOXII~
ELEC'TR('~ C.',PTO~
40O n g
P I PER(~*'YL
BffrOXl ~t.
K_
~ELPAR $394 run ,~"
16 ng PARATHIGN
i~LPAR P526 nm ~
FIG. 3---Simultaneous response of five detectors to parathion and piperonyl butoxide.
of nitrogen purge gas, the characteristic voltage profile of the Tracor Ni

63 ECD is not altered significantly.
Retention of gas-tight seals over a wide temperature range (125 to
270~ was a problem. Teflon ferrules and O-rings are satisfactory to 230
to 240~ only. Bowman and Beroza [30] wrapped the end of the column,
inside the swagelok nut, with asbestos string soaked in a high-temperature
liquid phase, for example, Dexsil 300. Carbon ferrules are commerically
available for high-temperature seals. Another high-temperature (400~
ferrule known as Vespel [31] has recently appeared on the market. This
product withstands repeated handling and is reusable.
The choice of liquid phases for GLC column packings to be temperature
programmed (125 to 270~ with multidetectors is limited because of
column bleed. OV-17 is used in our system because of minimal bleed and
the retention data published by Bowman and Beroza [32] for organo-
phosphorus pesticides and their metabolites (138 compounds) under
temperature programming conditions. In addition, any bleed that does

occur is minimized for the ECD because of the small volume (5 to 10
ml/min) of carrier gas diverted to this detector.
Some preliminary results that illustrate the analytical potential of the
multidetector system are presented in Figs. 4 to 9. Aliquots of three
,4-
. . f " ~ - ~ S 594
--J .3-
O
_>
-J _ / ~ ~ P 5z6
ff .2
CECD - NH 3
.,
ECD
I I I I I I I I
0 5 IO 15 20 25 30 55
T I M E - MINUTES
F I G . 4---Background responses for five detectors operating simultaneously with tempera-

ture programming.
eluates of increasing polarity from a Florisil column cleanup for an extract

of fruits from a market basket survey [33] were analyzed. The sample was
fortified (mg/kg) with the following pesticides:
Diazinon 0.1 Endosulfan 0.02

Malathion 0.1 p,p'-DDT 0.04
Aldrin 0.01 dieldrin 0.02
carbaryl 25
The five detector responses for a 5 /zl injection of Eluate 1 (hexane)

containing the equivalent of 50 mg of samples are shown in Fig. 4. The
two pesticides of interest to us are Aldrin at 0.01 m g / k g and p , p ' - D D T at
0.04 mg/kg. They are indicated by the ECD major response peaks at
0
r'-
m
O
E1
5- .5
z
9 4- C)
0
S 394 "11
z
J 3
0 " --
9 3- ~'F'" 0
> I z
NH 3 "0
o m
> (,t)
i
.-I
.._1
o
m
30
m
ECD
c
m
ECD
0
I I I I ; 1 I I I I I I I
m
5 I0 15 20 25 30 35 15 20 25 30 rn
z
T;ME - M)NUTES TIME -MINUTES z
FIG. 6--Responses o f five detectors to a 30 percent methylene
F I G . S~Responses of five detectors to a hexane eluate .from a Florisil chloride in hexane eluate from a Florisil column o f an extract
column of an extract from a spiked composite sample o f fruits. The J~'om a spiked composite sample o f fruits. The equivalent o f 50
equivalent of 50 mg of sample was injected. mg of sample was injected.
VV
0.2
O.I
(/3
F-
.J O-W
O
> 1"~.~,..,,,.~% EC D
_J
.O
/
NH
I I ~/ I I I I I
O 5 Of 15 20 25 30 35
TIME - MINUTES
FIG. 7--Responses of the electron capture and electrolytic conductivity OV mode) detectors
to a 50 percent ethyl acetate in hexune eluate from a Florisil column o f an extract from a
spiked composite sample of fraits. The equivalent o f 100 mg of sample was injected.
approximately 10 and 14 min. Other peaks are present but have not been
identified.
An important consideration on the operation of the CECD-NH 3 under
multidetector conditions is brought out in its chromatogram. Usually, the
first half minute of sample effluent bypasses the detector system by
manually operating a bypass valve. However, we do not do this because of
automation problems. These data indicate bypassing was not necessary for
this type of sample eluate.
Figure 5 shows the chromatograms for Eluate 2 (30 percent methylene
chloride in hexane) run under the same conditions as Eluate 1. We expect
to find dieldrin at 0.02 mg/kg and endosulfan I at 0.02 mg/kg in this
MCLEOD A N D COFFIN ON PESTICIDE RESIDUE SCREENING 193
S :594
.5-
.4-
,.•••
L
.I
P 526
0
I I i j I I I
5 IO 15 20 25 50 55
TIME- MINUTES
FIG. 8--Responses of the $394 and P526 flame photometric detectors to a 50 percent
ethyl acetate in hexane eluate from a Florisil column of an extract from a spiked composite
sample of fruits. The equivalent of lO0 mg of sample was injected.
eluate. The two major peaks at approximately 11 and 12 min of the ECD
response are from these pesticides. Note the unknown compound in the
CECD-NH 3 chromatogram, that was also present in Eluate 1. All other
detector chromatograms are free of response peaks except for electronic
noise. The third eluate chromatograms have to be displayed in three
figures because of their extensive and elevated backgrounds for the ECD,
CECD-NH 3, and FID. This eluate was 50 percent ethyl acetate in hexane
and could be expected to contain most of the sample coextractives.
Diazinon, malathion, carbaryl, and endosulfan II should appear in this
eluate. The equivalent of 100 mg of sample in 5 gl was injected.
The ECD and CECD-NH 3 responses are shown in Fig. 6. The ECD
results are off scale and it is not possible to detect diazinon or malathion;
5-
4-
6q
o> 5 -
.J
_J
-~.2-
I-
FID
O~
I I I I I I I I
O 5 IO 15 20 25 30 35
TIME - MINUTES
FIG. 9--Response of the flame ionization detector to a 50 percent ethyl acetate in hexane
eluate from a FloHsil column of an extract from a spiked composite sample of fruits. The
equivalent of 100 mg of sample was injected.
carbaryl shows on this detector as an off scale but discrete peak at 10

min.; endosulfan II can be detected as the last major peak on scale at
approximately 13 to 14 min. The CECD-NH 3 responded to diazinon, the
minor peak at approximately 8 min and to carbaryl, the major peak at 10
min. Whether carbaryl p e r se was being chromatographed was not
determined; response might be due to the formed isocyanate.
Figure 7 shows the chromatograms for $394 and P526. Diazinon
appears at approximately 8 min and malathion at approximately 11 min,
both are major peaks. The other major (5.5 min) and minor peaks in the
chromatograms are of unknown origin.
The FID response chromatogram (Fig. 8) indicates that a large number
of diverse compounds from sample coextractives are present. Carbaryl is
detected by the FID at the concentration used to fortify the sample and is
the shorter of the two major peaks at approximately 10 min. These data
for the FID can be used as an indication of the degree of cleanup
achieved.
Acknowledgment
The authors wish to thank D. C. Smith for her cooperation in supplying
sample extracts and to David Lewis for technical assistance.
References
[1] Gunther, F. A., Pure andApplied Chemistry, Vol. 21, 1970, p. 355.
[2] Compendium on Registered Uses of Pesticides in Canada, Information Canada, Ottawa,
1971.
[3] Beroza, M. and Bowman, M. C., International Symposium on Identification and
Measurement of Environmental Pollutants, National Research Council of Canada,
Ottawa, 1971.
[4] McCully, K. A. and McLeod, H. A., International Symposium on Identification and
Measurement of Environmental Pollutants, National Research Council of Canada,
Ottawa, 1971.
[5] Frehse, H. in "Pesticide Chemistry Series," Proceedings, 2nd International IUPAC
Congress, Gordon and Breach Science Publishers, 1971, p. 113.
[6] McLeod, H. A. and Wales, P. J., Journal of Agricultural and Food Chemistry, Vol. 20,
1972, p. 624.
[7] Morley, H. V. and McCully, K. A. in Methodicum Chimicum, Vol. L G. T. Verlag,
Ed., Stuttgart, 1973, Chapter 11.1, p. 850.
[8] Krejci, M. and Dressier, M., Chromatographic Reviews, 1970, p. 131.
[9] Lovelock, J. F., Analytical Chemistry, Vol, 35, 1963, p. 474.
[10] Burchfield, H. P. and Storrs, E. E., Biochemical Applications of Gas Chromatography,
Academic Press, 1962, p. 60.
[11] Karem, A., Analytical Chemistry, Vol. 36, 1964, p. 1416.
[12] Westlake, W. E., Advances in Chemistry Series, Vol. 104, 1971, p. 73.
[13] Coulson, D. M., Journal of Gas Chromatography, Vol. 4, 1966, p. 285.
[14] Riva, M. and Carisano, A., Journal of Chromatography, Vol. 36, 1968, p. 269.
[15] Bendix Process Instruments Division, Ronceverte, W. Va.
[16] Bowman, M. C. and Beroza, M., Analytical Chemistry, Vol. 40, 1968, p. 1448.
[17] Moseman, R. F. and Aue, W. A., Journal of Chromatography, Vol. 63, 1971, p. 229.
[18] Griee, H. W., Yates, M. L., and David, D. J., Journal of Chromatographic Science,
Vol. 8, 1970, p. 90.
[19] Versino, B. and Rossi, G., Chromatographia, Vol. 4, 1971, p. 331.
[20] Bergstedt, L. and Widmark, G., Chromatographia, Vol. 2, 1969, p. 529.
[21] Hutzinger, O. and Jamieson, W. D. in "Pesticide Chemistry Series," Proceedings, 2nd
International IUPAC Congress, Gordon and Breach Science Publishers, 1971, p. 7.
[22] Biros, F. J., Advances in Chemistry Series, Vol. 104, 1971, p. 132.
[23] Finnigan Corp, Sunnyvale, Calif., Model 6000 GC/MS Interactive Data System.
[24] Oaks, D. M., Hartmann, H., and Dimick, K. P., Analytical Chemistry, Vol. 36, 1964,
p. 1560.
[25] Wessel, J. R., Journal of the Association of Official Agricultural Chemists, Vol. 51,
1968, p. 666.
[26] Methods, l l t h ed., Association of Official Agricultural Chemists, 1970, p. 479.
[27] Camoni, I., Candolfo, N., Ramelli, G., Sampaolo, A., and Binetti, L., Bollettino Dei
Laboratori ChimiciProvinciali, Vol. 18, No. 5, 1967, p. 579.
[28] Brandenberger, H., Pharmaceutica Acta Helvetiae, Vol. 45, 1970, p. 394.
[29] McLeod, H. A. and Wales, P. J., CIC-ACS Joint Conference, Toronto, May 1970.
[30] Bowman, M. C. and Beroza, M., Journal of the Association of Official Agricultural
Chemists, Vol. 54, 1971, p. 1086.
[3l] Markey, S. P. and Simons, S. L,, Analytical Chemistry, Vol. 45, 1973, p. 818.
[32] Bowman, M. C. and Beroza, M., Journal of the Association of Official Agricultural
Chemists, Vol. 53, 1970, p. 499.
[33] Private communication, Mrs. D. Smith, Health Protection Branch, Health and Welfare
Canada, Ottawa, Ontario, Canada, 1973.
L. M. R e y n o l d s j a n d T. C o o p e r I
Analysis of Organochlorine
Residues in Fish
REFERENCE: Reynolds, L. M. and Cooper, T., "Analysis of Organoehlorine Residues

in Fish," Water Quality Parameters, ASTM STP 573, American Society for Testing and
Materials, 1975, pp. 196-205.
ABSTRACT: The analyses of fish for organochlorine residues (OC pesticides, PCBs,
PCTs) present some special cleanup problems due mainly to the difficulty of removing
coextracted oil. The oil causes erratic elution patterns of the residues from many
chromatographic column adsorbents.
Our overall approach to the extraction, cleanup, pre-GLC separations and GLC
analysis, as well as the quantitation of PCBs are described. The use of deactivated
Florisil in our cleanup procedure solved many of the earlier problems, and has given
consistently excellent cleanup (less than 0.01 g remains from 0.5 g fat) and recovery
(above 90 percent) of residues from fish and other biological tissues. The Florisil adsorp-
tion column is designed to tolerate up to 0.5 g of oil or fat, but with the initial Soxhlet
extraction, an appropriate aliquot can be subjected to the column cleanup. An addi-
tional cleanup step involving Florisil column partitioning is described for coping with
much larger fat samples, especially where very low residue levels are to be determined.
Our approach to PCB quantitation and the use of reference standards are discussed.
The proposed use of a 1:1 mixture of Aroclor 1254 and 1260 as reference for most biolo-
gical samples is preferred.
KEY WORDS: water quality, fishes, pesticides, chlorine organic compounds, environ-
mental tests, gas chromatography
W h e t h e r fish is v i e w e d as a f o o d s o u r c e or a s p o r t i t e m to m a n , o r as a
b i o l o g i c a l species t h r e a t e n e d by e x t i n c t i o n , t h e analysis o f fish for t o x i c
m a t e r i a l s , e s p e c i a l l y t h e o r g a n o c h l o r i n e (OC) r e s i d u e s is a very i m p o r t a n t
e x e r c i s e . T h e n u m b e r o f O C c o m p o u n d s t h a t a r e k n o w n to b e t o x i c or
h a v e u n f a v o r a b l e effects o n fish, a n d w h i c h o f necessity m u s t i n t e r e s t t h e
a n a l y t i c a l c h e m i s t , is i n c r e a s i n g . B e s i d e s t h e O C p e s t i c i d e s a n d poly-
c h l o r i n a t e d b i p h e n y l s (PCBs), t h e list n o w i n c l u d e s c h l o r i n a t e d t e r p h e n y l s ,
n a p h t h a l e n e s , d i o x i n s , b e n z o f u r a n s , a l i p h a t i c h y d r o c a r b o n s , etc. T h i s
p a p e r will d e a l m a i n l y w i t h t h e a n a l y s e s o f O C p e s t i c i d e s a n d P C B s in fish
by e l e c t r o n c a p t u r e - g a s l i q u i d c h r o m a t o g r a p h y ( E C - G L C ) . H o l d e n [l]Z
'Senior research scientist and technologist, respectively, Pesticide and Trace Analytical
Lab, Department of Applied Chemistry, Ontario Research Foundation, Sheridan Park,
Ontario LSK 1B3, Canada.
196

REYNOLDS AND COOPER ON ORGANOCHLORINE RESIDUES IN FISH 197
recently gave an excellent review paper on the analysis of fish for

organochlorine residues; hence the background literature and principles of
the various analytical techniques will not be given here. Of the basic steps
involved in OC analysis by EC-GLC---extraction, cleanup, identification,
quantitation, and confirmation--attention will be focused mainly on the
cleanup of fish extracts and the quantitation of PCBs. These are two of
the more difficult undertakings which contribute greatly to inaccuracies or
differences of results or both between analysts.
Cleanup of Biological Tissue Extracts

To date the main cleanup techniques involve adsorption chromato-
graphy, liquid-liquid partitioning, volatilization, low temperature precipi-
tation of lipids, and more recently Gel Permeation Chromatography
(GPC) has been applied [2]. The latter technique, which depends on
molecular size (molecular weight and shape) for the separation of fats
from OC residues, appears promising. It has been automated to cleanup
23 sample extracts per run [3], and our laboratory plans to have a look at
the commercial model.
In the Ontario Research Foundation (ORF) laboratory, wildlife tissues
form the bulk of our samples, and although our main multi-residue
methodology [4] involving cleanup by cold precipitation [5] and Florisil
column chromatography gave adequate cleanup for most substrates, there
was need for improvement in the cleanup of fish and fish products. We
compared a number of different methods and considering the various
factors, decided that the Canadian Food and Drug Directorate (FDD) [6]
and the U.S. Food and Drug Administration (FDA) [7] approaches were the
most promising. Both procedures are very similar, employing Florisil
column chromatography and elutions with different solvent systems.
Deactivated Florisil (regular activated at 300~ and then deactivated with
2 percent HzO) and Florisil PR (pesticide grade activated at 140~ are
used by the FDD and FDA methods, respectively.
Osadchuk and Bruns [6] of the FDD evaluated the cleanup properties
of Florisil, alumina, and silica gel and concluded that although all three
adsorbents exhibited strong similarities in their elution patterns, Florisil
appeared to be the best for general screening work. These authors also
found that deactivated Florisil gave superior cleanup for fats and oils as
compared to activated Florisil. After a number of preliminary experiments
we decided to adopt the FDD approach. Furthermore, during the summer
we had problems with our PR grade Florisil because of high and varied
laboratory humidity, causing inconsistent Florisil separations. With the
deactivated Florisil, this problem was minimized. A further problem
encountered with the PR grade Florisil was the presence of interfering
impurities (apparently phthalate esters from the containers) in certain
batches, making it necessary to cleanup the Florisil before using it. To

prepare the 2 percent H20 deactivated Florisil, the regular grade was first
heated at 300~ and then deactivated.
Gas Chromatographic Conditions

The GLC conditiohs used for these studies were as follows:
Instrument--Hewlett-Packard Model 5700 equipped with linear Ni 6a
electron capture detector and Model 7671A automatic sample injector.
C o l u m n - - 3 % OV-101/5% OV-210 on Chromosorb W high perfor-
mance 80/100, 1A in. by 6 ft glass spiral, 4 mm inside diameter, carrier 5
percent methane in argon at 60 ml/min, and temperature of 215~
Temperature of injector and detector, 215~ and 300~ respectively.
Recorder--Hewlett-Packard Model 7123A, 1 mV full scale with chart,
1/2 in./min.
Attenuation--1 to 4096.
Deactivated Florisil Column Cleanup and Separation

A chromatographic glass column (2.5 cm inside diameter by 30 cm) is
packed with 15 cm (~28 g) of Florisil (regular grade heated to 300~ and
then deactivated with 2 percent HzO), topped with 1 cm ('~1/2 in.)
NazSO4, and washed with 100 ml hexane (recoverable). The sample
extract (the equivalent of up to 0.5 g oil or fat) in about 5 ml (hexane) is
placed on the column and eluted at a rate of about 5 ml/min as follows:
(1) 130 to 135 ml hexane: Fraction I, and (2) follow with 300 ml 30
percent CH~CI2 in hexane: Fraction II.
The residues eluted in the two fractions are given in Table 1. When this
2 percent deactivated Florisil cleanup method was applied in triplicate to
0.5 g of cod liver oil, corn oil, egg lipids, and 3 g fish tissue (rainbow
trout), almost complete removal of lipid material was obtained.
Triplicate recovery trials for 10 organochlorines were carried out on the
2 percent deactivated Florisil cleanup and separation method with rainbow
TABLE 1--Elution of organochlorines from 2 percent deactivated florisil.

Fraction I Fraction II
PCT TDE (DDD)

Heptachlor Eluted Heptachlor epoxide Eluted
Aldrin by BHC (a-. [~-, 7-) by
PcB f
Mirex
HCB
DDE
Hexane
DD f
MeOCI
Dieldrin
Endrin
30% CH2CI~
in Hexane
trout tissue and cod liver oil as the substrates. The results are given in
Table 2 and indicate that 93 percent or better recoveries were obtained for
fish and fish oil when normal sample aliquots are analysed. With this
method the usual preliminary cleanup prior to Florisil, that is, solvent
partitioning or low temperature precipitation or both, is avoided. Ac-
cordingly, higher recoveries, cleaner extracts, and shorter time of analysis
are attained. The technique is versatile and should be used according to
the needs of the analysts. For example, if one is interested in PCB and
DDT, but not in dieldrin and endrin, pure hexane can be used for
elution, thereby avoiding the elution of any fat. For organophosphates
(OP) and more polar compounds, more polar elution systems can be
applied.
TABLE 2--Recovery data, 2 percent deactivated FlorisiL a
Fish (trout) Cod Liver Oil
OC ppm added Recovery, % b ppm added Recovery, % b
HCB 0.18 94 1.00 93

Lindane 0.28 98 1.60 104
Heptachlor 0.35 99 2.00 100
Aldrin 0.35 98 2.00 101
Heptachlor
epoxide 0.50 100 2.80 104
p,p'-DDE 0.71 96 4.00 101
Dieldrin 0.71 100 4.00 103
p,p'-DDD 1.06 100 6.00 104
p,p'-DDT 1.77 100 10.0 103
PCB (1:1)c 17.7 95, 98 100.0 100, 101
a3 g fish tissue and 0.5 g cod liver oil were used.

bRecovery data are the averages of triplicate analyses.
c PCB is a 1:1 (1254:1260) mixture with recoveries based on peaks 141 and 166.
Analyses of Fat or Oil Samples for Low Levels of OC Residues

In order to have the capability of coping with much larger fat samples
(up to 8 g fat or oil) where very low residue levels (~o5 ppb) are required,
we investigated the Florisil column partition cleanup method of Porter and
Burke [8]. This method involves distribution of the fat or oil on
unactivated Florisil by slurrying and then eluting (partitioning) the
residues into 150 ml of 10 percent HzO in acetonitrile. As reported by
Porter and Burke, we found that the method removed the bulk of the fat
(100 mg remained from the original 8 g). However, with this method we
recovered only 60 percent of the PCBs, although the recovery of the more
polar OC pesticides was good. After experimenting with different combi-
nations, we obtained >90 percent recovery of PCBs and other OCs using
200 ml of 5 percent H20 in CH3CN for the partitioning step. We applied
this modified method to the cleanup of 8 g cod liver oil and 8 g corn oil,
and the results are shown in Table 3. About 300 mg of the original 8 g of
oil remained in the OC fraction. We therefore completed the cleanup of
the remaining lipids by following up with the 2 percent deactivated
Florisil technique.
TABLE 3--Cleanup by Florisil partition column, a
Weight Number Mean Weight Standard

Substrate Applied, g Samples Eluted, g Range Deviation
Cod liver oil 8 8 0.27 0.26-0.28 <0.001

Corn oil 8 11 0.30 0.27-0.33 0.014
aColumn eluted with 200 ml of 5 percent H20 in acetonitrile.
The recovery results of the preceding combined procedures as applied to

cod liver oil and corn oil are given in Table 4. Excellent cleanup and
recovery are obtained when the combined Florisil column partition and
adsorption systems are used with up to 8 g oil.
TABLE #--Recovery data: combined Florisil systems Jbr low residues, a
Cod Liver Oil Corn Oil
OC ppm added Recovery, % b ppm added Recovery, % b
HCB 0.60 93 0.60 86

Lindane 1.00 102 1.00 98
Heptachlor 1.25 100 1.25 89
Aldrin 1.25 105 1.25 87
Heptaehlor
epoxide 1.75 101 1.75 92
p,p'-DDE 2.50 102 2.50 98
Dieldrin 2.50 96 2.50 94
p,p'-DDD 3.75 95 3.75 96
p,p'-DDT 6.25 93 6.25 95
PCB (1:1) c 62.5 92 62.5 92
a 8 g oil cleaned-up by combined partition: adsorption column systems.

bRecovery data are the averages of triplicate analyses.
cpCB is a 1:1 (1254:1260) mixture with recoveries based on peaks 141 and 166.
It should be emphasized that, in general, the partition column is not

necessary since the 2 percent deactivated Florisil column will cope with
most samples. For example, it can cleanup 10.0, 5.0, 2.5, and 1.67 g fish
or other substrate if the fat content of the sample is 5, 10, 20 or 30
percent, respectively.
PCB Quantitation
As was mentioned earlier, the quantitation of PCB is one of the main
reasons why analysts sometimes differ in their PCB results for a given
sample. The problem is a very complex one:
(a) there are a great number of components involved;
(b) the response factors of the different components may differ;
(c) the standard references used by different laboratories are likely
different;
(d) field samples may weather differently, and components may degrade
at different rates; and
(e) environmental samples are likely to contain mixtures rather than a
single formulation.
Burke [9] and Jensen [10] have recently elaborated on some of these
problems.
Many methods have been used or proposed for PCB quantitation
[11-20], but because of the complexity of the problem, no single approach
is ideal for all samples. For example, PCB quantitation of air and possibly
water samples (where the possibility of any special pattern is unlikely)
might best be done by perchlorination and measurement of the decachlor-
biphenyl. However, for many reasons, this technique would not be feasible
for large numbers of biological samples.
Since it may not be possible to get an accurate and true PCB result
without estimating each component separately (this would require pure
standards of individual components and ability to separate them by GC),
and since, even if accurate results were available, the lack of toxological
data would preclude the proper use of such data, our approach to PCB
quantitation (with biological samples as the main substrate) has been
governed or influenced by the following.
(1) Obtain the best estimate with minimum time and number of steps
(some of the more recent proposals are tedious and the results are not
necessarily more accurate).
(2) The approach should be consistent so that:
(a) comparisons can be made between samples, labs, countries, time
periods, etc.; and
(b) corrections can be made in the future (by applying a factor) if a
more accurate calculation method is found.
In the past, based on visual observation of the GC pattern or profile, we
have calculated most of our wildlife samples with Aroclor 1260 as
reference. The average of the calculations for peaks 141 and 166 are
usually reported [11]. As can be seen in Fig. 1, Aroclor 1254 has very
little of peaks 252, 302, and 320; hence whenever these peaks showed up
in samples, thereby giving a characteristic fingerprint, we used 1260 as
reference. However, it is obvious from the mixtures, for example, ratio of
5:1 (1254:1260), that it takes only a small proportion of 1260 to give
fair-sized peaks at 252, 302, 320. By going over the various standard
mixtures and calculating 166:252 ratios and comparing these to the actual
Ioe
J
1254
,97
e4
*os
*z~ 141
5"1
197 z~2
384 449
zsz ,
, I.I
IS4 Zl3 .302
59 zo
It2 les Zlt
,449
Jlllllllllllllllllllllllllllllllilll
0 5 I0 15 20 2,5 30 35
Minutes
FIG. 1--Gas chromatography profiles of Aroclor 1254, 1260, and some mixtures. Ratios
correspond to mixtures of 1254:1260. Peak identification numbers refer to retention times
relative to I, 1-dichloro-2,2-b~ (p-ehlorophenyl) ethylene (DDE) •
ratios found in samples, we have concluded that samples which indicate

1260 (presence of peaks 252, 302, and 320) are in most cases mixtures of
1254 and 1260. Of course, samples which do not show the 252, etc. peaks
(that is, show the 1254 pattern as some fish samples do) should still be
estimated as 1254. If the sample appears to be a mixture, as indicated by
the 166:252 ratio, two approaches can be used:
(a) assume a 1:1 ratio (1254:1260) for the sample and use a 1:1
standard mixture as reference (average for peaks 141, 166), and
(b) calculate 166:252 ratio for the sample and compare with those for
standard mixtures from Table 5 (or from a prepared curve) to get a
closer approximation of the exact proportion and the appropriate
factors for peaks 141 and 166 to convert the data.
Results should be reported as PCB concentration, giving the reference
ratio used.
TABLE 5--Ratio definition and conversion factor table.
1254:1260 Conversion Factors for 1:1 Ratio b

166:252 a
Ratio % 1254 Ratio Peak 141 Peak 166
1254 100 11.6 1.36 1.21

5:1 83.3 3.81 1.16 1.10
4:1 80 3.29 1.19 1.12
3:1 75 2.90 1.13 1.08
2:1 66.7 2.37 1.08 1.05
1:1 ,50 1.81 1.00 1.00
1:2 33.3 1.49 0.91 0.96
1:3 25 1.38 0.87 0.92
1:4 20 1.34 0.86 0.91
1:5 16.7 1.30 0.81 0.87
1260 0 1.16 0.80 0.87
a The peak height (or area) ratio of 166:252 determines the proportion of 1254:1260 and
also the appropriate conversion factor.
bThe conversion factor 09:
peak height of 1:1 mixture concentration of particular mixture

f = peak height of particular mixture x concentration of 1:1 mixture
Results should be reported as PCB concentration, giving the reference

ratio used.
From the correction factors of Table S it should be noted that:
(a) the largest possible difference between two estimates for a given
sample is by a factor of 1.70---this would be the case when one
formulation (1260 or 1254) is used as reference instead of the other;
(b) if a 1:1 ratio is used when 1260 should have been the reference,
then the result would be high by a factor of 1.25; and
(c) if a 1:1 ratio is used when 1254 should have been the reference,
then the result would be low by a factor of 1.36.
The two calculation methods outlined are simple and more realistic
than methods which depend merely on visual inspection of GC chromato-
grams, and should be consistent. The calculation based on a 1:1 assump-
tion is simpler but might be less accurate. However, we are more inclined
to favor this system since simplicity, repeatability, and comparability are
presently the most important requirements. Furthermore, as just dis-
cussed, the 1:1 ratio would lead to lower maximum errors than with
unmixed standards.
Preliminary PCB results from a fish check sample program organized
by Holdrinet [21] suggest that good agreement between analysts is possible
when a simple (direct peak height comparison) quantitation method is
standardized. Our proposed PCB quantitation methods could be adopted
easily by most residue laboratories.
The main criticism that might be leveled against the proposed quanti-
tation methods is that a particular GC peak may contain more than one
PCB isomer and the EC responses could be different. However, we feel
that such derived errors are minor compared to the contribution from
other sources, for example:
(a) the analytical methodology including inconsistency in the reference
standards and use of the incorrect reference standard, and
(b) possible errors introduced from long and tedious quantitation
methods including some assumptions that are made.
Quantitation of the lower chlorinated PCB mixtures were not included
because:
(a) in biological samples (including fish), the lower chlorinated PCBs
are not usually found being more biodegradable (1254 and 1260 are
most frequently detected in biologicals); and
(b) when the lower PCBs are found in samples (for example, paper-
board and other packaging materials), it is usually the 1242 or
lower types, and the quantitation of these is generally not influenced
(interferred with) by the presence of 1254 and 1260.
A collaborative study on field weathered samples using both proposed
quantitation methods should be appropriate to evaluate these two
approaches.
Acknowledgments
The technical assistance of Brenda Wheeler and Maria Reisinger is
gratefully acknowledged.
This study was supported in part by funds from the Province of Ontario
through the Ministry of Industry, Trade, and Tourism.
REYNOLDS AND COOPER ON ORGANOCHLORINE RESIDUES IN F I S H 205
References
[1] Holden, A. V., Proceedings, International Symposium on Identification and Measure-
ment of Environmental Pollutants, Ottawa, June 1971, p. 233.
[2] Stalling, D. L., Tindle, R. C., and Iohnson, J. L., Journal, Association of Official
Analytical Chemists, Vol. 55, 1972, p. 32.
[3] Tindle, R. C. and Stalling, D. L., Analytical Chemistry, Vol. 44, 1972, p. 1769.
[4] Vermeer, K. and Reynolds, L. M., CanadianField-Naturalist, Vol. 84, 1970, p. 117.
[5] McCully, K. A. and McKinley, W. P., Journal, Association of Official Analytical
Chemists, Vol. 47, 1964, p. 652.
[6] Osadchuk, M. and Bruns, G., Proceedings, The Pesticide Residue Analysis Seminar,
W. Canada, Edmonton, May 1971.
[7] Mills, P. A., Bong, B. A., Kamps, L. R., and Burke, J. A., Journal, Association of
Official Analytical Chemists, Vol. 55, 1972, p. 39.
[8] Porter, M. L. and Burke, J. A., Journal, Association of Official Analytical Chemists,
Vol. 56, 1973, p. 733.
[9] Burke, J. A., Journal, Association of Official Analytical Chemists, Vol. 55, 1972, p. 39.
[10] Jensen, S., PCB Conference II, Stockholm, 1972.
[11] Reynolds, L. M., Residue Reviews, Vol. 34, 1971, p. 27.
[12] Risebrough, R. W., Rieche, P., and Olcott, H. S., Bulletin of Environmental
Contamination and Toxicology, Vol. 4, 1969, p. 192.
[13] Jensen, S., Johnels, A. G., Olsson, M., and Otterlind, G., Nature, Vol. 224, 1969,
p. 247.
[14] Koeman, J. H., ten Noever de Brau, M. C., and de Vos, R. H., Nature, Vol. 221,
1969, p. 1126.
[15] Armour, J. A. and Burke, J. A., Journal, Association of Official Analytical Chemists,
Vol. 53, 1970, p. 761.
[16] Rote, J. W. and Murphy, P. G., Bulletin of Environmental Contamination and
Toxicology, Vol. 6, 1971, p. 377.
[17] Collins, G. B., Holmes, D. C., and Jackson, F. ]., Journal of Chromatography, Vol.
71, 1972, p. 443.
[18] Berg, O. W., Diosady, P. L., and Rees, G. A. V., Bulletin ofEnvironmentul Contamb
nation and Toxicology, Vol. 7, 1972, p. 338.
[19] Beezhold, F. L. and Stout, V. F., Bulletin of Environmental Contamination and
[20] Webb, R. G. and McCall, A. C., Journal of Chromatographic Science, Vol. 11, 1973,
p. 366.
[21] Holdrinet, M., personal communication.
7", A . B e l l a r ~ a n d J. J. L i c h t e n b e r g t
Some Factors Affecting the

Recovery of Polychlorinated
Biphenyls (PCB's) from Water
and Bottom Samples
REFERENCE: Bellar. T. A. and Lichtenberg, J. J., "Some Factors Affecting the Re-
covery of Polyehlorinated Biphenyls (PCB's) from Water and Bottom Samples," Water
Quality Parameters, ASTM STP 573, American Society for Testing and Materials, 1975,
pp. 206-219.
ABSTRACT: During studies on analytical methods for PCB's in water and bottom sam-
pies, variable recoveries from dosed samples and apparent decreasing recoveries with
the age of the sample were observed. Losses from dosed river waters as high as 10 per-
cent in one day and greater than 40 percent in one week were common. Recoveries from
bottom samples varied widely with the sample pretreatment and the method of extrac-
tion. In an effort to define some of the factors affecting the recovery of PCB's from
water, the following parameters and conditions were studied: sample container, extremes
of pH, aging under ambient conditions, and several preservation techniques. Several
methods of sample preparation and extraction of bottom samples were studied. Environ-
mentally contaminated water and lake bottom samples and dosed natural samples were
studied.
The separatory funnel liquid-liquid extraction method was found to be the most
efficient for the extraction of PCB's from natural waters. The air-dried, moisture-added
soxhlet extraction procedure proved to be the most efficient for recovery of PBC's from
environmentally contaminated lake bottom samples. Formaldehyde was found to be
effective for preserving PCB's in dosed natural waters for at least two weeks.
KEY WORDS: water quality, water pollution, pesticides, toxicity, recovery, aquatic ani-
mals, polychlorinated biphenyls (PCB's)
Polychlorinated biphenyls (PCB's) have been recognized for several

years as ubiquitous environmental pollutants. These complex mixtures of
variably chlorinated biphenyls have occurred in nearly every environmental
media [1,2]. 2 Their presence in water, sediment, biota, fish, birds, and
animals have been reported [3-5]. While the PCB's are reported to be less
toxic than the chlorinated hydrocarbon pesticides, acute toxic effects to
aquatic life have been demonstrated [3,6]. Since they do exhibit acute
' Chemist and chief, Organic Analyses Section, respectively, Methods Development and
Quality Assurance Research Laboratory, National Environmental Research Center, U.S. En-
vironmental Protection Agency, Cincinnati, Ohio 45268.
206
9
BELLAR AND LICHTENBERG ON POLYCHLORINATED BIPHENYLS 207
toxic effects and, like the chlorinated pesticides, they are biologically
magnified [7], it is important to maintain surveillance of the PCB levels
occurring in the environment.
The PCB's are chemically similar to many of the organochlorine
pesticides; therefore, the same analytical procedures are applied to both.
Being complex mixtures, the PCB's present a serious interference problem
when determining many pesticides. Therefore, special separation and
cleanup procedures are required for analyses when both occur in a given
sample. Much work has been published on the separation and the
determinative aspects of the analysis [8-10]. However, little has been
reported on factors that might affect the extraction and recovery of PCB's
from environmental samples.
During our studies on water, we noted variable recoveries from dosed
samples and an apparent decreasing recovery with the age of the sample.
In an effort to define some of the factors affecting the recovery, the
following parameters were investigated: the sample container, extremes of
pH, aging under ambient conditions, and several preservation techniques.
Liquid-liquid extraction and extraction with urethane foam plugs were
studied. During our studies on recovery of PCB's from bottom samples,
several methods for preparation and extraction of bottom samples were
compared. We were particularly interested in the newer column extraction
procedures since they appear to be becoming very popular. Environ-
mentally contaminated samples and dosed natural samples were studied.
Experimental
Apparatus--The high frequency mechanical dispersion instrument used
in one phase of the bottom sample extraction study was the Super Dispax
Tissumizer, Model STD 182N (Tekmar Company, Cincinnati, Ohio). 3
Reagents--Reference Standard PCB's Aroclors 1242, 1254, 1260, and
1016 (Monsanto Chemical Company, St. Louis, Mo.) prepared in acetone
solution. Formaldehyde---37% aqueous solution.
Procedure---The samples were collected in 1-qt to 6-gal glass containers
at various times over a period of one year.
The primary liquid-liquid extraction procedure employed for water
samples was that recommended by the U.S. Environmental Protection
Agency (EPA) for organochlorine pesticides [11]. In addition, the semi-
automatic magnetic stirring (Vortex) system of Kawahara et al [12] was
tested. The urethane foam procedure for extraction of PCB's from water
was that published by Gesser et al [13]. A modification of Gesser's
procedure, that of soaking the foam plugs in the sample with occasional
shaking, was also tested.
3Mention of products and manufacturers is for identification only and does not imply
endorsement by the Methods Development and Quality Assurance Research Laboratory of
the U.S. Environmental Protection Agency.
Several methods for extraction of bottom materials were investigated.

The soxhlet extraction procedure of Breidenbach et al [I4], column
elution procedures of Hesselberg and Johnson [15] and Southeast Environ-
mental Research Laboratory [16], the high frequency dispersion procedure
of Johnson and Starr [17], the mechanical shaking procedure of Goerlitz
and Brown [18] and Shell Chemical Company [19], and the blender
extraction method of Muth [20] were tested.
All bottom sample extracts were cleaned up in the same manner. The
standard Florisil column procedure [11] and the semi-micro silica gel
procedure of Leoni [9] were employed. When necessary, elemental sulfur
was removed from the extracts using mercury according to the method of
Goerlitz and Law [21]. Table 1 lists the conditions for preparation and
extraction of bottom samples.
T A B L E 1--Extraction procedures tested for recovery of PCB's from bottom samples.

Preliminary Dry Mat- Water Extracting
Method Ref Drying ter, % Added Solvent
Soxhlet [14] air dry at room >95 10% hexane-acetone (9:1)

temperature,
grind--mortar and
pestle
Shake I [191 air dry at room >95 slurry hexane-isopropyl
temperature, alcohol (3:1)
grind--mortar and
pestle
Shake II [18| none ... none acetone with hexane
added later
Blender [20] Na2SO, added ... none acetonitrile-acetone
(1:1)
Column I [15] partial air dry at ,,,70 none hexane
room temperature,
mix with NarSO,
and grind
Column II [16] partial air dry at ~50-70 none hexane-acetone (1:1)
room temperature,
mix with N%SO4
and grind
High frequency dis- [17] none ... none acetone
persion
Dosing Procedures--Water Samples (880 ml) in 1-qt sample bottles

were individually dosed by pipetting the PCB's in acetone solution (1
pg/ml) and shaking vigorously to achieve uniform mixing.
Bottom Samples Approximately 400 g of an air dried sediment (98.7
percent dry matter) was ground with a mortar and pestle and passed
through a 20 mesh screen. The sediment was then placed in a 1-qt wide
mouth glass bottle. Distilled water (200 ml) was added and mixed to form
a slurry. Aroclor 1242 (50.0 /~g) and Aroclor 1254 (50.0 /ag) in 1 /ag/~
acetone solution was added to the slurry with continual mixing. The bottle
was sealed and allowed to equilibrate for 20 h with occasional shaking.
Appropriate aliquots of the dosed sample were taken and treated as
required by the extraction methods to be tested. The high frequency
dispersion device, Column II, and soxhlet extraction were selected for
testing recovery on dosed bottom samples.
Quantitation Techniques--Two methods were used to reduce the
electron capture gas chromatographic data:
(a) When a dosed or environmentally occurring PCB equivalent to or
closely resembling a standard Aroclor was present, a simple calcula-
tion was made by summing all the peak heights of the unknown
and comparing it to the sum of the peak heights of the reference
Aroclor.
(b) When mixtures of PCB's, not representing a single Aroclor were
present, the method of Webb [10] was used to determine the PCB
concentrations and the most likely Aroclor represented.
All results for bottom samples were calcualted on a dry weight basis.
The dry weight was obtained by oven drying an aliquot of the sample at
approximately 105~ overnight.
Gas chromatography and mass spectrometry techniques were used to
confirm the identity of the PCB's in the environmentally contaminated
samples.

Water Samples
Initial studies to determine the cause of low and variable recoveries of
PCB's from dosed natural waters centered around the bottle cap liners.
Teflon lined caps which are used routinely are considered the best choice
when collecting samples for organic analysis. It was thought that the
Teflon may be sorbing some of the PCB's. However, no PCB's were
recovered from Teflon liners after soaking in hexane for 1 h. Sub-
sequently, a comparison of recoveries using Teflon lined caps and
aluminum lined caps was made. The samples were stored under ambient
conditions in the laboratory, up to 11 days. Figure 1 shows that there is a
tendency toward higher recoveries when using aluminum lined caps. In
each case, samples stored with aluminum cap liners yielded greater
recoveries than those stored with Teflon lined caps.
Sample Preservation
Figure 2 shows the results of aging on recovery of PCB's from dosed
natural river waters. Samples held at ambient conditions in the light
showed variable and often low recoveries with a general decrease in
100
~ALUMINUM CAP LINER
u ~ TEFLON CAP LINER
90 84
>
0 80-
ix
\\ \~AROCLOR1242
~70- -- ~ %. ~ ~'~'~..~..~OCLOR 1016
60" -- " " ~ "~. ~ , ~ . ~ "~-..~..... AR.~OCLOR 1016

AROCLOR 1242
50.
0 2
I
4
I 6
I 8
I10
, I : I
12
DAYS AFTER DOSING
F I G . l - - E f f e c t o f cap liner on recovery o f PCB's f r o m dosed river water (dose o f 1 t~g p e r

sample).
AROCLOR 1016
100
90-
w
>
80.
_ ~ A R O C L O R 1242
uar 70" AROCLOR I016
60"
.... PRESERVED
NON- PRESERVED
50,
I
2
, I
4 6 1 ,[ , ,I i
DAYS AFTER DOSING
F I G . 2--Effect o f aging on recovery o f PCB's f r o m dosed river water at ambient tempera-

ture, preserved versus non-preserved (dose o f l I~g p e r sample).
recovery with time. Recoveries ranged from 102 percent to a low of 67

percent over a period of 15 days. The recoveries from samples preserved
with formaldehyde were quantitative over the time period, averaging 105
percent and ranging from 92 to 116 percent. Figure 3 depicts typical
losses of the isomers of Aroclor 1016 that occur after aging a dosed
sample for 15 days. The greater losses occur in the isomers that contain
three or fewer chlorine atoms. The greatest losses occur in peaks 37 and
40 which represent PCB's containing three chlorine atoms. All natural
water samples tested showed a trend toward reduced recovery with the age
of the sample. The rate of loss appeared to be affected by the variation in
physical, chemical, and biological characteristics of the samples.
Table 2 compares the recoveries of Aroclor 1016 obtained from dosed
river waters that have been preserved with formaldehyde up to 15 days to
the recoveries obtained at time zero (<8 h after dosing). A statistical
AROCLOR 1016
STANDARD
....... AROCLOR 1016 DOSE
AGE 15 DAYS
(69% RECOVERY)
37
28
32 ~0
47 5458
I I I 1 I I
FIG. 3--Electron capture gas chromatogram of reference standard and recovered Aroclor
1016.
TABLE 2--Recovery of Aroclor 1016 from river

waters at time zero versus samples preserved
and aged (dose, 1 ~g/sample).
Recovery, %
Time Zero a Preserved b
109 92
107 107
107 110
98 111
107 109
116 93
111 116
102 111
92 104
109 105
111 108
100 111
99 95
lOS ...
Mean 105.4 105.5

No. of samples 14 13
Standard deviation 6.4 7.6
Variance 40.4 57.8
Coefficient of variation 6.0 7.2
Blank value (average) 6.0 6.0
Corrected mean 99.4 99.5
aTime zero = < 8 h after dosing.

bpreserved with formaldehyde {15 ml of 37%
solution).
comparison of the data shows very good agreement between the time zero
and preserved samples.
The pH of the sample had no effect on the extraction efficiency from
dosed samples over the range of 2.7 to 10.5. That is, the average
recovery at time zero for the pH levels listed in Table 3 was quantitative.
The accuracy and precision was essentially equivalent to that obtained for
the untreated samples shown in Table 2. The limited data obtained
suggest an apparent effect on the recovery as the sample ages at pH 5.3
and 7.3. Recoveries at these pH levels decreased with time although the
decrease was not as great as noted in other studies. Recoveries at pH 2.7,
8.8, and 10.5 were near quantitative throughout the time period studied.
The effect of storing samples at ambient temperatures in the light,
dark, and under refrigeration was studied. The results are listed in Fig. 4
and compared to results obtained for samples preserved with formal-
dehyde. Samples stored in the dark yielded higher recoveries throughout
the period of study than those stored in the light. Refrigerated samples
gave slightly better recoveries than those stored at ambient temperature in
TABLE 3---Recovery o f Aroclor 1016 from dosed river water at

various p H levels (dose, 1 ~g/sample).
pH Age, days Recovery, %
2.7 2 98
2.7 6 102
2.7 14 91
5.3 2 107
5.3 6 89
5.3 14 86
7.3 2 109
7.3 5 87
7.3 14 84
8.8 2 107
8.8 6 106
8.8 14 95
10.5 2 107
10.5 6 105
10.5 14 100
NOTE--
Blank value ~ 7%.
Day zero value (mean recovery) = 107%.
,ooL %
FORMALDEHYDE ADDED
90,
REFRIGERATED
A M B I E N T IN DARK
80
AMBIENT IN LIGHT
~'70
ud
m
60.
.50,-
I I I I I
DAYS AFTERDOSING
FIG. 4--Recovery o f Aroclor 1016 from dosed river water when stored at ambient tem-
perature (in light and the dark), under refrigeration, and preserved with formaMehyde (dose
q]" I pg per sample).
the d a r k . However, the t r e n d o f d e c r e a s i n g recovery was evident u n d e r all

t h r e e conditions. S a m p l e s preserved with f o r m a l d e h y d e gave t h e highest
a n d least v a r i a b l e recoveries a n d showed no d e c r e a s i n g t r e n d in recovery
with the age of t h e s a m p l e .
T h e use of t h e s e m i - a u t o m a t i c l i q u i d - l i q u i d e x t r a c t i o n system o f
K a w a h a r a et al [12] .was c o n s i d e r e d . A p r e l i m i n a r y e v a l u a t i o n showed it to
be inferior to the s e p a r a t o r y funnel s h a k e out. Therefore, it was n o t tested
f u r t h e r . A t t i m e zero, the m e a n recovery u s i n g this system was 84 p e r c e n t
with a r a n g e o f o f 77 to 92 percent.
Polyurethane F o a m s
T h e use o f u r e t h a n e f o a m plugs as r e p o r t e d by Gesser et al [13]
a p p e a r e d to show p r o m i s e as a m e a n s for c o n c e n t r a t i n g a n d isolating
P C B ' s . T h u s , we e v a l u a t e d t h e t e c h n i q u e on dosed distilled a n d n a t u r a l
river water. T a b l e 4 lists the d a t a o b t a i n e d using the c o l u m n p r o c e d u r e o f
G e s s e r a n d by s o a k i n g the plugs in t h e s a m p l e . T h e c o l u m n m e t h o d
w o r k e d well on distilled w a t e r a n d was m o r e efficient t h a n t h e s o a k i n g
p r o c e d u r e . However, t h e c o l u m n m e t h o d was not at all s u i t a b l e for use on
TABLE 4---Recovery of Aroclor lO16 from distilled water using
polyurethane foam plugs (dose, 1 ~g/sample).
Recovery, %
Column Elution Soaking
84 61
79 89
100 77
95 58
91 77
71 95
109 106
91 109
100 86
86 50
98 77
76 50
91 97
88 60
94 75
95 114
84 66
99 98
114 99
100
Mean 91.8 82.2
No. of samples 19 20
Standard deviation 10.7 20.0
Variance 114.6 399.0
Coefficient of variation 11.6 24.3
natural waters. Suspended material in the samples plugged the column.

We were unable to pass the total volume of a l-liter natural river water
sample through the column without adding external pressure, in which
case channeling occurred. Soaking the plugs in dosed river water worked,
however, the accuracy and precision was poorer than that obtained with
distilled water. Coating the plugs with SE-30 did not improve the
efficiency. These results coupled with the high background material from
the foam which had significant adverse effects on the gas chromatographic
(GC) column and the detector were the bases for rejecting the method.
As a result of the foregoing studies, the separatory funnel liquid-liquid
extraction technique was shown to be the most efficient for recovery of
PCB's from natural waters. Therefore, this method was used for analyzing
all wastewater samples described next.
Wastewater samples
Several industrial wastewater samples were collected and analyzed for

environmental levels of PCB's. In one case, additional aliquots of the
sample were then dosed with 5 /ag/liter of Aroclor 1242 and again
analyzed. Table 5 presents the results and statistical data derived from the
TABLE 5---R ecovery of PCB's from environmentally contaminated industrial waste samples,
concentration of Aroclor 1242 (/ag/liter).
Replicates 870 870 a 832 833
1 2.17 7.99 58.7 13.7
2 1.92 7.74 63.0 13.8
3 1.86 7.42 64.8
4 2.27 6.97 66.9
5 1.86 6.42 60.3
6 1.70 6.79 66.3
7 1.73 6.75 61.7
Mean 1.93 7.15 63.1
No. of samples 7 7 7
Standard deviation 0.214 0.575 3.07
Variance 0.046 0.330 9.46
Coefficient of variation 11.11 8.03 4.87
a Dosed with 5 /ag/liter of Aroclor 1242.
samples. The results show good precision for two levels of environmental
contamination. The precision for the dosed samples was equally good. The
recovery for the dosed sample averaged 103 percent.
Bottom Samples
Bottom samples collected from a large m a n - m a d e lake in Ohio were

found to be contaminated with Aroclor 1242 and 1254. Samples collected
from three locations in the lake were selected in order to evaluate seven
extraction procedures. The total quantity of sample available to us limited

the numbers of analyses that could be made. Nevertheless, useful informa-
tion was derived from the results. Table 6 lists the results of our study on
the lake bottom samples. The mechanical dispersion apparatus was not
available at the time and no comparison of this technique could be made
TABLE 6---Recovery of PCB's from environmentally contaminated lake bottom samples.
Dry Arocior Aroclor

Sample a Matter, 1242, Recovery, b 1254, Recovery b
Method Ref Area % /ag/kg % /ag/kg %
Soxhlet [14] 3 96.7 c 26.2 100 12.5 100

Shake I [19] 3 96.7 15.3 58.4 7.4 59.2
Blender [20] 3 37.3 5.6 21.4 9.4 75.2
Shake I1 [18] 3 37.3 1.0 3.8 2.7 21.6
Soxhlet [14] 5 96.4 c 54.3 I00 24.7 100
Column 1 [15] 5 7b.9 6.4 11.8 8.9 36.0
Soxhlet [14] 8 98.4 c 0 403 100
Shake I [19] 8 98.4 0 377 93.5
Column 1 [15] 8 98.4 0 343 85.1
Column I [15] 8 70.2 0 318 78.9
Blender [20] 8 70.2 0 343 85.1
Blender [20] 8 56.0 0 246 61.0
Shake II [18] 8 70.2 0 321 79.7
Shake II [18] 8 56.0 0 258 64.0
a Samples from Areas 3 and 5 were muck; samples from Area 8 were sandy muck.
b Recovery for soxhlet procedure considered to be 100%.
c 10% water added prior to extraction.
on these samples. The results obtained show that the air-dried, 10 percent
moisture-added soxhlet procedure provides significantly greater efficiency
than any of the other methods tested.
In addition, a study of the recovery of Aroclor 1242 and 1254 from
dosed bottom samples was carried out using three methods--mechanical
dispersion, column II, and the soxhlet. Results of this test (Table 7) show
the superiority of the air-dried moisture-added soxhlet extraction pro-
TABLE 7--Recovery of PCB's from dosed bottom samples
(dose 237 ~g/kg total PCB's). a
Dry Average
Matter, /ag Recovery,
Method Ref % Recovered %
1 2
High frequency dispersion [17] 52.5 b 210 88.7
Column II [16] 89.7 208 187 83.5
Soxhlet [14] 98.4 230 226 96.2
a Mixture of Aroclors 1242 and 1254, 118.5 tag/kg of each.

b Sample lost due to malfunction of dispersion apparatus.
cedure over the other extraction procedures as far as bottom sediments are
concerned.
We acquired a sewage treatment plant sludge which was found to
contain PCB's. Aroclors 1242 and 1260 were identified in the sample. Two
extraction techniques were employed in the analysis--mechanical
dispersion and soxhlet extraction. The results are listed in Table 8. In this
case, equivalent results were obtained with the two methods.
TABLE 8--Recovery qf PCB's from environmentally contaminated treatment plant sludge.

Aroclor Recovered(mg/kg)
Sample No. Method Dry Matter, % 1242 1260
831-A high frequency 18.1 34.4 14.2
dispersion
831-B soxhlet 93.2 34.4 12.5
The results of these studies show the air-dried, 10 percent moisture-

added soxhlet extraction procedure to be the most efficient method for
recovery of PCB's from bottom samples. This is consistent with the results
reported by others for recovery of certain organochlorine pesticides from
soils [22-25]. The better efficiency of this procedure is probably due to the
smaller particle size achieved when pulverizing the dry sediment and to
the longer solvent contact time provided by the soxhlet method as reported
by Saha et al [26] and Chiba and Morley [27]. Nash et al [28] evaluated
soxhlet, shake, and column procedures somewhat different from those
that we studied and concluded that his soxhlet and shake procedures gave
near equivalent recoveries for nine pesticides from soil. In this case, the
samples were not air dried or ground with a mortar and pestle prior to
soxhlet extraction.
The cleanup procedures employed, the standard Florisil and the micro
silica gel columns, were adequate for all industrial waste and sludge
samples. However, all bottom samples required further treatment with
mercury to remove sulfur. All procedures which required a water wash of
the extracts, the shake, Column II, blender, and the high frequency
dispersion methods, produced severe emulsions and additional manip-
ulative steps not required by the soxhlet procedure.
The high frequency dispersion technique looks promising, at least, for
sludge samples and should be evaluated further. However, some problems
have been encountered in its use. Small stones, pea size and even smaller,
are a hazard. They are thrown at terrific speeds into the wall of the
container and will shatter glassware. Further, even small particle size sand
causes excessive wear on the stator and rotor, in which case frequent parts
replacement is required.
Conclusions
The separatory f u n n e l liquid-liquid extraction procedure is the m e t h o d
of choice for recovery of PCB's from n a t u r a l waters. The air-dried soxhlet
extraction procedure is the m e t h o d of choice for recovery of P C B ' s from
b o t t o m a n d sludge samples. F o r m a l d e h y d e effectively preserves P C B ' s in
dosed n a t u r a l water samples, at least up to two weeks.
Acknowledgments
The authors wish to t h a n k James O ' D e l l for his assistance in collection,

p r e p a r a t i o n , and extraction of the samples used in this study. T h e G C - M S
confirmatory analyses were performed by J.W. Eichelberger a n d L.E.
Harris.
References
[1] Peakall, D. B. and Lincer, J. L., Bioscience, Vol. 20, 1970, p. 958.
[2] Gustafson, C. G., Environmental Science and Technology, Vol. 4, 1970, p. 815.
[3] Duke, T. W., et al, Bulletin of Environmental Contamination and Toxicology, Vol. 5,
1971, p. 171.
[4] Veith, G. D. and Lee, G. F., Water Research, Vol. 5, 1971, p. 1107.
[5] Veith, G. D. and Lee, G. F., Proceedings, 14th Conference on Great Lakes Resources,
Intern.ational Association of Great Lakes Resources, 1971, pp. 157-169.
[6] Zitko, V., Bulletin of Environmental Contamination and Toxicology, Vol. 5, 1970,
p. 279.
[7] Sanders, H. O. and Chandler, J. H., Bulletin of Environmental Contamination and
[8] Armour, J. A. and Burke, J. A., Journal of the Association of Official Analytical
Chemists, VoI. 53, 1970, p. 761.
[9] Leoni, V., Journal of Chromatography, Vol. 62, 1971, p. 63.
[10] Webb, R. G. and McCall, A. C., Journal of Chromatographic Science, Vol. 11, 1973,
p. 366.
[11] "Methods for Organic Pesticides in Water and Wastewater," U.S. Environmental Pro-
tection Agency, National Environmental Research Center, Cincinnati, Ohio, 1971,
[12] Kawahara, F. K. et al, Journal of the Water Pollution Control Federation, Vol. 39,
1967, p. 572.
[13] Gesser, H. D. et al, Analytical Letters. Vol. 4, 1971, p. 883.
[14] Breidenbach, A. W. et al, "The Identification and Measurement of Chlorinated Hydro-
carbon Pesticides in Surface Waters," U.S. Department of the Interior, Federal Water
Pollution Control Administration, Publication WP-22, 1966.
[15] Hesseiberg, R. J. and Johnson, J. L., Bulletin of Environmental Contamination and
[16~ "Pesticide Residue Methods for Sediment and Fish," Method SP-8/71, Southeast En-
vironmental Research Laboratory, Environmental Protection Agency, Athens, Ga.
[17] Johnson, R. E. and Starr, R. I., Journal of Agricultural and Food Chemistry, Vol. 20,
1972, p. 48.
[18] Goerlitz, D. F. and Brown, E., Techniques of Water Resources Investigations of the
United States Geological Survey. Book 5, Chapter A3, 1972.
[19] "Chlorinated Pesticide Residues in Water, Soils, Crops, and Animal Products," Method
PMS-911/67, Manual of Methods .for the Determination of Residues of Shell Pesticides,
Shell Chemical Company. 1967.
[20] Muth, J., unpublished work, September 1971.

[21] Goerlitz, D. F. and Law, L. M., Bulletin of Environmental Contamination and ToM-
cology. Vol. 6, 1971, p. 9.
[22] Williams, I. H., Journal of the Association of Official Analytical Chemists, Vol. 51,
1968, p. 715.
[23] Woolson, E. A. and Kearney, P. C., Journal of the Association of Official Analytical
Chemists, Vol. 52, 1969, p. 1202.
[24] Nash, R. G. and Harris, W. G., Journal of the Association of Official Analytical
Chemists, Vol. 55, 1972, p. 532.
[25] Teasley, J. I. and Cox, W. S., Journal of Agricultural and Food Chemistry, Vol. 14,
1966, p. 519.
[26] Saha, J. G. et al, Journal of Agricultural and Food Chemistry, Vol. 17, 1969, p. 877.
[27] Chiba, M. and Morley, H. V., Journal of Agricultural and Food Chemistry. Vol. 16,
1968, p. 916.
[28] Nash, R. G. et al, Journal of the Association of Official Analytical Chemists, Vol. 56,
1973, p. 729.
A. D. Thruston, Jr?
Liquid Chromatography of
Carbamate Pesticides
REFERENCE: Thruston, A. D., Jr., "Liquid Chromatography of Carbamate Pesti-

cides," Water Quality Parameters, ASTM STP 573, American Society for Testing and
Materials, 1975, pp. 220-226.
ABSTRACT: A commercial liquid chromatograph was evaluated and found useful for
analysis of carbamate pesticides. Liquid chromatography retention times for 23 carba-
mate pesticides are given. The ultraviolet detector required 20 to 1500 ng for the pesti-
cides studied to give a 25 percent full-scale recorder response.
KEY WORDS: water quality, liquid chromatography, carbamates, pesticides, environ-
mental tests
Most carbamate pesticides cannot be analyzed directly by gas chroma-

tography unless conversion to more suitable derivatives is carried out
[L2], 2 because they are thermally unstable. Techniques, such as semi-
quantitative thin-layer chromatography [3,4] or quantitative ultraviolet
spectrometry [5], vary in selectivity and sensitivity.
Liquid chromatography (LC) is a relatively new analytical tool that
offers selectivity and moderate sensitivity for analysis of these heat-labile
compounds. LC separation and detection of Sevin (carbaryl) and its
hydrolysis product, l-naphthol, from plant extracts have been reported [6],
but no comprehensive list of retention times and sensitivities of various
carbamate pesticides has been published.
A liquid chromatograph is similar to a gas chromatograph; each has an
injection port, a packed column, and a detector. A liquid mobile phase
under pressure is used in LC instead 6f a carrier gas as in gas
chromatography. Many compounds that are not volatile enough or too
heat-labile for analysis by gas chromatography will partition between the
LC stationary and mobile phases.
Two DuPont stationary phase columns were evaluated for analysis of
carbamate pesticides--Permaphase ODS (for nonpolar compounds) and
Permaphase E T H (for more polar type carbamates).
'Research chemist, Analytical Chemistry Branch, Environmental Protection Agency,
Southeast Environmental Research Laboratory, Athens, Ga. 30601.
220

THRUSTON ON CARBAMATE PESTICIDES 221
Experimental
Liquid Chromatograph
A DuPont Model 820 liquid chromatograph equipped with an ultra-
violet photometric detector that measures the absorbance at 254 nm was
used. The following columns and conditions were employed:
1. 1 m by 2 mm inside diameter stainless steel column, packed with
Permaphase ODS (octadecyl silane). Mobile phases were 6 and 30
percent methanol in water. A pressure of 1000 psi at 50~ main-
tained a flow of I ml/min.
2. 1 m by 2 mm inside diameter stainless steel column, packed with
Permaphase ETH (ether). Mobile phases were hexane, 1 percent
isopropanol/hexane, and 4 percent isopropanol/hexane. A pressure
of 400 psi at 40~ maintained a flow of I ml/min.
Solvents
Spectrograde hexane, isopropanol, methanol and methylene chloride.
Standard Carbamate Solutions

Standards (Table 1) were prepared as isopropanol solutions.

Table 2 lists retention times (Rt) and sensitivities for 17 carbamates on
Permaphase ODS. The first 12 eluted as sharp symmetrical peaks within
10 min with 6 percent methanol in water as the mobile phase; Fig. 1
shows a chromatogram of five of these carbamate pesticides and 1-
naphthol. The last five in Table 2 had excessive retention times, which
were shortened by changing the mobile phase to 30 percent methanol in
water.
Figure 2 shows chromatograms of seven of the more polar carbamates
on the Permaphase ETH column. A change in mobile phase polarity from
1 percent isopropanol in hexane to 4 percent isopropanol in hexane causes
significant changes in retention times.. Table 3 summarizes retention times
and minimum sensitivities of nine polar carbamates.
For these carbamates, the sensitivity of the ultraviolet detector ranges
from 20 to 1500 ng, a level suitable for pesticide residue analysis. This
work was done with pure solutions to obtain standard retention times.
Natural sample extracts were not studied systematically.
The LC technique was applied to a study of the kinetics of Sevin
degradation by micro-organisms. Aqueous samples of 4 to 8 /~1 were
injected into the LC (Permaphase ODS column using the preceding
TABLE l p C h e m i c a l names and sources o f the carbamate pesticides.
Common or Trade Name Chemical Name Source
Baygon | (aprocarb) 2-isopropoxyphenyl-N -methyl carbamate Chemagro Corp.

Furadan| 7-(2,3-dihydro-2,2-dimethyl)-benzo- Niagara Chem. Div.,
(carbofuran) furanyl-N-methyl carbamate FMC Corp.
Matacil| (aminocarb) 4-dimethylamino-3-methylphenyl- Chemagro Corp.
N-methyl carbamate
Mobam| 4-benzothienyl-N-methyl carbamate Mobil Chem. Co.
Sevin@ (carbaryl) 1-naphthyl-N-methyl carbamate Union Carbide Corp.
Landrin 3,4,S-trimethylphenylmethyl-carbamate Shell Development
Corp.
UC 10854 3-isopropylphenyl-N-methyl carbamate Union Carbide Corp.
UC 8454 l-(5,6,7,8-tetrahydro)-naphthyl- Union Carbide Corp.
N-methyl carbamate
Carbanolate 3,4-xylyl-6-chloro-N-methyl carbamate Upjohn Co.
RE 5305 3-sec,butylphenyl-N-methyl carbamate Chevron Chem. Co.
Mesurol | 4-met hy[thio-3,S-dimethylphenyl- Chemagro Corp.
N-methyl carbamate
Zectran | 4-dimethylamino-3,S-dimethyl-phenyl- Dow Chem. Co.
N-methyl carbamate
Sirmate| 2,3-isomer 2,3-Dichlorobenzyl-N-methylcarbamate FDA a
Sirmate, 3,4-isomer 3,4- Dichlorobenzyl-N-metbylcarbamate FDA a
Bux| 3-(1-Methylbutyl) phenyl Chevron Chem. Corp.
methylcarbamate
Azak| (terbutol) 2,6-Di-tert.-butyl-4-methylphenyl- Hercules Powder Co.
N-methylcarbamate
Benomyl (Dupont Methyl 1-(butylcarbamoyl)-2- E. I. DuPont de
Fungicide 1991) benzimidazole carbamate Nemours and Co.
IPC isopropyI-N-phenyl carbamate PPG
CIPC isopropyl-N-(3-chlorophenyl) carbamate PPG
Dimetilan| 3-(1-N,N-dimethylcarbamoyl-5-methyl)- Geigy Chem. Corp.
pyrazolyt-N, N-dimethyl carbamate
Temik| 2-methyl-2-methylthio propionaldehyde- Union Carbide Corp.
0-methylcarbamoyl oxime
Swep methyl-N-(3,4-dichlorophenyl) carbamate Niagara Chem. Div.,
FMC Corp.
Barban 4-chloro-2-butynyl-N-(3-chlorophenyl) Gulf Res. & Dev. Co.
carbamate
Thiram bis(dimethylthiocarbamoyl) disulfide E. I. DuPont de
Nemours and Co.
Mylone@ (dazomet) 3,5-dimethyl- 1,3,5-tetrahydrothiadiazine- Union Carbide Corp.
2-thione
Lannate| (methomyl) methyl N-[(methylcarbamoyl)oxy]- E. I. DuPont de
thioacetimidate Nemours and Co.
aFood and Drug Administration, Washington, D.C.

2 4 6
.0_
\
r-
e
I
o
Minutes
FIG. 1--Chromatogram of carbamate pesticides on Permaphase ODS column: 6 percent
methanol/94 percent water mobile phase. (1) Furadan, (2) Matacil, (3) Sevin, (4) Landrin,
(5) l-naphthol (hydrolysis product o f Sevin). and (6) Mesurol.
conditions) without prior clean-up or extraction. Sevin was observed in

these samples in the 11 to 33 mg/liter range based on comparison with
standard solutions.
Considerably lower detection limits can be obtained by concentrating
the sample. For example, a liter of tap water, spiked with 2 /ag/liter of
swep (methyl dichlorophenyl carbamate) was extracted with methylene
chloride [3] and concentrated to 200 /A. Figure 3 shows a distinct swep
peak in the chromatogram of a 5-/al injection of the concentrate on a
Permaphase ETH column, 40~ 1 percent IPA/hexane, 1 ml/min.
Conclusions
Liquid chromatography shows promise for analysis of carbamate pesti-
cide residues. The technique provides separations with speed and reso-
lution comparable to those of gas chromatography. Ambient or near
ambient temperature can be used with heat-labile compounds. Sensitivity
is 2 to 3 orders of magnitude less than that of electron capture gas
chromatography but is still adequate for many compounds.
3 (a) (b)
i
2 4
.e
C
m
_c
I
I
6 4 [ 6 6
Minutes
FIG. 2--Chromatogram of carbamate pesticides on Permaphase E T H column: (a) 1 per-
cent isopropanol/99 percent hexane mobile phase (1) CIPC, (2) Dimetilan, (3) Temik, and
(4) barban; (b) 4 percent isopropanol/96 percent hexane mobile phase (1) swep. (2) barban,
(3) Mylone. and (4) Lannate.
TABLE 2--Retention times (R t) and sensitivities o f various carbamates on Permaphase ODS

column, 50~ methanol~water mobile phase, lO00 psi, l ml/min flow rate,
ultraviolet detector.
Minimum Amount
Chemical 6% M e O H / 30% MeOH/ to give 25%
Name H~O, R t (min) H20, R t (min) FSD (ng)
Baygon 1.50 250

Furadau 1.60 300
Matacil 2.00 50
Mobam 2.10 25
Sevin 2.60 100
Landrin 3.25 1000
UC 10854 3.40 1000
UC 8454 3.50 1000
Carbanolate 3.60 1000
1 -naphthol a 4.30 250
RE-5305 7.20 1000
Mesurol 7.65 200
Zectran 8.30 200
Sirmate, 2,3-isomer ... 2.~ 1500
Sirmate, 3,4-isomer ... 2.60 1500
Bux ... 3.50 1500
Benornyl b ... 6.10 1500
Azak ... 6.35 1500
aHydrolysis product of 5evin.

bBroad skewed peak.
.o_
c
E
6
Minutes
FIG. 3--Chromatogram of swep, extracted from water spiked at 2 ~g/liter level: (1) sol-
vent peak and (2) swep.
TABLE 3---Retention times (R t) and sensitivities of various carbamates on Permaphase E T H

column. 40~ 0 to 4~ isopropanol/hexane mobile phase, 400 psi, 1 ml/min flow rate,
ultraviolet detector.
Minimum Amount
Chemical Hexane 1% IPA/Hexane 4% IPA/Hexane to Give 25%
Name R t (min) R t (min) R t (min) FSD (ng)
IPC 1.52 1.50 ... 250

CIPC 1.92 1.50 ... 500
Dimetilan 2.10 1000
Temik 2.66 1.68 100
Swep 3.00 1.80 20
Barban 7.20 2.96 400
Thiram ... 2.90 50
Mylone 9 3.82 100
Lannate 9 .. 4.88 500
References
[1] Ha|den, E. R., Jones, W. M., and Beroza, M., Journal of Agricultural and Food
Chemistry, Vol. 17, 1969, p. 56.
[2] Butler, L. I. and McDonough, L. M., Journal of the Association of Official Analytical
Chemists. Vat. 53, 1970, p. 495.
[3] Abbott, D. C., Blake, K. W., Tarrant, K. R., and Thomson, J., Journal of Chromo.
tography, Vol. 30, 1967, p. 136.
[4] EI-Dib, M. A., Journal of the Association of Official Analytical Chemists, Vol. 53,
1970, p. 756.
[5] Aly, O. M., Journal of the American Water Works Association. Vol. 59, 1967, p. 906.
[6] "Chromatographic Methods 820 MT," Information Sheet, DuPont Instruments,
Wilmington, Del., 13 Feb. 1970.
K . L. E. K a i s e r ~
Uncoated Teflon as Support and

Stationary Phase for Liquid/Solid
Gas Chromatography
REFERENCE: Kaiser, K. L. E., "Uncoated Teflon as Support and Stationary Phase for
Liquid/Solid Gas Chromatography," Water Quality Parameters, ASTM STP 573,
ABSTRACT. The use of Teflon, 30-60 mesh, as a combined support and stationary
phase for liquid/solid gas chromatography has been studied. The Teflon columns have
separation properties similar to porous polymer supports. They exhibit short retention
times at relatively low operating temperatures, durability, and low bleed. The chroma-
tograms suggest that pure Teflon columns are suitable for the separation of hydro-
carbons, alcohols, phenols, polychlorinated biphenyls, esters, and free fatty acids as well
as for direct aqueous injections, such as the determination of methanol in water.
KEY WORDS~ water quality, gas chromatography, tetrafluoroethylene resins, environ-

mental tests
Over the past decade, gas chromatography has become one of the most
useful techniques in the fields of organic and analytical chemistry. With
the advent of suitable interfacing systems, the combined gas chromato-
graphy-mass spectrometry (GC-MS), gas chromatography-infrared spectro-
scopy (GC-IR), and gas chromatography-nuclear magnetic resonance
spectrometry (GC-NMR) have developed to be important tools for the
analyst, especially for environmental and physiological problems, where
trace amounts of materials are to be investigated.
With the development of GC techniques, a large variety of stationary
phases, support materials, and column treatments have been probed for
many individual analytical problems. Quite recently, there has been a
trend from the customary liquid phase coatings on diatomaceous supports
to porous polymer phases (PP) which combine support and stationary
phase properties in a single substance. Their nature offers the advantage
of avoiding support coating with the subsequent problems arising from
active sites, column bleed, and long retention times. Porous polymers
instead have low bleed, are usually quite temperature resistant, and
'Research Scientist, Environment Canada, Canada Centre for Inland Waters, Burlington,
Ontario L7R 4A6, Canada.
227

because of their working principle are advantageous for the GC of many

different classes of chemicals. This fact has been recognized and made
advantage of for direct aqueous injections of samples containing ppm
amounts of methanol and other alcohols [1]. 2
This paper describes the application of Teflon powder, 30-60 mesh, as
combined stationary phase and support, similarly to porous polymer
materials, with respect to the GC analysis of hydrocarbons, alcohols, fatty
acid esters, free fatty acids, phenols, polychlorinated biphenyls, and
hydroxybenzoic acids in organic and aqueous media.
Properties of Teflon as Support

First application of Teflon as support for gas liquid chromatography is
described by Landault and Guiochon [2]. The authors recommend Teflon
as a useful support because of good thermal properties, high chemical
resistance, and a low content of active sites which will irreversibly absorb
small quantities of volatilized compounds. With polyglycols as stationary
phase, H E T P (height equivalent of a theoretical plate) values of 0.24 to
0.52 cm were accomplished versus 0.10 cm for a equally-sized column
with chromosorb as support. Uncoated Teflon columns were, however, not
investigated for their properties. Teflon was suggested as especially useful
support for the GC of polar materials. Similar observations were made by
Gunther and Jaglan [3], who found Teflon as a superior support for the
GC of organophosphorus pesticides and their metabolites. Kirkland [4]
reported on a study with fluorine containing polymers as solid supports in
GC with the application of various liquid phases such as squalane,
Carbowax 400, and diglycerol. In general, he recommended polytetra-
fluoroethylene supports as giving good peak symmetry for the GC of
highly polar compounds. His investigation also included liquid phase
loadings of 0, 2, and 5 percent, however, for Teflon-C and KeI-F with
n-butanol as solute and Carbowax 400 as stationary phase at 75~ an
optimum loading of about 20 percent was determined. In similar experi-
ments, Conder [5] determined the efficiency of GC columns with di-n-
nonyl-phthalate (DNP) at loadings from 0 to 20 percent on Teflon
supports. Thus, at 86~ a H E T P minimum was observed at about 10
percent loading. The same study also proved the activity of Teflon support
towards the retention of solutes such as ethanol at 86~
Experimental
Apparatus and Materials
The gas chromatograph was a Tracor MT-220 instrument with vertical
U-columns, and flame ionization detector at 250~ The injector block,
KAISER ON TEFLON COLUMNS 229
fitted for on-column injection was also kept at 250~ As carrier gas,
Helium U H P grade at pressures from 10 to 44 psi was used. Chemicals
were of analytical or better grade. Recording instrument was a Hewlett-
Packard recorder, Model 7128 A with 1 mV equal to full scale deflection
(F.S.D.) at a basic electrometer attenuation of 1 x 1. The exact atten-
uation employed is given with each chromatogram.
Column Preparation
U-shape glass columns of 1.8 and 1.2 m length of 0.25 in. outside
diameter were cleaned with chromic acid, rinsed with distilled water, and
air dried. Chromosorb T a (Johns-Manville), prepared from Teflon-C 4,
mesh size 30-60, was put into a wide neck Erlenmeyer flask and shaken
by hand at room temperature for about 15 min to dislodge the lumps.
This procedure allowed for electrostatic charge of the Teflon granules and
resulted in an easily flowing Teflon powder. It was then thinly spread onto
glassine weighing paper and still present conglomerates were picked out
and discarded. The powder was then poured from both ends into the glass
columns while being vibrated. For coiled columns a similar procedure can
be employed together with a slight suction of about 50-mm of mercury on
one end and constant vibration. Filling of the column was continued,
until, after about 10 min of vibrating no further uptake of material was
observed. Both ends were plugged with some Pyrex, prepurified glass
wool. Care was taken not to press the glass wool onto the Teflon material.
Column Conditioning
The columns were placed in the GC oven and with a helium stream of
approximately 20 m l / m i n heated at a rate of 5~ to a final
temperature of 250~ Some columns, envisaged for use at higher
temperature were conditioned by further cycled heating between 250~
and a m a x i m u m of 325~
Injection of Organic Solutions

To test the possible use of uncoated Teflon columns for the gas
chromatographic separation of hydrocarbons, a mixture of neat n-alkanes
was injected to the chromatograph. As can be seen from the resulting
chromatogram, Fig. 1, the hydrocarbons separate well and give rise to
quite symmetrical peaks under the conditions of strong temperature
p r o g r a m m i n g of 20~ At similar conditions, even n-hexatriacontane
3Registered trademark, Johns-Manville Products Corp., Celite Division.
'Registered trademark, E. I. duPont de Nemours & Co., Inc.
90
80
70 10
12
6O
t~
u)
u_
50
zLU 1112
0 40 L
m
uJ 14
Q.
13 1 1~16
30-
17 19
20-
10-
0-
; ; i i J i i r f i J i
0 2 4 6 8 0 2 4 6 8 10 12 14 min.
A 1.8m
150 ~, isothermal
10 psi He, 7 m l . m i n 4
B 1.2 m
110~ 250,~ 20~ -1
45 psi He, 2 5 m l . m i n "1
FIG. l~N.alkanes.
(n-C36) will elute from such a column within 15 min retention time.
However, as is evident from the comparison of hydrocarbon chromato-
grams of Fig. 1, with those obtained on common stationary phases such as
silicones or Apiezon, peak widths appear to be wider on the Teflon phase.
This system also needs a strong temperature programming in order to
separate low molecular weight hydrocarbons as clearly as higher ones. On
the other hand, this fact can be made use of in probing for molecular
weight ranges of unknown materials and to provide preliminary data of
those materials for more detailed studies with other stationary phases.
Figure 2 represents a gas chromatogram of a mixture of neat n-
alkanols obtained under similar conditions. As with the hydrocarbons,
there is no complete baseline separation, due mainly to some tailing of the
eluted alcohols. Yet, this effect seems to be less pronounced in this case,
although alcohols are well-known to tail on many liquid phases. The
C Clo
80
C+8
70 84
60
c5 5O
o~
t~
z 40
Iii
orr
iii
o_ 3O
20-
10- C12
O-
i i i i i i i i i i
0 1 2 3 4 5 6 7 8 9 min.
1 141 n - a l c o h o l s
1.8 m. 40 psi He, ~.,, m l . m i n "~
100 ~ 2 2 0 ~, 20~.min -+
FIG. 2--N-alcohols.
employed conditions allow for an equally rapid chromatographic study of

those solutions.
In Fig. 3, a chromatogram of a mixture of fatty acid methyl esters in
ethylbenzene is given. They are structurally related to long chain hydro-
carbons and alcohols. Temperature programming of 110 to 220~ at
20~ appears to be sufficient for the separation of those fatty acid
esters ranging from caprinic acid (Cx0) to behenic acid (C2z). All esters
elute with less than 10 rain retention time.
Figure 4 reproduces the chromatogram of a polychlorinated biphenyl
(PCB) mixture in hexane as solvent. Aroclor 1242 with 42 percent by
weight of chlorine is theoretically equivalent to a trichlorobiphenyl. In
fact, however, it contains at least 20 isomers of mono- to tetrachloro
biphenyls. Since the chromatographic separation on Teflon appears to
depend less on a dissolution of the solute in a stationary phase and rather
on absorbance on the surface and screening by pores in the Teflon [5], it
is quite interesting to note the separation of the chemically strongly-
70
60
Cll
50
~d
u~ 40
l--
Z
rr 30 012
uJ
20 02
10
x
od
cq~
i i i i i i 1 i i i J
0 1 2 3 4 5 6 7 8 9 10 11 min.
1.2m, 40 psi He, 25 m l . m m -1
110o- 220 ~ 10~
1141 fatty acid methyl esters in ethylbenzene
FIG. 3---Fatty acid methyl esters.
related PCB isomers into several groups of increasing molecular weights.

On close examination one can also observe a very small peak, designated
"O" in Fig. 4, which is due to unsubstituted biphenyl. The other peaks of
stronger intensities are mainly due to monochloro-biphenyl (designated
"1"), di-chloro- (2), trichloro- (3), and tetrachloro biphenyl (4). Even so,
the PCB's are chemically somewhat related to fluoroethylene derivatives,
Teflon columns without addition of any liquid phase, separate PCB's
preferentially by physical differences of the isomers. Yet the magnitude of
this effect depends on the type and polarity of the solutes. For example,
polyfluoro hydrocarbons, as was shown by Jequier and Robin [6], exhibit
retention times on uncoated Teflon which are significantly longer than
those of the parent hydrocarbons, and therefore dissolution of the fluoro
compounds in the upper layers of the Teflon support has been assumed.
For the PCB's the observed resolution indicates a separation, almost
entirely based on their molecular weight differences or the degree of
chlorination.
The gas chromatography of unesterified fatty acids has been investi-
gated by various authors. Metcalfe [7] reported on the use of polyester
columns treated with phosphoric acid. The columns so obtained were able
t o separate free long-chain saturated and unsaturated fatty acids up to a
KAISER ON T E F L O N C O L U M N S 233
80
3
70
60
ct 5O 2
I.E
I--
z 40
W
or r
W
a. 30 4
20
10
• 1
i i i i , ~ i l
0 1 2 3 4 5 6 7 rain.
1.8 m, 40 psi He, 25 ml. min "1
100~ 200 ~ 20 ~min'l
11.ll 20~ Aroclor 1242 in hexane
FIG. 4--Aroclor 1242.
chain length of Czz. Retention times experiences were in the order of 30

min or more for Cz0 acids at column temperatures of 220~ and above.
Other authors used basically similar systems with acid treated support
materials. Byars and Jordan [8], for example, report the separation of Cz
to Cla free fatty acids on a 1.5-m column, packed with Carbowax 20
M-terephthalic acid at 170 to 270~ Kirkland [4] used Carbowax 400 on
40-60 mesh Teflon-C for the separation of water, formic acid, and acetic
acid at 125~ Untreated TeNon, however, provides a reasonably good
stationary phase for the GC of free fatty acids with chain lengths of C o to
C16 as is shown in Fig. 5. Hexadecanoic acid elutes under a strong
temperature program within 6 rain. Slight tailing is evident, yet it can be
extrapolated that even longer free fatty acids can be separated at those or
similar conditions.
The gas chromatography of phenols has equally found interest in the
past years. They usually tend to give rise to strong peak tailing on a
variety of supports and stationary phases. Figure 6 shows the chroma-
togram of a phenol mixture in benzene in Chromosorb T at two different
234 WATER QUALITY P A R A M E T E R S
70-
60-
50-
C14
40-
c5
h"
30-
t--
C16
Z
UJ
o
20
UJ
&.
06
lO ~
x
o ----.
I I I I I I
0 2 3 4 5 7 min.
1.2 m, 40 psi He, 25 ml.min "1
100 o- 280 ~ 30~
3 141 free. fatty acids in ethanol
FIG. 5--Fatty acids.
conditions. Trace A shows the chromatogram of phenol, m-cresol, o-ethyl

phenol, and p-ethyl phenol, programmed from 45 to 125~ at 10~
Trace B is run at 85~ isothermal with the same mixture. Both chromato-
grams are run with the same attenuation of 8 • 103. The lyophobe
character of Teflon is quite well documented in the Trace B, where solvent
and all four phenols elute at 85~ within 3 rain from the 1.2-m column at
25 ml He/rain.
Related to phenols are hydroxy benzoic acids. These compounds with
two functional groups, of course, need yet stronger chromatographic
conditions. Thus, Fig. 7 shows the GC trace for ortho-, meta-, and para-
hydroxy benzoic acids in benzene. This chromatogram clearly shows the
differences in retention times between ortho- and meta-hydroxy and
between meta- and para-hydroxy benzoic acids. The first two are eluting
close together while para-hydroxy benzoic acids elute considerably later.
Since the three acids have the same set of functional groups, their
chromatographic behavior appears to be related to stereochemical effects
rather than to molecular weight and chemical functions.
This effect, of course, can be expected to be more dominating in
determining relative retention volumes with increasing polarity of those
KAISER ON T E F L O N COLUMNS 235
80
P
70 B
A
60
pE
50
i1,
zn l 40 pE
o
cr
l.lJ
&.
30
20
10
joE
x
L.__
1 i / i
0 1 3 4 5' 7' 8' o t 2 min.

1.2 m, 40 psi He, 25 m l . min 1
Phenols in benzene
A: 45 ~ - 125~ 10~ -1
B:85, ~ isothermal
FIG. 6----Pheno~.
substances. The chromatograms ot less polar compounds such as the fatty

acid esters, alkanes, and PCB's, as can be seen from the foregoing
figures, are more determined by their physical parameters such as simply
molecular weight differences.
To conclude the part on substances in organic solvents, Fig. 8 repre-
sents the gas chromatogram of a polarity mixture. Ethanol, nitromethane,
benzene, and pyridine are eluting in this sequence at 50~ column
temperature. Although ethanol and benzene have quite similar boiling
points and ethanol being more polar then benzene, it elutes far ahead of
benzene which clearly implies preferential elution of the compound with
the lower molecular weight. Nitromethane which has a higher boiling
point of 101~ lies intermediate, concordant with its molecular weight.
Pyridine, however, though very close to benzene in its molecular weight
elutes as last compound, distinctly separated from benzene. This chroma-
togram reconfirms the prior conclusion:
1. Uncoated Teflon as stationary phase as well as support allows
60-
50-
ri 40-
Us
m - HBA
I--
z 30-
orr o - HBA
w p - HBA
20-
10-
O-
x
,.__)
i J i i i -i
o 1 2 3 4 5 6 7 rain.
1.8 m, 100~ 200 ~, 20~ "1
44 psi He, 6 0 m l . min "1
1 I.II benzene solution
FIG. 7--Hydroxy benzoic acids.
separation of volatile compounds at temperatures below those for most

other column materials.
2. Teflon exhibits a very low polarity, thus, primarily separating
compounds of low polarity by their differences in molecular weight.
3. Teflon is well suited for the GC of strongly polar compounds, such
as phenols, free fatty acids, and aromatic acids at comparatively low
temperatures and short retention times.
Direct Aqueous Injections

Derived from the conclusions just mentioned, it was only a short step to
investigate the behavior of Teflon columns at the conditions of direct
aqueous injections. For the trace analysis of organic materials in water,
such as methanol, ethanol, ethylenglycol, and propylenglycol recently very
sensitive and rapid GC procedures have been produced by Fox [1]. The
columns applied were porous polymers of the poly (diphenylphenylene
oxide) nature, commercially available under the trade name TENAX. s
With this method, methanol, for example, can be determined in a range
of 1000 to approximately 0.S ppm by direct injection of the aqueous
solutions at less than 100~ It could be shown that Teflon can be used as
s A K Z O Research Laboratories, Arnheim, The Netherlands.
KAISER ON TEFLON COLUMNS 237'
80 ETHANOL
70
60
BENZENE
50
o5 PYRIDINE
I-
z 40
u.I
o
cr
w NITROMETHANE
Q,.
3o
20
10
i i i
0 1 2 min.
1.8 m, 50 ~
20 psi He, 65 ml.min "1
1 IAI mixture
FIG. 8---Polarity mixture.
chromatographic support and phase for the same type of problem at even
lower temperatures.
Figure 9 shows three sets of three consecutive injections of solutions of
methanol and ethanol in water. Part A of this figure shows the traces of
three 2 ~1 injections of 50 ppm methanol "as carbon" in water at 40~
As can be seen, all injections give rise to very fast and sharp eluting peaks
of methanol. Water itself does not lead to any appreciable deflection at
the attenuation applied. The difference in peak heights of these peaks is
mainly due to the time each injection lasts. With peak half widths of a
few seconds as experienced here, manual injection is likely to produce
these aberations. However, integration over the peak areas should produce
consistent and accurate results. Part B shows three consecutive injections
of methanol and ethanol in water at the concentrations given. Under the
conditions used, both compounds give rise to distinct, though not com-
pletely separated, peaks. Part C of Fig. 9 shows the GC traces of three
injections of 12-ppm methanol as carbon at 70~ through a 1.2-m Teflon
column. At the low attenuation of 8 x 1 and 5 /A injection, the FID
A
70 B
60
50
u= 40
i-
z
uJ
O
~ 3o
EL
20
10
x
co
L
i J i i r i i i
0 2 4 0 2 6 0 2 4 6 8 min.
A 1.8 m, 40 ~
20 psi He, 55 m l . m m -~
2 141 50 ppm CH3OH
B 1.8 m, 40 ~
20 psi He, 55 ml.m~n -~
2 141 50 ppm CH3OH, 70 ppm C2HsOH
C 1 2 m 70 ~
40 psi He, 25 ml m~n "~
5 t41 12 ppm CH3OH
FIG. 9---Methanol, e t h a n o l in water.
response to the eluting water is now visible as quite flat mound of low
intensity on which is superimposed the sharp methanol peak. As can be
seen from the time scale on Fig. 9, depending on the instrument settings
and separations required, a performance of a single analysis takes about 1
to 2 min and the procedure is thus well suited for serial analyses where
many samples are to be determined with least waiting and servicing time
in between analyses.
To confirm again the versatility of Teflon columns for GC, Fig. 10
shows two gas chromatograms each of 6/~1 of distilled water, programmed
from 50 to 250~ at an attenuation of 2 x 1. Trace A stems from a double
distilled water which was stored in a well-used Nalgene bottle. In this case
a strong peak is eluted at about 180~ which is almost absent from the
same water redistilled with the addition of alkaline potassium perman-
ganate. Obviously, the sample from the Nalgene bottle contains a volatile
FIG. lO--Distilled water.
material which appears to stem from the container, since the same water
prior to storing does not elute a material with the same retention time.
The material has been tentatively'identified by combined GC-MS as 2, 6
di-(t-butyl) 4 methyl phenol, a commonly used antioxidant for plastic
materials.
Finally, Fig. 11, shows a van Deemter diagram with the number of
theoretical plates of a 1.8-m column versus the carrier gas flow at
different temperatures. These plots are taken with n-hexadecane at 140 to
200~ in 20~ steps. As can be seen, with the lowest flow of approxi-
mately 7 ml He/min, obtainable with reasonable accuracy and stability on
this instrument, the highest number of theoretical plates was observed at
180~ (the solid line in Fig. 11). At all temperatures investigated, the
Teflon columns appear to have a plateau-like portion with only little
variation in the number of theoretical plates from where a strong increase
is observed at extremely low-flow parameters. At high flows the number of
plates drops dramatically to very small values.
240 W A T E R QUALITY PARAMETERS
FIG. l l--N-hexadecane.
Conclusion
In conclusion, the main characteristics of Teflon columns which do not
contain any liquid stationary phase include:
1. Teflon columns separate nonpolar compounds by differences in
molecular weights.
2. Teflon columns are useful for GC of polar substances at relatively
low temperatures.
3. Teflon columns are well suited for organic trace analyses, especially
with direct aqueous injections.
References
[1] Fox, M. E., in this symposium.
[2] Landault, C. and Guiochon, G., Journal of Chromatography, Vol. 9, 1962, pp. 133-146.
[3] Gunther, F. A. and Jaglan, P. S., Journal of Chromatography, Vol 46, 1970, pp.
108-109.
[4] Kirkland, J. J., Analytical Chemistry, Vol. 35, 1963, pp. 2003-2009.
[5] Conder, J. R., Analytical Chemistry, Vol. 43, 1971, pp. 367-370.
[6] Jequier, W. and Robin, J., Chromatographia, Vol. 4, 1971, pp. 59-65.
[7] Metcalfe, L. D., Nature, Vol. 188, 1960, pp. 142-143.
[8] Byars. B. and Jordan, G., Journal of Gas Chromatography, Vol. 2, 1964, p. 304-305.
M. E. F o x I
Applications of Direct Aqueous

Injection Gas Chromatography and
Freeze Concentration for the
Determination of Organic
Compounds in Water and Waste
Waters
REFERENCE: Fox, M. E., "Applications of Direct Aqueous Injection Gas Chroma-

tography and Freeze Concentration for the Determination of Organic Compounds in
Water and Waste Water," Water Quality Parameters, A S T M STP 573, American
Society for Testing and Materials, 1975, pp. 242-2S0.
ABSTRACT. Direct aqueous injection gas chromatography is shown to have practical

applications in the analysis of low molecular weight volatile organic compounds in water
and waste waters. Many such compounds cannot be extracted or concentrated by more
conventional solvent methods due to evaporation losses. Detection limits below 1 rag/
liter are possible. Freeze concentration may be used to lower this limit by a factor of
10 to 20 for more dilute solutions. Applications are described including the analysis
of methanol and aircraft deicer components in domestic sewage.
KEY WORDS: waste quality, environmental tests, gas chromatography, organic com-
pounds, waste water
The application of direct aqueous injection gas c h r o m a t o g r a p h y for the

d e t e r m i n a t i o n of volatile organic c o n t a m i n a t e s in n a t u r a l waters a n d waste
waters has obvious attractions. Foremost a m o n g these are simplicity,
speed of analysis, a n d the avoidance of composition changes of u n k n o w n
m a g n i t u d e which m a y a c c o m p a n y c o n c e n t r a t i o n a n d extraction pretreat-
m e n t s [1]. 2
M a n y workers have discarded this a p p r o a c h after experiencing severe
peak tailing, m e m o r y effects, a n d c o l u m n bleed. W o r k in recent years,
however, has greatly reduced the p r o b l e m of m e m o r y peaks or ghosting by
d e s i g n i n g fully swept inlet zones a n d p u r g i n g or periodic c l e a n i n g of the
'Chemist, Toxic Substances Unit, Canada Centre for Inland Waters, Burlington, Ontario
L7R 4A6, Canada.
242
9
FOX ON ORGANIC COMPOUNDS 243
column inlets [2,3]. The problems of peak tailing have been alleviated by
the addition of phosphoric acid, sililating agents, or other materials to
block reactive sites on the columns [4, 5]. Porous polymer beads have been
widely used to avoid the liquid phase stripping effects of water injections
but have usually imposed severe molecular weight restrictions. The most
common application has been the analysis of low molecular weight (C2-C5)
free fatty acids [4,5]. A recently developed polymer bead material (Tenax
G.C.) can be used without thermal degradation problems at temperatures
up to 400~ for a wide range of higher molecular weight compounds [6].
The remaining intractable drawback to direct aqueous injection gas
chromatography is a minimum detection level that is commonly not lower
than 1 mg/liter. This minimum detection level is eminently suitable for
contaminated wastewater analyses but is inadequate for the typical
concentrations of contaminants after dispersal in a large receiving body of
water.
The use of freeze concentration as a means of preconcentrating the
organic components of a dilute aqueous solution without selectivity or the
introduction of contaminants has been successfully demonstrated [7,8].
Unfortunately, for most natural waters, the effect of increasing inorganic
salt concentration on the mechanism of ice formation causes entrainment
of organics and usually limits the effective concentration factor to 15 to 25
times. A concentration factor of at least 100 times would be desirable for
many trace contaminant determinations. In addition, the equipment
currently available for freeze concentration can not operate efficiently with
small samples. Thus, long processing times are involved.
This paper will describe the successful application of direct aqueous
injection gas chromatography to two specific problems in wastewater
analysis and a preliminary attempt to use freeze concentration and direct
aqueous injection gas chromatography for the analysis of an effluent
plume in a receiving water body.
Residual Methanol in Sewage

In pilot plant studies currently in progress at the Canada Centre for
Inland Waters and elsewhere, methanol is added to sewage as a supple-
mentary carbon source in the biological denitrification of sewage. A rapid
and specific analysis for methanol in sewage down to less than 1 ppm was
required.
A porous polymer column packing material (Tenax G.C.) was chosen to
avoid the liquid phase stripping effects of water. At column temperatures
of over 100~ resolution of the water and methanol peaks was not
possible.
When the column temperature was lowered to 70~ however, the water
response appeared as a very low flat-topped mound with the methanol
appearing as a normal sharp peak on top of the mound. The duration of

the water response under these conditions for a 5 ~1 injection is in the
order of 50 to 60 s. It was found that other low molecular weight organic
compounds (up to C 3) could be differentiated from methanol by retention
times. Higher molecular weight compounds, either added or which may
have been present in sewage samples, were either retained in the
removable injection port liner or eluted through the column so slowly that
significant baseline deflections were not noted. Thus, a practical analysis
with little sample pretreatment (filtration and acidification) was accom-
plished with a speed of 50 or more samples per hour [9].
For this analysis, peak heights were found to give sufficient precision
(standard deviation = 1.2 at 50 ppm). An example of a typical set of
analyses with a 19 ppm standard and distilled water blanks is shown in
Fig. 1.
Aircraft Deicer in Sewage

At Canadian airports, during a five-month winter period, prodigious
quantities of deicer are sprayed on the wings and tails of aircraft. A
typical application is 80 gal per aircraft of which approximately three
quarters drains directly onto the ground. Sewage treatment plants
receiving this airport runoff are subjected to a very high organic carbon
loading with consequent disruption of normal treatment efficiencies.
Studies were initiated at the Canada Centre for Inland Waters and
elsewhere to determine the maximum loading of airport winter runoff
which could be tolerated without disturbing the normal operation of a
sewage treatment plant. The study required an analytical method for the
specific detection of aircraft deicer residues at various stages of the
treatment process. The expected concentrations were <1 to 500 mg
carbon/liter. Two types of deicer were encountered. One consisted of a
mixture of ethylene glycol plus propylene glycol in the ratio of 2:1 as a 50
percent solution in water, with added traces of corrosion inhibitor and
thickening agent. The other deicer was simply a 50 percent solution of
ethylene glycol with the trace additives. The analysis described is for the
first type.
A direct aqueous injection method based on the same Tenax G.C.
column was attempted. Complete resolution of the ethylene glycol and
propylene glycol peaks in a reasonable analytical time was not feasible.
However, with a column temperature of 160~ the two major components
eluted together as a single peak suitable for quantitative measurement.
Thus, the analysis could be reported as milligrams carbon per liter based
on the original ethylene glycol propylene glycol ratio of 2:1. In this
application the use of peak areas was found to be necessary to achieve a
suitable analytical precision. Figure 2 shows a typical set of duplicate
~3
ta
09
w w cr
Ill
E a. j . ~--
fflu_ I-.-
a.j
Z n....._.... I.D.
--"~0
r~ w
UJ 0 ~0
CO
Z -,6 ~
0
13,.
09
UJ
rr
OC
W
0
OC
o
W
CC
~ 1/ L..-./ I/12 12 I F X / 3 / ' L I 3 / ' L _ _ _ _ M ~ d % - ~ L

1 2 3 4 5 6 7 8 9 10 11
RUN NO.
i
!213 14
6 ; 20 mi~
FIG. l--Typical set of duplicate methanol in sewage analyses. Isothermal at 70~
analyses for aircraft deicer in sewage from pilot plant scale treatment
studies. As in the methanol determination described earlier, the analysis
time is quite fast, being in the order of I min per sample. When deicer
concentrations in pilot plant sewage reactors were determined by this
method a good agreement with the actual loading figures was obtained
(Table 1).
However, with occasional more detailed chromatograms programmed
from 112 to 170~ at 15~ a selective loss of ethylene glycol with
increasing residence time in the biological reactors was observed (Fig. 3).
Further investigation revealed that, although both components would
R1F R I E
"-'-" --'-- R4F
R2F R4E
"-"" ~ R5E
9 Ro_,~.r
X X ! C~
X X X
DIST.
WATER I
o
I I I I
0 5 10 15 min.
PEAK gg~176176176 g ges176176o o

AREA ~ t , , , ~ ~ m ~u~',o~'~'~ N~
FIG. 2 - - A typical set o f duplicate aircraft deicer in sewage analyses. Isothermal at 160~
TABLE 1--Glycol analyses from a series o f reactors compared to actual loading values.
Reactor No. 1 2 3 4 5
Glycol m g C/liter added 36 108 180 254 360

Glycol m g C/liter analyzed 35 102 174 276 368
Deviation from true value --2.7% --5.5% --3.3% +8.6% +2.2%
FOX ON O R G A N I C C O M P O U N D S 247
.,,r
s
-.1
X ,,J o
o 0
O J
U.I :h,
I.-- (.9 I--
O
IJJ D
z w I--
ILl
J>.-
r 5a o
cc >
IJJ "1"
cO I-- rr
Z I..iJ z
121 g .
O u.I I.-- 2
13..
00 U_
t21
ILl V-
CC z rr "~
ILl
,m
rr w u
ILl a z
Q
rr
O
o
u.I
rr ~ / ~ DIST. ~L I ,
U
t 1 1
F I G . 3--Aircraft deicer before and after biological degradation. Temperature programmed
from 112 to 170~ at 15~
degrade to zero concentration in the reactors, the lower raolecular weight

component, ethylene glycol, always degraded at a faster rate. This
observed effect indicates increasing error in the analysis with increasing
reaction time since the calculations are based on the original 2:1 ethylene
glycol, propylene glycol ratio. In practice, corrections were not deemed
necessary since significant error only occurred when the mixture had
almost completely degraded.
At this stage of the deicer treatment studies, an interest in glycol
degradation products developed. Fish toxicity tests showed that, even
though quite concentrated fresh deicer solutions were not toxic, some low
concentration, degraded solutions exhibited significant toxicity. Identifi-
cation of the early eluting degradation products observed in the tempera-
ture programmed chromatogram (Fig. 3) was felt to be desirable.
Using the analytical conditions employed for the methanol in sewage
analysis, five peaks corresponding to low molecular weight degradation
products of the deicer were observed (Fig. 4). These peaks were not
observed in sewage samples to which no deicer had been added. Three of
A
RSE H R5E
oo
T-
R3F
v
w
o9
Z
0
n
of)
W
OC
OC
w
0
0 DIST. I
0
111
n"
f t t t
i ! "' I !
0 5 10 15 20 min.
FIG. 4--Aircraft deicer degradation products. Isothermal at 70~
these peaks were tentatively identified as methanol, acetaldehyde, and

ethanol by comparison of retention times and incremental additions. No
other likely candidates with the same retention times were found. The two
remaining peaks were assigned as ethane and propionaldehyde with less
certainty. After tests with the identified degradation products it was
concluded that fish toxicities were due to other, unidentified sources.
Kraft Pulp Mill Effluent Plume

In this study of the fate of organic compounds in a pulp mill effluent
plume dispersing into a large receiving body of water, the main emphasis
has been on the identification of material recovered on a macroreticular
resin. Since complete or substantial losses of low molecular weight volatile
compounds can be expected, an attempt has been made to supplement
this information by direct aqueous injection gas chromatography. Unlike
the previously-mentioned examples, these samples are substantially diluted
with clean lake water. To offset this dilution, freeze concentration was
employed. High levels of dissolved inorganic salts prevented the achieve-
ment of a concentration factor of more than 15 times. Figure S shows
chromatograms of three of these samples run at 70~ in which methanol
(A) (c)
cc)
EC. x 10 EC, x 10
X
(B)
W ECx8
111
09
Z
o
O.
CO
uJ
cc
O:
uJ
0 DIST.
o DIST. WATER DIST.
WATER WATER
uJ
O:
__/7.
FIG. 5--Kraft mill effluent plume samples after freeze concentration. Isothermal at 70~
appears to be the only volatile component. In Fig. 6 a temperature

programmed chromatogram reveals several other components present at
very low concentrations. Identification of some of these components is
expected by means of gas chromatography-mass spectrometry.
I.U
03
Z
o
13..
03
UJ
n.-
cr
LU
O
n-
O
uJ
cr
I l
70 ~ C 230 ~ C (E H.)
FIG. 6---Kraft mill effluent plume sample after freeze concentration. Temperature pro-
grammed 70 to 230~ at ll~
Acknowledgment
The author wishes to thank R. N. Dawson, P. M. Sutton, B. Jank, and
P. Guo, all of the Environmental Protection Service, Environment Canada
for their interest in this work and their cooperation in providing treated
samples of sewage from pilot plant treatment studies.
References
[1] Baker. R. A., Air and Water Pollution. Vol. 10, 1966, p. 591.
[2] Dressman, R. C., Journal of Chromatographic Science, Vol. 8, 1970, p. 265.
[3] Geddes, D. A. M. and Gilmour, M. N., Journal of Chromatographic Science, Vol. 8,
1970, p. 394.
[4] Mahadeven, V. and Stenroos, L., Analytical Chemistry, Vol. 39, 1967, p. 1652.
[5] Henkel, H. G., Journal of Chromatography, Vol. 58, 1971, p. 201.
[6] van Wijk, R., Journal of Chromatographic Science, Vol. 8, 1970, p. 418.
[7] Baker. R. A. in Microorganic Matter in Water. ASTM STP 448, American Society for
Testing and Materials, 1969, p. 65.
[8] Kammerer, P. A., Jr. and Lee, G. F., Environmental Science and Technology. Vol. 3,
1969, p. 276.
[9] Fox, M. E.. Environmental Science and Technology, Vol. 7, 1973, p. 838.
Donald M a c k a y ) W. Y. Shiu, 1 and A. W. W o l k o f f 2
Gas Chromatographic
Determination of Low
Concentrations of Hydrocarbons in
Water by Vapor Phase Extraction
REFERENCE: Mackay, Donald, Shiu, W. Y., and Wolkoff, A. W., "Gas Chromato-
graphic Determination of Low Concentrations of Hydrocarbons in Water by Vapor
Phase Extraction," Water Quality Parametem, A S T M STP 573, American Society for
ABSTRACT." A novel system for the measurement of low concentrations of hydro-

carbons in water is described. The apparatus introduces a vapor sample of the hydro-
carbon to a gas chromatograph following transfer of part of the hydrocarbon from the
aqueous solution to a vapor phase. The thermodynamic basis for predicting the vapor-
liquid equilibration is given. The method is capable of analyzing hydrocarbon concentra-
tions in parts per billion range; however, the sensitivity depends on the hydrocarbon
solubility and vapor pressure.
KEY WORDS: water quality, gas chromatography, hydrocarbons, vapor phases, envi-
ronmental tests, thermodynamic properties
T h e m e a s u r e m e n t o f low c o n c e n t r a t i o n s o f dissolved a n d emulsified

h y d r o c a r b o n s in water is o f i m p o r t a n c e in pollution research, p e t r o l e u m
e x p l o r a t i o n , a n d b i o c h e m i c a l studies. C o n c e n t r a t i o n s o f interest r a n g e
from a b o u t 30 p a r t s in 10 a2' the c o n c e n t r a t i o n of m e t h a n e in t h e oceans
[ 1 ] ) to several p a r t s in 106, the solubility by dissolution a n d " a c c o m m o -
d a t i o n " of a l k a n e s in water [2], A n a l y s i s by gas c h r o m a t o g r a p h y is an
obvious choice; however, the p r i n c i p a l p r o b l e m is t h a t of i n t r o d u c i n g
sufficient h y d r o c a r b o n onto the c o l u m n to give a d e q u a t e d e t e c t o r
response. A flame ionization d e t e c t o r n o r m a l l y requires a b o u t 10 -9 g
h y d r o c a r b o n to give a r e a s o n a b l e p e a k for q u a n t i t a t i v e p u r p o s e s . A t a n
a q u e o u s c o n c e n t r a t i o n o f one p a r t p e r 109 , it is t h e n necessary to
i n t r o d u c e t h e a m o u n t o f h y d r o c a r b o n n o r m a l l y c o n t a i n e d in 1.0 ml o f
water. V a r i o u s m e t h o d s have been used to a c c o m p l i s h this.
' Associate professor and postdoctoral fellow, respectively, Department of Chemical En-
gineering and Applied Chemistry, Institute of Environmental Sciences and Engineering,
University of Toronto, Toronto, Ontario MSS 1A4, Canada.
Research scientist, Canada Centre for Inland Waters, Burlington, Ontario, Canada.
The italic numbers in brackets refer to the list of references appended to this paper.
251
9
McAuliffe [3] injected the water sample directly and adsorbed the water
on an Ascarite pre-column. Extraction of the hydrocarbon by an
immiscible solvent such as hexane has been used [4]. A recent ingenious
method has been developed by McAuliffe [5] in which the hydrocarbon is
partially partitioned into the vapor phase by equilibration of the aqueous
sample with helium in a 50-ml gas syringe, the vapor then being
transferred to a gas sampling valve and then to the column. By injecting
gas samples from repeated equilibrations it is possible to calculate the
amount of hydrocarbon in the original sample. This method has signifi-
cant advantages in that qualitative separation of hydrocarbons from water
soluble organic and inorganic compounds is obtainable, and the method is
capable of detecting very low concentrations (that is, one part on 1012) of
volatile hydrocarbons in water.
This paper describes a method for determination of dissolved hydro-
carbons in water using a novel apparatus based on McAuliffe's method.
Also given is a method of predicting the fraction of the hydrocarbon
partitioning into the vapor phase.
Theoretical
If a quantity of water containing a single dissolved hydrocarbon is equili-
brated with vapor in a temperature (T~ and a total pressure (Pr)
atmospheres, the moles of hydrocarbon in the vapor phase (M~) and
liquid phase (ML) will be given by Eqs 1 and 2. The sum of My and ML
is the total amount of hydrocarbon (Mo) originally present in the liquid.
The fraction (F) of the original hydrocarbon now present in the vapor
phase is given by Eq 3.
My = V o y 273 PT/22400 ( T + 273) (1)
ML = VL X/18 (2)
F = M v / ( M v + ML) = M v / M o (3)
where V6 and VL are the volumes (ml) of the vapor and liquid phases,
respectively, and x and y are the mole fractions of the hydrocarbon in the
liquid and vapor phases, respectively.
The equilibrium distribution of the hydrocarbon between the vapor and
liquid can be determined by equating the fugacity (f) in the two phases as
shown in Eq 4 in which 7 is the liquid phase activity coefficient of the
hydrocarbon in water and Ps is the hydrocarbon saturation vapor
pressure. It can be assumed that the vapor phase fugacity coefficient, ~, is
unity. Substitution of Eqs 1, 2, and 4 into Eq 3 gives Eq 5. It may be
MACKAY ET AL ON LOW CONCENTRATIONS OF HYDROCARBONS 253
seen that F is essentially independent of the concentration and total

system pressure, but does depend on temperature.
f = xyPs = yePPT (4)
F = V6 273/22400 (T + 273) (5)

(VG 273/22400 (T + 273)) + VI./18 Ps Y
If all the gas phase is injected on to the gas chromatography column,

the peak area obtained will be proportional to MoF. The amount of
hydrocarbon remaining in the liquid will then be Mo(1-F). In the second
equilibration the same equations apply and the amount of hydrocarbon
sampled will be MoF(1-F) and the amount remaining Mo(1-F) 2. In the
nth equilibration, the amount of hydrocarbon in the vapor will be
MoF(1-F),-'. That is, each peak is (l-F) times smaller than the pre-
ceeding peak. This relationship was first observed by McAuliffe [5].
When only part of the vapor is sampled, an analogous set of equations
can be derived in which F is replaced by a modified distribution ratio F'
defined as FVs/V6 where Vs is the vapor sample volume and Vo is the
total volume of the vapor phase. In the first equilibration the amount in
the sample is FMoVs/V6 and the total amount remaining in the liquid
and unsampled gas is Mo(l - FVs/V6). In the second equilibration the
amount in the sample M o ( F V s / V 6 ) ( 1 - FVs/V6) and in the nth
equilibration the amount in the sample is Mo(FVs/V6)(1 - FVs/V6),-'
or MoF'(1 - F')n-l.
In both cases of partial or total sampling a plot of the logarithm of the
areas of successive equilibrations versus equilibration number gives a
straight line of slope (l-F) or (1-F') and intercept (at n = 1) MoF or
MoF'. Thus after suitable corrections for instrument sensitivity Mo, the
original number of moles in the liquid may be determined from the value
of the first equilibration and the slope. Essentially, this is summing the
peak areas of an infinite number of equilibrations.
It is possible to determine F and hence Mo from only two equilibra-
tions; however, in practice, the accuracy can be improved by performing
about five equilibrations and determining the best straight line by a least
squares fit.
From Eq 5 the value of F can be predicted for various compounds. The
activity coefficient, y, may be estimated from liquid hydrocarbon solubility
data of McAuliffe [3] or Leinonen et al [6]. The activity coefficient is
essentially the reciprocal of the mole fraction of hydrocarbon in a
saturated aqueous solution. For solid hydrocarbons such as naphthalene,
the procedure is more complex as has been described by Prausnitz [7] and
Tsonopoulos and Prausnitz [8]. Some predicted values of F are shown in
Table 1. It is apparent that high values of F (approaching unity), which
TABLE 1--Predicted values of F and F"for the system.

VG = 54.0. VL = 65.0, Vs = 18.0 ml, and T = 25~
Vapor Pres-
sure (atm) Solubility,
Compound x 10+2 mg/liter y = 1/x F F"
n-octane 1.78 0.66 9.6 x 106 0.99 0.33

2,2,4-trimethylpentane 6.4 2.44 2.6 x 106 0.99 0.33
benzene 12.5 1780 2.4 x 103 0.163 0.0545
toluene 3.74 515 9.9 x 103 0.185 0.0616
o-xylene 0.86 204 2.9 x 104 0.133 0.0442
cumene 0.59 50 1.3 x 105 0.372 0.124
naphthalene 0.011 33 6.6 x 104 0.0044 0.00147
biphenyl 0.007 7.2 1.2 x 106 0.034 0.0113
cyclohexane 13.16 55 8.5 x 104 0.869 0.29
hexane 20.53 9.5 5.0 x l0 s 0.982 0.327
decane 0.23 0.052 1.5 x 108 0.992 0.331
are d e s i r a b l e from an a n a l y t i c a l viewpoint, are o b t a i n e d for h y d r o c a r b o n s

o f high v a p o r p r e s s u r e a n d low solubility (high activity coefficient). F o r
the m e t h o d to be feasible, F ' has to be at least a b o u t 0.05 in o r d e r t h a t a
r e a s o n a b l e slope is o b t a i n e d .
I t is noteworthy t h a t the solubility o f a h y d r o c a r b o n can be d e t e r m i n e d
directly from t h e slope (1-F') since all the t e r m s in Eq 5 a r e k n o w n except
the activity coefficient (and hence the solubility). This leads to the
possibility of d e t e r m i n i n g solubilities without c a l i b r a t i n g the gas c h r o m a -
t o g r a p h for response to the h y d r o c a r b o n . U n f o r t u n a t e l y , a s m a l l e r r o r in
the slope tends to b e reflected in a large e r r o r in the solubility p a r t i c u l a r l y
when the slope is very high or low, t h a t is, F t e n d s to zero or unity.
Experimental
T h e a p p a r a t u s is shown s c h e m a t i c a l l y in Fig. 1. A n a p p a r a t u s s i m i l a r in
p r i n c i p a l b u t very different in design has been d e s c r i b e d by W a s i k a n d
Brown [10]. All p a r t s were s t a n d a r d 316 stainless steel fittings. T h e b a l l
valve h a d an orifice o f 0.406 in.; in earlier e x p e r i m e n t s p r o p e r m i x i n g was
not o b t a i n e d with a ball valve o f s m a l l e r orifice. A short length o f 1/16-in.
stainless steel t u b i n g e x t e n d e d to the ball valve. This allowed the u p p e r
c h a m b e r to be fully swept by c a r r i e r gas. T h e u p p e r a n d lower c h a m b e r
volumes were 18 cm 3 a n d 100 cm 3, respectively. T h e a p p a r a t u s was
c o n n e c t e d by m e a n s o f " S w a g e l o k " Q u i c k Connects to a I-Iewlett P a c k a r d
M o d e l 57S0 G a s C h r o m a t o g r a p h e q u i p p e d with a flame ionization detec-
tor, a n d a h e a t e d (130~ gas s a m p l i n g valve (Model No. 19021A) which
was m o d i f i e d by r e p l a c i n g one of the s a m p l e loops by two q u i c k
c o n n e c t o r s which couple to the a p p a r a t u s . It is i m p o r t a n t to ensure t h a t
the design of the gas flow system is such t h a t there are no d e a d spaces in
which some o f the s a m p l e can be t r a p p e d .
MACKAY ET AL ON LOW C O N C E N T R A T I O N S OF H Y D R O C A R B O N S 255
IN
QUICK CONNECT"
FI'[TINGS TO GAS
CHROMATOGRAPH
GLASS UPPER CHAMBER

BETWEEN STAINLESS
STEEL FLANGES
BALL VALVE
- - S T A I N L E S S STEEL
LOWER C H A M B E R
I inch
----- VALVE
FIG, 1--Diagram of equilibration apparatus.
Approximately 65 cm 3 of the water sample to be analyzed was drawn

into the sample cylinder and the bottom valve closed. With the top valves
closed and the middle ball valve open, the apparatus was shaken
vigorously for 15 min by a wrist-action shaker, alternately inverting the
apparatus at 45 ~ to the horizontal to ensure proper mixing in all parts of
the apparatus. The system was allowed to stand with very mild shaking to
permit the water to drain back into the lower chamber then the ball value
was closed. The glass section enables any water trapped in the upper
chamber to be seen and shaken out. It is essential to avoid introducing
water to the gas liquid chromatograph. The apparatus was connected to
the gas sampling valve, which was then opened followed quickly by the
top valves so that the contents of the upper chamber were swept onto the
column.
After sufficient time, these valves were closed, the gas sampling valve
closed, and the ball valve opened. The procedure was then repeated.
Because the volume of the upper chamber was relatively large (18 cm3),
severe peak broadening was observed for those peaks with long retention
times. One method of reducing this effect is to trap the hydrocarbon on a

short (20 cm) column cooled by liquid N2; then introduce the sample by
replacing the coolant by boiling water.
Peak areas were determined using a Hewlett-Packard Model 3371B
Electronic Integrator. Areas were corrected for detector response by
making several injections of the pure component and averaging the
results.

Some typical solubility determinations of saturated solutions of hydro-
carbons in water are shown in Fig. 2. Since it is difficult to prepare and
maintain unsaturated solutions of accurately known concentration, satu-
rated solutions were used. Table 2 gives the results and a comparison with
10 5
BENZENE
TOLUENE
i0 ~ ~
{/3
Z
0 -..~XANE
c.)
<
n,-
<
Y
<
L.U
0..
lo
"~-~..~ RA DECAN E
EQUILIBRATION NUMBER
FIG. 2--Typical plots of peak area versus equilibration number.
MACKAY ET AL ON LOW CONCENTRATIONS OF HYDROCARBONS 257
TABLE 2--Solubility determination (rag/liter).

Experimental Solubility Literature
Solubility
Benzene 1769 1780 [3]
Cyclohexane 55.8 50.2 61.7 55[3]
n-Hexane 16.2 16.2 9.5 [3] 18.3 [I1]
Toluene 517 515 [3]
n-Decane 0.182 1.22 0.0052 [2]
n-Tetradecane 0.0259 0.0069 [12]
literature values for solubilities obtained from measurement of the slope

only. Calibration of the flame ionization detector gives a similar set of
results.
Solubilities below 20 mg/liter are suspect due to the possibility of
emulsified or colloidal hydrocarbon being present. This explains the
discrepancy in the results for n-hexane, n-decane, and n-tetradecane.
The method is believed to be capable of determining aliphatic hydro-
carbon concentrations in the parts per billion range. Determinations of
the solubility of a crude oil in water have enabled concentrations of
individual hydrocarbons in this range to be determined easily. It is
apparent from such determinations that when crude oil is contacted with
water, the principal hydrocarbons dissolved are the aromatics and the
lower alkanes. There is very little evidence of dissolution of the higher
alkanes. In determinations of this type, a plot of the logarithm of total
peak area versus equilibration number is not straight since the peak areas
are the sum of many individual component areas each with a charac-
teristic and generally different slope. Solubilities are best calculated by
summing the peak areas of as large a number of equilibrations as is
deemed necessary.
Hydrocarbons such as naphthalene are not well suited to analysis by
this technique since F is very low (little of napthalene transferring to the
vapor). F can be increased by heating the apparatus. It can be shown, for
example, that for benzene F increases from 0.09 at 25~ to 0.27 at 60~
This is due to the increase in hydrocarbon vapor pressure Ps which is
greater than the increase in solubility (that is, decrease in activity
coefficient).
The method has the advantage that sensitivity can be increased using
larger volumes of water, and improved accuracy obtained by increasing
the number of equilibrations. The apparatus is simple, easily operated,
and overcomes many of the difficulties introduced by extracting the
hydrocarbon into an organic phase. Sample handling is reduced and there
is no possibility of contamination from impure liquid extractant.
It is applicable to systems in which the hydrocarbon is present as a
mixture or in emulsified or colloidal form provided that the hydrocarbon
can be evaporated. Reliable analyses are possible for alkanes down to 10

ppb and for toluene to 75 ppb. The effect of increasing the system
temperature and application of the method to chlorinated hydrocarbons
are currently under investigation.
It is suggested here that the techniques of vapor and liquid extraction
are complementary. Vapor phase extraction will be preferred when the
dissolved hydrocarbons are reasonably volatile and insoluble, that is, the
group Psy is large. There is the risk of loss of hydrocarbon by evaporation
during liquid extraction of such systems. Mackay and Wolkoff [9] have
shown that the rate of evaporative loss in such systems is often greater
than is generally appreciated.
Liquid phase extraction will be preferred when the hydrocarbons are
involatile and may be present as particulate high boiling residues. There is
always a possibility in environmental samples that such material may be
present and would not be detected by vapor phase extraction.
In general, hydrocarbons below Cas are amendable to vapor extraction,
except if they have fused aromatic structures. The equations presented
here enable the feasibility of vapor extraction to be determined in
advance. It is hoped that this technique will be more widely used in the
future in the analysis of appropriate environmental samples.
Acknowledgments
The authors gratefully acknowledge the financial support of the
National Research Council of Canada, Environment Canada, and the
Institute of Environmental Sciences and Engineering of the University of
Toronto.
References
[1] Swinnerton, J. W., Linnenbom, V. J., and Cheek, C. H., Environmental Science and
Technology, Vol. 3, 1969, p. 836.
[2] McAuliffe0 C., Science, Vol. 158, 1969, p. 478.
[3] McAuliffe, C., Journal of Physical Chemistry, Vol. 70, 1966, p. 1267.
[4] Peake, E. and Hodgson, G. W., Journal of the American Oil Chemists Society, Vol.
43, 1966, p. 215.
[5] McAuliffe, C., Chemical Technology, 1971, p. 46.
[61 Leinonen. P. J., Mackay, D., and Phillips, C, R.. Canadian Journal of Chemical
Engineering. Vol. 49. 1971, p. 288.
[7] Prausnitz, J. M., Molecular Thermodynamics of Fluid-Phase Equilibria. Prentice-Hall
Inc., Englewood Cliffs, N.J., 1969, p. 385.
[8] Tsonopoulos, C. and Prausnitz, J. M., Industrial and Engineering Chemistry Funda-
mentals, Vol. 10, 1971, p. 593.
[9] Mackay, D. and Wolkoff, A. W., Environmental Science and Technology, Vol. 7,
1973, p. 611,
[10] Wasik, S. P. and Brown, R. L., Proceedings, Joint Conference on the Prevention and
Control of Oil Spills. American Petroleum Institute, March 1973, p. 223.
[11] Nelson. H. D. and deLigny, C. L., Recueil, Vol. 87, 1968, p. 528.
[12] Franks, F., Nature, Vol. 210, 1966, p. 87.
M. T. S t r o s h e r j a n d G. IV. H o d g s o n t
Polycyclic Aromatic Hydrocarbons

in Lake Waters and Associated
Sediments: Analytical
Determination by Gas
Chromatography-- Mass
Spectrometry
REFERENCE: Strosher, M. T. and Hodgson, G. W., "Polyeyelle Aromatic Hydrocar-

bons in Lake Waters and Associated Sediments: Analytical Determination by Gas
Chromatography.Mass Spectrometry," Water Quality Parameters, A S T M STP 573,
ABSTRACT." Marine and fresh water sediments exhibit a wide variety of polycyclie
aromatic hydrocarbons. These range from the two-ringed biphenyl and naphthalene
species to the seven-ringed coronene member, and include a variety of intermediate
aromatics exhibiting isomeric differences as well as alkylated derivatives. Analytical
methods were developed for this spectrum of aromatics involving extraction and purifi-
cation techniques followed by a gas chromatographic separation combined with mass
spectrometry for identification. In this manner, a total of 27 polycyelic aromatics were
detected in Great Lakes sediments along with three aromatics and three dicarboxylic
acid esters in waters of the Great Lakes. Limits of detection for aromatics were:
1 ng/liter in water, based on 30-liter water samples and 2 ng/g of dry sediment when
extracted from 100 g of wet sediment.
KEY WORDS: water quality, sediments, aromatic hydrocarbons, polycyclic compounds,

environmental tests, mass spectroscopy, gas chromatography
Polycyclic a r o m a t i c h y d r o c a r b o n s exist in m o s t g e o l o g i c a l e n v i r o n m e n t s
a n d h a v e b e e n d e t e c t e d by a w i d e r a n g e o f a n a l y t i c a l m e t h o d s . O c c u r -
r e n c e s a r e c o m m o n in a n c i e n t s e d i m e n t a r y r o c k s [1]. 2 B l u m e r [2] s h o w e d
t h a t c o m m o n c o n s t i t u e n t s o f soils w e r e t h e t w o b e n z y p y r e n e i s o m e r s , a n d
in a d d i t i o n , twelve o t h e r a r o m a t i c c o m p o u n d s w e r e d e t e c t e d , i n c l u d i n g
p y r e n e , p e r y l e n e , b e n z p e r y l e n e , a n d c o r o n e n e , all o f w h i c h c o m m o n l y
o c c u r in r e c e n t s e d i m e n t s . K e r n [3] a n d M e i n s c h e i n [4] r e p o r t e d polycyclic
a r o m a t i c s in m a r i n e a n d n o n m a r i n e s e d i m e n t s . O r r a n d G r a d y [5]
'Professional associate and director, respectively, Environmental Sciences Centre (Kan-
anaskis), University of Calgary, Calgary, Alberta T2N 1N4, Canada.
259
9
identified perylene in modern basin sediments off California while Peake

et al [6] also found perylene as the dominant aromatic in Black Sea
sediments. Perylene occurred in an alkylated series in Beaufort Sea
sediments [7] and these findings were of considerable interest in con-
nection with the alkylated homologeous series of perylene found in
petroleum [8]. Polycyclic aromatics were long known as air pollutants and
recent work has demonstrated significant advances in analytical tech-
nology [9-11].
Techniques for the separation of these aromatic structures varied from
liquid-solid chromatography [1] to the more sophisticated thin layer
chromatographic methods employed by Sawicki et al [12,13) and Peake et
al [6,7]. Separation/identification techniques included liquid chroma-
tography-ultraviolet (UV) spectrophotometry [14], as well as thin layer
chromatography-UV spectrophotometry [15], thin layer chromatography-
spectrofluorometry methods as reported by Sawicki et al [13], and thin
layer-gas chromatography [9l. Identification of aromatics was achieved by
spectrofluorometry [16], nuclear magnetic resonance [17], mass spectro-
metry [10,18,19], and gas chromatography [11,20,22]. Pering et al [23]
used a method based on gas chromatography and mass spectrometry for
determining polycyclic aromatics in meterorite samples, but the range of
detection was limited to four-ringed pyrene molecules.
The object of the present study was to develop a basically simple
method of sample preparation for polycyclics from waters and sediments
to be followed by combined gas chromatography/mass spectrometry
(GC/MS) designed to accommodate the entire range of polycyclic
aromatic hydrocarbons in this environment. Previous studies on recent
sediments showed that such polycyclic structures ranged in size from
double-ring compounds to seven-ringed members, many of which exist in
various isomeric and alkylated forms. The following methods were
developed to accommodate investigations of such polycyclic aromatic
hydrocarbons in fresh waters and fresh-water sediments in Canada.
Experimental
The laboratory procedure developed in the present study consisted of
three steps: an extraction process, a column chromatography clean-up
technique, and finally analysis by GC/MS. Various extraction methods
were used in the past to remove organic material from sediments and
water. The procedures outlined here were specifically employed to reduce
possible contamination as well as to provide rapid efficient methods of
extraction. A polytron homogenizer/disintegrator (Willems, Model PT
10-35) effected complete extraction of large quantities of sediment in a
matter of minutes, with no contamination to samples. Extraction of waters
in separatory funnels proved to be the quickest and most efficient method
STROSHER AND HODGSON ON POLYCYCLIC AROMATICS 261
for removal of the aromatics immediately after sampling to minimize

bacterial degradation as well as contamination. A column chromatography
technique was developed as a rapid and efficient method of providing a
relatively clean fraction of the polycyclic aromatic compounds for analysis
by G C / M S . These methods are outlined in detail as follows.
Reagents used in the analytical procedures were examined for purity by
fluorescence and G C / M S . Nanograde benzene and hexane from Mal-
linckrodt Chemical Works exhibited no contamination in quantities
required by these procedures. Acetone and methanol, 99 mole percent
pure from Fisher Scientific, were selected for their high purity as well.
Reagent grade hydrochloric acid was found suitable for use in the
extraction procedures of waters. All glassware was thoroughly cleaned in
chromic acid and the use of plastics was minimized to reduce possible
contamination by plasticizers.
Water samples were extracted in four-liter separatory funnels imme-
diately upon sampling. To obtain reasonable sensitivity, 30 liters of water
per sample were extracted. The waters were acidified to pH 6.5 with 6 M
hydrochloric acid to ensure complete extraction of organic matter. Each
portion of wat6r was extracted with benzene (1/100, volume/volume
(V/V)); the combined extracts were then reduced in volume in a rotary
vacuum evaporator to minimize losses of the lower boiling aromatic
material.
Cores of sediments were obtained in plastic coring tubes and refrig-
erated immediately to near freezing until extraction procedures com-
menced. Upon sectioning, samples of wet sediments ranging from 25 to
100 g were extracted with the polytron homogenizer/disintegrator, using
first acetone then benzene-methanol (3/1, v/v) in sufficient quantities to
ensure the complete extraction of organic matter. This required an
average of 200 to 300 ml for each of the acetone and benzene-methanol
extractions. The efficiency of the homogenizer was such that complete
extraction of a 100-g sediment sample was achieved in 3 to 4 min.
Separation of solvent from extracted sediment was achieved by filtration
using pre-extracted filter paper; filter papers from all suppliers showed
traces of plasticizers; hence the necessity for preextraction with benzene-
methanol. Combined extracts of sediments were reduced in volume in the
rotary evaporator.
The complete extracts of waters and sediments contained a wide variety
of organic material including aliphatic hydrocarbons, porphyrins, chlorins,
and carotenoids which interfere with the analysis of aromatic hydro-
carbons. A simple clean-up technique was adopted to remove the majority
of these interferring compounds using neutral activity I alumina as an
adsorbent. It was introduced in slurry form with hexane into 1-cm glass
columns to a depth of about 10 cm. Concentrated extracts were dissolved
in hexane then introduced to the wet adsorbent and eluted with this
solvent to remove aliphatic hydrocarbons. A second eluant was benzene

which removed the aromatic material with sufficient purity for the
G C / M S analysis. A further elution with methanol removed the porphyrin,
chlorin, and carotenoid pigments.
Instrumentation
A Finnigan Model 1015 gas chromatograph mass spectrometer was used
in the analysis of the polycyclic aromatic hydrocarbons. It comprised a
Model 1700 Varian gas chromatograph coupled by a Gholke separator to
a quadrupole mass spectrometer. The GC column was 6 ft by 1/8 in.
stainless steel packed with 3 percent DEXSIL 300 on Chromosorb W,
A / W , 80-100 mesh. Conditions were as follows: Helium carrier gas flow
= 25 c3/min, injection port temperature = 250~ separator temperature
= 250~ and column oven temperature was programmed at 4~
from 150 to 325~ Mass spectometer conditions were: electron energy =
70 eV and total current = 450 gA. A suitable amount of each sample in
benzene was injected on the gas chromatographic column at the initial
150~ temperature, then programmed at 4~ to the maximum
325~ temperature and held there for approximately 20 min. All of the
aromatics of interest were eluted in this manner.
Results
A mixture of 20 polycyclic aromatic hydrocarbon standards simulating
an aromatic fraction of a fresh water sediment extract was used to
establish the appropriate separation/identification techniques for use in
the G C / M S system. These aromatics ranged from biphenyl (154 m/e) to
coronene (300 m/e) and included some varieties in alkylated and isomeric
forms as found in similar environments by previous investigators [2-6].
The variety of aromatics found in sediments required different gas
chromatographic separation conditions in the GC/MS system than those
employed by conventional gas chromatography because of the lower
carrier gas flow rates required in the coupled G C / M S system. The
combined system required compensation in column temperature to effect
proper resolution of compounds, as indicated by results obtained on
column packings with liquid phases such as SE-30, 5E-52, OV-1, and
OV-17, where good separations of the lower boiling aromatics were
obtained but higher molecular weight aromatics remained unresolved
because of the temperature limitation of those column packings.
A chromatographic column with a 3 percent DEXSIL 300 liquid phase
was adopted because of its stability at the required higher temperatures.
Figure 1 demonstrates the capability of this column to separate the
mixture of standard aromatics. This chart displays the total ion current
output of the mass spectrometer when used as a detector for the gas
2.
4,2-BENZANTHRACENE
CHRYSENE
TRIPHENYLENE
BENZOFLUORENES
4
>-
BIPHENYL
I--
z
IM
I.-
z PHENANTHRENE
b.J
>
m
..1
I PYRENE BENZPYRENES
3.
2 , 3 - BENZO-
FLUORANTHENE
PERYLENE
2 0 - METHYL
CHOLANTHRENE
2-METHYL
ANTHRACENE
9-METHYL
NTHR
I I 1 I I I I
450 ~ '170~ 490 ~ 210 ~ 230 ~ 250 ~ 270* 290 = 340* 325* HOLD
ELUTION TEMPERATURE
FIG. 1 - - G C / M S total ion current display o f a mixture o f standard polycyclic aromatic

hydrocarbons.
chromatographic effluent. Good resolution with minimal column bleed

was attained throughout the entire temperature range. The 20 polycylics
injected resulted in 15 peaks, 12 of which represented single compounds.
The remaining three peaks contained two or more compounds each.
These multicompound peaks were resolved by repetitive oscillograph
scanning over their respective GC elution periods. For example, Peak 1 of
Fig. 1 included three benzofluorene isomers. In order to demonstrate the
presence and order of these three isomers, measurements were made of
the relative abundances of fragment and doubly-charged ions of each
isomer. Rapid oscillograph scanning of the mass range from 70 to 320
m / e for the duration of the GC peak revealed the necessary differences in
the ionic abundance to determine the order of isomer elution within the
peak. Table 1 lists these ions for the three isomers along with their
relative abundances. The preceding shoulder of the GC peak was
identified with the 1,2-benzofluorene isomer with the remainder of the
large peak exhibiting the 3,4-benzofluorene compound first, followed by
the 2,3-benzofluorene isomer. Similarly, the multicomponent Peak 2
which appeared at 250~ was resolved by the rapid scanning technique to
show three aromatics in sequence: 1,2-benzanthracene, chrysene, and
TABLE 1--Mass spectra of benzofluorene isomers.
Relative Abundance
m/e 1,2 2,3 3,4 Isomers
217 19 17 18
216 100 100 100
215 84 101 77
214 6 7 8
213 21 24 22
189 8 8 11
187 3 6 6
108 11 11 17
107.5 15 18 23
107 7 8 14
106.5 11 11 19
94.5 14 17 25
93.5 7 9 11
triphenylene. The abundances of their respective fragmentation ions

confirmed the identification as shown in Table 2, for the ion abundances
of the three compounds.
Particular attention was given to the third GC peak (280~ that
contained more than one compound. It contained the two benzpyrene
isomers, one of which is the known carcinogenic isomer benz(a)pyrene, the
TABLE 2--Mass spectra of three polycyclic aromatics--228 m / e .
Relative Abundance
m/e 1,2 Benzathracene Chrysene Triphenylene
229 20 22 20
228 100 100 1130
227 16 17 12
226 36 39 32
202 7 8 7
200 6 9 6
114 14 11 22
113 20 19 25
112 8 8 10
101 12 14 14
100 10 11 12
other being ben(e)pyrene which has recently been suspected as being

carcinogenic as well. Table 3 shows that the major differences in the two
isomers occur in the abundance of the doubly charged ions, 126 and 125
m / e along with the differences in their respective singularly charged ions
at 252 and 250 m/e. The mass spectrometric ionization of the two isomers
is sufficiently different to permit the identification of one or both isomers
qualitatively as well as quantitatively [10]. While Monteiro and Reed [24]
demonstrated a computer method of analysis of mixtures to simplify
TABLE 3---Mass spectra of benzpyrene isomers.
Relative Abundance
role Benz(a)pyrene Benz(e)pyrenc
253 23 22
252 100 100
251 10 9
250 26 36
126 29 22
125 23 27
124 10 13
quantitative measurements of all multicomponent occurrences, the current

study was restricted to demonstrating the ability to distinguish qualita-
tively the different polycyclics in a mixture.
Water from the Great Lakes contained few polycylic aromatics and they
appeared only to be lower molecular weight species. Figure 2 demonstrates
a typical analysis of a 30-liter lake water sample with five identified
components. Methylnaphthalenes, biphenyl, and methylanthracenes were
detected in quantities ranging from 3 to 600 ng/liter. There were no
detectable amounts of other polycylic aromatics in the waters; abundances
BIS (2-ETHYL HEXYL)

PHTHALATE
BIS (2- ETHYL HEXYL

AOIPATE
DI-n-BUTYL PHTHALAT
>-
I-
z
ILl
Z
LU 2-METHYL ANTHRACENE
>
I--
-.I
rw
f I f I I I
450" d80 ~ 2'10 ~ 240 = 270 = 300* 355*
ELUTION TEMPERATURE
FIG. 2 - - G C / M S total ion current display of water extract from Lake Ontario.
were evidently less than 1 ng/liter. The major constituents of aromatic

fractions of the lake waters were identified as dicarboxylic acid esters
(plasticizers) [25]. Detected in the water were: di-n-butyl phthalate,
bis-(2-ethylhexyl) adipate, and bis-(2-ethylhexyl) phthalate at levels
ranging from 0.01-7.0 pg/liter.
Analytical blanks of the water and sediment analysis produced no
detectable compounds, and it was therefore concluded that the methods
contributed less than 1 rig/liter of polycylics or plasticizers. A possible
source of contamination was noted in the water sampling system where
100 m of plastic tubing was used to obtain each lake water sample,
however, the variable occurrences of plasticizers detected in the lake
waters indicate their existence in the waters rather than basic contami-
nation by the sampling process. Sediment samples were obtained by
Benthos coring methods which employed plastic coring tubes. These
coring tubes were analyzed by the methods just outlined and showed
plasticizers identical to those found in sediment samples; hence plasti-
cizers detected in sediment samples could not be interpreted as indigenous
to the lake samples.
Lake sediments cored to a depth of 110 cm showed a total of 27
identified aromatics in the range from biphenyl to coronene. Table 4 lists
TABLE 4---Occurrences of polycycHc aromatics in sediments.
Elution
Aromatic m/e Temperature, ~
Biphenyl 154 161
Anthracene 178 186
Phenanthrene 178 188
2-Methyl Anthracene 192 200
q-Methyl Anthracene 192 207
Tetrahydro Pyrene 206 212
Fluoranthene 202 215
q, 10-Dimethyl Anthracene 206 217
Pyrene 202 220
Benzofluorenes (3 isomers) 216 229
1.2 Benzanthracene 228 250
Chrysene 228 250
Triphenylene 228 250
Methyl Chrysene 242 258
Dimethyl Chrysene 256 270
2.3-Benzofluoranthene 252 274
Methyl Benzofluoranthene 266 278
Benz(e)pyrene 252 280
Benz(a)pyrene 252 280
Perylene 252 283
Methyl Benzpyrenes 266 284
Methyl Perylene 266 286
20-Methyl Cholanthrene 268 288
Benzperylene 276 308
Coronene 300 325 (hold 12 min)
these compounds along with their gas chromatographic elution tempera-

ture and molecular weights. Figure 3 displays the total ion current output
of the mass spectrometer scanning an aromatic fraction of a typical
sample of lake sediment taken from the sediment-water interface. Detec-
tion limits of individual aromatics averaged 2 ng/g of dry sediment, and
total concentrations of aromatics in the sediment reached a maximum 54
/ag/g of dry sediment with perylene, benzpyrene, and benzofluoranthene
structures (all 252 re~e) being common constituents. These three com-
pounds were also detected as methylated derivatives along with various
other alkylated aromatics, suggesting perhaps that reducing conditions
and conditions suitable for transalkylation exist in these sediments. Figure
4 illustrates reconstructed mass spectral responses for some of the
alkylated aromatics of interest.
There is a general possibility of increased carcinogenicity with increasing
alkylation of these polycyclics. However, little is known of this activity in
specific cases and only further examination of individual isomers can
determine which are carcinogenic. Four known carcinogens were com-
monly detected in these sediments, benz(a)pyrene, 20-methyl cholanthrene,
2,3-benzofluoranthene, and 1,2-benzanthracene. In addition two other
PLASTICIZER
4,2 - BENZANTH RACENE

CHRYSENE
2 , 3 - BENZOFLUORANTHENE
METHYL BENZ(E) PYRENE

~.. CHRYSENE
I"" DI-METHYL BENZPERYLENE
CHRYSENE ERYLENE
PLASTICIZER 2 0 - METHYL
IJ,J HOLANTHR~
I-
Z UORA CORONENE
I
_J
w
METHYL
\
9,'~0 o DIMETHYL
ANTHRACENE ANTHRACENE
BIPHENYL
PYRENE
I I I I I I I t
91 5 0 = `170 ~ `190~ 2`10 ~ 230 ~ 250 ~ 270 ~ 290 ~ 5'10 ~ 3 2 5 ~ HOLD
ELUTION TEMPERATURE
FIG. 3--GC/MS total ion current display of sediment extract taken from the sediment-
water b~tert'ace of Lake Erie.
Z06
i
100
03
lS~
It i" ;i
]go
2O0
]H
~4z
9,10 DIMETHYL ANTHRACENE
300
METHYL CHRYSENE
200 300
zs~
!
lOO
tJ 200
OlMETHYL CHRYSENE
300
zsa
!
100
Jli 200
iJl;; METHYL BENZPYRENE
300
2S2
41~T
M E T H Y L PERYLENE
~o 200 300
rL
ZS3 ~6a
t2;' 2ag
..... 20-METHYL CHOLANTHRENE
[ I i" tl h
100 200 300
m/e
FIG. 4---Reconstructed mass spectra of some alkylated po~cyclic aromatic hydrocarbons

commonly.tbund in lake sediment extracts.
polycyclic aromatics detected in the sediments are thought to have some

carcinogenic activity; these are chrysene and benz(e)pyrene. One other
substance of interest in this fraction was elemental sulfur in the octagonal
$8 form (rn/e 256). Its elution temperature ranged from 195 to 210~
causing some interference in detection of methyl anthracenes, especially in
ion current displays. However, careful examination of mass spectral
responses at particular aromatic elution intervals is adequate to show the
existence of those aromatics despite the interfering ions.
The method described in this paper has been applied to explore the
presence of polycyclic aromatic hydrocarbons in a suite of sediment cores
a n d water s a m p l e s o f the G r e a t Lakes. T h e results o f this e x t e n d e d study

will be p u b l i s h e d elsewhere.
Summary
A c o m b i n e d gas c h r o m a t o g r a p h - m a s s s p e c t r o m e t e r m e t h o d was devel-
o p e d for the d e t e r m i n a t i o n o f polycyclic a r o m a t i c h y d r o c a r b o n s in waters
a n d associated sediments. T h e necessary s e p a r a t i o n for m o s t c o m p o u n d s
was achieved by gas c h r o m a t o g r a p h y ; however m a s s s p e c t r o m e t r y was
essential for c o m p l e t e identification o f a few unresolved c o m p o u n d s . T h e
high degree o f m u l t i p l e ionization d i s p l a y e d by t h e polycyclic a r o m a t i c s
e n h a n c e s the possibility o f m a s s s p e c t r o m e t r i c s e p a r a t i o n o f these c o m -
p o u n d s t h a t were unresolved b y the gas c h r o m a t o g r a p h i c m e t h o d alone.
In this m a n n e r , a total o f 27 a r o m a t i c c o m p o u n d s were d e t e c t e d in t h e
s e d i m e n t s along with t h r e e a r o m a t i c s a n d t h r e e dicarboxylic acid esters in
the water.
Acknowledgments
T h e a u t h o r s t h a n k W . M. J. S t r a c h a n for the c o o r d i n a t i o n a n d
collection of the w a t e r a n d s e d i m e n t s a m p l e s from the G r e a t Lakes. This
work was s u p p o r t e d by f u n d s from E n v i r o n m e n t C a n a d a , C a n a d a Centre
for I n l a n d W a t e r s , B u r l i n g t o n , O n t a r i o , u n d e r C o n t r a c t No. 0 1 G R .
KW412-2-1052.
References
[1] Hodgson, G. W., Hitchon, B., Taguchi, K., Baker, B. L., and Peake, E., Geochimica
Et Cosmochimica Acts, Vol. 32, 1968, pp. 737-772.
[2] Blumer, M., Science, Vol. 134, 1961, pp. 474-475.
[3] Kern, W., Helvetica Chimica Acts, Vol. 30, 1947, pp. 1595-1599.
[4] Meinschein, W. G., Bulletin of the American Association of Petroleum Geologists, Vol.
43, 1959, pp. 925-943.
[5] Orr, W. L. and Grady, J. R., Geochimica Et Cosmochimica Acta, Vol. 31, 1957,
pp. 1201-1209.
[6] Peake, E., Casagrande, D. J., and Hodgson, G. W., "Fatty Acids, Chlorins, Hydro-
carbons, Sterols and Carotenoids in Selected Samples from a Black Sea Core," Amer-
ican Association of Petroleum Geologists, Black Sea memoirs, 1971, in press.
[7] Peake, E., Strosher, M., Baker, B. L., Gossen, R., McCrossan, R. G., Yorath, C. J.,
and Hodgson, G. W., Proceedings, International Geological Congress, Montreal, Aug.
1972, Sec. 5, 1972, pp. 28-37.
[8] Hodgson, G. W., Baker, B. L., and Peake, E., Proceedings, Seventh World Petroleum
Congress, Vol. 2, 1967, pp. 117-128.
[9] Brocco, D., Cantuti, V., and Cartoni, G. P., Journal of Chromatography, Vol. 49,
1970, pp. 66-69.
[10] Lao, R. C., Thomas, R. S., Monkman, J. L., and Pottie, R. F., "Mass Spectrometric
Identification and Measurement of Polycyclic Aromatic Hydrocarbons Found in Air
Pollutants," presented at International Conference on Measurement and Identification
of Environmental Pollutants, Ottawa, June 1971.
[11] Zoccolillo, L., Liberti, A., and Brocco, D., Atmospheric Environment, Vol. 6, 1972,
pp. 715-720.
[12] Sawicki, E., Stanley, T. W., Elbert, W. C., and Phaff, J. D., Analytical Chemistry,
Vol. 36, 1964a, pp. 497-502.
[13] Sawicki, E., Stanley, T. W., and Johnson, H., Microchemical Journal, Vol. 8, 1964b,
pp. 257-284.
[14] Popl, M., Dolansky, V., and Mostecky, J., Journal of Chromatography, Vol. 59,
1971, pp. 329-334.
[15] Martinu, V. and Janak, J., Journal of Chromatography, Vol. 65, 1972, pp. 477-485.
[16] Van Duuren, B. L., Analytical Chemistry, Vol. 32, 1960, pp. 1436-1442.
[17] Keefer, L. K., Wallcave, L., Loo, J., and Peterson, R. S., Analytical Chemistry,
Vol. 43, 1971, pp. 1411-1416.
[18] Nounou, B., International Journal of Mass Spectrometry and Ion Physics, Vol. 4,
1970, pp. 219-234.
[19] Robinson, C. J., Analytical Chemistry, Vol. 43, 1971, pp. 1425-1434.
[20] Gump, B. H., Journal of Chromatographic Science, Vol. 1, 1969, pp. 755-760.
[21] Bhatia, K., Analytical Chemistry, Vol. 43, 1971, pp. 609-610.
[22] Frycka, J., Journal of Chromatography, Vol. 65, 1972, pp. 432-434,
[231 Pering. K. L. and Ponnamperuma, C., Science, Vol. 173, 1971, pp. 237-239.
[24] Monterio, L. F. and Reed, R. I., Journal of Mass Spectrometry and Ion Physics,
Vol. 2, 1969, pp. 265-285.
[25] Blum, W., "Analysis of Dicarboxylic Acid Esters (Plasticizers)," Finnigan Instruments
Corporation Application Tips, Vol. 42, 1972.
A. E. George, j G. T. Smiley, ~ D. S. Montgomery, 1
and H. Sawatzky j
A Gas Liquid-Gas Solid

Chromatographic Method for the
Identification of Sources of
Oil Pollution
REFERENCE: George, A. E., Smiley, G. T., Montgomery, D. S., and Sawatzky, H.,
"A Gas Liquid.Gas Solid Chromatographic Method for the Identification of Sources of
Oil Pollution," Water Quality Parameters, A S T M STP 573, American Society for
ABSTRACT: A two-step gas chromatographic fingerprinting technique has been de-
veloped for the identification of petroleum that may be conveniently applied to oil spills.
The first step consists of a gas chromatographic separation on nonpolar silicone rubber
(SE-30) which separates according to boiling point. Five arbitrary 20 ~ cuts are made,
then further separated by gas chromatography on columns of lithium chloride supported
on diatomaceous silica (Chromosorb A). The advantage of this inorganic packing is its
high thermal stability that permits the separation of high-boiling oil components not
readily affected by weathering. It also has the added advantage of causing no "bleed-
ing" problems that can complicate further analyses involving mass spectroscopy. The
simultaneous use of the flame ionization detector and the Melpar sulfur detector pro-
vides highly characteristic fingerprints, some of which are shown. This method has been
applied to two heavy crude oils, and two fuel oils involved in oil spills from the Arrow
and Irving Whale to demonstrate the pofential of the method.
KEY WORDS. water quality, gas chromatography, crude oil, fuel oil, water pollution,
environmental tests
Pollution emanates from crude oil or fuel oil as a consequence of ships

discharging their tank washings or bilges at sea, but occasionally is the
result of collisions or accidental spillages. Leaks from pipelines, storage
tanks, and uncontained oil during undersea drilling operations can also be
sources of poUufion. In the last few years, slicks appeared off the coasts of
Nova Scotia, Australia, California, Alaska, England, and Florida. These
mysteriously appearing slicks could be caused naturally by oil seeping
through fissures in the ocean floor or by ships sunk during war time.
The problem of oil spillage in Canadian waters is becoming increasingly
' Research scientist, technologist, head, and research scientist, respectively, Fuels Research
Centre, Department of Energy, Mines and Resources, Ottawa, Ontario K1A 0G1, Canada.
271
9
serious. According to a report prepared for the federal Department of the

Environment, major oil spills that would seriously affect the coastal
environment of western Canada can be expected repeatedly if tankers
begin transporting Alaskan oil to United States refineries. The oil
spillages which drifted to the western coast of British Columbia were early
indications of the validity of this forecast.
Work associated with various aspects of oil spillage has been increasing
steadily for several years by the major oil companies and government
agencies. One problem of growing interest is the identification of pollution
samples. In the past, the analysis of beach samples of oil has been largely
in terms of oil resins and asphaltenes, wax content, and elemental
analysis. The characteristics that help distinguish one oil from another
have been found to include volatility, amount and relative proportion of
trace metals, and a significant difference in sulfur and nitrogen content. A
Canadian Association, The Oil Slick Group (consisting of 200 scientists),
is exploring the use of neutron activation analysis to establish the origin of
an oil slick [1].2 In the United States, Gulf General Atomic has been
investigating a similar neutron activation system since 1968, and work is
being done at the University of Lund in Sweden to "tag" tanker loads
with isotopes of iodine.
In a previous report [2], it was shown that the positive identification of
the crude oil source of an unknown oil washed upon a beach presents
many difficulties when one considers the variety of crude oils trans-
shipped throughout the world. The position is further complicated by the
effects of exposure on oil from the time of discharge to the arrival of the
pollution on the coastal areas. There are various pitfalls in trying to draw
conclusions from the conventional methods of oil source identification.
A number of techniques exist for pollutant identification. These have
been used with varying degrees of success [3-13]. All these methods
require sample clean-up and sometimes other pretreatment, such as
ashing, before analysis. Also, in many cases, comparatively large samples
are required for analysis.
Among the analytical methods already known to yield properties
significant for the recognition of oil from various pollution sources, the
gas chromatographic "fingerprint" method is recommended as the most
suitable of the methods already mentioned for a quick examination of a
pollutant. A direct gas chromatographic analysis of an oil sample has
several advantages: very small amounts in the order of 50 /al are quite
enough for analysis, no sample pretreatment is required, analysis is
relatively rapid, and the fingerprinting method is the most dependable
among the known procedures.
However, the gas chromatographic procedures that have been used are
2The italic numbersin brackets refer to the list of referencesappendedto this paper.
GEORGE ET AL ON GAS LIQUID-GAS SOLID CHROMATOGRAPHY 273
not sufficiently discerning to distinguish between very similar oils.

We have developed a two-step gas chromatographic procedure that is
superior to the single-step method. Also, our method is capable of dealing
with very high boiling materials.
Experimental
Samples
Five samples were investigated:
1. Lloydminster crude oil;
2. Lathom crude oil;
3. Bunker C from the Arrow cargo;
4. Weathered Bunker C from the Arrow incident collected from the
beach on Crichton Island, Nova Scotia, on 21 April 1970 (the
wreckage was on 4 Feb. 1970); and
5. Weathered Irving Whale Bunker C.
The method of fingerprinting comprises two steps:
Simulated Distillation and Preparative Step--A "Varian Aerograph"
Model 2100 gas chromatograph was employed throughout the whole
investigation. In the simulated distillation and preparative step a glass
column (5 ft by 0.25 in. outside diameter) was used. It was packed with
10 percent silicone rubber SE-30 on acid-washed Chromosorb W,
Dimethyl dichlorosilane-treated (DMCS), 60 to 80 mesh. The temperature
was programmed at 4~ from 50 to 300~ and then held isother-
mally at this temperature. The chart speed was 0.2 in./min. The carrier
gas was helium and the flow rate was 200 ml/min. The effluent was split
and one third of it was diverted into the detector and the main stream to
collection system.
Firstly, to establish a calibration curve, a 0.9-gl sample of n-paraffin
mixture C10-Ca6 (10 percent solution in ethylbenzene) was chromato-
graphed starting from 50~ up to 300~ of column temperature. Cali-
bration of the column was repeated three times to ensure that the column
did not change during the course of investigation.
The same column, without changing any of the conditions was used for
direct chromatography of each of the five samples under investigation
(Figs. 1 to 5). In this case the dual detector Melpar-Flame Ionization was
used. A 30-/zl sample of the crude oil was directly injected, using a 100-gl
Hamilton syringe, without overloading the SE-30 column. The injection
port temperature was kept at 300~ a~,d the initial 3 in. of the column
were filled with acid-washed Chromosorb W to trap out heavy residue and
material boiling above 500~ and thus protect the column. The effluent
not passing into the detector was led into traps; each containing 60 mg of
acid-washed Chromosorb W, 60 to 80 mesh. This amount of Chromosorb
~AI I [213[4151B~
AJKOV,T'S INOEX
IO00 ~ "1600 2400 3600
" 1 ," ~ , , ,
i~ 200 2so '~' ' ~,,~' ' ~ , ~ " ~,~6 . . . . 5'oo

BOILING POINT, ~
FIG. l--Sample: Lathom crude oiL Column: silicone rubber (SE-30) on Chromosorb W.
~-AI I [2tSI4r 51B~
FID
,1ELPAR
~ KOVAT'S INDEX
I000 1600 2400 3600
150 200 250 300 350 400 450 500
BOILING POINT, ~
FIG. 2--Sample: Lloydminster crude oil. Column: silicone rubber (SE-30) on Chromo.
sorb W.
GEORGE ET A L O N G A S L I Q U I D - G A S S O L I D C H R O M A T O G R A P H Y 275
12151.1,1.
FIO
KOVAT 'S INDEX
400
l 9 ! . . . . . J 9 i , , , / , 9 9 t, !
#o 2'C,o ' z~o ~ ~so 400 4So
BOILING POINT , *C
FIG. 3---Sample: Arrow cargo. Column: silicone rubber (SE-30) on Chromosorb W.
e-,I,[2 I ~ 14[ 51,-~
KOVAT 'S INOEX
/ ,~oo . . %00 . . . . . ~?o. .... ~oo .... ?Ioo

,30 '2~ 2~ 36o ~o 4~ 430
BOILING POINT "C
FIG. 4 ~ S a m p l e : weathered Arrow. Column: silicone rubber (SE-30) on Chromosorb W.
W was used to maintain the pressure differential needed to get the

required split ratio. Five successive 20~ were collected in the boiling
range of 250 to 350~ These fractions were then rechromatographed on
the analytical lithium chloride-diatomaceous earth column to obtain the
fingerprints.
Separation According to Type---An inorganic column was used in this
step. It consisted of a glass U-tube (5 ft by 0.25 in. outside diameter)
packed with lithium-chloride (LiC1) on diatomaceous silica (Chromosorb
A) of 60 to 80-mesh size (1:1 by weight). The packing was prepared by
<-.1.12 I+1.10 J+--,

FID
KOVAT'S INDEX
~o 2bo 2~ ~o ~so 4bo +~o

aOILING POINT, "C
FIG. S--Sample: weathered Irving Whale. Column: silicone rubber (SE-30) on Chromo-
sorb W.
covering the chromosorb with an aqueous solution of the salt and

evaporating the mixture to dryness. The dry mixture was then fired at 700
to 750~ for 30 min. in a muffle furnace. The chromatograph used was a
Varian 2100 fitted with both flame ionization and Melpar sulfur detectors.
The flow rate was 85 ml/min of helium.
The Chromosorb W from each collection tube that contained a fraction
was packed into a glass capillary tube under nitrogen. The capillary tubes
and their contents were then injected with a Hewlett-Packard solid injector
on to the salt-containing column at room temperature. The column oven
was heated to 50~ and then programmed at 4~ The column was
calibrated with amounts of normal alkane mixture that gave detector
responses comparable to the responses obtained during the chromato-
graphy of the petroleum fractions.
Detectors
In this investigation the flame photometric detector with a sulfur-
selective filter (394 /an) was used in conjunction with the flame ionization
detector to obtain two fingerprints simultaneously, and in this way to
affect a significant improvement in the identifying power of the method.
Assigning Kovat's Indices to the Chromatograms

To compensate for fluctuations in chromatographic conditions, and
make the results from the analytical step comparable in different labora-
tories, the retentions were expressed in terms of Kovat's indices [14].
Retentions on the oil chromatogram were expressed relative to the
retentions of n-alkanes as reference compounds.
Discussion
Though gas chromatography is, in principle, the best and most
promising tool in the field of oil spill identification, all previous attempts
to obtain identification chromatograms for crude and fuel oils suffered
from the polar stationary phases being unable to stand high column
temperatures. This difficulty precludes the possible use of the heavy
residue for a dependable fingerprint. Usually these heavy ends are most
promising for providing characteristics least changeable under weathering
conditions.
In some cases single chromatograms, obtained by using nonpolar
relatively thermally stable chromatographic columns, can be used to
establish the source of spillage. However, if the oils are quite similar, and
overlapping between a large number of fingerprints occurs, this one-step
analysis will not be adequate. In many paraffinic oils [15], the chroma-
togram consists of a number of peaks standing out on a broad "envelope"
representing abundant numbers of incompletely separated components.
More efficient separation is a necessity for a dependable fingerprint. The
peaks representing the profiles of normal paraffins have been used [16] as
the main criteria in the chromatograms to differentiate between oil spills
and to identify them, but normal paraffins are known for their suscepti-
bility to biodegradation [17,18] and more stable compounds would be
more useful for identification purposes.
The approach used in this investigation to offset these disadvantages
was to first use gas chromatography to obtain speed and the advantage of
being able to use small samples of oil spills without any pretreatment. To
increase the resolving power of the method an analytical step, which
separates according to type, was added to the chromatography on
nonpolar silicone rubber columns as used by most investigators.
Simulated Distillation
The time required for true boiling point (TBP) distillation---6 to 100
h--precludes its use for quick examination of a pollutant. Gas chroma-
tography is gaining wide acceptance [19,20] as the quickest reproducible
method of distillation and the most accurate method of determining initial
and final boiling points of hydrocarbon materials. This simulated distil-
lation was used as a preparative step.
Gas-Solid Chromatography
In previous publications [21-23], we have shown that high-boiling
hydrocarbons and sulfur compounds can be separated according to type
on porous silica, coated with lithium chloride, at temperatures no higher
than for comparable separations on gas-liquid chromatography. From the
results obtained we recommended the use of salt columns for efficient

chromatographic characterization of oil spills. Lithium chloride on
Chromosorb A and lithium chloride on Porasil F are, so far, the best
combinations tested in our laboratory. The fact that these inorganic
column packings allow characterizations at the high boiling ranges should
considerably imporve the fingerprinting technique because these ranges
are least affected by changes caused by evaporation. Also, the inorganic
gas chromatographic columns have the added advantage that there are no
"bleeding" problems that can complicate mass spectroscopy, hydrodesul-
furization, and various new detectors. Efficient separations can be
achieved on salt columns as evidenced by mass spectrometry. The analysis
of a distillate fraction of Athabasca bitumen separated isomers of both the
benzothiophenes and the naphthalenes [21].
The boiling ranges of the arbitrary simulated distillation cuts chosen for
fingerprinting and their corresponding Kovat's indices are shown in Table
1. The width, in terms of Kovat's indices, of the particular arbitrary cut
that is rechromatographed on the supported lithium chloride (LiC1)
column is shown in Figs. 6 to 17 by two dashed lines to indicate the limits
of the nonspecific interaction region. We have discussed specific and
nonspecific interactions on the salt columns in detail in a previous report
[24]. The material eluting before the lower limit of the nonspecific
FIO
r I
I
800 I000 1200 1400 1600 1800 2000 2200 2400
K OVAT'S INDEX
FIG. 6--Lathom, 290 to 3IO~ LiC1 on Chromosorb A.

I FID
KOVAT'I$NDEX
FIG. 7--Lathom, 310 to 330~ LiCl on Chromosorb A.
TABLE 1--Boiling ranges and Kovat's Indices corresponding to the

.fingerprinting fractions.
Fraction No. Boiling Range, ~ Kovat's Index Range
1 250-270 1383-1495
2 270-290 1495-1620
3 290-310 1620-1760
4 310-330 1760-1905
5 330-350 1905-2060
interaction region is believed to contain nonplanar cyclic saturates, that

within the limits mainly saturated alkanes, whereas the region that lies
beyond the higher limit represents the specific interaction caused by
aromatic structures or heteroatoms.
Dual- Trace Fingerprinting
Oils contain many types of sulfur compounds that are very resistant to
degradation. The aromatic sulfides, benzothiophenes, dibenzothiophenes,
and other thiophenes are very stable [17]. Because these compounds vary
MELPAR
e6o K)bO ~ZbO p4bo I s o o l e ~ o z6oo z~oo z,ioo zsoo

KOVAT'S INOIEX
FIG. 8--Lloydminster, 290 to 310~ LiC! on Chromosorb A.
FID
MELPAR
I000 1200 1400 1600 1=800 2 0 0 0 2200 2400

KOVAT'S INDEX
FIG 9--LIoydminster, 310 to 330~ LiCI on Chromosorb A.
GEORGE ET AL ON GAS LIQUID-GAS SOLID C H R O M A T O G R A P H Y 281
i/
1200 1400 1600
!
KOVAT' S INDEX
1800
~
2000 2200
FIG. 10--Arrow cargo, 270 to 290~ LiCl on Chromosorb A.
/
\
1200 ' 14()0

y ,I
1600
NOVA1"'$ INDEX
. . . . .
1800 EO00
FIG. 1 l - - W e a t h e r e d Arrow. 270 to 290~ LiCI on Chromosorb A.

FID
\
MKL~R
1400 16'00 leO0 2000 2ZO0 2400 2600

KOVAT'S INOEX
FIG. 12--Arrow cargo, 310 to 330~ LiCl on Chromosorb A.
considerably for different oils in both type and amount, they should be
ideal for identification purposes.
A sulfur-selective gas chromatographic detector such as the Melpar
flame photometric detector (FPD), can trace a sulfur "fingerprint" for oils
in a manner analogous to the carbon "fingerprint" obtained with the
flame ionization detector (FID). Because the FPD response varies for
different types of sulfur-containing compounds and is affected by the
nature of the hydrocarbon components, this detector gives unique and
reproducible chromatograms for different oils even, in many cases, for
those of very similar sulfur content. The latter can be true even when the
FID carbon traces are very similar. For these reasons, the Melpar detector
is valuable in fingerprinting oils and surpasses the quantitative micro-
coulometric sulfur detector. In some cases, the sulfur compounds are
largely absent from the lower-boiling fractions of crude oil so that
evaporative weathering has a smaller effect on the FPD fingerprint than
on the FID fingerprint.
The microorganisms which digest oil attack normal paraffins prefer-
entially. Consequently, we are inclined to think that the FID chromato-
jl
~,'oo ' ,ebo ' ;88o ' z~o " =zoo" 2;oo"
KOVAT 'S INDEX
FIG. 13---Weathered Arrow, 310 to 330~ LiCI on Chromosorb A.
gram of a biologically aged paraffinic crude oil will become like a

naphthenic oil but that the Melpar sulfur chromatogram will remain
relatively unchanged [24].
Fingerprints of the Lathom and Lloydminster Crude Oils

All of the flame ionization traces of the Lathom fractions (two of them
shown in Fig. 6 and 7) show two envelopes of peaks. The first envelope of
peaks generally ends fairly abruptly near the limit of the nonspecific
interactions. Then there is a valley between the two envelopes. If the
boiling ranges were narrower, the valleys probably would be nearer to the
base line.
In all the chromatograms of the Lathom fractions, there is considerable
material with negative specific interaction which indicates the probable
existence of substantial amounts of nonplanar saturated cyclic hydrocar-
bons. It seems that, as the boiling points of the fractions increase, the
initial materials that elute involve greater negative specific interaction.
Possible substitution on the cyclic structures explains this observation.
~JIID 1
!IV ....
~'oo" ~'oo' ~o
KOVAT'
INDEX
S
e6oo22bo'24'oo'2~od2,r
FIG, 14---Arrow cargo. 330 to 350~ LiC1 on Chromosorb A.
In Fig. 6, normal hexadecane and heptadecane appear quite prominent.

In Fig. 7, normal octadecane and, to a much lesser extent, nonadecane
are very evident. The second envelope of peaks in the chromatograms of
these Lathom fractions is due to aromatic hydrocarbons and sulfur
compounds. Probably the sulfur compounds are mostly substituted benzo-
thiophenes. The tail on the sulfur chromatograms might be due to dialkyl
and alkyl aryl sulfides.
As can be seen in Fig. 8 and 9, the Lloydminster fractions differ
considerably from the Lathom fractions. The first envelope of peaks
declines much sooner in the Lloydminster fractions. In fact, octadecane
appears to be in the second envelope in the 290 to 310~ fraction. In
general, the Lloydminster chromatograms are more spread out than the
Lathom for both the flame ionization and the Melpar sulfur traces. As the
boiling point of the fractions increases, there appears to be considerable
sulfur-containing material with specific interaction lower than for the
Lathom fractions. Also the amount of material involving negative specific
interaction increases.
FID
I I I & & *
\
MELPAR
L300 1500 1700 1900 2100 2300 2500 290c
KOVAT'$ INDs
FIG. 15---Weathered Arrow. 330 to 350~ LiCI on Chromosorb A.
Chromatography o f the Arrow and Irving Whale Samples on the Silicone

R u b b e r (SE-30) Column
The chromatographic FID traces obtained during simulated distillation
on the nonpolar silicone rubber column are shown in Figs. 3 to 5. It can be
seen that the major peaks from the weathered samples still match the ones
from the cargo oil. However, the lower-boiling material has been reduced
by evaporation and the peaks have been quite subdued. Even in the high-
boiling region, where losses by evaporation are not as significant and
material would be more suited for comparison, the peaks are still
subdued. In fact, the main part of the chromatogram of the weathered
Irving Whale Bunker C is more similar to the Arrow cargo (Figs. 3 and 5)
than to the weathered Arrow Bunker C. Thus, a single chromatographic
separation according to boiling point is not sufficient for dependable
fingerprinting. However, if comparisons are made with chromatograms
obtained from the salt column, the situation becomes quite different.
Fingerprints o f the Arrow and Irving Whale Samples on the Salt Column
Though having poorly resolved chromatograms on the silicone rubber
I000 1200 1400 1600

KOVAT'S
I 1800
tNOEX
2000 2200
FIG. 16--Weathered Irving Whale, 270 to 290~ LiCl on ChromosorbA.
column, the weathered Arrow and Irving Whale Bunker C oils show
significant differences on the salt column. A large portion of the FID
trace of the Irving Whale fractions lie in the negative interaction range
(Figs. 16 and 17), indicating a stronger naphthenic nature than the Arrow
fractions. The latter, on the other hand, show prominent straight-chain
paraffin peaks, for example, normal hexadecane (Fig. 11), normal hepta-
decane, normal octadecane, and normal nonadecane (Fig. 13), then
normal eicosane (Fig. 15) which are missing in the equivalent cuts of the
Irving Whale sample. In both samples, however, the nonplanar saturated
cyclic content gradually rises with boiling point.
The weathered Arrow fractions (Figs. 11, 13, and 15) show two groups of
peaks on the FID trace, the first of which ends near the upper limits of
the nonspecific interaction range. This tendency is less noticeable in most
of the Irving Whale chromatograms, for example (Fig. 17) with the
exception of the 270 to 290~ fraction (Fig. 16).
Also the FPD trace shows no sulfur in the area between the dashed
lines over the whole boiling range of the weathered Arrow samples. In the
GEORGE ET AL ON GAS LIQUID-GAS SOLID C H R O M A T O G R A P H Y 287
FtD
MELPAR
12bo i~oo le'oo l a b o z6oo ~oo z,ioo

KOVAT'S INDEX
FIG. 17--Weathered Irving Whale, 330 to 350~ LiCI on Chromosorb A.
Irving W h a l e fractions, sulfur is represented in this region and increases

considerably from 290~ up to the end of the fingerprinting range, for
example, (Fig. 17). Thus, this two-step method can clearly differentiate
between two weathered bunker fuel oils which would have appeared
similar after one-step chromatography.
On the other hand, the cargo samples and weathered samples of the
A r r o w Bunker C oil show considerable differences on the silicone rubber
chromatograms due to weathering as previously discussed, but their salt
column fingerprints are very similar (Figs. 10 to 15) giving all the peaks at
the same retentions as expressed by Kovat's indices. The minor differences
present do not affect the high efficiency of the method for identification
purposes.
Reproducibility
The whole procedure (simulated distillation, collecting fractions, and

rechromatography according to type on the salt column) was repeated for
both the cargo and weathered samples of the Arrow Bunker C oil.
Chromatograms were reproducible for both the carbon and sulfur traces.
Automating the Method

The procedure can be automated to a large extent to facilitate the
handling of a large number of pollutant samples at the same time. This
would allow wider and faster comparisons and identifications.
Acknowledgment
The authors are grateful to the Inland Waters Branch of the Depart-
ment of the Environment for supporting this program financially. They
also thank T. M. Potter for technical assistance, A. Y. McLean of the
Nova Scotia Technical College for samples of weathered oil from the
Irving Whale and Arrow spillages, and V. O. Juba of the Husky Oil Co.
Canada for a sample of Lloydminster crude oil.
References
[1] "More Weapons for Oil Spills," Chemical Engineering, Vol. 77, No. 15, 1970, pp.
40-42.
[2] George, A. E., Smiley, G. T., Montgomery, D. S., and Sawatzky, H., "New Gas
Chromatographic Method for the Identification of Sources of Oil Leaks and Spills,"
Divisional Report FRC 72/54-RBS, June 1972.
[3] Kawahara, F. K., Environmental Science and Technology, Vol. 3, 1969, p. 1.50.
[4] Thruston, A. D. and Knight, R. W., Environmental Science and Technology, Vol. 5,
1971, p. 64.
[5] Done, J. N. and Reid, W. K., Separation Science, Vol. 5, 1970, p. 825.
[6] Matthews, P. J., Journal of Applied Chemistry, Vol. 20, 1970, p. 87.
[7] Mattson, J. S., Analytical Chemistry, Vol. 43, No. 13, 1971, p. 1872.
[8] Mattson, I. S., Mark, H. B., Kolpack, R. L., and Schutt, C. E., Analytical Chemistry,
Vol. 42, No. 2, 1970, pp. 234-238.
[9] Cadberg, S. R. and Skarstedt, C. B., Meddn Havsftsbelab Lysekil. VoL 96, 1970,
p. 10.
[10] Simard, R. G., and Hasegawa, i., Bandaruk, W., and Headington, C. E., Analytical
Chemistry. Vol. 23, 19S1, pp. 1384-1387.
[11] Brunnoek, I. V., Duekworth, D. F., and Stephens, G. G., Journal of the Institute
of Petroleum, Vol. 54, No. 539, 1968, pp. 310-325.
[12] "The ARROW Incident," compiled at the Atlantic Oceanographic Lab., Bedford In-
stitute, Dartmouth, Nova Scotia, July 1970, prepublication edition.
[13] Bryan, D. E., Quinn, V. P., Hackleman, R. P., and Lukens, H. R., U.S. Atomic
Energy Commission, Report GA9889, 21 Jan. 1970.
[14] Journal of Gas Chromatography, Vol. 6, 1968, p. 1 (ASTM Committee E-19).
[15] Journal of the Institute of Petroleum, Vol. 56, No. 548, March 1970, pp. 107-117
(The Institute of Petroleum Standardization Committee)
[16] Ramsdale, S. J. and Wilkinson, R. E., Journal of the Institute of Petroleum, Vol. 54,
No. 539, 1968, pp. 326-332.
[17] Litvinenko, S. N., Grigor'eva, G. P., Sanina, N. G., Mabhort, A. M., and Tikhonruk,
1. F., Khimiia I Tekhnologiia Topliv I Masel, 1971, pp. 502-505.
[18] Atlas, R. and Bartha, R., "Biodegradation of Petroleum by Two Marine Bacterial
Isolates," ACS National Meeting, Washington, D.C., 13-16 Sept. 1971.
[19] Eggertsen, F. T., Groennings, S., and Hoist, J. J., Ana(ytical Chemistry, Vol. 32,
1960, p. 904.
[20] Green, L. E., Schmauch, L. J., and Worman, J, C., Analytical Chemistry, Vol. 36,
No. 8. 1%4. p. 1512,
[21] Sawatzky, H., Smiley, G. T., George, A. E., and Clugston, D. M., "Gas Chromato-
graphic Separation of Sulphur Compounds from Athabasca Bitumen," ACS National
Meeting, Los Angeles, 28 March-2 April 1971.
[22] George, A. E., Smiley, G. T., and Sawatzky, H., "Evaluation of Lithium-Chloride-
Diatomaceous Silica Systems for Gas Chromatography of Petroleum Sulphur Com-
pounds," Mines Branch Research Report R-249, Jan. 1972,
[23] Sawatzky, H., George, A. E., and Smiley, G. T., "The Evaluation of Lithium Chloride-
Coated Porous Silica for the Gas Chromatographic Separation of Petroleum Fractions,"
Division of Petroleum Chemistry, ACS Meeting, Dallas, 8-13 April 1973.
[24] Adlard, E. R., Creaser, L. F., and Matthews, P. H., Analytical Chemistry, Vol. 44,
1972, p. 64.
Michael Gruenfeld ~
Quantitative Analysis of Petroleum

Oil Pollutants by Infrared
Spectrophotometry
REFERENCE: Gruenfeld, Michael, "Quantitative Analysis of Petroleum Oil Pollutants

by lnfirared Speetrophotometry," Water Quality Parameters, A S T M STP 573. American
ABSTRACT: The accuracy and sensitivity of infrared spectrophotometry are evaluated

for the quantitative analysis of water dispersed oils, by single point analysis. Absorbance
versus concentration (Beer-Bouguer Law) plots are prepared for viscous and nonviscous
crude and processed oils in Freon 113, carbon tetraehloride, and in a mixture of these
solvents. Absorbances at 2930/cm are measured in 10 and 100-mm path length cells,
with and without ordinate scale expansion. Solution concentrations in the range 0.5 to
40 rag/100 ml oil in solvent yield linear plots that pass through the origin. The concen-
tration 0.05 mg/100 ml oil in solvent yields a recognizable absorption band at approx-
imately 2930/cm when measured in 100-mm path length cells with ordinate scale
expansion • This is considered the practical detection limit of these oils by the
infrared (IR) technique. Stability of oil absorptivities following solution storage, and use
of IR absorptivities for oil identification are also examined briefly.
KEY WORDS: water quality, oils, infrared spectrophotometers, spectrophotometry,
environmental tests, water pollution
I m p r o v e d m e t h o d s for the q u a n t i t a t i v e analysis of water dispersed

p e t r o l e u m oils are u n d e r investigation by the I n d u s t r i a l W a s t e T r e a t m e n t
Research L a b o r a t o r y of the National E n v i r o n m e n t a l Research Center
(NERC), C i n c i n n a t i . Such methods are needed to evaluate the per-
f o r m a n c e of oil-water separator devices, to measure the dispersed oil con-
c e n t r a t i o n in water below surface floating oil slicks, to m o n i t o r oil
c o n c e n t r a t i o n s in effluent waters of various industrial plants a n d water-
craft, a n d to s u p p o r t studies of the effect of water dispersed p e t r o l e u m
oils on the biosphere. T h a t low c o n c e n t r a t i o n s of water dispersed oils
adversely affect living organisms is well known: for example, Jacobson [1] 2
has shown that kerosene extracts in water, in the parts-per-billion range,
'Supervisory chemist, U.S. Environmental Protection Agency, Industrial Waste Treatment
Research Laboratory, National Environmental Research Center, Edison, N.J. 08817.
290

GRUENFELD ON ANALYSIS OF PETROLEUM OIL POLLUTANTS 291
upset the chemotactic response of Nassarius Obsoletus to oyster and

scallop tissue.
Most methods that are currently used for the quantitative analysis of
water dispersed oils can be classified as gravimetric or spectroscopic pro-
cedures. The determinative step of gravimetric methods is simple weigh-
ing, while the determinative step of spectroscopic methods is the measure-
ment of radiation absorption by dissolved oils. Solvent extraction is
commonly used by gravimetric and spectroscopic methods to isolate and
concentrate water dispersed oils for quantitative measurement. Solvent
evaporation (stripping), which precedes weighing in gravimetric methods,
has a drawback because the more volatile petroleum fractions are lost. A
gravimetric method by the American Public Health Association [2]
illustrates yet another drawback: questionable sensitivity and accuracy are
achieved by weighing minute oil residues in "large" (approximately 125
ml) distilling flasks. Harva and Somersalo [3] conclude that the spectro-
scopic methods are more accurate and sensitive---a justifiable conclusion.
Infrared spectrophotometry (IR) is the most commonly used spectro-
scopic technique for the quantitative analysis of water dispersed oils. In an
American Petroleum Institute procedure [4] dispersed oils are extracted
from water with carbon tetrachloride; a mechanical shaking apparatus
and a 5-liter extraction flask are used. The absorption band maxima of oil
at 2850 and 2930/cm are measured, using 10-mm path length quartz
cells. Oil concentrations are determined by comparing the sum of these
absorbances to the sum of absorbances derived from a prepared carbon
tetrachloride solution containing an accurately known concentration of the
oil. The latter solution is the standard solution, and the determination is
performed by single point analysis. These terms are discussed later. In
this method a blend of hydrocarbons (37.5 percent isooctane, 37.5
percent cetane, and 25 percent benzene) that is thought to approximate
the IR absorptivity of an average petroleum oil is used to prepare the
standard solution when a portion of the dispersed oil is not available.
In an IR method by Beckman Instruments, Inc. [5], 100-ml of
dispersed oil in water samples are extracted with 2-ml carbon tetra-
chloride, using hand-held separatory funnels. The absorption band max-
imum of oil at 2930/cm is measured as in the previous method, using
10-mm path length near IR silica cells. The 2850/cm band is not used.
Reference solutions are prepared as oil in water dispersions having known
oil content, and each of these dispersions is extracted with 2-ml carbon
tetrachloride. The extracts are then measured at 2930/cm, and a concen-
tration versus absorbance plot is derived. This plot is used to quantitate
dispersed oil in water samples.
The two preceding methods utilize the same extraction solvent, but they
differ in most other respects. The method by the American Petroleum
Institute uses a large oil extraction flask and a mechanical shaker; salt
and acid are added; extraction is performed with successive 100-ml

portions of carbon tetrachloride; two IR absorption band maxima are
measured; and comparison is made to only a single standard solution. The
Beckman Instrument method uses a small hand-held separatory funnel;
one 2-ml portion of carbon tetrachloride is used to extract a 100-ml
sample volume; acid or salt are not added; one IR absorption band
maximum is measured; and a concentration versus absorbance plot
derived from several oil-in-water reference solutions is used for sample
determination. These methods thus differ in the type of apparatus used,
sample and solvent volumes, addition of salt and acid, and the method of
IR measurement and data handling. Gruenfeld [6] recently investigated
some of these parameters in detail. He examined the influence of salt and
acid, and compared the extraction efficiencies of carbon tetrachloride and
Freon 113 (1,1,2-trichloro-l,2,2-trifluoroethane). Freon 113, like carbon
tetrachloride, can be used for IR measurement of oil at 2930/cm.
Gruenfeld [6] extracts l-liter dispersed oil in water samples with four
consecutive 25-ml portions of solvent, using 2-liter hand-held separatory
funnels. Use of Freon 113 is of special interest because it is far less
poisonous to exposed personnel than carbon tetrachloride. According to
Sax [7], carbon tetrachloride is highly toxic (10 ppm TLV) when inhaled
or absorbed through the skin, while Freon 113 is much safer (1000 ppm
TLV).
The present paper describes a study of the accuracy and sensitivity of
the IR technique, when used for the quantitative determination of
petroleum oils by single point analysis. Carbon tetrachloride, Freon 113,
and a mixture of these solvents are used. The detection limit of oils by IR,
the stability of oil absorptivities during prolonged solution storage, and
the utility of these absorptivities for oil identification are also examined.
Four oils were used: No. 2 Fuel Oil, which is a low viscosity distillate oil
(2.4 cSt at 100~ Number 6 Fuel Oil, which is a high viscosity residual
oil (2300 cSt at 100~ South Louisiana Crude Oil, which has a low
viscosity (4.8 cSt at 100~ and Bachaquero Crude Oil, which has a
moderately high viscosity (1070 cSt at 100~ IR measurements were
made in 10 and 100-ram path length silica cells, at approximately
2930/cm. The oils, solvents, final solution volumes, and cells were selected
in order to correlate this work with the previously reported investigation
by Gruenfeld [6].
Atwood et al [8] reported a somewhat similar, though more limited
study of IR for the quantitative analysis of oils. A blend of hydrocarbons
(isooctane, hexadecane, and benzene) was used to represent "typical" oils,
and carbon tetrachloride solutions of this blend were measured in 10 and
100-mm path length quartz cells. No actual oils, or solvents other than
carbon tetrachloride were used. An effort was not made to establish
whether concentration versus absorbance (Beer-Bouguer Law) plots of oils
are linear and pass through the origin; that is, whether oils can be quanti-
tatively determined by single point analysis.
Experimental
Apparatus
A Perkin Elmer Model 457A infrared grating spectrophotometer 3 was
used for the determinations. Solution absorbances were measured in
10-mm (Beckman Instruments, Inc., Catalog No. 580015) and 100-mm
(Fisher Scientific Co., Catalog No. 14-385-930F) path length ceUs. These
are rectangular silica and cylindrical Supracil cells, respectively. Cell
holders obtained from the Perkin Elmer Corporation (Catalog No. 186-
0091) were used for the 10-mm path length cells, while holders obtained
from International Crystal Laboratories (Catalog No. R 100-22 with Teflon
gaskets as spacers) were used for the 100-ram path length cells.
Reagents
The oil solutions were prepared in spectroanalyzed carbon tetrachloride
(Fisher Scientific Co., Catalog No. C-199), Freon 113 solvent (E. I.
DuPont De Nemours and Company, Inc.), and in a 98 percent Freon
113/2 percent carbon tetrachloride (by volume) solvent mixture. Freon 113
is a DuPont refrigerant. It is 1,1,2-trichloro-l,2,2-trifluoroethane, of
specified purity. This isomer of trichlorotrifluoroethane is available from
several manufacturers, under a variety of trade names.
Procedure
Oil solution concentrations were adjusted to yield absorbances that were
within the ordinate scale range of the IR chart paper. Measurements were
made with matched 10 and 100-mm path length cells, without ordinate
scale expansion, and with ordinate scale expansion x,5. Measurements
without scale expansion required oil concentrations of 2 to 40 rag/100 ml
and 0.5 to 4.0 mg/100 ml for measurements in the 10 and 100-mm path
length cells, respectively. Measurements with ordinate scale expansion ><5
required concentrations of 0.5 to 4.0 mg/100 ml and 0.05 to 0.4 mg/100
ml for measurements in the 10 and 100-mm path length cells, respectively.
All the solutions contained accurately known oil concentrations. The
reference cell was filled with solvent from the same reagent bottle that was
used to prepare the oil solution. Absorbances derived from measurements
without scale expansion were read directly from a nonlinear absorbance
type chart paper. These absorbances were measured as vertical distances
between the 2930/cm absorption band maximum of oil and a baseline
Mention of trade names or commercial products does not constitute endorsement by the
U.S. Government.
drawn tangent to absorption minima adjoining the band maximum (Fig.

1). Absorbances that were determined by using ordinate scale expansion
><5 required use of a linear transmittance type chart paper, and calculation
by special equation (Fig. 2). The solvents used were Freon 113, carbon
tetrachloride, and 98 percent Freon 113/2 percent carbon tetrachloride
(by volume). Absorbance versus concentration (Beer-Bouguer Law) plots
of the oils in the solvents were derived.
0.10
0.20
0.30
0.40
O,SO
0,60
0.70
0.80
0.90
1.0
I I I I I I
3200 3000 2800
WAVENUMBER CM "!
FIG. l--lnfrared absorption band of No. 2 Fuel Oil dissolved in Freon 113 (0.034% w/v),
using lO-mm path length silica cells; Freon 113 is in the re(erence beam. Absorbance at
2930/cm is determined as the d(fference between Points A and B.
The utility of IR absorptivities as a parameter for identifying weathered

oils was also briefly investigated. Diverging slopes of Beer-Bouguer Law
plots illustrate differences in oil absorptivities. Eiomml~* values (absorb-
ances measured in 10-mm path length cells, normalized to 1 percent
weight/volume dissolved oil) of the oils were calculated and evaluated for
stability, following loss of volatile oil components as a consequence of
G R U E N F E L D ON A N A L Y S I S OF P E T R O L E U M OIL P O L L U T A N T S 295
100- (i) a (2)
90.
80-
Z
7o,
iz
~ 60.
so"
' 1
2930cm" 2930cm "1
5B
Absorbance = log~o5B + D -- C
B = % T of the unexpanded baseline

C = % T of the expanded baseline
D = % T of the expanded peak m a x i m u m
FIG. 2--1nfrared absorption band of No. 2 Fuel Oil dissolved in Freon 1i3: (1) without
ordinate scale expansion, and (2) with ordinate scale expansion •
solvent distillation (stripping). An oil solution was stripped free of solvent,

following IR measurement in 10-ram path length cells. The solvent was
discarded and the oil residue weighed. The residue was then redissolved,
diluted to the previous volume (100-ml), and the solution measured again
by IR. The new Et0~m~~ absorptivity was compared to the original value.
The solvent stripping procedure of the American Public Health Associa-
tion [2] was used.
A brief investigation was also made of the stability of oil absorptivities
following prolonged solution storage under normal room light and tem-
perature conditions. Oil solutions were prepared in carbon tetrachloride,
Freon 113, and in the 98 percent/2 percent solvent mixture. South
Louisiana Crude and No. 2 Fuel Oils were used; solution concentrations
were 2 and 10 mg/100 ml. Following the initial absorbance measure-
ments, the solutions were stored for eight days on an exposed laboratory
shelf, in clear-glass tightly sealed 100-ml volumetric flasks. The absor-
bance measurements were then repeated and compared with the original
values.
Results and D i s c u s s i o n s
The major purpose of this study was to assess the utility of IR for the
quantitative determination of petroleum oils, by single point analysis.

Solutions of four representative oils in two solvents, and in a mixture of
these solvents, were used to prepare the appropriate absorbance versus
concentration (Beer-Bouguer Law) plots. Quantitative determination by
single point analysis requires linear plots that pass through the origin. Use
of single point analysis usually offers a considerable time saving because
only one standard solution is used for each analysis, rather than a
Beer-Bouguer Law plot derived from several solutions. The term standard
solution designates a solution containing an accurately known concen-
tration of oil with the same identity as the dispersed oil in the water
sample. The following equation is used for single point analysis
ax
C~ = C~.
As
where
Cx = the unknown oil concentration of the sample extract used
for IR measurement;
AxandAs = the absorbances of the sample extract and standard
solution, respectively; and
Cs = the standard solution concentration used for IR measure-
ment.
The measurements were made in cells with identical path lengths, and
using identical ordinate scale expansion settings. The four oils selected for
study were thought to be fairly representative of viscous and nonviscous
crude and processed petroleum oils. The choice of solvents was determined
by the previously cited publications, and by consideration of oil solubility,
IR absorptivity, and solvent toxicity.
Carbon tetrachloride is used in the previously described IR methods by
the American Petroleum Institute [4], Beckman Instruments, Inc. [5], and
Atwood et al [8]. As previously indicated, this solvent is highly toxic.
Freon 113 is used in a gravimetric procedure by the American Public
Health Association [2], and is much safer than carbon tetrachloride.
Freon 113 is evaluated in the present study for IR measurement of oils.
Gruenfeld [6] compared the ability of these solvents to extract dispersed
oils from water. He found them equally effective for extracting small
quantities (less than 200 ppm) of water dispersed oils. Although Freon 113
is adequate for extracting low concentrations of water dispersed oils, it
does not readily dissolve (cut) undispersed viscous oils such as No. 6 Fuel
and Bachaquero Crude oils. Freon 113 is therefore not recommended for
preparing IR standard solutions of viscous oils. This problem is solved by
first dissolving a sufficient amount of viscous oil in carbon tetrachloride to
yield an accurately known concentration approximating 20 mg/ml. This
solution (2.0-ml) is then diluted to 100-ml with Freon 113, to yield a final
98 percent Freon 113/2 percent carbon tetrachloride mixture that contains
GRUENFELD ON ANALYSIS OF PETROLEUM OIL P O L L U T A N T S 297
approximately 40-mg oil. A fresh portion of the 98 percent/2percent

solvent mixture is used for further dilutions. These standard solutions will
be discussed further.
The oils used in the present study were dissolved directly in the solvents,
to yield solutions having accurately known concentrations. These solutions
simulate solvent extracts of actual dispersed oil in water samples. Beer-
Bouguer Law plots were prepared and evaluated for compliance with the
criteria for single point analysis; that is, plots that are linear and pass
through the origin. Solutions of the four oils in the two solvents and in the
solvent mixture were measured by IR. A range of concentrations was
examined in 10 and 100-mm path length cells, with and without ordinate
scale expansion. Linear plots that pass through the origin were obtained
for the four oils in carbon tetrachloride (Fig. 3) and in 98 percent Freon
1.1-
1.0-
0.9-
0.8-
0.7-
0.6-
Z
O
- o.s- KEY
9 NO. 2 FUEL OIL
9 NO. 6 FUEL OIL
BACHAQUERO CRUOE OIL
0.4- 9 SOUTH LOUISIANA CRUDE OIL
0.3
0.2
0.1
0
r" lb 2b 3b 4~ sb 6b
CONCENTRATION ( m g / l O O ml) OIL IN SOLVENT
FIG. 3--Oil solutions in carbon tetrachloride measured in lO-mm path length cells with-
out ordinate scale expansion.
113/2 percent carbon tetrachloride (Fig. 4), and for the two less viscous
oils in Freon 113 (Fig. 5). Number 6 Fuel and Bachaquero Crude oils
were not readily soluble in Freon 113. Therefore, they were dissolved
initially in carbon tetrachloride, and then diluted further with Freon 113,
by the previously described procedure, to yield a final 98 percent Freon
113/2 percent carbon tetrachloride solvent mixture. The potential absorp-
tivities of viscous oils in Freon 113 are thought to match their absorptivi-
ties in the solvent mixture, because Beer-Bouguer Law plots of the
nonviscous oils in Freon 113 yield slopes that match the slopes of these
oils in 98 percent Freon 113/2 percent carbon tetrachloride (Figs. 4, 5,
and 6). Preparation of standard solutions in 98 percent Freon 113/2
1.0-
0.9-
0.8-
0.7-
0.6
Z
0.5 KEY
9m 9 NO. 2 FUEL OIL
9 NO. 6 FUEL OIL
* BACHAQUERO CRUDE OIL
0.4 9 SOUTH LOUISIANA CRUDE OIL
0.3-
0.2-
0.1-
10 20 30 40 50 60
CONCENTRATION ( m g / I O 0 mi) OiL IN SOLVENT
FIG. 4--Oil solutions in 98 percent Freon 113/2 percent carbon tetrachloride measured in
lO-mm path length cells without ordinate scale expansion.
1.0-
0.9-
0.8-
0.7-
0.6- KEY
// m NO. 2 FUEL OIL
UISANA CRUDE OIL
O.S-
0.4-
0.3-
0.2-
0,1-
lb 2b 3'0 4'0 s'0 go

CONCENTRATJON (mg/lOO ml) OIL IN SOLVENT
FIG. S---Oil solutions in Freon 113 measured in lO-rnm path length cells without ordinate
scale expansion.
percent carbon tetrachloride is recommended whenever Freon 113 is used

as the extraction solvent for quantitating dispersed oil in water samples.
Use of IR for the quantitation of water dispersed oils at concentrations
below 1 ppm, by single point analysis, was also examined. Appropriate
Beer-Bouguer Law plots were derived from measurements in 100-mm path
length cells without ordinate scale expansion, using solutions of the four
oils in carbon tetrachloride (Fig. 7), in 98 percent Freon 113/2 percent
carbon tetrachloride (Fig. 8), and solutions of the two less viscous oils in
Freon 113 (Fig. 9). While some of the plots exhibit deviations from
linearity above 0.7 absorbance, all the plots are linear below this value
and pass through the origin. In some cases, portions of an oil used for
I.O
0.9
0.8
0.7-
0.5 ~ KEY
~O / u FREON-I13
0.3
0.2
0.1"
110 210 310 4'.0 5'.0 6'.0

CONCENTRATLON ~g/100 m~) Oft. iN SOLVENT
FIG. 6--Solutions of No. 2 Fuel Oil measured in lO0-mm path length cells without
ordinate scale expansion.
measurements in 10 and 100-mm path length cells were inadvertently

taken from different drums. This caused the slopes of some plots deriving
from measurements in lO-mm path length cells (Figs. 3, 4, and 5) to
differ from slopes of plots deriving from measurement of the same oils in
100-mm path length cells (Figs. 7, 8, and 9). Use of Freon 113 or 98
percent Freon 113/2 percent carbon tetrachloride in 100-mm path length
cells results in a sluggish response of the IR instrument recorder pen;
Freon 113 has a higher absorptivity at 2930/cm than carbon tetrachloride.
The IR instrument gain setting should be increased sufficiently for these
measurements to yield a properly shaped absorption band at approxi-
mately 2930/cm (Fig. 1). Linear Beer-Bouguer Law plots that pass
1.0
/ /
0.9-
//
0.8-
//
0.7-
0.6-
/
/
/
/
i 0.5- KEY
9 NO. 2 FUEL OIL
9 NO, 6 FUEL OIL
9k BACHAQUERO CRUDE OIL
0.4- 9 SOUTH LOUISANA CRUDE OIL
0.3-
0.2-
0.1-
o i:o s 31o 41o s~o 6~o

CONCENTRATION (mg/100 ml) OIL IN SOLVENT
FIG. 7 - - 0 i l solutions in carbon tetrachloride measured in IO0-mm path length cells with-
out ordinate scale expansion.
through the origin were also obtained for measurements of the four oils in
carbon tetrachloride, using 10-mm path length cells with ordinate scale
expansion ><5 (Fig. 10). But, considerable deviation from linearity resulted
from the measurement of Bachaquero Crude oil in 100-mm path length
ceils with ordinate scale expansion ><5 (Fig. 11).
Accurate quantitative determination of water dispersed oils by single
point analysis can be accomplished in the concentration range 2 to 40
rag/liter (ppm) oil in water, by using 10-mm path length cells without
ordinate scale expansion (Figs. 3 to 5). This concentration range assumes
use of the extraction procedure by Gruenfeld [6], whereby l-liter oil in
water samples are extracted with four 25-ml portions of solvent. Improved
1.0.
0.9-
0.8-
0.7
0.6"
0.5' KEY
9 NO. 2 FUEL OIL
9 NO 6 FUEL OIL
BACHAQUERO CRUDE OIL
0,4 9 SOUTH LOUISIANA CRUDE OIL
0,3 84
0.2"
0,/
f.o 2'.0 3'.0 4'.0 s'.o f,o

CONCENTRATION (rag/T00 ml) OIL IN SOLVENT
FIG. 8---Oil solutions in 98 percent Freon 113/2 percent carbon tetrachloride measured in
lO0-mm path length cells without ordinate scale expansion. A higher than normal IR instru-
ment gain setting was used.
1.0
0.9.
0.8 9 9
0.7 ~
0.6"
<
0.5" KEY
/ J NC~2 FUEL O'L

LOUISIANA CRUDE O'L
0.4
0.3
0.2
0.1'
1:o s 3'.0 4'.0 s'.o 61o

CONCENTRATION (rng/lO0 rnl) OIL IN SOLVENT
FIG. 9 - - 0 i l solutions in Freon 113 measured in lO0-mm path length cells without or-
rlinate scale expansion. A higher than normal IR instrument gain setting was used.
sensitivity can be achieved by using less solvent. Accurate quantitation of

oils in the concentration range 0.5 to 3 ppm oil in water can be achieved
by single point analysis when using 100-mm path length cells without
ordinate scale expansion (Figs. 7 to 9), or 10-ram path length cells with
ordinate scale expansion x5 (Fig. 10), The measured absorbances in
100-ram path length cells should zzot exceed 0.7. Use of 100-ram path
length cells with ordinate scale expansion ><5 yields nonlinear Beer-
Bouguer Law plots (Fig. 11). While accurate quantitation of oils by single
point analysis is not possible under these conditions, useful information is
gained about the detection limit of the IR technique. These measurements
0.10 -
0.09 -
0.08"
0.07'
0.06
0.05-
0.04 - KEY
9 N O , 2 FUEL OIL
9 N O . 6 FUEL OIL
.it 6 A C H A Q U E R O CRUDE OIL
0.03 - 9 S O U T H L O U I S I A N A CRUDE O I L
0.02 -
0.01 9
o l'o 2:o 3'0 4:0 s'o 6:0

CONCENTRATION (rag/100 m l ) OIL I N S O L V E N T
FIG. lO---Oil solutions in carbon tetrachloride measured in lO-mm path length cells with
ordinate scale expansion •
0.11 "1
0.t0
0.09.
0.08.
0.07
0.06
z
==
o
0,05
0.04
0.03
0.02
0.01
0 0~, 0:2 0'.3 0:4 o'.s 0:6 o7

CONCENTRATION (mg/lO0 ml) OIL IN SOLVENT
FIG. 11--Carbon tetrachloride solutions o f Bachaquero Crude Oil measured in lO0.mm

path length cells with ordinate scale expansion x5.
yield a recognizable absorption band at approximately 2930/cm (Fig. 1)

for an oil solution containing 0.05 mg/100 ml oil in solvent. This is
equivalent to 0.05 ppm dispersed oil in water when the extraction
procedure by Gruenfeld [6] is used. This detection limit of the IR
technique can be improved further by reducing the volume of extraction
solvent.
Consideration of the Beer-Bouguer Law plots demonstrates that the
lines diverge and, therefore, that the oils have different absorptivities.
These absorptivities appear to remain reasonably stable, despite loss of the
more volatile oil components (Table 1). They are solvent dependent,
however, as demonstrated by the diverging Beer-Bouguer Law plots that
1~0.
0.9.
0.8'
0.7-
0.6-
~ 0.5"
m KEY
/~/ 9 IN CARBON TETRACHLORIDE
FREON-113
0.4"
0.3
0.2"
O.1
lb ~'o 3'0 4'o s'o 6'0

CONCENTRATION (mg/lOO ml) OIL IN SOLVENT
FIG. 12--Solutions o f No. 2 Fuel Oil measured in lO-mm path length cells without
ordinate scale expansion.
1.O
0.9"
0.8-
0.7-
0.6"
O.5- KEY
m /i e ]N CARBON TETRACHLOR,DE
EON 113
0.4-
0.3-
0.2-
0+1-
;~ 2'o 3'o 4'o s'o +'o

CONCEN'[RATION (mg/100 ml) OIL IN SOLVENT
FIG. 13---Solutions of South Louisiana Crude Oil measured in lO-mm path length cells
without ordinate scale expansion.
are obtained from the same oil in different solvents (Figs. 12 and 13): No.
2 Fuel and South Louisiana Crude oils yield absorptivities in Freon 113
that differ from their absorptivities in carbon tetrachloride. These
differences are also apparent when comparing Figs. 3 and 4, and others.
IR absorptivities are thus a promising parameter for "passive tagging" oils
because they differ from oil to oil, and yet remain reasonably stable. Their
solvent dependence may also be useful for distinguishing among similar
oils. The term "passive tagging" describes a procedure whereby a
weathered oil residue, that is, an environmental pollutant, is correlated
(matched) with an unweathered portion of the same oil.
The stability of solution absorbances, and therefore oil absorptivities,
TABLE 1--Effect of solvent distillation on oil absorptivity. (No. 2 Fuel and South Louisiana
Crude Oils are in Freon 113 solution; No. 6 Fuel and Bachaquero Crude Oils are in 98%
Freon 113/2% carbon tetrachloride solution).
Before Distillation After Distillation a
Oil in Solvent Absorptivity b % Loss Absorptivity %

Oil (mg/100 ml) (El0 mm 1%) (by weight) (El0 mml%) Change
No. 2 Fuel 104 21.4 11.5 21.1 1.4

14.5 23.0 19.6 8.4
South Louisiana 101 22.4 3.0 21.6 3.6
Crude 10.5 14.3 21.7 3.1
No. 6 Fuel 10.6 23.8 0.0 23.8 0.0
Bachaquero 103 16.1 4.4 15.3 5.0
Crude
a Solvent distilled (stripped) from 100-ml solutions having known oil content, using the
procedure of the APHA [2]. The weighed residues are rediluted to 100-ml and the solution
absorbances measured at 2930/cm.
bE10 mm 1% values are absorbances at 2930/cm, measured in 10-ram path length cells,
normalized to 1% (weight/volume) dissolved oil.
following p r o l o n g e d solution storage was also e x a m i n e d . Oil solutions in

c a r b o n t e t r a c h l o r i d e , F r e o n 113, and 98 p e r cen t F r eo n 1 1 3 / 2 p e r c e n t
c a r b o n t e t r a c h l o r i d e were stored u n d e r n o r m a l r o o m light an d t e m p e r a -
ture conditions, in c l e a r glass flasks for eight days. T h e a b s o r b a n c e s
following storage m a t c h e d those prior to storage, within 1 percent. P r o m p t
solvent e x t r a c t i o n o f dispersed oil in w a te r samples is t h er ef o r e recom-
m e n d e d . Oil t r a n s p o r t and storage in c a r b o n t e t r a c h l o r i d e or F r e o n 113
solution should p r e v e n t biological d e g r a d a t i o n and evaporative losses t h a t
can occur in the water m a tr i x .
References
[1] Jacobson, S., personal communication, Woods Hole Oceanographic Institution, Woods
Hole, Mass., 7 June 1972.
[2] Standard Methods for the Examination of Water and Wastewater, 13th ed., American
Public Health Association, New York, 1971, pp. 254-256.
[3] Harva, O. and Somersalo, A., Suornen Kemistilehti, Vol. 31(b), 1958, pp. 384-387.
[4] Manual on Disposal of Refinery Wastes. Vol. IV, Method 733-58, American Petroleum
Institute, 1958.
[5] Infrared Application Note 68-2, Beckman Instruments, Inc., Mountainside, N.J., 1968.
[6] Gruenfeld, M., Environmental Science and Technology. Vol. 7, 1973, pp. 636-639.
[7] Sax, I. N., Dangerous Properties oflndustrial Materials, 3rd ed., Reinhold, New York,
1968, pp. 535, 1192.
[8] Atwood, M. R., Hannah, R. W., and Zeller, M. V., Infrared Bulletin No. 24, The
Perkin-Elmer Corp., Norwalk, Conn. 1972.
O:J
~ m
O
m
O
CQ
m a
m l
N. H. F. Watson, j G. F. Carpenter,2 a n d M . M u n a w a r I
Problems in the Monitoring

of Biomass*
REFERENCE: Watson, N. H. F., Carpenter, G. F., and Munawar, M., "Problems

in the Monitoring of Biomass," Water Quality Parameters, A S T M STP 573, American
ABSTRACT. Two general approaches for the estimation of standing crop for biomass
determinates have been attempted in fresh waters. One involves identification and
enumeration of organisms and subsequent extrapolation to biomass from volumetric or
weight factors. Several direct methods are available for determining biomass, all of
which give results affected by sample size but do not discriminate between types of
organisms present. Some examples from current Great Lakes research are presented.
KEY WORDS. water quality, biomass, aquatic biology, environmental tests, nekton,
plankton
P e r h a p s t h e m o s t visible i m p a c t of t h e e u t r o p h i c a t i o n o f a w a t e r b o d y is
the increase in b i o m a s s ( m a t e r i a l o f living origin). This a c c u m u l a t i o n has
resulted from i n c r e a s e d rates o f p r o d u c t i o n associated with i n c r e a s e d
p h o t o s y n t h e s i s o f algal stocks. M a n y o f the adverse consequences o f c u l t u r a l
e u t r o p h i c a t i o n in lakes (algal b l o o m s , h y p o l i m n i a l oxygen depletion) c a n be
t r a c e d directly to this a c c u m u l a t i o n o f algal biomass. Therefore, the
c u m u l a t i v e progress o f e u t r o p h i c a t i o n m a y well be d e s c r i b e d by a p e r i o d i c
survey o f s t a n d i n g stocks.
W h i l e it m a y be a r g u e d effectively t h a t biological water quality p a r a m e t e r s
should include some m e a s u r e of the type o f o r g a n i s m s p r e s e n t as well as the
q u a n t i t y , it is our c o n t e n t i o n t h a t m u c h c a n be inferred a b o u t the c h a n g i n g
t r o p h i c status of lakes from p e r i o d i c surveys o f the s t a n d i n g crop b i o m a s s .
*This paper was sponsored by C.I.C., Analytical Chemistry Division, and Environment
Canada, Canada Centre for Inland Waters, Burlington, Ontario L7R 4A6, Canada.
t Great Lakes Biolimnology Laboratory, Fisheries and Marine Service, Environment
Canada, Canada Centre for Inland Waters, Burlington, Ontario L7R 4A6, Canada.
2Industrial Biotest Laboratories Inc., Northbrook, Ill. 60062.
311

This paper will deal with the consideration of approaches to the determi-
nation of adequate biomass measures and will suggest field and lab
procedures which will ensure practical achievement of this goal.
Blomass Estimation
There are several problems associated with achieving an adequate biomass
estimate. Most of these are inherent in the distribution of living organisms in
the lake ecosystem. Some types of biomass estimation currently in use are
difficult to convert from one type of unit to another. The whole load of living,
moribund, and dead organisms produced in the water column, washed in
from the drainage basin, plus resuspended sediment is referred to as seston.
This material is particulate in nature, although a considerable amount of
colloidal or dissolved organic material may be produced by the living
material.
The size of this biological particulate material ranges from less than a
micron for the smallest algae and bacteria up to the largest free swimming
organisms called nekton. This nekton fraction in fresh waters is largely made
up offish.
The suspended semi-motile portion (termed plankton) still covers several
orders of magnitude of linear dimension. Some of the animal members of
this plankton assemblage possess considerable sensitivity to light and current
and can avoid many types of gear more or less effectively. However, most
members of the planktonic assemblage, whether plant or animal, appear to
be distributed in a non-random clumped manner when distribution is
examined in either a horizontal or vertical sense. The fine-structure of
horizontal patchiness is not well-known in large lakes, but evidence for
vertical microstratification is available from optical density data (Fig. 1). The
example is from small lake studies by Baker and Brook [1],3 but we have
used similar transmissometer curves on a recent Lake Superior Cruise to
locate concentrations of seston in the epilimnion and in the lower part of the
thermocline.
Samples taken for biomass estimates must therefore be taken in such
quantity and in such a pattern as to accommodate this spatial variability.
Estimates of abundance and variability suggest sample sizes of approxi-
mately 500 liters may be necessary for adequately sampling zooplankton;
phytoplankton samples require considerably smaller samples.
Procedure
It is perhaps appropriate here to describe the types of procedures which
are currently used to assess biomass. They are of two major types: estimates
from counts or from assays of constituents and direct biomass deter-
minations.
WATSON ET AL ON M O N I T O R I N G OF B I O M A S S 313
0i 10 20 TEMP ~
n
0 1.0 2.0 O.D. co

I , _ @-
o.g.
'~
5-
E
"1-
iii
D
10-
./... Predominantly
OSCILLATORIA
REDEKEI
15-
/J
vv
FIG. 1--An example of vertical microstratification of phytoplankton demonstrated by
optical methods and confirmed by spot sampling from Baker and Brook [1].
Many plankton examinations have been designed to identify the numbers

of individuals in component species of phytoplankton or zooplankton
populations. Where counts of subsamples have been deemed representative
of the sample, attempts have been made to convert numbers to biomass by
transforming numbers to cell volume for phytoplankton or to dry weight
biomass for zooplankton. These operations involve assigning each cell type to
a simplified geometric shape, and making the appropriate measurements to
define volumes assuming a density of 1.0 or slightly greater, or directly
estimating dry weights from weights of individual organisms. It is obvious
that the accuracy of such methods is limited by the ability to choose adequate
geometrical shapes to describe the algal cell (Table 1), accurate linear
measurements of cells, adequate dry weight estimates of organisms, and
above all, to an adequate estimate of numbers in the plankton sample.
Several methods exist for determining plankton biomass estimates based
on the assay of some particular compound found in the sample. Thus,
chlorophylls have been used as a measure of phytoplankton biomass:
chlorophyll a because of its role as a universal photosynthetic transducer of
light energy, chlorophyll b in the aquatic system restricted to green algae,
and chlorophyll c in other algal groups. Breakdown products such as
TABLE l--Examples o f alternative computations o f cell volume using different approxima-

tions to simple geometric shapes for the same cells. Computations are based on the
same linear dimensions.
Subjective Volume Selected

Geometric Computed After Comparing Actual
Species Simulation ~3 Volume Shape to Geometric Shape
Scenedesmus b(iuga 2 cylinders 72 48
Scenedesmus bijuga 2 prolate
spheroids 48 .. -
$. bicellulaHs 2 cylinders 42 28
S. bicellularis prolate speroid 28
Chlamydomonas globosa oblate spheroid 250
C. globosa prolate 294
spheroid 294
phaeophytins and phaeophorbides also have been measured and their

concentrations attributed to the degredative process of digestion after
grazing [2].
Astaxanthin concentration has been suggested as a measure of zooplank-
ton (especially copepod) biomass in the plankton and sediments of Lake
George, N.Y. [3]. Several authors have recently assayed the amount of l i v i n g
biomass by t h e concentration of ATP or various enzyme systems in
particulate fractions [4].
Most of these methods use some form of optical density measurement
usually at one or more wavelengths on specifically extracted particulate
samples. While it is true that very low concentrations of specific compounds
can be detected and quantified in such assays, the fact remains that these
parameters are of doubtful value as biomass measures, and only give relative
biomass information because there is often a tenuous relationship, if any,
between the concentration of the parameter and biomass measures such as
dry weight. Concentrations of these substances change relative to biomass
because of physiological changes in the concentrations of these compounds in
organisms, or changes in species composition of the seston. Thus,
Glooschenko et al and other investigators [5,6] report diel changes in
chlorophyll a content of the seston. We can demonstrate, for 1970 data,
seasonal differences between the surface chlorophyll content of Lake Ontario
and estimates of phytoplankton biomass derived from counts and volume
conversions (Fig. 2). There is a reasonably close agreement between
chlorophyll a and estimated phytoplankton biomass during the spring
months when diatoms predominate in the samples. On the summer cruises,
chlorophyll a estimates were low while phytoplankton biomass was maximal.
At that time species composition was different, with large numbers of green
and blue-green algae present. Without better direct measures of biomass, it
is impossible at this stage to assess any of these estimated methods very well.
WATSON ET AL ON MONITORING OF BIOMASS 315
FIG. 2--Seasonal distribution o f phytoplankton biomass estimated from counts and vol-
ume conversions, chlorophyll a, primary productivity, and composition of phytoplankton
.from Lake Ontario, 1970. (Biomass and species composition from Munawar and Nauwerek
[5]; chlorophyll a from Glooschenko et al [2]; and primary productivity from Glooschenko
et at [6].)
Direct measures of assessing the weight of seston in the water columns

have been employed for a considerable period of time. Birge and Juday [7]
reported on the particulate dry weight and ash-free dry weight of Wisconsin
lakes in the 1930's, and Rawson [8] reported on the standing crop of net
plankton in these biomass units. Questions have been raised over the
sampling methods of these early attempts (pump and centrifuge, net hauls).
Pumps have been repeatedly shown to destroy some plankton organisms
passing through their impellers, and larger organisms have been shown to
evade the inlet currents while centrifugation may not be an adequate method
of sedimentation. Net hauls may strain all the zooplankton from the water
column sampled, but only part of the phytoplankton samples will be retained
in the net. A variable unknown fraction of smaller organisms, the
nannoplankton, will pass through the net.
Membrane and glass fiber filters have been devised with pore sizes to
effectively trap most biological organisms. These filters with effective pore
diameters down to 0.45/,an have been introduced in limnological sampling of
particulate matter over the past 20 years, especially for the study of primary
production rates with C 14. Concomitantly, they have been used in the
measurement of chlorophyll a concentration where most workers now use
glass fiber filters. Robertson and Powers [9] have weighed glass fiber filtered
samples to quantify particulate matter in the Great Lakes. However, they
removed zooplankters from the filters so their values are for phytoplankton
biomass only.
Similar filtered samples have been routinely analyzed as part of the water
quality surveillance program at C.C.I.W. for particulate phosphorus,
nitrogen, and carbon from specific depths by either pump or bottle cast for
several years. Most of these samples have been small, 300 ml or so, and have
not taken into consideration a complete vertical profile of the water column,
and probably represent values for only part of the planktonic seston because
of the size of the samples.
In addition, the Great Lakes Biolimnology Laboratory (formerly Fisheries
Research Board Detachment) has for several years undertaken intensive
biological surveys of the species composition and abundance of zooplankton
and phytoplankton in the Great Lakes. Additionally, we have made surveys
of chlorophyll concentrations at different times of year. Since one of the
ultimate aims of this work has been to develop effective methods for biomass
surveys, attempts have been made to report the data in biomass as well as
numerical units. In addition to the portion of the net haul in which
zooplankton was identified and counted, aliquots were filtered on glass fiber
filters, dried and ashed. Duplicates were also taken for analysis of particulate
carbon or chlorophyll a concentration. For each sample, there were estimates
of crustacean zooplankton concentration, ash-free dry weight, and chloro-
phyll a concentration [10]. Numbers were converted to biomass using
estimates of the weights of individual organisms to give a value for total
crustacean biomass, and relations between ash-free weight and particulate

carbon were derived (Fig. 3).
Our biomass estimates are not ideal for several reasons, although the
information available is useful and consistent irternally (Fig. 4).
The plankton net biomass does not give a total biomass, because it
neglects the contribution of the variable amounts of nannoplankton which
passed through the net. We can estimate the relative changes in this from the
ratio of plankton net chlorophyll a to total chlorophyll a determined for each
cruise. However, our phytoplankton biomass estimates expressed as chloro-
phyll a concentrations are relative rather than absolute, so that it is
impossible to calculate a total biomass figure or partition the net biomass
directly into phytoplankton and zooplankton fractions.
These preliminary observations have at least given us a feeling for the types
of problems involved in sampling biomass, and we would like to suggest on
the basis of our past experience a system of sampling and analysis which we
feel will provide adequate estimates of total biomass concentrations in the
150
t00
E
Z
O
rn
rr
<
O
50
" r = 0 860
Syx : 16.80
I I I I I l
0 510 100 150 200 250 300 350
LOSS OF WEIGHT ON IGNITION mglm 3
FIG. 3--The relationship between ash-free dry weight and particulate carbon in a series
o/" samples from central Lake Erie, 1971.
~E 100 1000
o~
E PLANKTON NET BIOMASS co
(ASHFREE DRY WT.) ~ E
~ 10 100 E
I co
O / J \ C~-U.S~T.A.gEAN N
O~ 1.0- 10
o
t-
uJ
z
0.1 ! t J I I I I ~1 n I I 1
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
MONTHS
FIG. 4--A comparison of plankton net dry weight, estimated crustacean dry weight
(from counts x weight conversion factors), and plankton net chlorophyll from Lake Ontario,
1970.
water column and estimates of the component zooplankton and phyto-

plankton fractions.
The method involves taking pairs of samples at each station. One of each
pair should be a vertical haul with a plankton net of sufficient diameter and
length to ensure efficient straining of the water column. The second sample
should be made to the same depth as the plankton haul with an integrating
water sampler. At C.C.I.W., we have successfully used a sampler similar to
the one described by Schr6der (Ref 11, Fig. 8) to obtain integrated samples
to a depth of 20 m and are working on one which should sample down to
50 m.
A suggested protocol for handling these samples is as follows:
Measured portions of both these samples should be filtered on fiberglass
filters (large zooplankton should be picked off integrator filters), dried in a
dessicator, and analyzed for ash-free dry weight or particulate carbon. In
addition, a fraction of the integrator sample should be passed through a seive
of the same mesh size as the plankton net used, and each fraction dried on a
G F / C filter to estimate net and nannoplankton concentrations. Biomasses
should be expressed as concentrations (per cubic meter), although net and
integrator dimensions would allow areal abundance (per square meter)
units as well. Addition of the nannoplankton values to plankton net biomass
would allow a total biomass to be calculated, and subtraction of net
phytoplankton values from the plankton net value would produce a
zooplankton biomass value.
The quantities of material collected in the samples should be sufficient for

the performance of duplicate analyses or alternative use of the samples, such
as preservation for later identifications or counts and chlorophyll a or other
assays. Other major advantages of this procedure are that a large enough
sample is taken to overcome the effects of horizontal and especially vertical
microdistributions o f planktonic organisms and that the subsampling and
analysis procedures should allow absolute values o f total biomass to be
obtained within defined limits o f sampling and analysis error. T h e methods
involved are comparatively rapid machine analyses giving direct values rather
than laborious counting techniques. We hope to refine the scheme in the next
year and to integrate it into a continuing surveillance p r o g r a m to provide
basic information on the quantity of biological material in the Great Lakes.
Acknowledgments
The authors wish to acknowledge the direction and continuing interest of

R. A. Vollenweider and A. Nauwerck in the early stages of this investigation
and the assistance of L. Devey and I. F. M u n a w a r in the identification and
counting of many of the zooplankton and phytoplankton samples.
References
[1] Baker, A. L. and Brook, A. J., Archiv Fiir Hydrobiologie, Vol. 69, 1971, pp. 214-233.
[2] Glooschenko, W. A., Moore, J. E., and Vollenweider, R. A., Limnology and Ocean-
ography, Vol. 17, 1972, pp. 597-605.
[3] Pierce. A. C., thesis, Department of Biological Science, State University of New York,
Albany, 1972.
[4] Holm-Hansen, O., Limnology and Oceanography, Vol. 14, 1969, pp. 740-747.
[5] Munawar, M. and Nauwerck, A., Proceedings, 14th Conference on Great Lakes Re-
search, International Association for Great Lakes Research, 1971, pp. 69-78.
[6] Glooschenko, W. A., Moore, J. E., Munawar, M., and Vollenweider, R. A., Journal of
the Fisheries Research Board of Canada, Vol. 31, 1974, pp. 265-274.
[7] Birge, E. A. and Juday, C., Ecological Monographs, 1934, pp. 440-474.
[8] Rawson, D. S., Journal of the Fisheries Research Board of Canada, Vol. 10, 1953,
pp. 224-237.
19] Robertson, A. A. and Powers, C. F., Special Scientific Report, Great Lakes Research
Division, University of Michigan, Vol. 30, 1967, pp. 1-8.
[10] Watson, N. H. F. and Carpenter, G. F., Journal of the Fisheries Research Board of
Canada, Vol. 31, 1974, pp. 309-317.
[11] Schr~Sder, R., Archly Fiir Hydrobiologie, Vol. 66, 1969. pp- 241-243.
T. C. H u t c h i n s o n ~ a n d P. M . S t o k e s ~
Heavy Metal Toxicity and

Algal Bioassays
REFERENCE: Hutchinson, T. C. and Stokes, P. M., "Heavy Metal Toxicity and Algal
Bioassays," Water Ouality Parameters, ASTM STP 573, American Society for Testing
ABSTRACT: Algal bioassays for heavy metals can detect low levels in the environment,
for example, 0.01 ppm for silver. Algae respond to increasing levels of heavy metals
such as copper, nickel, mercury, silver, or cadmium by reduction of growth rate.
Occasionally. the response to nontoxic metals is an increase in growth rate. At very
low concentrations some potentially toxic metals may be necessary micronutrients. Algal
species differ quite markedly in their sensitivity to heavy metals.
Combined effects of two or more metals at toxic concentrations may he synergistic
(for example, copper-nickel) or antagonistic (for example, cadmium-selenium). The
critical concentrations for toxicity of a particular metal may be different at different
times during the growth of an algal culture, as welt as being dependent upon other
chemical and physical conditions. Algal cells appear to markedly concentrate metals
from solution, even at concentrations of these metals in the medium which do not
apparently inhibit cell division. Bioassays provide the only direct method for assessing
the biological availability of metals in solution. Algae isolated from metal-polluted lakes
appear to have evolved specific metal tolerances. These "tolerant" algae actually accu-
mulate more of the metals concerned than do their "nontolerant" relatives. Correlations
between fish toxicity tests and algal bioassays may allow the relatively expensive fish
testing schemes to be replaced by simple and cheaper algal bioassays.
KEY WORDS: water quality, algae; bioassay, heavy metals, environmental tests
O f t h e v a r i o u s p a r a m e t e r s to be c o n s i d e r e d in a d i s c u s s i o n o f w a t e r
q u a l i t y , M c K e e a n d W o l f [1] 2 d i s t i n g u i s h b e t w e e n criteria, objectives,
requirements, a n d standards. W h i l e t h e l a t t e r t h r e e i m p l y v a r y i n g d e g r e e s o f
d e f i n i t e rules, r a n g i n g f r o m t h e a i m s u n d e r ideal c o n d i t i o n s i m p l i e d in
o b j e c t i v e s , to t h e a b s o l u t e n e s s o f t h e s t a n d a r d , t h e t e r m criterion s i m p l y
designates a means by which a judgment can be made. When data are being
a c c u m u l a t e d to serve as y a r d s t i c k s o f w a t e r q u a l i t y w i t h o u t r e g a r d for legal
a u t h o r i t y , t h e t e r m " c r i t e r i o n " s e e m s m o s t a p p r o p r i a t e . I t is in t h i s c o n t e x t
Associate director and professor of Botany, and associate professor of Botany, respective-
ly, Department of Botany and Institute of Environmental Studies, University of Toronto,
Toronto, Ontario MSS IA4, Canada.
320

HUTCHINSON AND STOKES ON HEAVY METAL TOXICITY 321
that algal bioassays for heavy metals will be discussed.

It is our belief that living organisms provide a very sensitive index of their
environment and that their responses to their environment can be intelligen-
tly interpreted. Basically, bioassays utilize the measurable response of living
organisms to environmental variables, including their chemical and physical
surroundings. When a substance is toxic, the most usual expression of
toxicity is in terms of (1) minimal lethal dose (MLD) which designates the
minimum concentration necessary or observed to kill one or more of the test
species, or (2) tolerance limit median (TLm) which is the concentration
which will kill S0 percent of the tested organisms. In both of these
parameters, the time limit has to be specified and is rarely more than 96 h.
Other environmental conditions need also to be specified and perhaps
standardized, but regrettably this is rarely done. Such variables as pH,
temperature, diurnal fluctuations in light, etc., would be included here.
Contamination of aquatic environments by heavy metals occurs from a
number of sources including specific industrial effluents, mine streams,
agricultural run off, deposition of airborne particles, and solubilization of
existing metal in rocks or sediments by acidification of waters, for example,
by sulfur dioxide (SO2) solution from industrial sources. Many of the metals,
especially the heavy metals, are known to be toxic, that is, poisonous. The
sublethal but damaging doses of these are ill defined. Acute versus chronic
toxicity is not often distinguished, and standardized interpretable yardsticks
for assessing concentration in different systems are lacking. Studies in our
laboratories have indicated that in certain situations heavy metals can be
assayed by unicellular green algae, but that results of such assays cannot be
simply extrapolated to field situations, and in some instances reduction of
growth is not an adequate indication of danger levels. Perhaps this paper will
introduce more problems than it solves, but at least it is hoped that it will
establish the value of a bioassay approach and its complexity and stress that
technical solutions can and should be sought.
Experimental Details
A field situation with which we are familiar is the presence of mines and
smelters close to a number of small lakes in the Sudbury, Ontario, region
[2, 3]. Water from five of the lakes, four of them on a transect running out
from the Coniston smelter, was tested for its effect on the growth of four
species of algae.
Assays of Lake Water

The water samples were collected in June and September of 1970 in 2S-liter
acid-washed polyethylene Carboys. The water was taken from random points
around the lake edges. The samples were then taken to the laboratory and
stored at 5~ until the bioassays were run. pH and conductivity data were
recorded very shortly after the sampling.
For the bioassays four unicellular green algae were selected for use, chosen
to provide a range of algal types and because of their differing growth
characteristics. They were taken from the botany departmental culture
collection and comprised Chlorella vulgaris, Scenedesmus acuminata,
Haematococcus capensis, and Chlamydomonas eugametos.
The algae were raised in stock cultures and transferred to fresh nutrient
solution for 8 days prior to an experiment; so that they were in logarithmic
phase when introduced to the metal-containing flasks. Experiments were run
using the algae in the lake waters alone, with no additions, to test the ability
of these waters to support growth under controlled environmental conditions
in growth chambers. Repeat experiments were performed with the pH of all
cultures adjusted to 6.8 prior to commencement of the bioassays, to
determine the differences between the lake water in supporting growth in the
absence of inherent pH differences. A third series was run in which the pH
was adjusted to 6.8 and a nutrient medium was added, to eliminate both pH
effects and nutritional difference between the lake waters. The assumption
was that excess nutrients would be then available for the short-term growth
studies. Nutrients such as phosphate and nitrogen would not then be
limiting. A modified Bold's Basal Medium (BBM) was used for this purpose
at one-tenth recommended strength. The modification was that no EDTA
chelate was added, so that comlflications caused by the presence of
introduced organic chelates would be minimized. Iron was added as ferric
sulfate at 0.05 mg/liter. Heavy metals were omitted from the micronutrient
solutions and boron was added as H3BO 3 at 0.05 mg/liter (Table 1). In a
fourth series of experiments the lake waters were bioassayed using nutrient
additions only, but without adjustment of the pH. This examined the
potential toxicity of the lake waters in the absence of nutritional stress, but
with the original pH differences intact. All bioassays were carried out using
three replicate 125-ml Erlenmeyer flasks under standardized conditions.
Experiments were run from 10 to 14 days, depending upon the algal species.
Further details are given in Ref4.
Assays of Heavy Metal Toxicity

The four test species just described were used in bioassays designed to
examine the relative toxicity of various heavy metals under controlled
conditions. These bioassays were carried out using synthetic media, that is,
BBM nutrient media at one-tenth normal strength, with ferric sulfate as an
iron source, and with heavy metals omitted. The metals were introduced as
inorganic salts, either as the nitrate or the sulfate. In the case of lead, both
nitrate and acetate were used. For each metal a range of concentrations was
used. Levels of O (control), 0.05, 0.1, 0.5, 1.0, and 5.0 ppm were used in a
TABLE 1--Growth media used in experiments.
"Complete" Medium for Inoculum "Modified" Medium for

and General Maintenance of Culture Growth Experiments
Macronutrients, mg/liter
NaNO3 250 250
KHzPO4 175 175
CaCI22HzO 25 25
MgSO47HzO 75 75
K2HPO4 75 75
NaCI 25 25
Chelating Agent
EDTA 50 none added
Semi-micronutrients, mg/liter
FeSO47HzO 4.98 (Fe -----1.0 ppm) 0.25 (Fe = 0.05 ppm)
H3BO4 11.42 (B = 1.6 ppm) 0.57 (Bo = 0,08 ppm)
Micronutrients, mg/liter
ZnSO47HzO 8.82 (Zn = 2 ppm) none added
MoO3 0.71 (mo = 0.5 ppm) none added
Co(NO3)26H20 0.49 (Co = 0.08 ppm) none added
MnCI24H20 1.44 (mn = 0.35 ppm) none added
CuSO45H20 1.57 (Cu = 0.4 ppm) none added
Vitamins, ng/liter
Thiamin 0.1
Biotin 0.5
Baz 0.5
pH
Initial pH adjusted to 6,8 (after sterilization) with same as "complete"
0.1 NHCI or 0.1 NNaOH
first screening a n d t h e n these levels refined d e p e n d e n t u p o n results. T h u s , if

all growth ceased at c o n c e n t r a t i o n s between 0.05 a n d 0.1 p p m , t h e n a second
e x p e r i m e n t was performed u s i n g a narrower range, for example, 0.005, 0.01,
0.025, 0.05, 0.075, 0.1 p p m , etc. Very careful washing t e c h n i q u e s were used
in the p r e p a r a t i o n of these cultures to m i n i m i z e trace c o n t a m i n a n t s .
Distilled, deionized water was used to m a k e u p the m e d i u m . Growth was
recorded either after 10, 12, or 14 days or at 2-day intervals u s i n g
s u b - s a m p l e s . Duplicate samples were t a k e n from each of the three replicate
flasks. Initial inocula in all experiments were c o u n t e d a n d designed to b e g i n
at approximately 5/104 c e l l s / m l of culture. Metals e x a m i n e d i n c l u d e d
mercury, c a d m i u m , silver, copper, nickel, cobalt, selenium, lead, a n d b a r i u m .
The c o m b i n e d effects of two metals was e x a m i n e d in a n u m b e r of instances
to d e t e r m i n e whether interactions occurred, with relative increases or
decreases in toxicity over predicted values, t h a t is, synergisms a n d
antagonisms (or ameliorations) were assayed.
Cultures were grown at 1200 ftc, with diurnal temperature fluctuation of

20 to 25~ A 16-h light, 8-h dark day was provided. Flasks were shaken once
a day to prevent clumping of cells.
Algal Isolate from Metal Polluted Lakes

Water samples were collected from Baby and Boucher Lakes in June 1970,
and green and blue-green algae obtained after centrifugation. Algal cells are
quite uncommon in these two polluted lakes, so that large volumes of water
were required for screening. From these scarce algal isolates grown on
nutrient agar and in aqueous culture, two frequently occurring isolates were
chosen for further study. These were obtained in pure culture.
From Bably Lake the green unicellular alga Chlorella fusca Shihira et
Krauss vat. vacuolata Shihira et Krauss 1965 was selected (referred to as
Chlorella-lake strain), and from Boucher Lake Scenedesmus acutiformis var.
alternans Schroeder 1897 (referred to as Scenedesmus-lake strain). For
comparison, two commonly used laboratory strains of Chlorella and
Scenedesmus were used, that is, Chlorella vulgaris Beijerinck 1890
(Chlorella-laboratory strain) and Scenedesmtis acuminatus (Lag) Chodat
1902 (referred to as Scenedesmus-laboratory strain).
For isolation of the lake algae, Baby and Boucher lake water was collected
in acid washed Nalgene containers and brought to the laboratory within three
days. Fifty milliliters was then centrifuged at 2500 rpm for 10 min. The
supernatant was poured off and the l-ml residue was streaked onto agar
plates containing complete BBM. The plates were incubated in light until
growth was visible. Microscopic examination was made at this preliminary
stage. Individual colonies of algae were removed into fresh liquid media and
the procedure repeated until unialgal cultures were obtained. Bacteria were
eliminated by periodically plating on BBM-agar and removing single colonies
into liquid. Stocks were maintained on slants under low light at 25~
All growth experi~ments were carried out in 125-ml Erlenmeyer flasks with
50 ml of medium. Cultures were shaken once a day during sampling. The
temperature was maintained at 25~ with 1200 ft-c illumination, on a 16-h
light, 8-h dark cycle. Flasks were plugged with cotton and maintained in a
sterile condition.
Prior to an experiment the appropriate strain was inoculated from slants
into a flask of complete liquid medium. Before inoculation of each
experiment, the liquid culture containing potential inoculum was streaked
on agar plates to check for bacterial contamination. After 8 days of growth,
the cell concentration was determined. One milliliter of this inoculum was
then added to each experimental flask. The initial count in experimental
flasks at time 0 was between 0.03 and 0.06 x 106 cells/ml.
Counting--Cell concentrations in all experiments were determined by
counting in a Levy Ultra Plane haemocytometer 1/20-mm 2 by 1/10-mm
deep. Each treatment was run in triplicate and each flask was sampled every
48 h. Thus, mean values were determined from six counts.
Criteria for Growth---Cell number was routinely used as an estimate of
growth. Dry weight of washed cells (dried at 90~ was plotted against cell
number. During exponential growth there was a linear relationship between
cell number and dry weight. Subsequently, cell number ceased to increase,
while dry weight continued to rise for several more days. Over the range of
time used in these experiments, cell number was considered a reasonable
estimate of growth.
Lake Water--After preliminary centrifuging to remove particles, lake
water was filtered through a millipore membrane filter of 0.4S-tam pore size.
For supplemented lake water medium, the nutrients of the modified
medium were added to sterilized lake water.
Medium Used--Modified BBM, see Table 1.
Determination of Metal Levels in Harvested Algae--Preparation of Samples

for Atomic Absorption Analysis
The samples for analysis were washed, and dried in 3/8-in.-diameter test
tubes and then digested by means of a 1:1 nitric acid:perchloric acid mixture
(BDH Analar grade chemicals). One milliliter of acid was added initially per
sample and the samples were left to pre-digest overnight at room
temperature. Two more milliliters of acid were then added per sample and
these were then placed on a sand bath at 325~ for 5 h. Samples were then
allowed to cool overnight and diluted to 10 ml with distilled water in
volumetric flasks. Acid blanks were similarly prepared.
Atomic Absorption--Samples were analyzed with a Unicam SP 90A

(Series 1) atomic absorption spectrophotometer and a Perkin Elmer 165
Recorder. Westinghouse hollow cathod lamps were used. Copper was
analyzed at a wavelength of 324.75 and nickel at 232.0/am.

Bioassays of Lake Waters from Around Sudbury
The concentration of heavy metals in some of the lakes assayed is given in
Table 2. It is shown that concentrations of greater than 0.1 ppm copper
occured in Baby, Kelley, and Boucher Lakes and concentrations greater than
0.5 ppm nickel occurred in Alice, Baby, Boucher, and Kelley Lakes. These
are all levels which are highly detrimental to fish and have been recorded as
algicidal for several species [1, 5, 6, 7]. The lakes differed, however, in both
the absolute concentrations of each metal and also in their relative ratios.
Fuller details of the chemical composition of these lakes are given in Ref8.
Figures 1 and 2 show examples of the growth responses for selected algae
Sudbury Lake Waters -- Chlorello vulgaris
9 + BBM, pH adjusted to 6,8

L40
[] + BBM, not adjusted
[] -- BBM,
120
tO0
w
~ Bo
~ GO
40
2O
CONTROL BOUCHER ALICE BABY KELLEY LONG
FIG. 1--Histogram of the bioassays (growth) o f Chlorella vulgaris in lake waters collected
ttt,tlr S u d b u ~ . Data are shown .for natural lake water, lake water with the p H adjusted to
6.8. and bt lake water with supplemental nutrients.
TABLE 2--Concentration o f some heavy metals (ppm) and p H values f o r some o f

the lakes in the Sudbury area samples collected September 1970.
Lake pH Ca Cu Ni Ag Cd Fe
Boucher 6.7 19.0 0.13 2.53 0.002 0.020 0.31

Alice 6.3 11.2 0.06 6.36 0.001 0,006 0.23
Baby 4.05 3.6 0.52 2.65 0.003 0.003 0.28
Kelley 5.9 60.8 0.16 0.95 0.008 0.008 0.73
Long 6.6 S.0 0.03 0.12 0.037 .,. 0.30
in each of the lake waters. The hatched blocks (Fig. 1) represent growth in
unaltered lake water. Boucher Lake is close to the Falconbridge smelter (0.5
mile) and the remaining four lakes are a series at increasing distances from
the Coniston smelter. In all waters growth was severly reduced compared
with the controls grown in nutrient media (1/10 BBM). Long Lake, which
was the most remote from the smelters, gave the best growth, but this was
only 25 percent of control. The series with only nutrients added are shown as
the clear histograms. Performance was overall improved by the addition of
FIG. 2--Bioassays qf the growth qf.fbur algae in lake water .from Sudbury with the pH
adjusted to 6.8 and with supplementary nutrients.
nutrients but generally this was not a major improvement and did not
approach that of the controls (Long Lake 30 percent of control). The growth
in Alice and Baby Lakes, in particular, continued to be very poor. In a third
series pH was adjusted to 6.8 and nutrients added. The differences between
the lakes persisted, but Long Lake was now 95 percent that of the control.
Growth in Baby and Alice Lakes remained very poor. Clearly the major
check to growth in these lakes, except for Long Lake, is not one of nutrient
deficiency. The small but significant increase in growth in Baby Lake water
as a result of pH adjustment from 4.1 to 6.8 may suggest a partial
amelioration of heavy metal toxicity, since heavy metals such as copper and
nickel are less soluble, and therefore less available for uptake, at higher
pH's.
These initial experiments, then, indicate that the lake waters close to the
Coniston smelter are toxic to algal growth. The scarcity of the phytoplankton
flora in these lakes in the field appears to correlate with the bioassay studies.
Chemical analyses of the lake waters suggest possible causes, particularly
heavy metal toxicity. In addition to copper and nickel, levels of iron in two of
the lakes are also high. It should be noted that copper is used very commonly
as an algicide so that its potential inhibiting effects are considerable.
A comparison of species response to each of the lake waters when nutrients
were added and the pH adjusted is shown in Fig. 2. It is seen that the pattern
of response is distinctly species specific. Thus, Scenedesmus grew best of the
four species in Baby Lake and Chlorella the worst. In contrast, in Boucher
Lake Chlorella performed the best and Haematococcus the worst. In Kelley
Lake the differences in response between the species were most marked. It is
of interest that Kelley Lake is the only one of the five with both high heavy
metal concentrations and high organic content, via sewage input. For
Chlorella at least, the organic-metal complexes formed may well ameliorate
the heavy metal toxicity. The results of the species comparison suggest that
careful selection and study of a range of assay species may well allow quite
sensitive indicator species to be developed.
Response of the Algae to IndividuaI Metals

The response with time of Chlorella vulgaris to a range of copper
concentrations is shown in Fig. 3. Ceils continued to divide activity at 0.04
ppm copper, but those grown at 0.1 ppm and higher levels failed completely
within a week. At 0.2 ppm copper the cells appeared to be initially inhibited,
then showed slow growth, followed by final death. It is not apparent whether
the cells growing after 3 days were a few resistant ones or whether the entire
inoculum was initially inhibited. Examination of the cells revealed marked
anatomical changes and chloroplast disruption by the twelfth day.
Figure 4 shows comparative data for Chlorella grown in media-containing
silver. It appears that silver was even more toxic than copper. Cells were
unat~le to grow at 0.06 ppm and even at 0.01 ppm growth appeared to be
initially retarded.
Figures 5 and 6 show the growth of Chlorella in mercury and cadmium-
containing media, respectively. Both were highly toxic. Growth in mercury
appeared to be progressively reduced, with cessation at 0.2 ppm whereas for
cadmium (and for silver) there appeared to be more of a cut-off point. In
cadmium, all concentrations above 0.05 ppm had an inhibitory effect. Lead,
both as inorganic nitrate and organic acetate, was nontoxic. Growth was, in
fact, somewhat stimulated at concentrations up to 10 ppm lead. However,
there are severe problems in keeping lead in solution at 10 ppm and above in
the absence of a chelate, which complicates the picture.
Using a second alga, Scenedesmus, as an example, Fig. 7, shows growth in
nickel solution. Nickel, for Scenedesmus, was less toxic than copper, or
indeed any of the previously considered metals. Growth ceased at 0.5 ppm
but at 0.25 ppm was almost as good as in the control. It will be recalled that
four of the five lakes used as test lakes for bioassay experiments had nickel
levels greater than 0.5 ppm, and several also had copper levels greater than
Laboratory Chlorella COPPER
~I in modified BBM + Cu ppm Cu

4b o
. X ~ X -04
1.0
..J
_J
uJ
,o 0.1
.04
.01
48 96 ;44 216 264
TIME, H O U R S
FIG. 3 - - G r o w t h ofChlorella vulgaris in nutrient solution with copper additions. The solid
circh's marked on the base line represent the growth in 0.4 p p m o f copper.
0.1 ppm. It is at least possible, therefore, that either of these metals, copper
or nickel, by itself, could account for the poor growth encountered in the lake
water bioassays when these same test species under identical conditions for
growth were used and with nutrients added.
The problem of interpretation of single metal bioassays is compounded by
the species differences referred to previously. Figure 8 shows the growth of
the four test species in various copper-containing media after 10 days of
growth. Chlorella survived at 0.05 ppm but was killed by 0.1 ppm,
Chlamydomonas grew well at 0.1 ppm but was killed at 0.3 ppm,
Haematococcus showed a gradual decline in cell number, with no survivors
at 0.3 ppm, and Scenedesmus grew quite poorly from copper levels of 0.1 to
0.5 ppm but nevertheless survived. Only at 0.7 ppm were cells finally killed.
A similar type of pattern of response is shown for the test species in nickel
(Fig. 9), although this time Scenedesmus appears the most sensitive and
330 WATER OUALITY PARAMETERS
S t L VE R Chfore I Io
X 0 ppm
0 .01 "
13 . 0 5 "
Z~ .10 "
9 -50 "
9 1.00 .
I00
x
dz
IO
I-0 12
2 6 8 I0 ~4
GROWTH, DAYS
FIG. 4---Growth qfChlorella vulgafis in nutrient with silver additions,
Chlamydomonas the most resistant. These results recall the differences in

response of the test species to the lake water bioassays shown in Fig. 2 and
emphasize that a deeper knowledge of species responses to particular metals
may allow quite sensitive and specific indicator organisms to be developed for
testing effluents for metals, etc. The problems of interpreting environmental
chemical conditions from single species bioassays without a thorough
knowledge of the species response is also emphasized.
MERCURY Chore]lo vulgoris
X 0 ppm
0 .05
n .10
Z~ ,15
+ . 20
9 "30
I000 9 .40
9 .50
.5
I[
I00
x
d
z
IO
2 4 6 8 - I0
GROWTH , DAYS
FIG. S---Growth qf Chlorel|a vulgaris in nutrient solution containing mercury.
Adaptation of Algae to Metals in the Environment

The choice of algal species for bioassay is partly based on the rationale that
algae are the primary producers in aquatic ecosystems. It might, thus, be
reasonable to use indigenous species for bioassays. A number of algae from
the Sudbury lakes have been used to assay metals in a series of comparisons
with closely allied laboratory strains. Since the lakes from which the algae
were isolated are clearly contaminated by metals, Table 2, then it may be
CADMIUM ChlorellQ
X 0 ppm
o -01
n -03
- 05
9 .I0
9 -15
9 -20
+ 9 25
i<
UJ
2 4 6 8 I0 12 14
GROWTH, DAYS
FIG. 6--Growth ofChlorella vulgaris in nutrient solution containing cadmium.
supposed that any algae isolated must be tolerant at least to those metals
which occurred in the lakes.
The response to copper of an isolated lake strain of Scenedesmus and the
laboratory strain are shown in Figs. 10 and 11, respectively. The lake strain
showed a progressive decrease in growth but even at 0.4 ppm Cu was growing
well. In contrast, the laboratory strain failed to grow at concentrations above
0.04 ppm, that is, at a copper concentration often-fold less.
Similar results for other algae, for example, strains of Chlorella from other
lakes have been obtained [8]. It is perhaps of particular importance to note
that tolerance to nickel appeared to exist to a marked extent in algae isolated
from nickel-containing lakes and that tolerance to silver also existed in these
same species, even though silver is (a) not present in significant concentra-
tions in the lake, and (b) is an exceedingly toxic heavy metal (see Fig. 4). This
has a great many theoretical aspects of interest, such as common
mechanisms of metal tolerance, but this will be reported elsewhere.
5.0 NICKEL Ni ppm

Loborotory sScene
m u . ~de
s ".05
in modified BBM + Ni ./~r~./,~/- .25
I'C ~, 7~" ~';"
///
~o i I/////
_J
i
04[ //
N\
" ' " -/X.

"x%.
"x
~/X
9015 I I i I I I
48 96 144 216 264 312
T I M E , HOURS
FIE;. 7--Growth o f S c e n e d e s m u s a c u m i n a t a in nutrient solution containing nickel.
One's choice of test organisms, therefore, will be dependent upon the aims
of the assay. This is an obvious truism, but the literature is replete with
fundamental failures in this respect because of the lack of knowledge of the
species used. The suggestion that Daphina magna be the test organism of
choice for bioassays, made by the U.S. Environmental Protection Agency, is
a response to the problem because it was coupled with major research into
the response of that species of zooplankton to a wide range of environmental
variables. However, while for many conditions it may be a particularly useful
test species and is a rather well-researched animal, it would be foolish to
expect it to be the most sensitive species for, say, DDT, NTA, mercury,
copper, and lead.
Whatever the choice of test organism, the ability of algae to adapt rapidly
to high levels of toxic substances should be noted, and test organisms
checked regularly for tolerance levels. In the ideal world, tests of an unknown
solution should also include a set of control tests at different concentrations
of the suspected toxic substance.
COPPER
X X Chlorello (.94)
[3 [3 Sceneclesrnus (3"3)
0 0 Hoemotococcus (,88)
Z& ~ Chlomydotrlonos (2-0)
I00
I
q0 ,20 -30 .40 ,50 -60 -70
CONG. PPM,
FIG. 8---Growth of.tbur algae in bioassays of copper-containing nutrient solutions after

IO-days growth. Values in brackets were relative starting cell counts.
Interference in Bioassay by Organic and Inorganic Materials

One of the distinctions between biological assays and chemical measure-
ments is that one should be able to distinguish biologically available forms of
metals from nonavailable forms by bioassay, while chemical analysis would
not make this distinction. As an example of this, the water from Baby Lake
was used as medium for copper assay by Chlorella. The results are shown in
Table 3.
The growth in lake water plus nutrients was measured with different
amounts of copper, and in a parallel series of experiments distilled water
with nutrients was used. The copper, as measured by atomic absorption, is
shown in the right-hand column. In the defined medium, cell concentration
of 0.06 x 106 were reached with 0.15 p p m copper. In the lake water, the same
concentration occurred with 0.04 p p m added copper. The total amount of
copper in the lake water when measured chemically was 0.54, but only 0.11
NICKEL
X X Chlorello (.74)
rl rt Scenedesmus f3-3)
0 0 Hoemotococcus (-88)
Zl Zl Chlomydomonos (2-0)
IOO
..I
2E
6
z
J
._1
w
I'~ .Io .2o .30 .4o -50 -60 .~o

CONC., PPM
FIG. q---Growth ot'.tbur algal species in bioassays qt" nickel-containing solutions. Values in
braekets are the relative starting cell counts.
TABLE 3---Bioassay q f copper by lake Chlorella.

Total Copper
Amount Growth Measured in Solution
Copper Added, Cell No./106 at by Atomic Absorp-
Medium ppm 10 Days tion, ppm
Lake water -t- nutrients 0 0.13 0.52
Lake water q- nutrients 0.04 0.06 0.54
Distilled water + nutrients 0 0.28 0.00
Distilled water -t- nutrients 0.04 0.24 0.04
Distilled water + nutrients 0.15 0.06 0.15
p p m was available biologically, t h a t is, the response to 0.04 p p m was t h a t

equivalent to 0.15 p p m c o p p e r in distilled water.
R e a s o n s for t h e n o n a v a i l a b i l i t y o f m e t a l s in w a t e r i n c l u d e the presence o f
o t h e r o r g a n i c a n d i n o r g a n i c substances. F o r e x a m p l e , when m o r e t h a n o n e
m e t a l is p r e s e n t in high c o n c e n t r a t i o n the c o m b i n e d effects m a y not be
s i m p l y additive a n d c a n n o t be p r e d i c t e d without prior investigation. T h e
relative nontoxicity o f Kelley a n d B o u c h e r l a k e w a t e r shown in Figs. 1 a n d 2,
CU
t COPPER
6-0 Boucher Loke Scenedesmonos
in m o d i f i e d BBM + Cu
4-0
t, ~ 9 ' 15
~" p" ~0 .20

,/ _ ~'Os ~ -40
I-0
0-8
0-~
1o
0
- 0`r
6
z
J
.z 0`~
0`1
0 48 96 144 216 264

TIME , HOURS
FIG. I O - - G r o w t h response o.f an isolated strain o f Scenedesmus .from a m e t a l p o l l u t e d

htke to a nutrient solution containing c o p p e r at several concentrations.
despite their high levels of copper and nickel, perhaps can be partially
ascribed to the high organic content of Kelley Lake, with the likelihood of
organo-metal complexes such as fulvic and humic acids, citrates, etc., and
the relatively high base metal content of both lakes. Our unpublished data
strongly implicate calcium as an ameliorating factor for heavy metal toxicity.
The Assay of Metals in Combination

Experiments were designed to test the effects of metals by themselves and
in combination. This is a practical problem since metals in combination are
the normal field situation, as seen in the Sudbury lakes. Similar techniques
for determination of growth were used. The data are presented here in
histograms, with results expressed as percentage of control after 10 days of
growth.
For copper-nickel combinations with Chlorella, all combinations showed
COPPER
2-0
Loborotory Scenedesmonos ppm Cu
I-0 in modified BBM + C u J "004
0-8
O-6
0-4
~.~
w
u .06
~O~ -I0
-04
\ ,. ~O "15
902 \ // ~0 "20
140
901 i I I I I
0 48 96 144 216 264
TIME HOURS
FIG. 1 1 - - G r o w t h response or" a laboratory strain q# Scenedesmus to nutrient solutions

containing copper at several concentrations.
synergistic effects (Fig. 12). For example, 0.05 ppm copper reduced growth
to 95 percent, while 0.05 ppm nickel was stimulatory. In combination,
however, the growth was reduced to 38 percent of control. The effect of 0.05
ppm copper with 0.1 ppm nickel was more dramatic. The latter was not
inhibitory and the former only reduced growth to 95 percent. In combina-
tion, growth was completely inhibited.
Similar results were obtained for Haematococcus (Fig. 13) in which
striking synergism can be seen when the effects of 0.05 ppm copper and
nickel separately are compared with the combined presence of both metals in
solution.
High levels of copper and nickel are present in combination in the Sudbury
lakes and in many other comparable situations. Thus on the basis of
chemical analyses, combinations of metals should be assayed. Water quality
standards need to take account of this.
Not all combinations of metals are synergistic, however. Another pair of
metals--cadmium and selenium--in combination showed antagonisms, that
is, the toxic effects of one metal was ameliorated by the presence of the other.
This is shown in Fig. 14 which illustrates the ~rowth of Chlorella with
140- Chlorello vul~aris __ Copper/Nickel Synergism
i2ol
I00
3:
0
n."
"~ 8 0
,_J
0
r
I--
z 60
o
-L
(.,,)
la.
o
40
2o I -
o "---3
Cu 0 "05 -I0 0 .O5 .10 0 "05.10
Ni o o o 'O5 "O5 -O5 -IO .IO -IO
METAL CONC- PPM
FIG. 12--Growth ot'Chlorella vulgaris in nutrient solutions containing nickel and copper
.~ep.rately and together.
14C
Hoem(ztococcus copensis Copper / Nickel Synergism
12r
IOC
Z=
0
QE
8C
d
er
m
I,-
4C
2(:
Cu 0 -05 .lo 0 -O5 "10 0 -05 -I0

Ni 0 0 0 -05 -O5 -05 -IO ,io -Io
METAL CONC" PPM
FIG. 13--Growth o.f Haematococcus capensis in nutrient solutions containing nickel and
copper both together and separately.
HUTCHINSON AND S T O K E S ON HEAVY METAL TOXICITY 339
140
Chlorello vulcjaris Cadmium/Selenium Synergism
120
-r
i-
~= I00
0
n-
O
J 8r
0
ne
t-
Z
o 6(;
tt.
0
4(2
2C
Cd 0 "05 "10 0 .05 "10 0 "05 "lO

Se 0 0 0 "05 -05 "05 910 -I0 "10
METAL CONC" PPM
FIG. 14--Growth of C h l o r e l l a v u l g a r i s in nutrient solutions containing cadmium and

seh'niunl both separately and together.
cadmium and selenium in combination. Cadmium is the more toxic metal.

0.05 ppm cadmium reduced the growth to 55 percent and 0.05 ppm selenium
reduced it to 75 percent. In combination, the growth was 79 percent of the
control. At 0.05 ppm cadmium and 0.1 ppm selenium, growth was
stimulated compared with the control. Haematococcus gave similar patterns
of amelioration or metal antagonism with combinations of cadmium and
selenium (Fig. 15).
Synergism and antagonism are, of course, only two of the possible
explanations for the differences between chemical and biological measure-
ments, but they are two which can be relatively readily checked. Chelation by
organic molecules is another important and complex issue not discussed
here.
The Time Factor in Assay Studies

5o far all data have been for rates of growth. Usually, a time is selected for
measurement when growth is still exponential in a culture, for example, 10
days from inoculation. As mentioned in the beginning of this paper, the
usual period of time used for toxicity tests is much less than this. Results of
toxicity tests will often vary depending upon the length of time elapsed since
the experiment commenced (namely, the time since metal was introduced).
This is seen, for example, in experiments with mercury using Chlorella and
140 Hoemolococcus copensts Cadmium / Selenium Synergism
120
I00--
o
o 80--
g,
Ilg
b-
60--
40--
20--
i
Cd O .O5 .IO O .O5 .lO O -05 .IO
Se O O O -O5 -O5 . 0 5 dO -IO -tO
METAL CONC. , PPM
FIG. 15--Growth of H a e m a t o c o c c u s capensis in nutrient solutions containing cadmium

and selenium both separately and together.
Haematococcus (Fig. 16). The first pair of graphs were made from data on
the growth of 4 or 5-day old cultures, and the second pair for 9-day old
cultures. For Chlorella. 0.2 ppm was much more toxic at 9 days than it was at
5 days, while for Haematococcus, the mercury was more toxic at 5 days than
it was at 9 days, when there appeared to have been some recovery. If one is
concerned with maintenance of an intact ecosystem then long-term tests
might have more meaning than acute toxicity tests. Again, the assay has to
be designed according to the aims, but certainly these results indicate that
time (duration) should be carefully specified. Quick may not be good, it
could be merely misleading (and quick).
Metal Uptake into Algal Cells

In terms of the seasonal changes in ecosystem, even 10 days is not very
long. One serious consequence of metallic pollution of waters is the potential
increased toxic levels of metal compounds which may accumulate in or on
cells. Laboratory studies on the uptake of metals indicate that high
concentrations in cells may be reached before any serious toxic effects are
manifest. Table 4 shows the uptake of copper and nickel by the lake isolate of
Scenedesmus. 0.1 and 0.3 ppm of copper did not seriously affect growth, yet
uptake into cells was quite high. A similar effect is seen for nickel at 1.0
ppm. There appears to be a relation between uptake and toxicity, however.
MERCURY MERCURY
X - - X Chlorella ( 5 days) X X Chlorella (9days)
0 0 Haematococcus (4days) 0 0 Hoemafococcus (9 days)
I000
.5IOC
x
x
IO
X~X~x
1.0 ~
-20 .40 -60
~ X - - X.20- - X -40
I
-60
CONC,, PPM
F I G . 16--Growth o f Chlorella and H a e m a t o c o c c u s in solutions containing a range of
mt,rcuQ, concentrations, grown.tbr two time periods.
Nickel is taken up much less, and is correspondingly less toxic. In both cases
the concentration factor is of the same order of magnitude irrespective of the
range of initial concentrations used. Uptake was linear with initial
concentration.
Concentration factors of metals of this magnitude in primary producers
would presumably result in a build-up in higher trophic levels. Consumers
will be exposed to the external concentration of the media, plus increased
levels in their food. Fish and invertebrates are often much more sensitive to
heavy metal toxicity than are green algae [6]. Added to this, the tolerant
TABLE 4---Uptake of" metals by lake Scenedesmus.
Concentration in
Original Concen- Cells at 6 Days,
tration in ppm/g dry Concentration Growth as
Element Medium, ppm weight algae Factor % Control
Copper 0.1 287.8 2878 99

Copper 0.3 885.1 2950 69
Copper 0.5 1238.5 2477 64
Nickel 1.0 92.8 93 76
Nickel 2.0 200.0 100 41
Nickel 3.0 300.0 100 29
algae take up much higher levels of copper and nickel than do laboratory
strains, so that with increasing levels of metals in their environments, the
problem will become compounded by adaptation of primary producers.
Danger levels in terms of the rest of the ecosystem might be reached long
before 50 percent reduction in algal growth was observed.
Conclusion
In conclusion, then, bioassays appear to be a potentially useful way of
determining the response of living organisms to heavy metal contaminants in
the aquatic environment. A great deal of work has gone into studies of
responses of animals to a variety of potentially toxic substances and this is
especially true of fish. Algal studies are less well documented. Algal
responses have significance in that they are critical primary producers in
most aquatic ecosystems and allow growth to be utilized as a criterion of
response. Unhealthy cells do not divide. They are easily cultured and
require a minimum of equipment. The species tested have shown considera-
ble sensitivity to low concentrations of a number of heavy metals in their
environment. It appears possible to differentiate specific responses by
different algae to the metals. This range of response allows assays of lake
waters, although combined effects of two or more metals may be either
additive, synergistic, or ameliorative. The studies of isolated resistant
strains demonstrate their ability to evolve heavy metal tolerant strains. This
could be a complicating factor in laboratory studies. These and the
"normal" strains show biological accumulation of these metals, often to an
extent which has possible implications for higher trophic levels. Metal
tolerance does not imply the ability to exclude the metal from the cell. The
algae tested have possible use in estimating the biological availability of
metals in water, and this is often advantageous compared with chemical
estimates of total metal concentration which may be almost irrelevant to
biological availability.
Test conditions for bioassays need to be rigorously stated. Not only is this
true for external conditions but threshold levels for metal toxicity appear to
differ during the growth of a culture.
Acknowledgments
The authors wish to thank the National Research Council, Canada, for
grants enabling them to carry out the studies and K. Krauter, J. Lindsay,
and J. Fitchko for laboratory assistance.
References
[1] McKee, J. E. and Wolf, H. W., Water Ouality Criteria, 2nd ed., Resource Agency,
California State Water Resources Control Board, 1963.
[2] Myslik, G., "Effect of Heavy Metal Pollution on Phytoplankton in Sudbury Lakes,"
masters thesis, University of Toronto, Dept. of Botany, 1973, (unpublished)
[3] Stokes, P. M., Hutchinson, T. C., and Krauter, K., Canadian Journal of Botany,
Vol. 15, 1973, pp. 2155-2168.
[4] Hutchinson, T. C., Water Pollution Research in Canada, Vol. 8, 1973, pp. 68-90.
[5] Doudoroff, P. and Katz, M., Sewage and Industrial Wastes, Vol. 25, 1953, pp. 802-839.
[6] Lisk, D. J., Advances in Agronomy, Vol. 24, 1972, pp. 267-327.
[7] Warnick, S. L. and Bell, H. L., Journal of the Water Pollution Control Federation,
Vol. 41, 1969, pp. 280-284.
[8] Stokes, P. M., Hutchinson, T. C., and Krauter, K., Water Pollution Research in
Canada. Vol. 8, 1973, pp. 178-201.
T. E . M a l o n e y I a n d W . E . M i l l e r 1
Algal Assays: Development

and Application
REFERENCE: Maloney, T. E. and Miller, W. E,, "Algal AssayB: Development and

Application," Water QualityParameters, ASTM STP 573, American Society for Testing
ABSTRACT: The development, refinement, standardization, and practical application
of an algal assay procedure are described. Results of experiments are presented to illus-
trate the use of the algal assay procedure to determine algal growth-limiting nutrients in
fresh waters, including lakes and streams; for the assessment of receiving waters to
determine their nutrient status and sensitivity to change; for the assessment of the
effects of changes in waste treatment processes on receiving waters; and for the evalu-
ation of materials and products to determine their potential effects on algal growth.
KEY WORDS. water quality, algae, bioassay, environmental tests, nutrients
The algal assay has become an extremely valuable test for evaluating water
quality, especially in relation to eutrophication. The use of algal assays is by
no means new; they have been used for m a n y years to predict and evaluate
the effect of nutrients and toxicants u p o n primary producers in the aquatic
environment. M a n y investigators have improvised algal assays to meet
specific needs, b u t these have offered no basis for comparison o f acceptably
reproducible results a m o n g laboratories or a m o n g samples obtained from
different geographic areas. Recognizing the need for an acceptable
standardized algal growth test, in early 1968 the National Euthrophication
Research P r o g r a m prepared a tentative procedure. The intended use o f this
procedure was to (1) identify algal growth-limiting nutrients; (2) determine
biologically the availability of algal growth-limiting nutrients; (3) quantify
biological response to changes in concentrations of algal growth-limiting
nutrients; and (4) develop a rational framework for application, with
judgment, of quantified parameters to practical problems.
In M a r c h 1968 a group of scientists, having a fundamental knowledge of
algal physiology and algal growth responses and having experience with algal
'Chief and chief, Physiology Section, respectively, Eutrophication and Lake Restoration
Branch. Pacific Northwest Environmental Research Laboratory, National Environmental
Research Center, U.S, Environmental Protection Agency, Corvallis, Ore, 97330.
344

MALONEY AND MILLER ON ALGAL ASSAYS 345
assays of various types, was brought together to carefully scrutinize the

tentative procedure and to recommend additions, deletions, and changes.
This group recognized three fundamentally different procedures: a static
"bottle test," a continuous-flow chemostat test, and an in situ test.
The three tests were described in the "Provisional Algal Assay Procedure"
(PAAP) [1] 2 which was published in February 1969. Shortly after publication
of the PAAP, a group of laboratories undertook a comprehensive research
program to improve and evaluate it. The first phase was concerned with
comparing the bottle test with the continuous-flow chemostat test for
assaying the algal growth-nutrient concentration relationships in natural and
enriched ~vaters. Each laboratory followed the same plan for the evaluation
using algal test species from a common source. When specific problems were
recognized, certain laboratories were assigned to investigate them after
agreement was reached on the procedure to be followed. Some of the specific
problems investigated were: (1) formulation of a culture medium to minimize
intracellular nutrient carryover in the inoculum; (2) determination of the
optimum age of the inoculum; (3) determination of the optimum sample
surface area to volume ratio in the test container; (4) determination of
optimum physical conditions, such as light intensity and temperature; (5)
determination of methods to control pH and to ensure the availability of
carbon dioxide; (6) determination of the best method or methods to measure
algal biomass; and (7) determination of the best method or methods for
sample preparation.
Soon after this study was initiated to compare the bottle test with the
continuous-flow chemostat test, it became apparent that the latter would be
much too difficult to carry out in laboratories having less sophisticated
equipment and relatively inexperienced personnel. Since it was felt that the
test should be designed for the broadest possible application, most of the
emphasis was placed on the refinement of the bottle test. In August 1971 the
"Algal Assay Procedure: Bottle Test" (AAP) [2] was published. Prior to its
publication an interlaboratory precision test, involving eight laboratories,
was undertaken to evaluate the capabilities of the bottle test. Each laboratory
used an identical protocol including the same size samples and test flasks,
an inoculum of the same age, and the same methods for sample preparation.
The algal test species and chemical reagents were provided from a common
source.
In the first interlaboratory test, algal growth responses in various
concentrations of the culture medium were evaluated, as well as growth
responses in the culture medium with important nutrients deleted or added
back in varying concentrations. In the second interlaboratory test, two
natural lake water samples were assayed; one from a common source and
another collected from a lake in the vicinity of each of the participating
laboratories. Each sample was treated prior to assaying by both autoclaving

and membrane filtration. A regression analysis of nutrient strength and algal
yield indicated that each laboratory in its use of the assay consistently and
significantly produced growth that was linearly related to nutrient strength.
The results of the interlaboratory precision test were published in October
1971 [3]. Following this, the bottle test has been practically applied in
numerous situations to assist in solving and understanding eutrophication
problems. These are described in this paper.
Experimental
Sample Preparation and Additions
All assays on waters and waste waters were conducted according to the
protocol described in the AAP [2]. All lake and river water samples were
treated prior to the various nutrient and wastewater additions by autoclaving
at 1.1 kg/cm z (15 psi) at 121~ (250~ for at least 30 min or 10 min/liter,
whichever was longer. After autoclaving and cooling, the water or wastewater
samples were allowed to equilibrate in a mixture of 1 percent carbon dioxide
in air to restore the carbon dioxide lost during autoclaving and to lower the
pH to its original level. Following this, the samples were passed through a
0.45/ma membrane filter.
When additions (spikes) were made to the various waters or waste waters,
they were as follows: nitrogen as NaNO~, phosphorus as K2HPO4, carbon as
NaHCO 3, and nitrilotriacetic acid (NTA) as the trisodium monohydrate salt.
Additions of the latter were calculated as the free acid. Secondary sewage
effluent was that resulting from settled primary sewage effluent subjected to
activated sludge treatment. Tertiary sewage effluent was that resulting from
alum precipitation of activated sludge effluent. Secondary effluent/nitrilo-
triacetic acid (SE/NTA) indicates that 5.0 mg NTA/liter was added to the
primary settled effluent prior to activated sludge treatment. Tertiary
effluent/nitrilotriacetic acid (TE/NTA) was SE/NTA subjected to alum
treatment.
Inoculation, Incubation, and Biomass Determination

All assays were carried out in 500-ml Erlenmeyer flasks containing 100 ml
of water or waste water. Each flask was inoculated with a unialgal culture of
Selenastrum capricornutum Printz to give a final concentration of 103
cells/ml. Stock cultures of the test alga were grown in AAP culture medium.
All test flasks were incubated at 24 ___2~ under 4304 lx (400 ft-c) ___10
percent of continuous cool-white fluorescent lighting and shaken on a
gyrotory shaker at 110 oscillations/min.
Cell counts were made with a Model B Coulter Electronic Particle Counter
equipped with a mean cell volume computer. 3 It had previously been

determined that there was a direct correlation between mean cell volume and
dry weight.

Chemical analyses of the waters and waste waters are shown in Table 1.
Figures 1 and 2 show the effects of the addition of nitrogen, phosphorus, and
nitrogen and phosphorus combined on algal growth in water from Lake
Lansing, Mich., and the Santee Reservoir, Calif., respectively. Growth with
these nutrient additions was compared to that in inoculated control flasks.
These results illustrate the use of the algal assay in determining algal
growth-limiting nutrients in lake waters. In Lake Lansing water, for
example, the addition of 0.05 mg phosphorus/liter increased algal growth
approximately four-fold while the addition of both 0.05 mg phosphorus/liter
FIG 1--El~ects of nutrient additions to water from Lake Lansing, Mich., on algal growth.
'Note--the use of trade names does not constitute endorsement by the U.S. Environ-
mental Protection Agency.
TABLE 1--Chemical content (rag~liter) o f waters and waste waters.
Total Inor- Total Solu- Total Solu-

Sample Total C ganic, N a ble, P Ortho-P ble, Fe
Lake Lansing 40.0 0.17 0.400 0.012 0.011

Santee Reservoir 26.0 0.07 0.160 0.070 0.017
Palouse River 12.0 <0.01 0.056 0.040 0.350
Snake River 35.0 0.16 0.007 0.005 0.017
Lake Michigan 21.0 0.25 0.006 0.003 0.038
Triangle Lake 5.0 0.06 0.005 0.004 <0.100
Waldo Lake 1.0 <0.01 0.003 0.001 <0.100
2% Secondary Effluent 3.0 0,36 0.077 0.075 <0.100
2%, Tertiary Effluent 3.0 0.43 0,003 0.003 <0.100
a(NO2 + NO3 + NH,).
FIG. 2--Effect o f nutrient additions to water from Santee Reservoir, Calif., on algal growth.
and 1.0 mg nitrogen/liter increased the amount of algal growth over 13

times. This indicated that while phosphorus was initially algal growth-
limiting, nitrogen became growth-limiting when sufficient phosphorus was
present. On the other hand, in the Santee Reservoir water, nitrogen was
definitely algal growth-limiting and only the addition of nitrogen resulted in
an increase in algal biomass.
When nutrient additions were made to water from the Palouse River in
Washington (Fig. 3) and to water from the Snake River in Idaho (Fig. 4), the
results showed that the former was nitrogen limited with respect to algal
growth and the latter phosphorus limited. As has been pointed out previously
[4], the algal assay could serve as a valuable method for monitoring streams
and establishing nutrient criteria for receiving waters as a basis for water
quality control.
When Lake Michigan water was inoculated with the algal test species, only
approximately O. 1 mg dry weight/liter of algal biomass was produced (Fig. 5).
,o2[_
I01
CONTROL
I.ON
E . . . . . 0.05 P
I- ............. o.oIP
"1" I0 o
(..9 - - I.ON + 0 . 0 5 P
hi
>-
n,-
9 ~
C)
i0-1 .// ~ ~ .......
10-2
I I I I I t I
0 4 8 12 16 20 24
DAYS
FIG. 3--Effect of nutrient additions to water ,from the Palouse River, Wash.. on algal
growth.
IO 2 --
101
~ I0 o
CONTROL
i 0.05 P
--- O.OIP
i0-1
iO-Z ~
I I I I I I
0 4 8 12 16 20 24
DAYS
FIG. 4--E.~'ect o f nutrient additions to water .from the Snake River. Idaho, on algal
growth.
When additions of 0.005, 0.010, 0.015, and 0.020 mg phospllorus/liter were

added to the water, the amount of algal biomass produced increased to
approximately 0.75, 2.0, 3.5, and 7.0 mg dry weight/liter, respectively.
These data not only show that Lake Michigan water was phosphorus limited,
but also indicates what the potential impact of additional phosphorus
discharges to the lake would be.
Table 2 shows the effect on algal growth of adding increments of
phosphorus back to alum treated waste water. These assays were conducted
in conjunction with a full-scale lake restoration demonstration project on a
highly eutrophic lake in northern Minnesota [5]. The project consists of
nutrient removal (primarily phosphorus) from municipal wastewater effluent
while permitting the treated effluent to continue to flow into the lake.
In 1970, a comprehensive research program was conducted at our
laboratory to evaluate the influence of NTA on eutrophication. The use of
the algal assay played an important part in this evaluation. Three of the
M A L O N E Y AND MILLER ON A L G A L ASSAYS 351
IO 3
IO z CONTROL -A/F- -
~ §
A ~ .~ + O.OlO P
._1 ......... §
+O.020P
E IO I
I--
-1-
(.9
LI.I ~ ~ ~
.........
9 ~
/
IO ~
)-
I:E /
1:3 /
/
iO-I
1 0 - 2 ~
0 4 8 12 16 20 24
DAYS
FIG. S---Effect of phosphorus additions to Lake Michigan water on algal growth.
TABLE 2--Algal growth stimulation by restoring

phosphorus to tertiary waste water.
P-Concentration Algal Dry Weight

(rag/liter) Cells/ml (mg/liter)
0 3 920 0.08
0.01 83 235 2.92
0.02 228 745 7.94
0.03 451 347 14.61
0.04 663 726 21.15
waters used in the algal assays were from Triangle Lake, Ore., a
mesotrophic lake; Waldo Lake, an ultra-oligotrophic lake; and Cline's
pond, a small highly eutrophic farm pond. Various additions were made
to each of these waters and the amount of algal biomass produced compared
to that in controls. The additions included 5.0 mg NTA/liter alone,
and secondary and tertiary waste water derived from raw waste water to
which 5.0 mg NTA/liter had been added. The results, shown in Figs. 6,
7, and 8, indicate that NTA had no stimulatory effect on algal growth.
lOS r , , i ~ , , i r , , 1 , , , i , , w I ~ , ,
O CONTROL & + 5 . 0 NTA

9 +2%SEC. EFF. 9 +2%S.E./NTA
O + 2% T E R . E F F . 9 + 2% T . E, / N T A
10 7
10 a !! ~ m
-- I
u lOSi
|04:
100 4 8 12 16 20 24
DAYS
FIG. b--E~i, ct qt" additions qt" N T A and waste waters on algal growth in Triangle Lake
water.
I0 e , ~ i , , , i ,,-,~ , i , , , I , T , i , , ,
G CONTROL ~x + 5 . 0 N T A
I~1 + 2 % S E C . E F F . 9 +2% S.E./NTA
O + 2% T E R . E F F . 9 + 2% T . E . / N T A
10 7
10 6
U
10 s
10 4
,-sT-r, , ,-v77, , ; , o,
1030 ' 24
4 8 12 16 20
DAYS
FIG. 7--E~'ect o f additions o f N T A and waste waters on algal growth in WaMo Lake water.
The secondary effluent was the only addition which stimulated algal
growth in these waters.
Conclusion
The need for extensive research to refine and standardize an algal assay
procedure has been shown. The uses of such assays to assist in the
solution of practical eutrophication problems have been illustrated. While
IO s , , i , , , i , , , i , , , I ~ , , i ~ , ~
0 C O N T H , OI, ~, + 5 . 0 N T A
m + 2% S E C . E F b ' . 9 + 2% S. E . /NTA
O + 2% T E R . ElZ'F. O +2% T.E./NTZ
10 z
106
u
E
/~ jI ----------r~
lO s
!
1030 4 8 12 16 20 24
DAYS
FIG. 8---El~'ct o/~"additions qf N T A and waste water on algal growth in Cline's Pond water.
the algal assay cannot be used by itself in evaluating and solving

eutrophication problems, combined with good chemical and physical
analyses and limnological investigations, it can be an extremely useful
procedure.
References
[/] "Provisional Algal Assay Procedure," Joint Industry/Government Task Force on Eutro-
phication, 1968.
[2] "Algal Assay Procedure: Bottle Test," Environmental Protection Agency, National Eutro-
phication Research Program, 1971.
[3] "Inter-Laboratory Precision Test--An Eight-Laboratory Evaluation of the Provisional

Algal Assay Procedure Bottle Test," Environmental Protection Agency, National Eutro-
phication Research Program, 1971.
[4] Maloney, T. E.. Miller, W. E., and Blind, N. L., "Use of Algal Assay in Studying
Eutrophication Problems," Advances in Water Pollution Research. 6th International
Conference, Jerusalem, 1972, Pergamon Press, Oxford and New York, 1973.
[5] "'The Shagawa Lake Project," EPA-R3-73-026, U.S. Environmental Protection Agency,
Office of Research and Monitoring, Ecological Research Series, 1973.
A. S. W. d e Freitas I a n d J. S. H a r t j
Effect of Body Weight on Uptake of

Methyl Mercury by Fish
REFERENCE: de Freitas, A. S. W. and Hart, J. S., "Effeet of Body Weight on Uptake

of Methyl Mercury by Fida," Water Quality Parameters, ASTM STP 573, American
ABSTRACT: An uptake rate of 1.10 ng mercury (Hg) per h per fish was obtained when
small golden shiners, Notemigonus crysoleucus, with a body weight of 2.5 g were ex-
posed to water at 13~ containing 0.1 x 10-3 /ag H g / m l . Results on methyl mercury
uptake from water by fish ranging in body weight from 2 to 120 g demonstrate that
smaller fish accumulate mercury in their body tissues at a taster rate per unit weight of
body tissue than larger fish. Mercury uptake followed first order mass action kinetics in
which the whole fish was treated as a single uptake compartment. The acquired body
burdens of mercury in the test fish varied proportionately with the 0.76 power of the
body weight. The observed body weight exponent of 0.76 is similar to the function one
would expect if uptake rate increased in proportion to metabolic rate. Therefore, factors
such as water temperature and fish activity may also effect uptake of methyl mercury
from water due to their direct effect on metabolic rate.
KEY WORDS. water quality, fishes, body weight, environmental tests, mercury
(methyl), uptake
The widespread presence of mercury in fish, predominantly in the form

of methyl mercury [1,2], z has been explained by most investigators
[1,2,3,4] as a food chain biomagnification process resulting from the
generalized contamination of the food web by methyl mercury. The
contaminating methyl mercury is assumed to originate in the sediments by
bacterial conversion of "inorganic" mercury to methyl mercury which then
finds its way into the water phase [5]. Methyl mercury from the water
phase moves into biota and subsequent bioaccumulation and food chain
transfer by aquatic organism then occurs. Information on mercury
dynamics in fish from uptake, clearance, and tissue distribution studies
[6,7,8,9] has led to a qualitative appreciation of some of the factors
involved in mercury bio-accumulation. A lack of quantitative information,
' Associate and principal research officer, respectively, Division of Biological Sciences,
National Research Council of Canada, Ottawa, Ontario K I A 0R6, Canada. Issued as
N.R.C.C. No. 14319.
356

DE FREITAS AND HART ON METHYL MERCURY IN FISH 357
however, on uptake of mercury, particularly from water, and its retention

index or clearance from fish has inhibited the development and validation
of predictive models for mercury accumulation. In this report, the effect
of body weight on uptake of methyl mercury from water has been
determined by subjecting fish ranging in body weight from 1 to 140 g to
aqueous solution of methyl mercury for periods up to 12 h.
Experimental
Fish were maintained in large 45 to 100 gal capacity fiberglass tanks
supplied with a continuous flowthrough of dechlorinated tap water. The
diet of Northern Pike, Perch, and Ling consisted of live minnows made
available ad libitum. Brown Bullheads were fed a diet consisting of live
worms and minnows. Small fish were fed pelleted trout diet, minced
worms, or Tetramin Goldfish food, depending on the species.
Studies on uptake of mercury from water were carried out in glass tanks
with a water capacity of about 20 liters, or fiberglass tanks with a water
capacity of about 120 liters. Each tank was equipped with a system for
controlled flowthrough of dechlorinated tap water. "Steady state" condi-
tions with respect to mercury concentration in the perfusate solution were
achieved by addition of a priming dose of mercury (2aaHg labeled) to the
test tank water followed immediately by continuous infusion of a zaaHg
labeled solution. The mercury infusion solution was allowed to mix with
the flowthrough water supply just prior to its entry into the test tank.
In a typical uptake test, about 50 to 80 fish ranging in body weight
from 1 to 120 g were placed in a 120 liter exposure tank (S to 7 g
fish/liter perfusate solution in tank) approximately 1 h after addition of
the priming dose of mercury.
Serial samples of perfusate solution were removed from the test tank at
30-min intervals throughout exposure period. The concentration of
mercury in the perfusate solution was estimated by measuring the amount
of radioactivity in the samples of perfusate solution using a Nuclear
Chicago standard type "Deep Well" gamma counter, Model 4233.
In all uptake tests, samples of 12 to 20 fish were removed after
exposure periods of 1, 2, 4, and up to 12 h for determination of mercury
taken up by the fish. The total amount of mercury taken up by the fish
was estimated by measuring the radioactivity present in the total carcass
tissues using the Model 4233 system. Each fish was homogenized and
samples of homogenized tissue (10 g) were placed in counting vials for
radioactive assay.
In order to quantitatively relate uptake rate to fish body weight it was
necessary to develop a suitable compartmental kinetic concept.
For this purpose, mathematical analyses were undertaken assuming a
two-compartment system and a one-compartment system (for example,
combining external adsorption and internal uptake). However, since the

former yield biologically unrealistic body weight exponents, it was con-
sidered that methyl mercury uptake could be better described by a single
compartment model following first order mass action kinetics as described
by the following equation
Yt = K . W a (1)
where
Yt ---- amount of Hg (~ag) taken up from the water in t h,
W = body weight of fish (g),
K ~- constant depending on magnitude of uptake and release rates,
and
a ---- body weight exponent.
The transfer coefficient, equivalent to the volume of water cleared of
mercury by 1 g of fish per hour, for any given body weight is represented
by the equation
Y
Tc -- (2)
W.t.C
where
Tc = transfer coefficient
C = concentration of mercury in the water (/ag/g).
The transfer coefficient can be related to body weight according to the

following function
K, W a- I
Tc -- (3)
t.C
A release or clearance rate term in the uptake equations was not

required since (1) clearance studies immediately following even short
periods of exposure to radioactively labeled methyl mercury in solution
demonstrated that essentially all mercury on the gill surface at 10 rain
post exposure moved into the body of the fish and was not lost from the
fish in the form of fast clearing compartment, 3 and (2) fractional
clearance rates of methyl mercury are so small that short-term uptake
experiments using isotopes would not be effected to any measurable
3The uptake behavior of inorganic mercury differs considerably from organic mercury, and
may require a more complex analysis in that a large proportion of the inorganic mercury on
the gill surface appears to be in fairly rapid exchange with the mercury in the water.
degree by o n g o i n g clearance. In this c o n t e x t it should also be e m p h a s i z e d

t h a t the c o n c e p t u a l i z a t i o n was not r e q u i r e d to yield " n e t " rates o f u p t a k e
b u t r a t h e r to establish t r a n s f e r coefficient values which could be b u i l t into
e q u a t i o n s which include c l e a r a n c e factors, in o r d e r to e s t i m a t e " n e t "
a c c u m u l a t i o n rates.

T h e use of a t r a n s f e r coefficient (Tc) assumes t h a t u p t a k e rate o f
m e r c u r y is directly d e p e n d e n t on m e r c u r y c o n c e n t r a t i o n in the water a n d
t h a t Tc is i n d e p e n d e n t of c o n c e n t r a t i o n . To check this a s s u m p t i o n t h r e e
g r o u p s o f m i n n o w s a n d suckers were e x p o s e d for 3 h to a r a n g e o f methyl
m e r c u r y c o n c e n t r a t i o n s at 14 to 16~
T h e results (Table 1) illustrate t h a t m e a s u r e d t r a n s f e r coefficients were
i n d e p e n d e n t o f c o n c e n t r a t i o n over a 100-fold range, covering levels in
water usually e n c o u n t e r e d in n a t u r e .
TABLE 1--The effect of methyl mercury concentration in water

on its uptake from water by fish. a
Methyl Mercury Fish Transfer
Concentration Body Coefficient b Uptake
in Water, Weight, (water to fish), Rate,
/ag Hg/ml g per h gg Hg/h/fish
0.01 x 10-3 2.1 4- .2 3.8 4. 0.4 0.08 x 10-3

(18)
0.10 x 10-3 2.5 -4- 1 4.2 + 0.3 1.10 x 10-3
(25)
1.00 x 10-3 2.2 4- .2 4.1 4- 0.4 9.02 • 10.3
(15)
a In this series of uptake tests, golden shiners (Notemigonus crysoleucus) were exposed for
2 h at a water temperature of 12 to 14~ Values in parentheses refer to the number of fish
used per test.
bTransfer coefficient values represent the weight of water in grams completely cleared of
mercury content by 1 g of fish in 1 h. Each value represents the average -4- standard error.
T h e effect of e x p o s u r e t i m e a n d b o d y weight on u p t a k e of methyl

m e r c u r y by a g r o u p o f 55 fish is illustrated in Fig. 1. T h e n fish consisted
o f 35 minnows ( N o t e m i g o n u s crysoleucus), b o d y weight range 1 to 12 g,
a n d 20 suckers ( M o x o s t o m a m a c r o l e p i d a t u m ) , b o d y weight r a n g e 10 to
120 g. It is clear from the d a t a t h a t the b o d y b u r d e n o f m e r c u r y increases
b o t h with t i m e o f e x p o s u r e a n d b o d y weight. Analysis o f the d a t a in t e r m s
o f the s i n g l e - c o m p a r t m e n t c o n c e p t u l i z a t i o n yielded Yt = 4.3 14~'76 a n d
}re = 8.3 W ~ for the 2 a n d 4-h e x p o s u r e p e r i o d s to water with a methyl
m e r c u r y c o n c e n t r a t i o n of 0.2 x 10-3 /~g H g / m l , respectively. W h e n
15
O.z..
~.r \X
>-~ lo
a:r-
tOt~
2 o
40 80 120
FISH BODY WEIGHT (g)
FIG. 1--Uptake o f methyl mercury in relation to fish body size. Uptake rates and transfer
coefficients calculated on the basis o f the single-compartment concept defined in the text
using a fish body weight exponent o f 0.75 (established by the data in Fig. 2) and a methyl
mercury concentration in water o f O.03 x 10 -3 I~g Hg/mL
calculated in terms of uptake per hour and a mercury concentration of

0.03 x 10-3 #g H g / m l water, the equation becomes Y = 0.16 W ~ No
specific significant species differences were observed when the data for
each species was analyzed separately.
To a first approximation, then, results on methyl mercury uptake from
water presented in Fig. 2 demonstrates that body burdens of mercury in
fish vary proportionately with the 0.76 power of the body weight. The
marked effect of a body weight exponent of 0.76 on reducing the transfer
coefficient in large fish compared to small fish is illustrated in Fig. 1 and
Table 1.
Although the one-compartment model defined by Eq 1 appears to
account for a substantial part of the observed variation in uptake rate
with size of fish using a body weight exponent of 0.76, it is a patently
simplistic model and the value of the exponent may vary slightly in
natural environments due to possible interspecies differences and seasonal
effects.
Further evidence for these relationships is given by the fact that uptake
rates for a number of other species of larger body size, including carp,
brown bullheads, and pike, not used in the calculation of the equations,
nevertheless showed close agreement with the computed values. Thus, a
600
A
"I- U hr. exposure
~L
!
o
x 400
hi
exposure
0
t,r
hl
)-
"1- " ~..~'~ 2 hr. exposure
W r - -
I I I I I I l
0 40 80 120
FISH BODY WEIGHT (g)
FIG. 2--Methyl mercury uptake by .fish at 15 to 16~ from dechlorinated tap water
containing 0.2 x 10-3 l~g Hg/ml. Species studied, Catosomus commersoni, Monostroma
macrolipidotum, Notemigonus crysoleucas, and Notropis cornutus.
variety of species covering a 200-fold range in body Weight tested over a

wide range of mercury concentrations showed uptake rates and transfer
coefficients predictable from body weight when the temperature was held
constant at 15 to 16~
The uptake rate of methyl mercury from water by fish of various body
weights ranging from 2 to 2000 g have been calculated for fish in water at
15 to 16~ containing methyl mercury at a concentration of 0.03 x 10 -3 /ag
Hg/ml and are presented in Table 2.
While the body weight exponent of fish tested at the temperature of
acclimation may be relatively stable especially over a sufficiently wide
weight range, those of fish moved to new conditions may show large
variations especially when the weight range is small. In the group of
minnows and suckers (1 to 12 g body weight) moved from 12 to 13~ tap
water and tested in either tap water or Ottawa River water at 10 and
TABLE 2--Effect offish body size on uptake from water of methyl mercury by various
species at water temperatures of l5 to 16~
Body Transfer Uptake c

Species Weight. Coefficient b Rate,
Studied a g (Tc), per h g Hg/h/fish
Experimental 2 4.39 0.26 x 10-3

Values 10 2.98 0.89 x 10-3
SO 2.03 3.05 x 10-3
100 1.72 5.16 x 10-3
200 1.06 6.36 x 10-3
300 0.94 8.46 x 10-3
Extrapolated 500 0.80 12.09 x 10-3
Values 700 0.72 15.12 x 10-3
1000 0.65 19.50 x 10-s
1500 0.57 25.65 x 10-3
2000 0.52 31.20 x 10-3
3000 0.46 41.40 x 10-3
a Species studied, Catostomus commersoni, Monostoma macrolipidotum, and Notemigonus

crysoleucas.
bTransfer coefficient values represent the weight of water in grams completely cleared of
mercury content by 1 g of fish in 1 h.
c Uptake rates calculated on the basis of the single compartment concept defined in the
test using Te values established by the data illustrated in Fig. 2. A mercury concentration in
the water of 0.03 x 10-3/ag/ml was used, to calculate uptake rates.
20~ the body weight e x p o n e n t (a) in t h e e q u a t i o n Y = K W a for these

fish varied as shown in the following.
Condition Number of Fish Exponent (a)
tap water, 10~ 42 0.69 + 0.03

tap water, 20~ 48 1.00 + 0.04
river water, 10~ 36 0.82 + 0.05
river water, 20~ , 40 0.57 + 0.05
mean 0.77 + 0.04
T h e ex p o n en t s were all significantly different, but t h e average value o f

0.77 for the e x p e r i m e n t was nearly identical to those o f fish with a wider
weight range previously a c c l i m a t e d and tested in t ap water at 14 to 16~
Several studies have d e m o n s t r a t e d an influence of both body weight
[3,10] and age [4] on the a c c u m u l a t i o n o f m e r cu r y by fish, b u t this is the
first one to d e m o n s t r a t e t h a t the u p t a k e rate and transfer coefficients are
p r o p o r t i o n a l to a power function of body weight. In fact, the evidence
presented suggests t h a t u p t a k e rate increases with increasing b o d y weight
in p r o p o r t i o n to some power function less t h a n unity, which is close to the
function one would expect if u p t a k e rate increased in p r o p o r t i o n to
m e t a b o l i c rate. In a n u m b e r of fish species m e t a b o l i s m M = K . W a,
where (a) varies f r o m 0,67 to 0.85 in different species [11]. Th ese
considerations raise the possibility t h a t u p t a k e of methyl m e r c u r y u n d e r
any given water quality c o n d i t i o n is m e t a b o l i c a l l y d e t e r m i n e d a n d t h a t

both m e t a b o l i s m and u p t a k e would be m o d i f i e d similarly by factors such
as t e m p e r a t u r e , activity, a n d body weight.
U n d e r the present test conditions, m e t a b o l i c rate would be expected to
be s o m e w h a t higher t h a n t h a t for resting fish and ventilation volumes
would p r o b a b l y reach at least 300 m l / k g / m i n , a c c o r d i n g to d a t a for
suckers t a b u l a t e d by Skelton [12]. This would c o r r e s p o n d to a b o u t 180
m l / h for a 10 g fish. A c c o r d i n g to the e s t i m a t e d transfer coefficient,
a b o u t 3 ml water were cleared of m e r c u r y per g r a m or a b o u t 30 m l / h for
a 10-g fish. This seems well within the r a n g e o f c a p a b i l i t y o f the fish with
an e s t i m a t e d ventilation o f at least 180 m l / h and removing only a b o u t 17
p e r c e n t o f the m e r c u r y p e r f u s i n g the gills.
I f u p t a k e of m e r c u r y increases with some m e t a b o l i c a l l y related power
function o f body weight less t h a n unity, then the fractional release rate
m u s t decrease with increasing weight to m a i n t a i n either c o n s t a n t or
i n c r e a s i n g m e r c u r y c o n c e n t r a t i o n with increasing body weight. S u p p o r t for
d e c r e a s i n g f r a c t i o n a l c l e a r a n c e with increasing weight has been f o u n d by
R u o t u l a [13] and by p r e l i m i n a r y studies of the authors. It is, therefore,
possible t h a t b o t h u p t a k e a n d c l e a r a n c e o f methyl m e r c u r y in fish are
strongly d e p e n d e n t on m e t a b o l i c rate.
References
[l] Jernel6v, A. and Lann, H., Oikos (Copenhagen), Vol. 22, 1971, pp. 403-406.
[2] Zitko, V., Finlayson, B. J., Wildish, D. J., Anderson, J. M., and Kohler, A. C., Journal
qf the Fisheries Research Board of Canada. Vol. 28, 1971, pp. 1285-1291.
[3] Johnels, A. G., Westermark, T., Berg, N., Persson, P. I., and Sjoslrand, B., Oikos,
Vol. 18, 1967, p, 323.
[4] Bache, C. A., Gutenmann, W. H., and Lisk, D. J., Science, Vol. 172, 1971, pp.
951- 952.
[5] Gillespie, D. C. and Scott, D. P., Journal of the Fisheries Research Board of Canada,
Vol. 28, 1971. pp. 1807-1808,
[6] MacLeod, J. C. and Pessah, E., Journal qfthe Fisheries Research Board of Canada, Vol.
30, 1973, pp. 485-492.
[7] Lockhart, W. L., Uthe, J, F., Kenney, A. R., and Mehrle, P. M., Journal of the
Fisheries Research Board qfCanada, Vol. 29, 1972, pp. 1519-1523.
[8] Weisbart, Melvin, Canadian Journal of Zoology, Vol. 51, 1973, pp. 143-150.
[9] Burrows, W. D. and Krenkel, P. A., Environmental Science and Technology. Vol. 7,
1973, pp. 1127-1130.
[10] Scott, D. P. and Armstrong, F. A. J., Journal of the Fisheries Research Board of
Canada, Vol. 29, 1972, pp. 1685-1690.
[11] Winberg, G. C., "Rate of Metabolism and Food Requirements of Fishes," Fisheries
Research Board of Canada, Translation Series No. 194, 1960, published originally in
Nauchnye Trudy Belorusskovo Gosudarstvennovo Universitete imeni V. I. Lenina, Minsk,
1956.
[12] Shelton, G., "The Regulation of Breathing," Fish Physiology. Vol. 4, W. S. Hoar and
D. J. Randall, Eds., Academic Press, New York and London, 1970.
[13] Ruotula, private communication.
Monitoring and Remote Sensing
J. E. H a g a n j a n d R. L. E s t e s j
Experiences in Operating a
Continuous Water Quality
Monitoring Network
REFERENCE: Hagen, J. E. and Estes, R. L., "Experiences in Operating a Continuous

Water Quality Monitoring Network," Water Quality Parameters, ASTM STP 573,
ABSTRACT: Experiences in operating a network of continuous water quality monitors

in the southeastern United States have demonstrated the utility of such networks for
pollution abatement monitoring. Operation and maintenance procedures and routine
calibration and quality control procedures produce highly reliable data. While local
cooperators are necessary for economic reasons, the vital role of the trained electronic
technician cannot be overemphasized.
Modifications and technical improvements to monitoring equipment are discussed.
Problems encountered have been primarily mechanical in nature, particularly pumps.
Computer programs and semiautomated data handling systems have been developed
to produce timely reports.
KEY WORDS: water quality, automatic control equipment, monitors, pumps, environ-
mental tests, pollution
Early efforts at automatic water quality monitoring in the Southeast

Region of U.S. Environmental Protection Agency (EPA) began in the
mid-1960's. Equipment which was commercially available at the time was
installed at several sites. While some of these early experiences were
disheartening, it was nevertheless valuable background for development of
instrumentation and for the present network. This experience was in part
responsible for the decision to prepare specifications for monitoring
equipment rather than to take off-the-shelf devices. Much of the testing of
prototype equipment was conducted by the Taft Sanitary Engineering
Center working through the Southeast Regional Office.
The present automatic water quality monitoring program began in 1968
'Chief. Monitoring and Data Support Branch, and electronics engineer, respectively, Sur-
veillance and Analysis Division, Region IV, Environmental Protection Agency, Athens, Ga.
30001.
367

with the purchase of 15 monitors which were built to agency specifi-

cations 2 by the Schneider Instrument Company.
From the outset, it was realized that continuous monitors were a
departure from prevailing practice in environmental monitoring. Con-
sequently, extra care was exercised in the inspection, maintenance, and
quality control program to ensure the collection of valid data. Data
processing initially consisted of visual scanning charts for violations of
water quality standards and manually tabulating hourly observations.
Reports were prepared manually and distributed to cooperators. Con-
version to a computerized data system resulted in serious interruption of
the data handling. That problem and the resulting backlog of unprocessed
data have now been corrected at considerable cost and effort.
During these several years of operating experience, many problems have
been encountered. Emphasis in this paper will be upon the solutions to
those problems in an effort to help others avoid them.
The System
At the outset, let it be clear that the assessment of the automatic water
quality monitoring program in Region IV is that it has been successful.
While the system has not grown or developed as rapidly as was originally
envisioned due to fiscal constraints, the scope and operation has shown
that:
1. automatic monitors can measure parameters of environmental
significance,
2. automatic monitors will operate reliably in the field with proper
attention,
3. automatic monitors can produce data of acceptable analytical
quality, and
4. systematic data handling can produce timely results without
telemetry.
The "system" consists of three principal components: hardware, per-
sonnel, and procedures. It is important to grasp this broad system
concept. Too often monitor systems are described only as hardware. Yet,
the personnel who calibrate and service that hardware are indispensable.
And the procedures by which monitoring information is reported and fed
back into the decision-making process must be the ultimate test of the
system's success.
Hardware
The on-site hardware typically consists of a submersible pump, piping

and valving, the monitor with its manifold, sensors, signal conditioning
2Mentink. A. F.. Spec~tications.for an Integrated Water Quality Data Acquisition System,
U.S. Department of the Interior. FWPCA, Jan. 1968.
HAGAN AND ESTES ON CONTINUOUS WATER QUALITY MONITORING 369
circuits and recorders, and a shelter. Electrical power must be supplied

for both pump and monitor operation.
The primary purpose of the shelter is to maintain a suitable environ-
ment for electronic equipment, to protect the equipment from vandalism,
and to provide a work space for personnel who service the equipment.
Structures presently in use vary from brick buildings with steel doors to
lightweight metal buildings and two-wheel camper trailers. Experience has
shown that the visually-accessible shelter suffers the least vandalism. The
trailer-mounted monitor offers the obvious advantage of being relatively
easy to move. While most monitors are installed for periods which would
justify fixed structure shelters, mobility may be very desirable at sites
which are subject to flooding.
Pumping systems have been the single most serious problem in the
existing network. Typical problems include:
1. remote or inaccessible pump site or both which makes maintenance
difficult,
2. physical damage to pumps by vessels,
3. premature impeller wear from sand or other suspended materials,
and
4. leaking seals causing electrical short.
An unfortunate experience with an inaccessible pump resulted in the
loss of several months of data. The pump was rigidly mounted and
accessible only from a boat. The pump failed during flood and it was
necessary to wait for the water to recede before retrieving the faulty
pump. This experience has led to the decision that all pumping systems
will be mounted on either floats or tracks and that they will be recoverable
from piling, dock, bridge, or similar structure which is accessible from
shore and not subject to prolonged flooding.
At one station in a busy harbor, an adrift vessel severely damaged the
shelter structure and pumping system. Although the equipment was
recovered, the station was out of operation for several m o n t h s while the
involved parties settled claims and rebuilt the supporting piling structure.
Such disasters cannot be completely eliminated, but consideration of
navigation hazards in the selection of monitor sites can minimize the
possibility and the severity of damage.
At another station, suspended solids were eroding pump impellers in
about two to three months. A skirt was fitted around the pump intake.
The upward velocity of the water was such that the suspended sand
particles would not be drawn into the pump. Yet, the settling well volume
was small enough not to adversely affect the sample. Unique site specific
problems require equally unique solutions.
Some improvements have been made on the monitor itself although
hardware modifications have generally been minor. At the start of the
monitoring program, two problems occurred quite frequently. One was

pressure increases in the water intake manifold, which would blow water
out onto the electrical wiring. This was corrected by installing a pressure
regulator and flow restriction in the manifold. The other problem mani-
fested itself only after several months of operation. Threaded plastic
plugs, which provided access to the sample manifold for routine weekly
cleaning, would wear and begin to leak. To resolve this problem,
compression plugs similar to drain plugs used in small boats were
installed. No further problems have been encountered with leaky mani-
folds.
Over a time span of approximately two years, the pH system would
suddenly start producing erroneous results. The cause of this was finally
traced to the reference electrode. This electrode would become plugged
and would cause the instrument to read extremely high or off scale. The
problem existed in either a dirty environment or when the temperature of
the sample decreased enough to cause salting out of the electrolyte. A
number of different corrective techniques were tried over a year of
operation. Finally, a new type of electrode became available on the market
that minimized this problem. This electrode, after a trial period, has now
been adopted and is in use throughout the monitoring network.
Personnel and Maintenance

The heart and success of any automatic monitoring program is the
service personnel who inspect, clean, calibrate, and maintain the hardware
system. In almost any monitoring situation, bad data are worse than no
data. And, again, the quality of the data produced by the hardware
system is highly dependent on the quality of service and calibration the
equipment receives. Two types of servicing are provided: weekly cleaning
and monthly calibration.
Because of the distance between monitor sites and, consequently, the
time and expense of travel from the central office, local cooperators or
contractors are utilized for weekly service. Experiences involving coopera-
ting private industry, state pollution agencies, Federal agencies, and paid
contractors have not indicated any preference based on performance.
Certainly, there is economic advantage in having cooperators who contri-
bute their services in exchange for data in which they have an interest.
During the weekly cleaning, the following activities are conducted:
1. check reading of probes before cleaning;

2. shut down pumps and clean monitor manifold and probes;
3. make minor repairs to pumping or piping system, probes, or
recorders;
4. change chart, if needed;
5. restart pump and thoroughly flush and backflush the system; and
6. calibrate dissolved oxygen (DO) probe and check calibration of other
probes.
Note that no adjustments except for DO are made on weekly inspec-

tions. These inspections are made by local cooperators or contractors who
are not trained electronic technicians. They simply record calibration data
and notify the project leader if serious troubles are encountered.
Once each month, or on special call, the station is visited by an EPA
electronic technician. He performs the complete cleaning and calibrating
routine previously described. In addition, he performs an electronics test
of the monitor circuit and recorder, makes calibration adjustments, and
when necessary, replaces electronic circuitry modules.
This procedure has resulted in the virtual elimination of downtime due
to electronic or recorder failure, and loss of data due to poor calibration.
Since the beginning of the monitoring program, the procedure for
calibration has changed drastically. Initially, all calibration was performed
using standard solutions and placing the probe to be calibrated into the
solution, making a correction adjustment and then going to the next
solution and repeating this process. Since the adjustments on the analyzers
are interdependent, the process must be iterated three or more times.
When a probe is changed from one solution to another, it requires time to
reach a stable reading. The length of time depends on the parameter
being measured, but averages about 10 min. This procedure required a
large amount of time and increased the likelihood of contamination of the
calibration standards.
A better method is now in use which features a minimum of chemical
standardization and a maximum of electronic adjustment. Briefly, the
procedure is as follows: the probe to be calibrated is placed in a standard
solution and the voltage output to the recorder is noted. This is repeated
for three calibration solutions for each sensor. After recording these
voltage values, the internal reference adjustments are set to produce these
voltages. When the voltages are set, then the analyzer is calibrated to
provide the meter readings that the standards would have read if they
were used. This method speeds the process by a factor of three, provides
greater accuracy, and keeps contamination of the standards to a
minimum.
During the summer months, several stations were showing degradation
of the DO content of the water sample delivered to the monitor as
compared to the stream. After trying increased sample flow to reduce the
retention time in the pipes, a disinfection and backflushing scheme was
developed. Once per month when the technician visits a station, liquid
bleach is placed in the sample line. This bleach is pumped up and down
the line by cycling the pump until it is free of debris. This debris consisted
of worms, algae, and other attached organisms. After performing this

backfiushing, the loss of DO between pump and monitor is insignificant.
The weeks that the cooperator visited the station, backflushing is
performed using only the sample water itself and not the bleach.
Much of the station down-time is due to mechanical malfunctions which
go undetected until the next regularly-scheduled service visit. In general,
down-time can be minimized by: (1) maintaining a stock of spare parts,
and (2) telemetry systems which sense and indicate equipment mal-
functions.
Previously-mentioned fiscal constraints have prevented the implemen-
tation of telemetry systems in the Southeast Regional network. Therefore,
the data system described here must be considered intermediate.
Data Processing
Automatic monitoring devices produce an incredible volume of data.

The ultimate success of the system cannot be fully judged until these data
have been reduced, analyzed, interpreted, and reported. The present data
system involves converting data from strip charts to computer format for
processing into the EPA water quality control information system.
Strip charts and quality control check sheets are mailed to EPA weekly
by the local cooperators. Charts are visually inspected and necessary
instrument calibration corrections are noted on the chart by the electronics
technicians. Charts are then delivered to data processing which digitizes
hourly values. Digitizer output is a punched paper tape. This tape is then
input through a remote terminal into the EPA computer system. Editing
and calibration corrections are made, a verification plot of data points
prepared, and data are written onto a disc file. Once each month, a
report is prepared from the disc file and distributed to all interested
parties including the local cooperator. Disc files are then merged into a
magnetic tape master file of hourly observations. Simultaneously, daily
summaries are computed and submitted for storage in the EPA Water
Quality Control Information System (STORET). The present operating
schedule permits a monthly report to be distributed by the 15th day of the
following month.
One additional piece of data subsequently obtained and stored into
STORET separately is flow. Flow, or stage, could be monitored at each
station; however, short-term water quality data utilization involving detec-
tion of standards violations is concerned only with concentration. Long-
term trend interpretation requires flow, but these data can be obtained
from U.S. Geological Survey within an acceptable time frame for long-
term trend analysis.
Procedures
Tying hardware, personnel, and data processing together into a system
calls for a procedure. Many individual steps in this procedure have been
mentioned, but the overall procedure has additional intrensic value.
Careful procedures result in good habits by cooperators and service
personnel. Procedures provide for quality control. Procedures provide
timely submission of strip charts, data, and reports. Deviation from
procedure provides an immediate warning of trouble in the system.
Cost
Present operating costs are estimated at $7100 per year including
personnel, travel, ADP support, utilities, and a paid local cooperator.
These estimates are based on a staff of one electrical engineer (half-time),
two electronics technicians (0.8 time), data clerk (half-time), utilities at
$100 per month, and local cooperator at $100 per month. Replacement
and repair are not included (see Table 1).
TABLE 1--Cost estimate summary.

Total Annual Rate
Capital Cost
Monitor 12 000 1200
Pumping system 500 250
Shelter 5000 500
Subtotal 1950
10% Repair and replacement 195
Annual capital cost 2145
Operation
Personnel 3950
Travel 5oo
ADP 250
Utilities 1200
Local cooperator 1200
Annual operation 7100
Total Annualized Cost 9245
The present staff of two technicians have operated up to seven sites at

once and that level of activity was used to estimate the costs. The
m a x i m u m load for two technicians would be about eleven sites in which
case the unit cost would be reduced somewhat.
Capital investment in equipment may run between $8000 to 12 000 per
monitor. Assuming an average life of ten years for each instrument, the
annualized capital cost is approximately $1200.
Pumping system costs about $500 and have a life of about two years.
Annual cost is $250.
Shelters and p u m p mounting facilities average about $2500 for shore-
based structures, or up to $5000 for piling-mounted structures. The
structures should be good for the life of the station.
A 10 percent repair and replacement rate would cost up to $195 per
year.
One station measuring hourly observations of four parameters for one
year will generate 35040 data observations at a total annualized cost of
$0.26 per observation.
D. H. Cullen j
Automatic Water Quality

Monitoring Within the
Saint John River Basin
REFERENCE: Cullen, D. H., "Automatic Water Quality Monitoring Within the Saint
John River Basin," Water Quality Parameters, A S T M STP 573, American Society for
ABSTRACT: Automatic water quality monitoring within the Saint John River Basin was
conceived in 1968, and its sponsors were the New Brunswick Government and the Water
Quality Branch. Initially, it was intended to monitor one trans-boundary stream which
was causing serious concern because of the indiscriminate dumping of potato processing
waste by a United States processor. The number of monitors was subsequently increased
to eight, and these have been located throughout the basin. The parameters being moni-
tored at present are: temperature, dissolved oxygen, pH, conductivity, and chlorides.
The paper outlines the various problems encountered to date in operating the moni-
tors. These include electronic difficulties, telemetry, pumps, probes, and operating
under winter conditions. The various solutions that were applied to overcome the
problems are also discussed.
Handling of the analytical information generated by the monitors is also outlined,
together with an assessment of automatic monitoring and its future use, as viewed by
the author.
KEY WORDS: water quality, monitors, environmental tests, automatic control equip-
ment. wastes
I n 1968 t h e r e was, on t h e b o r d e r b e t w e e n N e w B r u n s w i c k a n d M a i n e , a
mini incident whereby citizens dammed the waters of the Presquile River
as a p r o t e s t a g a i n s t a p o t a t o p r o c e s s i n g p l a n t in M a i n e d i s c h a r g i n g its
wastes to this t r a n s - b o u n d a r y s t r e a m . T h e wastes h a d d e t e r i o r a t e d t h e
w a t e r q u a l i t y o f t h e s t r e a m to t h e e x t e n t t h a t it killed s e v e r a l h u n d r e d fish
and created an undesirable aesthetic situation.
A s a r e s u l t o f this i n c i d e n t t h e N e w B r u n s w i c k G o v e r n m e n t a p p r o a c h e d
t h e D e p a r t m e n t o f E n e r g y , M i n e s , a n d R e s o u r c e s ( W a t e r Sector) to install
a Chief, Atlantic Region Water Quality Branch, Inland Waters Directorate, Environmental
Management Service, Department of the Environment, Moncton, New Brunswick E1C 8N6,
Canada.
375
9
a monitor in the stream to provide a constant check on water quality and

hopefully to avoid further incidents.
The Federal Department cognizant of its role in boundary waters and
having a desire to examine automatic monitors working under Canadian
climatic conditions decided to purchase eight units. This being the
number which it was felt could adequately provide the experience and
maintain a monitoring function over the entire Saint John River Basin.
The objectives initially set for the monitors are as follows:
1. to study the robot type automatic monitor under Canadian climatic
conditions;
2. to determine its potential as a tool in water management;
3. to automatically measure several water quality parameters (hourly or
more frequently), to record the data in printout form for instant
viewing, and to provide the information in easy-to-read format to
interested agencies;
4. to act as deterrent to polluters; and
5. to show water quality trends for water quality management and
public information purposes.
Monitor Purchase
In March 1970, eight Automatic Water Quality Monitors (AWQM)
were purchased and delivered to Fredericton, N.B., by Automated
Environmental Systems Ltd., of Woodbury, N.Y.
Parameter Selection
The parameters selected for the monitors were limited to those that
could be measured (in the concentrations to be found in the Saint John
River System) by the electro-chemical type of sensor. (Since at the time of
purchase wet chemistry types of monitors did not meet the requirements
of quick response.)
The parameters selected were dissolved oxygen, temperature, conduc-
tivity, pH, chloride, and turbidity. Other sensors were available, but they
were not considered to be significant for the Saint John River Basin.
Physical Arrangement and Operation of Monitors

The automatic monitoring system consists of three basic units; the field
monitor, the data acquisition center (central, which controls the system),
and the telemetry and address system. There is auxiliary equipment such
as a small computer and an alphagraphic unit which is used on a time-
shared basis for data reduction.
CULLEN ON AUTOMATIC WATER QUALITY MONITORING 377
FieM Monitor
The field monitor is designed to be self sufficient. Its makeup and
operation is explained in the following.
Shelter--Each field monitor is housed in its own shelter, six of the eight
units are in travel trailers 10 ft (3.05 m) wide and 20 ft (6.1 m) long. O f
the remaining two, one is in an Armco prefabricated building and the
other is housed in a small building which is located on top of the
Mactaquac Dam. All shelters are heated during the winter, air condi-
tioned in summer, and have an abundance of electric circuits. The travel
trailers have a work surface for laboratory and repair work, storage
cabinets, a 30 gal (110 liter) distilled water storage tank, and a sink. All
plumbing is within the shelter except the supply line from the submersible
pump. This supply line constantly feeds about 15 gal/min (gpm) (57
liters/min) of the water to be analysed to the flow chamber.
Field Monitor Operation--Water to be analyzed is continuously pumped
(via a submersible pump) through a flow chamber which houses the
parametric sensors; the transducer outputs are cabled to the signal
conditioners. The output of each sensor is continuously available in
engineering units at the output of the signal conditioners. There is also an
inherent scanning capacity in the monitoring system. This allows outputs
to be serially scanned to a common point for either display of the
parameter reading or for input to the telemetry package for transmission
to the data central.
Water Quality Parameter Sensors--These measure dissolved oxygen
concentration, hydrogen ion concentration (pH), temperature, conduc-
tance, dissolved chloride concentration, and turbidity. Each parametric
system has its own individual signal conditioner which amplifies, linear-
izes, and controls the 0 to 5-V direct current (d-c) positive output signal.
Certain parametric systems have automatic temperature compensation and
also some have multi-range change capabilities. Each monitor can accom-
modate ten sensors, thus allowing room for growth.
Equipment Status Sensors--These are subdivided into two groups:
environmental alarm and monitor status. The environmental alarm equip-
ment provides adjustable high or low limits or both for each parameter.
When a limit is exceeded, two simultaneous signals are produced: one to
the automatic sampler and the other to the status alarm circuit. On
receiving a signal the automatic sampler will collect three 2-liter bottles of
water. The monitor status alarm equipment is provided to indicate how
well the monitor is functioning. The present status checks are as follows:
1. Service Indicator (is the monitor being serviced),
2. Low Flow (pump out of service),
3. Door Alarm (shelter door open),
4. Drain Clog (water not escaping from flow chamber through drain
line, shelter probably flooded),
5. Shelter Temperature High (inside trailer too warm),
6. Shelter Temperature Low (inside trailer too cold), and
7. Sample Taken (as described under environmental alarm equipment).
When any of the equipment status sensors are signaled, an alarm
condition is indicated within the field monitor by a light which remains on
over the status point until the condition is cleared. Also a signal is
transmitted to the data acquisition center.
Data Acquisition Center (Central)

The central station controls and monitors the eight remote stations. A
single logic coupler at the central station is in communication with each of
the remotes via a signal grade telephone line. The logic coupler at the
central is programmed to interrogate the remotes twice per hour, once per
hour, or every two hours, as selected. When the selected time interval for
scanning arrives, the central station initiates a message to all of the
remotes. The first message is a station address unique for each remote.
The first station alone responds to the initial address transmitted and the
station will automatically turn itself on. The central will then advance the
remote through its various status and parametric readings. This same
sequence is repeated for remote Station 2 and so on. The programming of
the central is accomplished through switches and matrix panels.
When a station is addressed there is an automatic printout of the
station code, year, day, hour, and minute followed by the status and
parametric readings. This requires 2 min for each remote. The data
received is logged on punched paper tape in American Standard Code for
Information Interchange (ASCII) and on hard copy printout for instant
viewing (Table 1).
The central was designed with a capability of handling 30 scan points,
and at present 15 scan points are being utilized.
Telemetry and Address Systems

References previously made regarding telemetry refers to the system
which was in use prior to January 1973. In January the installation of a
new and improved telemetry system was begun, it was completed in May
1973.
The pre-1973 system utilized a line interruption of predetermined length
to address each remote station. Station 1, for example, was called by
interrupting the line for 0.5 s, Station 2 for 0.75 s, Station 3 for 1.13 s,
etc. Each station had an integrator and voltage comparator adjusted so
that it would recognize its pulse when transmitted.
When the address pulse was recognized by the remote it caused the
Station Number
Year
Day of Year
Hour
Minute
Service Indicator
Low Flow
Door Alarm
0
Drain Clog l-
ill
Shelter Temp. High =~
Shelter Temp. Low
Sample Taken
Conductivity Range (0-Low) (1-High)
Conductivity
pH
Dissolved Oxygen
Temperature
Chloride Range (0-Low) (1-High)
Chloride
Low calibration (Range 008-010)
High calibration (Range 169-173)
6Zl~ ONIHOIINO~ h117vno I=J31VAA OllVlAIOII7V NO N 3 7 7 r l O
electronics to be preset. Clock pulses transmitted from the central caused

each scan point to be scanned in turn. The output of each water quality
sensor was fed to a modulator. The modulator converted to 0 to 5-V
output to an equivalent pulse length. This pulse caused a loop inter-
ruption which was sensed at the central. Zero volts represented a 33.3-ms
pulse and 5 V represented a 100-ms pulse. Once received by the central
these pulses were converted back to voltages. The scan time for each
remote is 2 min.
Monitor Loeatlons
The monitors are located within the Saint John River Basin at the
locations shown on Fig. 1. Four of the monitors are at hydro dam sites
and measure the water quality in their respective headponds. These sites
are Mactaquac, Beechwood, and Grand Falls on the main stem of the
Saint John River and at Tinker on the Aroostook River, a trans-boundary
stream. One monitor is on the Saint John River about two miles below
Florenceville, N.B.; monitors are also located on the Meduxnekeag and
Presquile Rivers, both trans-boundary streams. The eighth unit is on the
Kennebecasis River about five miles below Sussex, N.B.
FIG. l--Saint John River drainage basin.

Field Monitor Services
Utilities
The monitors and the central are linked by the telemetry circuit. Each
monitor shelter is equipped with a telephone which is necessary for
communications between each monitor and central during calibration,
standardization, and trouble shooting. Each shelter has been equipped
with a 100-A electrical entrance.
Shelter heat at the hydro sites is maintained electrically, at all others it
is a combination of propane and electricity. Each shelter has an air
conditioning unit.
Water Intakes
The design of water intakes had to take into account the necessity of
removing or replacing the pumps during all seasons of the year. The most
critical period being during the winter season when ice cover can amount
to 40 in. (1 m).
Intakes for the monitors located at hydro sites are relatively simple
affairs. The submersible pump is suspended on a stainless steel cable from
a winch stand which is fastened to a part of the dam superstructure. By
this means the pump can be raised or lowered easily during periods of
open water. The intake pipe is nylon reinforced plastic and is protected
from the elements between the winch stand and shelter by insulation
which is covered by aluminum sheeting. Heating cables run inside the
covering to prevent freezing in the winter, should the pump stop.
The water outlet is similar in most cases to the inlet, with the return
water being allowed to free fall back to the river.
Water intakes for the other sites where trailers are located along river
banks are more complex. Several suggestions for intakes were investigated
before deciding on those now in use. Two of the installations utilize a
10-in. buried steel pipe, extending between a shelter and the low water
level of the river (Fig. 2). The submersible pump can be run down or
retrieved from the pipe during all seasons.
Between the shelter in which the pipe is terminated and the monitor
shelter, the intake is insulated and wrapped with electrical heating cables.
Water is returned to the river by the free fall method as indicated on
the drawings.
One site utilizes a wet well (Fig. 3). Here the water flows by gravity
from the river through a 3-in. plastic pipe to a 10-in. vertical pipe in
which is housed the submersible pump. From the wet well to the monitor
shelter the intake is located inside a buried 8-in. plastic pipe. The return
water line from the trailer is buried in a separate trench and goes directly
to the river.
PO
0
c
t'-
.<
"u
m
--4
m
"I1
FIG. 2 - - W a t e r i n t a k e a r r a n g e m e n t with s u b m e r s i b l e p u m p inside lO-in, steel pipe.

C
t'-
r"
m
Z
0
Z
C
--I
0
-.I
.-I
m
"-n
0
C
t-
O
Z
-4
0
"n
FIG. 3 - - W a t e r intake arrangement with submersible pump l o c a t e d in a w e t well. Z
Operational Problems and Applied Corrective Measures

Contrac~
The monitors were delivered by the supplier in March 1970. The
locating and utility installations were completed by November 1970. In
October 1970, the first contract for winterization of three water intakes
and shelter foundations was let. This contract was not completed until
May 1971. A second contract to winterize four more intakes and founda-
tions, as necessary, was let in October 1971. The contractor for this
second contract went bankrupt with the job about three quarters done,
this contract was not completed until September of 1972.
A contract was let for the conversion of the telemetry and address
system in August 1972, and this was completed in May 1973.
Telemetry and Address

The pre-January 1973 telemetry and address system was found to have
several drawbacks.
1. The transmission interruptions by which each station was addressed

were adjustable at the central, thus allowing the possibility of mis-
adjustments.
2. The transmission interruptions were controlled by timing capacitors
whose value would change with heat and age. The accuracy of the
length of the interruption was impossible to achieve using this
method. That is, line interruption for Station 7 was 5.71.
3. Distortion in the telephone lines or noise spikes would alter the
duration of the interruption.
4. All of the drift errors that were possible in the central were repeated
in the remote.
5. If a line interruption (address pulse) of long duration was broken by
a noise spike it was possible for two lower numbered stations to
answer at the same time.
6. The address pulses were detected in the remote by mercury wetted
mechanical relays that were sometimes sluggish and therefore
distorted the pulse.
7. The telemetry system is essentially a series loop, if one station opens
the loop it is open to all others. Therefore, if one station had a
power failure the loop was open making it difficult to determine
which station was causing the problem.
8. No quality or validity checks were incorporated within the telemetry
system.
9. All of the disadvantages of transmitting station address pulses could
be applied to the transmitted data.
The post-May 1973 telemetry system, which was designed, built, and
installed by Leigh Instruments Ltd., eliminates most of the afore-
mentioned drawbacks and has added quality and validity checks.
The central and remote now transmit ASCII. This is a binary code and,
therefore, any digit can be described with eight successive pulses. The
eighth pulse is called a parity pulse. This pulse can be checked after
reception of a digit to see if any of the other pulses have been altered.
Each station now has a unique ASCII address. The central transmits
the address of the selected station seven times. If the station receives five
of the seven transmissions it will preset the electronics for a scan. This
digital method eliminates the need for adjustment at the central and the
remotes, it also eliminates errors due to component drift, station selection,
transient voltages, and line distortion. Also, the mercury wetted relays
have been replaced by solid-state relays which respond faster and have a
longer life expectancy.
In the remote each parameter value is displayed on a digital voltmeter.
This number with its parity check is transmitted to the central.
In the event of a power failure at a remote, the loop circuit is fail safe.
That is, the loop will close instead of remaining open. At the central the
failing stations can be detected by looking at the data printout.
Provision now has been made at each remote to monitor the trans-
mission using a teletype. This can be a help in determining whether the
fault lies with the system or the telephone company. Since there are too
many numbers transmitted from a station to put them all on one line of
the teletype, a carriage return line feed code has been inserted after the
16th scan point. This carriage return line feed is detected as the number
13, 14, or 15. These numbers serve as a check on the digital transmission.
Two additional channels have been added to each station, one is a 50-mV
signal and the other is a 4.75-v signal. Both of these fixed signals are
measured and transmitted in exactly the same manner as the other
parameters. This provides a calibration check from the output of the
signal conditioners to the final printout at the central (Table 1).
Pumps
Pumps have been a major problem associated with AWQM operation.
The flow-through water monitoring system utilized, involves 24-h/day
pump operation. We have mainly used a 189 semi-positive dis-
placement, water-cooled submersible pump, that has been demonstrated
to neither aerate-deaerate the pumped water, with a 15 gpm capacity.
Several pump motors burned out during the initial monitoring operation
period because of the pump system design. This design had incorporated
a heat relay which would open when a pump would draw excessive
amperage. After the relay cooled down, it would close and the pump
motor would start again, with nothing being done about the original
cause. This was repeated until the p u m p motor burned itself out. This
problem was partially remedied by the installation of overload switches
which have to be reset manually after being opened. These, however, are
not fail-safe devices and burned out pumps are still a problem. Also,
continuous pumping in waters with excessive suspended matter wears out
p u m p parts.
For unexplained reasons a p u m p may operate at one site for six months
while its duplicate replacement may only last one day. At other times a
p u m p may operate up to a month without incident and, if stopped to
carry out necessary maintenance to a supply line or the flow chamber, will
not start again without overhaul.
We have, since April 1973, been working with the manufacturer of the
1-hp Prosser P u m p to determine how we can best utilize and maintain
these p u m p s considering the use they receive. To date we are satisfied we
are making progress with the manufacturer on modifying his p u m p for
our type of use.
Electronics
Long runs of unshielded wire in both the central and remotes have
caused our most serious electronic problem. Transient voltages from
transmit relays, light switches, and motor starting circuits were being
picked up and causing parameter shift registers to be reset or advanced at
the wrong time. (These problems have been corrected.)
Lightening has been the cause of some major failures. This has
necessitated the replacement of numerous components in the remote
circuitry. These lightening strikes have happened despite the fact that
trailer and electronic systems are grounded.
The chloride probe has been and continues to be a source of concern.
This probe has a voltage output that is logarithmic. The lowest range is 0
to 120 mg/liter of chloride, the normal chloride level in the Saint John
River Basin is around 20 mg/liter. The amplifier is relatively insensitive to
these levels. Also, linearizing the probe output and adjusting the slope
with the amplifier is a difficult job.
To overcome this problem with the chloride measurement, consideration
is being given to transmitting the nonlinearized signal back to the central
and using the computer to calculate the real value in engineering units.
Sensors
O f the sensors which were originally purchased with the system, two
have been a consistent problem; chlorides and turbidity. The chloride
problem was discussed in the previous section. The turbidity probe which
is a photometric sensor measures the amount of light which can pass
through a portion of water. It is composed of a light source window and a

light sensor window. An automatic cleaning devise is incorporated with
the probe and was designed to spray a jet of distilled water on the
windows at regular intervals. We have found this auto-cleaning mechan-
ism to be very unsatisfactory. The water jet will not clear the windows of
algae or even fine suspensions, so we have had to discontinue the turbidity
measurement. It may be possible to reinstate this probe if some better
cleaning arrangement can be designed.
Climatic
Weather conditions have, especially during the winter and spring flood
periods, played a disruptive role in the automatic monitor operation.
Winter snowfall in the northern area of New Brunswick can amount to
120 in. (3 m). The snow itself has on several occassions delayed propane
deliveries to the shelters. This has led to freezing conditions inside the
shelters which disrupts the operation of temperature sensitive components.
When the lack of heat has been accompanied by a p u m p failure, we have
experienced freezing of the water intake and drain lines. The freezing of
the exposed lines is difficult but not impossible to cope with, however,
with the trailer shelters there are internal water lines built into the floor
section. These plastic lines have frozen and cracked, requiring replace-
ment. This type of freezing should not occur since all intake water lines
should drain through the pumps, which do not have foot valves; however,
this draining does not always occur. Frequently, when the p u m p stops, a
technician can service it quickly, but it is discouraging and near disaster-
ous when this occurs during evenings and weekends.
The exposed water intake lines have not frozen while the p u m p is
operating, however, the return lines have been clogged by ice which backs
the water up into the shelter. This clogging occurred as a result of the
stalagmite-like formation of ice which happens at the free fall end of the
drain lines.
The trailer shelters were purchased as a package with the monitors. Our
first winter experience with the trailers revealed that their roofs were not
made to withstand very much of a snow load. The snow caused the metal
roof plates to separate causing a number of leaks.
Flooding of the Saint John River in the spring, especially the one in
May of 1973, caused concern, because monitors are located in areas which
could be flooded. Last spring wheels were installed on three trailers so
they could be removed if necessary.
Vandalism
This has been a very minor problem. One of the trailer units at
Apohaqui on the Kennebecasis River had one end damaged by boys

throwing rocks.
The trailer at Belleville on the Meduxnekeag River was broken into
twice, with the door lock having to be replaced both times. However,
nothing was taken and there was no internal damage.
Monitoring Servicing
Prior to the installation of the new telemetry system, remote calibration
and standardization would take up to 8 h. Much of the time was spent
adjusting scaling amplifiers to obtain the same reading at the central that
was being generated at the remote. This condition was directly related to
problems with the old telemetry system, which have been enumerated.
Therefore, it was seldom that all remotes were providing valid data at one
time.
With the revised telemetry system, a remote can now be calibrated and
standardized in 4 h or less. Our experience since May 1973 is indicating
that once-a-week calibration and standardization is adequate. However,
we are now aiming at a five-day interval between visits, unless an earlier
visit is indicated by the status checks read at the central.
For monitor servicing there are two electronics technicians. They have
received training in water chemistry which enables them to carry out the
necessary chemical analysis for system calibration. These technicians also
have the capability to carry out electronic adjustments and repairs, p u m p
servicing, and electrical maintenance, plus several other functions which
are necessary to maintain each remote year-round.
There is also an electronics supervisor who manages the A W Q M
system. He is required to handle the more complicated electronic
problems, to renovate, m a k e design changes, and generally to keep the
system operational.
Data Output
All monitor (punch paper tape) data are processed in a P D P / 8 L
computer with 8K memory. The present program assembles the data on a
24-h basis and provides a statistical summary. Copy of printout for
Meduxnekeag River at Belleville, Station 4, is shown on Table 2.
Graphs of daily and monthly results for each parameter will be
available by December through a program which is currently being written
to allow us to use an alphagraphic printer.
We have been assembling and distributing monitor data periodically
over the past two years. However, due mainly to our telemetry problems
we have had to caution users regarding its validity. Today, with the parity
check we have on each parameter measurement sent over the telemetry
CULLEN ON AUTOMATIC WATER QUALITY MONITORING 38{}
TABLE 2--Meduxnekeag River at Belleville, Station 4, daily report.
Dissolved
Oxygen, Tempera- CI,
H Min Conductivity pH rag/liter ture, ~ mg/liter
1 6 212 7.83 7.02 22.5 10.2

2 6 209 7.67 7.01 22.3 9.9
3 6 209 7.45 6.93 22.0 9.7
5 6 209 7.17 6.90 21.6 9.2
6 6 211 7.10 6.88 21.5 9.3
7 6 210 7.06 6.88 21.3 9.2
8 6 211 7.06 6.90 21.3 9.3
9 6 211 7.10 6.92 21.3 9.3
10 6 210 7.22 6.97 21.3 9.3
11 6 210 7.41 7.02 21.4 9.4
12 6 207 7.76 7.13 21.6 9.8
13 6 206 8.21 7.26 22.4 10.9
14 6 202 8.39 7.33 23.3 11.1
15 6 202 8.50 7.42 23.9 11.2
16 6 203 8.57 7.44 24.6 11.5
17 6 205 8.62 7.47 25.1 11.7
18 6 204 8.65 7.46 25.1 11.7
19 6 202 8.62 7.44 25.1 11.5
20 6 199 8.58 7.37 25.0 11.5
21 6 197 8.51 7.26 24.8 11.1
22 6 192 8.40 7.17 24.5 10.9
23 6 190 8.26 7.08 24.2 10.4
Summary Report
Minimum Maximum
No. of
Test Value H Min Value H Min Mean Readings
Conductivity 190 23 6 212 1 6 205 22

pH 7.06 8 6 8.65 18 6 7.92 22
Dissolved oxygen 6.88 7 6 7.47 17 6 7.15 22
Temperature 21.3 10 6 25.1 17 6 23.0 22
Chloride 9.2 7 6 11.7 17 6 10.4 22
Percentiles 10 % 25 % 50 % 75 % 90 %
Conductivity 197 202 206 210 211

pH 7.10 7.22 7.83 8.51 8.62
Dissolved oxygen 6.90 6.93 7.18 7.37 7.44
Temperature 21.3 21.5 22.4 24.6 25.1
Chloride 9.3 9.3 10.2 11.2 11.5
system, together with the high and low generated signals, we are now
convinced that 95 percent (or more) of the data are valid.
Assessment
1. The objectives originally established for the AWQM system have
been partially met. The outstanding item still to be assessed is its real
value as a tool in water management. This, we believe, will be known only
when management and user agencies have had more time to assess the
generated data.
2. Prior to the telemetry conversion the operation of the AWQM's was
an almost impossible job. The telemetry system which was initially
purchased was hopefully the only one of its type. As previously indicated,
when problems developed there was no easy way to troubleshoot the
system.
The new system has, to date, proven to be reliable, and there is now
time to study and make other improvements.
3. I would recommend to future purchasers of AWQM systems that
they buy them on a turn-key basis. That is, the supplier should install,
place entire system in an operational mode, and satisifactorily operate it
for a specific period of time before it is accepted. In this manner the
purchaser is not involved with the debugging of the system. The supplier
must also be required to provide plans, schematic's, parts list, etc., and a
suitable training course for those who will be responsible for maintaining
the system.
Future Outlook for AWQM's

As the concern for water quality grows so does the need for increased
surveillance of streams, lakes, and coastal environs. When this need for
information requires that it be collected daily or more frequently, then the
answer is probably in the use of AWQM's. With the type of sophistication
that is envisioned with future systems, whereby they will not only measure
those parameters that can be sensed by electro-chemical means, but also
employ wet chemistry techniques that will require only weekly servicing,
then their use for measuring and analyzing water parameters could be
near limitless.
D. A . J. M u r r a y , j D. P o v o l e d o , ~ a n d R. V. S c h m i d t ~
Field Analysis of Dissolved Gases

in Lake Waters by Gas
Chromatography
REFERENCE: Murray, D. A. J., Povoledo, D., and Sehmidt, R. V., "Held Analysis
of Dissolved Gases in Lake Waters by Gas Chromatography," Water Quality Param-
eters, A S T M STP 573, American Society for Testing and Materials, 1975, pp. 391-397.
ABSTRACT: Equipment for the analysis in the field of dissolved gases in lake waters by
gas chromatography is described. It is'portable and can be battery operated. Water
from the required depth is pumped continuously through a small glass stripping device.
It is equilibrated with flowing helium and the gases introduced into the gas chromato-
graph by a sample loop. Problems of oxygen and carbon dioxide invasion and degassing
of supersaturated gases are avoided. The method will measure from 2 to 1000 micro-
moles/liter of carbon dioxide.
KEY WORDS: water quality, gas chromatography, dissolved gases, sampling, environ-
mental tests
T h e p u r p o s e o f this work was to develop a p o r t a b l e b a t t e r y - o p e r a t e d gas

c h r o m a t o g r a p h , c a p a b l e o f p e r f o r m i n g a r a p i d analysis of the dissolved
gases in l a k e waters u n d e r a wide r a n g e o f t e m p e r a t u r e a n d w e a t h e r
conditions. This would facilitate the s t u d y o f p r i m a r y p r o d u c t i o n by
m e a s u r i n g the d a y - t o - d a y c h a n g e s in c a r b o n dioxide a n d oxygen con-
centrations.
M a n y gas c h r o m a t o g r a p h i c techniques for s e p a r a t i n g a t m o s p h e r i c gases
have been d e s c r i b e d . Some o f the m o r e recent p u b l i c a t i o n s are W i l h i t e
and Hollis [1] 2 a n d Carle [2] who used single columns, while F o r s e y [3],
Purves [4], Di L o r e n z o [5], a n d H e r b e r t a n d H o l d i n g [6] used two or m o r e
c o l u m n s . O b e r m i l l e r a n d C h a r l i e r [7] used two c o l u m n s a n d s u b a m b i e n t
t e m p e r a t u r e s to o b t a i n a r g o n m e a s u r e m e n t s . Weiss a n d C r a i g [8] have
p e r f o r m e d a c o m p l e t e analysis o f gases from sea water on b o a r d an
o c e a n - g o i n g ship using two gas c h r o m a t o g r a p h s .
' Technician. research scientist, and biologist, respectively, Fish and Ecosystem Toxicology
Section, Department of the Environment, Freshwater Institute, Winnipeg, Manitoba R3T
2N6, Canada.
391
9
Techniques for stripping gases from water samples have been described
by Williams and Miller [9], Swinnerton, Linnenbom, and Cheek [I0,11],
Gaunt and Shanks [I2,13], Walker and France [14], and Stainton [15].
None of these methods were satisfactory for continuous monitoring, and a
new stripping device was designed.
Experimental
Apparatus
A portable battery-operated gas chromatograph (Analytical Instrument
Development Inc., A.I.D. Model 512) with a microthermal conductivity
detector was used with a battery-operated recorder (Easterline Angus Co.,
Multirange Model 7400). The columns were 18 in. by 1/8 in. stainless
steel packed with Porapak Q, and 48 in. by 1/8 in. stainless steel packed
with Molecular Sieve 5A, placed in series. The sample loop was fitted with
a 1.0-ml stainless steel loop.
Operating Conditions
Oven temperature 48~
Detector temperature 45~
Detector current 10 mA
Helium flow through the columns 8 psi
A Masterflex peristaltic pump (Cole Parmer Instrument Co.) with two
No. 7014 heads was used to provide identical flow rates of water and
helium through the stripper at 25 ml/min. The pump was powered either
by 115 V ac or by 12 V dc from a car battery.
Stripping Chamber (see Fig. 1)

Continuous flows of water and helium were joined in the stripping
device where the helium bubbles through a glass frit and removes the
dissolved gases. A column of water 15-cm high was found to be satis-
factory [11].
Procedure
Lake water from the required depth was pumped at 25 ml/min to the
stripper by a Masterflex pump using a No. 7014 head (Fig. 2). A Y-piece
with one arm constricted by a 1-in. length of thermometer capillary tubing
was placed in the line just prior to the pump. This introduced a 1 percent
flow of 2 N acid which lowered the pH of the sample to about 3.0. A flow
of 25 ml/min of helium was metered into the stripper by a fine control
needle valve. A second No. 7014 add-on head to the peristaltic pump
sucked helium, enriched with dissolved gases, out of the stripper through
MURRAY ET AL ON DISSOLVED GASES IN LAKE WATERS 393
Icm. l.D.
"p HELIUM OUT
WATER OUT q
15•cms
- -
20
D
i:,
o
WATER IN
o
\
J GLASS FRIT
HELIUM IN
FIG. 1--Stripping chamber.
SAMPLE LOOP VALVE

HELIUM
DRYING
Tos
NEEOLE~--~L_~ ~
I SWITCH
RECORDER~
SAMPLE 2N ACID
FIG. 2--Flow diagram.
a drying tube and the sample loop. After about 5 min equilibration time,
the sample loop was injected into the gas chromatograph (Fig. 2, Position
2) where the Porapak Q column separated the carbon dioxide from the
composite oxygen, nitrogen, and methane peak. This then passed on to
the Molecular Sieve column where it was separated and the carbon
dioxide absorbed (Fig. 3). A polarity switch was activated between the
carbon dioxide and the oxygen peaks, since each half of the detector
functions alternately as a reference and as a sensor. The peak heights were
then measured and the concentrations calculated from calibration factors.
I - COMPOSITE PEAK
2- C02 x 64
3- OXYGEN x 4
4- NITROGEN x 6 4
5-METHANE x I
INJECT
5
MINUTES
FIG. 3---Chromatogram showing all gases present.
Calibration
Carbon Dioxide
Standard solutions of sodium bicarbonate were used to calibrate the
system and to check the operation at regular intervals. The peak height
response gave a linear relationship with concentration up to 1000
micromoles/liter, and a calibration factor was found. Samples analyzed
for carbon dioxide by the syringe technique of Stainton [15] were in good
agreement with those analyzed in the field. Regression analysis gave a
correlation coefficient of 0.997 for the response from standard solutions.
Oxygen
Simultaneous samples were taken from identical depths and analyzed by
the stripper/gas chromatography method and by the Winkler titration
method for oxygen. The latter samples were fixed in the field immediately
and titrated later in the laboratory. A linear relationship was found
between the two methods and a calibration factor was calculated. Re-
gression analysis gave a correlation coefficient of 0.990 for the response
from lake water samples against the Winkler results.
Discussion
The equipment described in this paper was tested on Lake 227 in the
Experimental Lakes Area of the Fisheries Research Board, southeast of
Kenora, Ontario. This lake has been extensively fertilized with orthophos-
phate and nitrate since 1969 [16], and is presently in a highly eutrophic
condition.
The chromatogram in Fig. 3 is typical of a 5-m sample showing all the
gases present. The results shown in Figs. 4 and 5 have been chosen to
illustrate typical depth profiles and diurnal curves. They were obtained
under generally clear weather conditions.
DEPTH PROFILE (,um/I)
200 400 600 800 1000

~ f I , , ,
i !
1\
60 4
Z/
or"
I,I
I--
LW
6-
CARBON
9 DIOXIDE ~ o
8-
FIG. 4---Typical depth profiles.
48- ./.1~ 318
42 \o OXYGEN .o - 312
<
E
36 \o - 306
30- - 300
ILl
t'~ E
24- - 294 v
t'~
Z Z
~ 18- >-
t'r"
<
(_)
12- 292
*.., CARBONDIOXIDE 276

6-
I I I I I I I I I
6 8 I0 12 2 4 6 8 10
AM NOON PM
FIG. S - - D i u r n a l curve, 0.5 m.
There was a blank of 30 micromoles/liter for oxygen which was due to

argon. This was the concentration of argon from 4 to 8 m depth in Lake
227 as measured in 1972 using subambient temperatures, which separate
oxygen and argon on a Porapak Q column. This blank was subtracted to
give true values for oxygen.
At the time of writing, the equipment is being used on a raft with a 2.5
kW, 115 V ac generator on shore and a long underwater cable. However,
the system may be operated independently within the battery life of the
individual components. There is a small hut on the raft to protect the
equipment and the operator from the rain and wind since this is a
semipermanent installation.
Alternating current from small generators are notably unstable, but the
A.I.D. gas chromatograph overcomes this to give stable conditions of
temperature and detector current, after an initial warm-up period. This is
important because peak height response is very sensitive to changes in
these parameters, as well as changes in flow rate. The two heads on the
peristaltic p u m p were used to give identical flow rates for helium and
water in the stripper, and the balancing flow of helium was set with a fine
control needle valve. The reliability of the whole system depends on the
reproducibility of all the variables, and careful control is necessary during
the calibration and the analysis of lake water samples.
This technique offers the advantages of a complete in situ analysis of
the dissolved gases in a lake water sample in 15 min, with no invasion

problems since the system is a closed circuit. Bubbles formed by super-
saturated air being released and sticking to the sides of the sample tubes
can cause erratic results for oxygen and nitrogen. Proper design and
placing of the components will alleviate this problem, and the use of
Teflon sample tubes helps the continuous movement of the entire sample.
The method could be extended to the analysis of methane, nitrogen,
and hydrogen sulfide with suitable calibration. Hydrogen sulfide produced
from the anaerobic regions is completely absorbed on the Drierite
column between the stripper and the sample loop.
Using a microthermal conductivity detector the system was sensitive
enough to detect and measure 2 micromoles/liter of carbon dioxide. This
could be further enhanced by using a larger sample loop.
Acknowledgments
The authors are indebted to many colleagues at the Freshwater Institute
and wish to thank them for criticism, advice, and encouragement.
References
[1] Wilhite, W. F. and Hollis, O. L., Journal of Gas Chromatography, Vol. 6, 1%8, pp.
84 88.
[21 Carle, G, C., Journal of Chromatographic Science, Vol. 8, 1970, pp. 550-551.
[3] Forsey, R. R., Journal of Chromatographic Science, Vol. 6, 1%8, pp. 555-556.
[4] Purves, M. J., in Gas Chromatography in Biology and Medicine, Ruth Porter, Ed.,
J&A Churchill, London, 1969, pp. 113-120.
[5] DiLorenzo, A., Journal of Chromatographic Science, Vol. 8, 1970, pp. 224-226.
[61 Herbert, R. A. and Holding, A. J., Journal of Chromatographic Science, Vol, 10, 1972,
pp. 174-175.
[7] Obermiller, E. L. and Charlier, G. O., Journal of Gas Chromatography, Vol. 6, 1%8,
pp. 446-447.
[8] Weiss. R. F. and Craig, H., Deep-Sea Research and Oceanographic Abstracts, Vol. 20,
1q73, pp. 219-303.
[9] Williams, D. D. and Miller, R. R., Analytical Chemistry, Vol. 34, 1%2, pp. 657-659.
[101 Swinnerton, J. W., Linnenbom, V. J., and Cheek, C. H., Analytical Chemistry, Vol.
34, 1962, pp. 483-485.
[I1] Swinnerton, J. W., Linnenbom, V. J., and Cheek, C. H., Analytical Chemistry, Vol.
34, 1%2, p. 1509.
112] Gaunt, H. and Shanks, C., Chemistry and Industry, (London), 1964, pp. 615-653.
[13] Gaunt, H. and Shanks, C., Chemistry and Industry, (London), 1%5, pp. 328-330.
[141 Walker, J. A. J. and France, E. D., Analyst (London), Vol. 94, 1969, pp. 364-368.
[/51 Stainton, M. P., Journal of the Fisheries Research Board of Canada, Vol. 30, 1973,
pp. 1441. 1445.
[16] Schindler, D. W., Armstrong, F. A. J., Holmgren, S. K., and Brunskill, G. J.,
Journal qr the Fisheries Research Board of Canada, Vol. 28, 1971, pp. 1763-1782.
S. Sizgoric j and A. L CarswelP
Underwater Probing with

Laser Radar
REFERENCE: Sizogoric, S. and Carswell, A. I., "Underwater Probing with Laser

Radar," Water Quality Parameters, A S T M STP 573, American Society for Testing and
Materials, 1975, pp. 398-413.
ABSTRACT: Recent advances in laser and electro-optics technology have greatly

enhanced the feasibility of active optical probing techniques aimed at the remote sensing
of water parameters. This paper describes a lidar (laser radar) that has been designed
and constructed for underwater probing. The influence of the optical properties of water
on the general design parameters of a lidar system is considered. Discussion of the
specific details in the choice of the constructed lidar is given. This system utilizes a
cavity-dumped argon-ion laser transmitter capable of 50-W peak powers, 10 ns pulses,
and megahertz pulse repetition rates at 10 different wavelengths in the blue-green (450
to 520 nm) region of the spectrum. The performance of the system, in probing various
types of water, is demonstrated by summarizing the results of initial laboratory and field
experiments.
KEY WORDS: water quality, optical radar, lasers, environmental tests, underwater
equipment
In recent years there has been an increasing interest in the use of lidar
(laser radar) as an active optical remote sensor. Improved lasers with high
powers, short pulses, and ever-increasing range of wavelength selection
and tunability provide suitable transmitter sources for lidar systems.
However, to date the lidar work has been almost exclusively concerned
with investigations of atmospheric phenomena such as aerosol scattering,
cloud studies, and smoke plume tracking [1-3]. 2
Lidar probing underwater is also quite feasible, but so far only a few
such studies have been reported in the literature [4-7] and the true
potential of underwater lidar systems is not yet clearly established. This
paper reports on a newly developed marine lidar unit that has been
designed and constructed to undertake an evaluation of lidar systems for
underwater remote sensing purposes [8].
a Project scientist and professor of physics, respectively, Department of Physics and
CRESS, York University, Downsview, Toronto M3J 1P3, Canada.
398

SIZGORIC AND CARSWELL ON LASER RADAR 399
Although an underwater lidar involves the same basic design principles

employed in the atmospheric lidar, there are a number of very important
additional considerations. It is well known that because of the optical
absorption properties of water [9] the available wavelength region for
operation of an underwater lidar system is limited to the blue-green
portion of the spectrum. Even within this relatively clear optical window
the absorption is such that lidar operation would almost always be at
ranges less than about 100 m. This fact necessitates the use of very short
transmitter pulses and rather wide-band electronic detection systems since
the total time of useful lidar return will only be a few hundred nano-
seconds. In addition the very rapid decrease in signal intensity with depth
requires a very wide dynamic range for both the optical and electronic
components of the lidar receiver.
The water, because of the very large amount of scattering, also degrades
the well collimated beam, spreading the illumination and decreasing
the light flux per unit area very rapidly with increasing penetration depth
[I0]. Thus, two of the best features of atmospheric lidar operation,
namely, high intensity compared to background (solar) light and good
space and time resolution, are very severely restricted in the underwater
application. In addition to the beam degradation caused by the volumetric
scattering in natural water systems, the surface wave structure will further
broaden the beam and decrease the obtainable signal [11]. Thus, there is
a need for more experimental data to obtain information on the practical
utilization of lidar systems for measuring underwater parameters of
interest.
Lidar Design Considerations

In designing a lidar system there are two basic configurations of
interest. These are schematically illustrated in Fig. 1. The first is
the monostatic or backscatter lidar sketched in Fig. la in which the
transmitter and receiver are located at the same position and are
collinearly directed along a single line of sight. The second (Fig. lb) is the
so-called bistatic lidar in which the source and receiver are spatially
separated. With the monostatic configuration the transmitter must be
pulsed to provide spatial information using the time of flight of the
backscattered signal. With the bistatic configuration the transmitter can
be either pulsed or continuous since the spatial location of the scattering
volume is determined by the intersection of the transmitter and receiver
beams as illustrated. In atmospheric work, the monostatic lidar is most
commonly used because of its convenience and relative ease of alignment,
although bistatic systems have been used on several occasions [12].
For underwater applications the substantial amount of beam spreading
and the very large near-field return may make the bistatic system a more
(a)
tro~~ receiver
tronsmifter receiver
....:. .....
. ' . "W Q t O C . ..:....ii.
:.- ".
FIG. I.--Possible lidar configurations f o r underwater studies: (a) backscatter lidar and
(b) bistatic lidar.
attractive configuration than the backscatter system, although for any

airborne lidar instrument the bistatic configuration would be ruled out.
For ship-based systems either configuration would be suitable and, in fact,
the recent work of Ivanov et al [6] utilized a bistatic configuration in
which the entire laser assembly was lowered to a depth of 5 m below the
sea surface. The backscatter arrangement of Fig. la is still the most
convenient and the most readily analyzed and is the one initially being
employed in the present program. In this configuration the received
power, Pr, can be expressed approximately as
SlZGORIC AND CARSWELL ON LASER RADAR 401
Pr =- PtCATle-~'1R f l e - a z R Tz (1)
(R t + R / n ) 2
where Pt is the transmitted power, A the area of the receiver, and C a

constant including numerical factors and the optical efficiency of the
system. R 1 is the distance (in air) from the transmitter to the water
surface, and R is the distance from the surface to the subsurface
scattering location of interest. Tt is the transmission coefficient of the
water surface for the incident beam, and at is the average extinction
coefficient of the downward traveling beam. For volumetric scattering, g is
the volume backscattering coefficient averaged over a scattering volume of
length CT/2n in the direction of beam travel where c is the velocity of
light, T is the pulse length, and n is the index of refraction of water. For
scattering from a submerged surface (for example, the lake bottom)
would represent the average surface reflectivity assuming that the surface
fully intercepted the transmitted beam. a 2 and T z are the attenuation and
transmission coefficients for the upward traveling beam.
The values for the downward and upward attenuation coefficients, a t
and tx2, will generally be different because of the effects of beam spreaa-
ing. The downward traveling beam will initially be rather well collimated
and the value to be used is the so-called " m o n o p a t h " attenuation co-
efficient [10,13]. However as the beam spreads and diffuses, the forward
scattering tends to replenish the beam and reduce the rate of attenuation.
In this case the "multipath" attenuation coefficient is appropriate. In
addition, it is also obvious that in any real situation the attenuations (up
and down) would not ever be constant, and in reality we would have to
write the attenuation as
where the integrals are over the total two way path distance and the point
values of a(r) would include beam spread effects and any spatial variations
of the water properties.
The transmission coefficients, T 1 and Tz would include the ordinary
reflective losses at the air-water interface and the loss of intensity because
of surface roughness arising from wave structure. These surface effects
would be values averaged over the diameter of the beam and would thus
depend on the beam size and water roughness, and would generally differ
for the downward and upward paths. As well as altering the values of the
transmission coefficients, the surface roughness would also add to the
broadening of the beam as it passes through this interface [11]. For some
applications, however, such as with shipborne equipment, it is possible to

optically couple the transmitter and receiver to the water using emersion
optics, and in this way these surface complications can be greatly
minimized.
From Eq 1 it is apparent that the scattered laser signal contains
information on the attenuation coefficient and the volume scattering
coefficient of the water albeit in a rather complicated form. Thus, this
signal should be utilizable to derive measurements of these quantities and
their variation with distance along the propagation path as is done with
atmospheric lidar systems.
It should also be pointed out that in the simple form of Eq 1 we are
considering only the intensity of the backscattered signal. The signal will
also contain a considerable amount of polarization information which can
also be used to measure the water properties [14]. Such measurements of
the polarization are already demonstrating their usefulness with atmos-
pheric lidar measurements [15,16].
It should also be noted that /3 includes both elastic and inelastic
scattering processes. For Raman scattering, the appropriate Raman
scattering cross section would be employed, and by measuring at the
appropriate wavelength shift from the incident radiation, this scattering
can be used to identify selectively and monitor specific Raman active
chemical species in the water in a fashion similar to that already
demonstrated with atmospheric lidars [I7,18]. The practical problem here,
of course, is that the Raman signals are several orders of magnitude below
those for the Mie and Rayleigh scattering.
When considering subsurface Raman measurements it is also necessary
to include in Eq 1 the wavelength dependence of the water absorption
since the transmitted and scattered beams will be at different wavelengths.
In Fig. 2, a sketch of the absorption coefficient as the function of
wavelength in the optical "window" of several water samples is shown. As
already noted, distilled water has minimum absorption in the blue-green,
centered at about 500 nm. In natural waters the overall absorption
increases and the center of the window moves toward the yellow portion of
the spectrum [9]. Included in Fig. 2 are solid lines representing the two
strongest emissions of the argon laser at 488 and 514.5 nm. The dotted
lines represent the approximate central positions of the Raman shifted
Stokes bands of the OH stretch (3450/cm) in liquid water arising from
these two laser frequencies. These Raman shifts are quite substantial and
show that it is necessary to have the primary transmitted wavelength well
toward the blue end of the window to ensure that the Raman lines are not
too heavily attenuated.
There are at present relatively few commercially available lasers which
will meet the necessary requirements for subsurface probing. The three
best systems are the frequency doubled neodymium laser operating at 530
1.2
Vineyard
l
I I
~ /.0 .sound ~
I I
.~ .8
.Chesopeoke
.6 .Boy
.2 -wofer
Distilled
-
0 t I
400
1
500
I I
600
II I ,
700
I
Wavelength (nrn)
FIG. 2--Absorption in the visible "window" for several water samples. The solid lines in-
dicate the major argon laser emissions at 488 and 514.5 nm. The dashed lines indicate the
approximate positions o f the band centers for the 3450/cm H20 Raman scattering obtained
using the argon lines.
nm [6, 7], the neon laser at 540.1 nm [4], and the argon ion laser which
operates at a n u m b e r of wavelengths between about 460 and 515 nm with
its two most intense emissions at 488 and 514.5 nm as shown in Fig. 2.
The typical operating parameters of these lasers are summarized in
Table 1. The neodymium laser is flash lamp pumped and operated in the
Q-switched mode, The neon laser is excited by electrically pulsing the low
pressure gas inside a discharge tube. The argon laser is a continuous gas
discharge with the laser output pulse being controlled by means of an
intra-cavity acousto-optic diffraction cell [19] to operate in the so-called
"cavity d u m p e d " mode.
The Marine Lidar System
Our system design has been based on the use of the argon laser
operating in this cavity dumped mode. This unit, although it has lower
TABLE 1--Typical operating parameters of lasers suitable for underwater lidar systems.
~. r PRF Peak Power AveragePower
Laser (nm) (ns) (Hz) (W) (W)
Neodymium 530 15 0.1-100 106-10' 0.05-0.2
(doubled)
Ne 540 5 100 104 0.005
Argon-Ion 460-515 0.2 0-107 102 0.5
10-1000
peak powers than either the neodymium or neon lasers, has high average
power and was chosen because of its greater all-round versatility for
underwater studies. The laser employed for the transmitter is a Spectra
Physics Model 164 argon laser with a Model 365 acousto-optic output
coupler. This unit at 488 and 514 nm delivers peak powers of up to 75 W,
pulse repetition frequencies variable from continuous wave (CW) to 107 Hz
and pulse widths variable from about 10 -e to 10-e s. Average power in the
cavity dumped mode of up to 0.4 W at these wavelengths is available. The
unit will operate on seven other argon lines in the blue-green spectral
range at lower power outputs.
Figure 3 shows a schematic diagram of the overall system, and Fig. 4
shows a photograph of the apparatus as mounted on its mobile carriage.
The laser transmitter is mounted horizontally and the output beam
(approximately 1.S mm diameter) is expanded to a diameter of about 2
cm by an adjustable collimator. The lidar can be operated in either a
parallel-beam configuration ~(~) in Fig. 3) or a coaxial-beam configuration
( Q in Fig. 3) for the transmitter-receiver geometry. The choice of the
geometry is dictated by the specific requirements of the experiment. The
parallel geometry provides a very strong suppression of the near field
backscattering when the water surface is close to the lidar unit. This is
achieved by adjusting the alignment so that the receiver does not see the
laser beam impingement spot on the surface nor the first few meters of
subsurface scattering.
LASs l"~,aNSM/T/'LrR R
/ ~ • / Pt,V...CE ' ~ Me'ot~'P,e t,evt~q

I cau.~roR ~ ~,srt,oY
irm~r,e'e ANO PULSe I--"1 p ~ o c ~ s ~ r ~ opncs

~--~ L~'L,Y CONrROL$ U (F,L,R . Ls POs .tc...)
eOLARO*~ I Ip'm" r ~
CAMERA ~ I ~.V a RANGE 6ArtN~ \
F I G . 3--Lidar system arrangement. (1) and (21 indicate alternative arrangements for the
transmitter beam geometry.
FIG. 4--Photograph of the marine lidar system.
The receiver is a 20-cm diameter Newtonian telescope with variable field

stop and processing optics which include a 1.0-nm bandpass filter and a
quarter wave plate and linear polarizer for analysis of the scattered signal.
A very high speed 5-stage photomultiplier (RCA Type C31024) is used as
the detector. The output is fed through a preamplifier to either a 250
MHz oscilloscope or to a "boxcar" signal averager. Triggering signals and
pulse delays are provided by the associated electronics with the primary
synchronization being from the output laser pulse itself which is detected
by a fast silicon photodiode mounted in the transmitter section.
The major limitation in the present system is in the frequency response
of the boxcar averager which cannot clearly resolve pulses which are
shorter than about 30 ns. This limits the range resolution of the present
system to about 4 or 5 m in water. A new signal averager to be installed
shortly will have subnanosecond time resolution and will provide lidar
operation limited only be the resolution of the optical pulse (1 to 2 m).
As shown in Fig. 4 the lidar is mounted on a mobile carriage complete
with its electronics and beam steering optics. Seen in this photograph (left
foreground) are the 45-deg mounted mirrors for downward direction of the
transmitter and receiver beams. These are mounted on an extendable
mount which protrudes over the side of the tank (for lab use) or the vessel
(for shipborne measurements). The vertical bars shown in this assembly
are to support a subsurface mirror-periscope assembly to permit horizontal
propagation of the lidar beam at depths ranging from zero to about 2 m
when working in the tanks of the Canada Centre for Inland Waters
(CCIW) at Burlington where the depth is only about 3 m.
Measurements
Some initial measurements with this lidar system have been conducted
at the large indoor CCIW tanks. In addition, in August 1973 the system
was taken aboard the CCIW research vessel Limnos and measurements
were made over a four-day period in Lake Erie at a position about 10
miles south of Port Stanley, Ontario.
In the indoor tank trials the main aim was to investigate lidar signals
from the volumetric backscattering of relatively stable and homogeneous
water samples and from diffusely scattering surfaces positioned at known
underwater distances to ascertain the useful operating range of the lidar
system. The targets used were 4-ft-square plywood sheets painted either
black or white.
Figure 5 shows a schematic trace of a typical signal return from a
submerged target. As shown in this figure the return contains an initial
broad peak from the volumetric scattering of water itself followed by a
narrow pulse from the target. The volumetric return initially increases
with range as the receiver field of view encompasses the transmitted beam.
After full overlap of the beams at Point P the volumetric signal falls off
according to Eq 1. The target return will have a range dependent
amplitude, h, as also calculated from Eq 1 using the reflectivity of the
surface in place of the volume backscattering coefficient. The width, w, of
the target return is dependent upon the time resolution of the system and
is a direct indication of the spatial resolution capability of the system.
I f one assumes in Eq 1 that aa = a2 ---- a constant then a plot of
In [(R 1 q- R / n ) 2 Pr] versus R
should produce a straight line whose slope is twice the attenuation

k~
Volumetric~ Torget
return ~ return
| | J m I
o l'O
UNDERWATERRANGER
FIG. S---Schematic diagram o f a typical lidar return .from a submerged surface. P is the
point at which the receiver beam fully overlaps the transmitter beam. W and h are the
measured width and height o f the target return.
coefficient. This plot can be obtained in two ways: once from the
volumetric scattering itself and again from the target pulse amplitude h as
a function of depth.
Figure 6 shows sample results obtained with the lidar operating at 514
nm for ranges up to about 17 m with the white target. The curves shown
are a superposition of the scattered signals obtained from the water itself
with no target (lowest trace), and from the target when located at several
different ranges. These traces are averaged outputs over approximately a
1-s averaging time when using a pulse rate of 10 s Hz. In all cases the
initial pulse of the volumetric scattering is seen to be identical. (The top
two traces have gain reductions of 10 and 20 to keep the target return on
scale and in these the volumetric return thus appears greatly reduced.)
The target return is seen to drop off very rapidly with increasing range,
with the maximum detectable range being about 20 m. It is apparent
from the figure that the width of the target return pulses corresponds to a
range of about 4 m, and as already mentioned this width is presently
limited by the resolving time of the boxcar averager. Since the target
return is, in fact, considerably narrower than this, this electronic limita-
tion is reducing the measured penetration range considerably (that is,
narrow small signals at greater ranges would not be detectable). Although
the width of the target returns corresponds to about 4 m, it is possible to
locate the position of the target to a better accuracy as can be seen from
the shapes of the target return peaks.
d.6.
15
9.25m
xO.05
~. I0 xO.I
0
k
11.?5 m
14.25m
0
0
16.YS m
0
0 L 0
0 4 8 12 16 20
UNDERWATER RANGE - m
FIG. 6---Sample backscattered signal plots as a function of range for white targets at
d({~'erent underwater distances. Note that the top two traces have ordinate scales com-
pressed by the factors shown.
SIZGORIC A N D CARSWEL L ON LASER RADAR
The data shown in Fig. 6 were obtained for the receiver without any
polarization optics. If a polarizer is inserted in the receiver and aligned
either parallel or perpendicular to the polarization direction of the linearly
polarized transmitter signal, a considerable variation in the volumetric
scattering intensity is observed. Figure 7 shows sample results with a
,,. II polorized
.• 2
--- s polarized
-/4
I~ / xx
I I I ~ ~-~ '
-0 4 ,9 12 16
UNDERWATER RANGE- m
FIG. 7 - - R e t u r n s ,from a target at 7 m as observed with receiver containing a linear pol-
arizhtg element,
target located at a range of about 7 m. The perpendicular to parallel ratio

(I2/Ia) for the volumetric scattering is found to be about 0.2 indicating
that this scattering strongly retains the polarization of the incident beam.
The corresponding ratio (14/13) for the target, however, is about 0.9
indicating an almost complete depolarization of the signal scattered by the
target surface. Such measurements are qualitatively in agreement with
polarization studies made by transmission measurements underwater.
The possibility of employing polarization to suppress the volumetric
return as already utilized for some underwater photographic application is
apparent from such results. The polarization measurements also appear to
indicate an increase of depolarization with penetration depth, an effect
consistent with the multiple scattering process and recently observed in

atmospheric cloud studies [16]. However, the exact magnitude of this
increase cannot be obtained until the higher resolution electronic system is
available.
Taking the data shown in Fig. 6 and range correcting it as just
described, it is possible to evaluate the attenuation coefficient. This has
been done in Fig. 8 for both the target echoes and the volumetric
,o3to\
| \ echoes
l ~O-woter$cottering
o-torget
102
k
I0 13 q
D \
13
I I I
6 I0
UNDERWATER
14
RANGE-rn
18
FIG, 8--Semilog plot o f the range corrected intensity of returns from both the targets and
the volumetric water scattering.
scattering. For the targets it is seen that a fairly good straight line is
obtained and from this a value of a = 0.25/m is derived. Simultaneous
transmissometer measurements over a 1-m path length using a wavelength
of 490 nm gave values in reasonable agreement with the lidar measure-
ment. The volumetric returns in Fig. 8 show a nonlinear plot which
appears to approach the slope of the target returns with increasing
penetration depth. It is believed that this effect is caused by the relatively

poor spatial resolution of the system which would tend to reduce the
intensity of the near field returns of the volumetric scattering. This feature
will be examined more closely with the improved detection system.
The trials on Lake Erie were conducted to test the system under real
field conditions. Measurements were made at 488 and 514 nm and for
both receiver polarizations (that is, parallel and perpendicular to the
transmitted polarization). Targets of the same geometry were lowered
from the ship and returns were recorded for various depths. As yet the
data have not been fully analyzed but, in general, the results reflect those
found in the tank measurements. Maximum penetration depths obtained
in Lake Erie were only about 10 m. This was caused by the greater
turbidity of the lake water (the value of a was generally measured by
transmissometer to lie between about 0.5 and 1.0/m) and by the signal
degradation caused by surface wave structure.
During the trials the lake surface was quite choppy with swells reaching
several feet. This introduced additional variability into the signal and
degraded the signal-to-noise ratio as anticipated. However, the degrada-
tion was not as large as originally feared even considering the rather small
beam sizes used in this experiment which would aggravate the beam
distortion problem. The lidar was directed into the water at an angle of 15
or 20 deg from the vertical to ensure that the specular surface returns did
not enter the receiver. If this was not done the noise in the return was
unacceptably high.
The system performed well for both day and night operation in spite of
the rigors of the trip. A large number of profiles similar to those of Figs.
6 and 7 were obtained at both wavelengths. Again the data was limited by
the bandwidth of the receiving electronics. It had been hoped when
planning the trip, that the lidar would be able to observe the thermocline
in the lake where a substantial discontinuity in the turbidity occurs.
However, during the measurement period the thermocline was at depths of
the order of 15 to 16 m which were beyond the present detection
capabilities of the lidar.
One other preliminary series of test has been undertaken with this
system as well. In the laboratory, a simple monocromator has been used
on the receiver and the Raman backscattered signals from several water
samples have been observed. It has been found that using the 488 nm
wavelength, rather good signals from the 3450/cm H20 band are obser-
vable at ranges of several meters in these initial tests. One difficulty
already encountered, however, is the stray light arising at the Raman
frequency from fluorescence in the water. This fluorescence signal is
virtually negligible in distilled water but is observable in tap water and is
very large in some natural water samples such that the Raman return is
markedly obscured. This work is continuing, and an effort will be made to
ascertain the potential of subsurface diagnostics of natural waters by

Raman lidar scattering.
Conclusion
Although our experience with the marine lidar unit is still rather
limited, the system has already demonstrated a useful potential. The
present system is capable of providing measurable signal returns at
depths up to 25 or 30 m depending on the attenuation. The most direct
application of this capability would be for depth sounding and bottom
profiling, and accuracies to within a fraction of a meter should be
obtainable.
Some spatial "structure" on the volumetric return has already been
detected in the Lake Erie data and such variations should provide
information on the scattering and attenuation as a function of depth.
Also, since the magnitude of the volumetric scattering depends on the
optical quality of the water, the relative intensity of this signal may serve
as a useful indication of water quality which could be obtained remotely
and rapidly.
The polarization of the backscattered signal is also of considerable
importance. There is already evidence that the depolarization arises from
multiple scattering processes and depends strongly on the turbidity of the
water [14]. Since polarization is a readily measurable quantity it should
prove to be another very useful monitor of water conditions. So far only
two components (parallel and perpendicular) of the polarization have been
monitored. The complete polarization information of the signal is con-
tained in the four components of the Stokes vector and measurements of
these should be even more informative. Such measurements are already
being made with our atmospheric lidar and should present no difficulty
for the marine system.
Finally, the potential usefulness of the Raman return is obvious. The
signal is not large but it is definitely quite measurable, particularly near
the water surface. This signal, as already indicated by other workers
[17.18,20,21], could be potentially useful for a variety of measurements
including pollutant identification and temperature measurement. Such
measurements will not be easy, in view of the many problems associated
with the weak Raman signals and the perturbing effects of fluorescence
and the stray background light. It may be that they will only become
viable by using tuned lasers to obtain the enhancement of resonant
scattering. However, at this stage pertinent experimental field data is still
lacking and the need for further measurements of this type is clear.
Acknowledgments
The authors wish to acknowledge the assistance of W. R. McNeil and
the financial support of the Canada Centre for Inland Waters and the
Canada Centre for Remote Sensing.
References
[1] Collis, R. T. H., Applied Optics, Vol. 9, 1970, pp. 1782-1788.
[2] Derr, V. E. and Little, C. G., Applied Optics. Vol. 9, 1970, pp. 1976-1992.
[3] Kildal, H. and Byer, R. L., Proceedings, IEEE, Vol. 59, 1971, pp. 1644-1663.
[4] Hickman, G. D. and Hogg, J. E., Remote Sensing of the Environment, Vol. 1, 1969,
pp. 47-58.
[5] Ivanov, A. P., Kalinin, I. I., Kozlov, V. D., Skrelin, A. L., and Sherbaf, I. D.,
lzvestiia, Atmospheric and Oceanic Physics, Academy of Sciences, USSR, Vol. 5, 1969,
pp. 212-215.
[6] Ivanov, A. P., Kalinin, I. I., Skrelin, A. L., and Sherbaf, I. D., Izvestiia, Atmospheric
and Ocean Physics, Academy of Sciences, USSR, Vol. 8, 1972, pp. 884-890.
[7] Levis, C. A., Swarner, W. G., Prettyman, C., and Reinhardt, G. W., Proceedings,
NASA Wallops Conference on Hydrographic Lidar, Sept. 1973.
[8] Sizgoric, S. and Carswell, A. I., Canadian Aeronautics and Space Journal, to be
published Dec. 1973.
[9] Fujisawa, A. and Nakao, S., Electronics and Communications in Japan, Vol. 54-B,
1971, pp. 77-83.
[10] Duntley, S. Q., Journal of the Optical Society of America, Vol. 53, 1963, pp. 214-233.
[11] Prettyman, C. E. and Cermak, M. D., Transactions, IEEE, Vol. GE-7, 1969, pp.
235- 243.
[12] Ward, G., Cushing, K. M., McPeters, R. D., and Green, A. E. S., Applied Optics,
Vol. 12, 1973, p. 2585.
[13] Tyler, J. E., Smith, R. C., and Wilson, W. H., Journal of the Optical Society of
America, Vol. 62, 1972, p. 83-91.
[14] Granatstein, V. L., Rhinewine, M., and Levine, A. M., Applied Optics, Vol. 11, 1972,
pp. 1870-1871.
[15] Carswell, A. 1., Houston, J. D., McNeil, W. R., Pal, S. R., and Sizgoric, S., Canadian
Aeronautics and Space Journal, Vol. 18, 1972, p. 335.
[/6] Pal, S. R. and Carswell, A. I., Applied Optics, Vol. 12, 1973, pp. 1530-1535.
[17] Melfi, S. H., Applied Physics Letters, Vol. 22, 1973, pp. 402-403.
[18] Inaba, H. and Kobayasi, T., Nature, Vol. 224, 1969, pp. 170-173.
[19] Maydan, D., Journal of Applied Physics, Vol. 41, 1970, pp. 1552-1559.
[20] Bradley, E. B. and Frenzel, C. A., Water Research, Vol. 4, 1970, pp. 125-128.
[21] Schwiesow, R. L., Proceedings, Conference on Sensing of Environmental Pollutants,
AIAA Paper No. 71-1086, Nov. 1971.
C. H. L a n g f o r d I
Fast Kinetic Spectrometry and

Automated Trace Analysis
REFERENCE: Langford, C. H., " F a s t Kinetic Spectrometry and Automated Trace

Analysis," Water Quality Parameters, A S T M STP 573, American Society for Testing
ABSTRACT: Mixtures of species may be analyzed by a method where differing rates of

reaction with a common reagent serve to differentiate components. The rate constants
are usefully regarded as the reciprocals of frequencies and the method may be called
"kinetic spectrometry." Literature on the stopped flow technique reports analyses of
mixtures of metal ions and of mixtures of amino-polycarboxylates using convenient
instrumentation. The prospects for automated direct analyses of metal complex species
present in natural waters are discussed.
KEY WORDS: water quality, spectroscopy, kinetics, analysis, environmental tests
Kinetic methods have occupied a respectable corner of analytical

chemistry which, like most scientific "corners," has enjoyed considerable
expansion in recent years. Two books review the general patterns of this
development [1,2]. 2 Most kinetic methods of analysis have been confined
to methods of measurement of reaction kinetics dependent on what are
now usually called "classical" chemical kinetics [3]. Classical kinetics
denotes processes with half lives longer than approximately 10 s where
"ordinary" methods of mixing reagent and sample can be used to initiate
a reaction [3]. But, the most significant developments of recent years in
aqueous solution kinetics are those in methodology for fast reactions. The
purpose of this paper is to suggest some of the new opportunities that fast
reaction techniques present. Additionally, it will be an intention to point
out some special features related to complex formation reactions of
significance to behavior of metal ions in environmentally interesting
systems [4].
There are two important pragmatic instrumental features of modern
fast reaction kinetic techniques that recommend their consideration in
1 Professor of Chemistry, Metal Ions Group, Chemistry Department, Carleton University,
Ottawa, Ontario K1S 5B6, Canada.
2 The italic n u m b e r s in brackets refer to the list of references appended to this paper.
414

LANGFORD ON KINETIC SPECTROMETRY AND TRACE ANALYSIS 415
automated analysis schemes. (1) They are "fast" (inherently). (2) Major
problems related to automated sample handling had to be solved before it
was possible to measure fast reactions. The main impediment is sensitivity.
Eigen [5] has observed that a method such as excess ultrasonic
absorption provides a new kind of "spectroscopy," a spectrum of time
scales of chemical relaxation. This spectroscopy can provide important
information of speciation in a solution analogous to (but distinct from)
that provided by electronic or vibrational excitation. Unfortunately, some-
thing like the excess ultrasonic absorption spectrum is not available when
solute concentrations are below (optimistically !) about 0.01 M. It is
necessary to have a suitable chemical amplifier before it becomes possible
to apply kinetic spectroscopy to interesting systems. Such an amplifier is
regularly provided in chemical analysis by the derivation of new, sensi-
tively detectable, species from the original species. This requires that the
kinetic technique is not a perturbation of the existing equilibria in a
solution (as in ultrasonic absorption) but the following of kinetics of net
chemical change. The only fast kinetic technique that meets this require-
ment is a flow-mixing technique. Thus, attention here is limited to the
continuous-flow and stopped-flow methods of following fast reactions.
That stopped-flow spectrophotometric kinetics can provide a kinetic
spectrum (that is, analysis of a mixture of species) in certain interesting
circumstances has recently been demonstrated. The most impressive work
is that of Margerum et al [6-8]. The first important study [6] established
that qualitative and quantitative analysis for up to 30 metal ions down to
10 -6 M concentration could be achieved by converting these ions into their
CyDTA (diaminocyclohexane-N,N,N 1, NX-tetraacetate) complexes then re-
acting in a stopped-flow apparatus with another metal ion or excess H §
or both. Kinetics were observed with time scans of milliseconds to minutes
and acidities of 10 -8 to 1 M [H*.] to achieve full differentiation. In the
Eigen sense, this is resolution of 30 peaks in a kinetic spectrum. The
observable was usually the copper complex of CyDTA so that the final
absorbance was related to total CyDTA and, hence, to total metal ions in
the particular experiment. The parameter characterizing the different
metal ions was the rate constant which is the kinetic spectroscopy analog
of a "peak."
In another study [7], the role of analyte and reagent was reversed.
Mixtures of aminopolycarboxylates (at the ppb level) were converted into
their Ni(II) complexes. These were then reacted with CN- and the rate of
formation of Ni(CN)42- monitored. The total absorbanee change gave the
total concentration of aminopolycarboxylates, but the rate constants
extractable from analysis of the kinetic behavior of the mixtures allowed
analysis of two and three, and even four, component mixtures of
aminopolycarboxylates.
The preceding examples clarify the power of kinetic spectrometry to
resolve interesting mixtures. Parallel progress exists in demonstrating the

automation of techniques in this area. Malmstadt et al [9] have designed a
stopped flow spectrometer with automated sampling having an instrument
cycle of 1 s. The apparatus was applied to phosphate determination from
0.1 ppm up. (The method might well be extended to mixtures of
phosphate, arsenate, and silicate.) The critically important problem of
automated data management for kinetic spectrometry on mixtures has
also been laced. Margerum's group [8] has developed an on-line (with
data collection under computer control) least squares data reduction
system for a small computer which was demonstrated to manage analysis
of three component mixtures (alkaline-earth-CyDTA complexes) with
favorable error parameters.
Our own interest in this approach centers on one aspect of kinetic
spectrometry which, as yet, has remained unexploited. The systems just
mentioned have so far required modification of the chemical species
present in a "raw" water sample to convert them into favorable species for
kinetic spectrometry. This reduces output information, especially with
respect to metal ions. If all metal ions must be converted to CyDTA (or
similar) complexes before analysis, we can determine the nature (and
oxidation state) of the metal ions from kinetic spectrometry but not the
state of complexation in the raw water sample. (One need only attend to
the controversy over lead toxicity to recognize the biochemical significance
of state of complexation.) Recent work on kinetics of complex formation
reactions [4] (see Ref 10 for a review) indicates that rates of complex
formation are significantly sensitive to ligands already present. Thus,
kinetic spectrometry contains the possibility of contributing more directly
than any other method on the immediate horizon to the development of
methodology for metal ion analysis in water that is specific to the nature
of particular complex species. We have recently been exploring an
example. Fe(CN)64- reacts with Fe 3§ species in aqueous media to produce a
product absorbing strongly at 6"75 nm. Rates of this reaction are sensitive
to pH in the absence of chelating ligands and differ for NTA (nitrilo-
triacetate), tartrate, and salacyclic acid chelates by a factor of 10.
Two remaining aspects deserve to be anticipated. As far as sensitivity is
concerned, it can be substantially improved. All methods so far use
absorption spectroscopic detection. Fluorescence is commercial [11].
Fluorescing chelates, for example, could be used with metal ions. Second,
for faster reactions, current instrumentation has become too sophisticated.
Prior to the introduction of stopped-flow technique, continuous-flow
methodology was developed [3]. A main reason stopped flow has taken
over in the laboratory is scarcity of sample. If direct species analysis of
"raw" water samples is desired, this is not the limiting problem. A step
"backward" to continuous-flow instrumentation promises simplicity and
reliability suitable for consideration for continuous monitoring.
LANGFORD ON KINETIC SPECTROMETRY AND TRACE ANALYSIS 417
References
[1] Yatsimirskii, K. B., Kinetic Methods of Analysis, Pergamon, Oxford, 1966.
[2] Mark, H. B. and Reichnitz, G. A., Kinetics in Analytical Chemistry, Wiley (Inter-
science), New York, 1968.
[3] Caldin, E. F., Fast Reactions in Solution, Wiley, New York, 1964.
[4] Langford, C. H., Inorganic and Nuclear Chemistry Letters, Vol. 9, 1973, p. 679.
[5] Eigen, M., Zeitschrift Fur Elektrochemie, Vol. 64, 1960, p. 115.
[6] Margerum, D. W., Pausch, J. B., Nysscn, G. A., and Smith, G. F., Analytical
Chemistry, Vol. 41, 1969, p. 233.
[7] Coombs, L. C., Vasiliades, J., and Margerum, D. W., Analytical Chemistry, Vol. 44,
1972, p. 2325.
[8] Wills, G., Woodruff, W. H., Frysinger, J. R., Margerum, D. W., and Pardue, H. L.,
Analytical Chemistry, Vol. 42, 1970, p. 1350.
[9] Javier, A. C., Crouch, S. R., and Malmstadt, H. V., Analytical Chemistry, Vol, 41,
1969, p. 239.
[lO] Langford, C. H. and Sastri, V. S., Biennial Reviews of Chemistry, Inorganic Chem-
istry, Series One. Vol. 9, Chapter 6, MTP--Butterworths, London, 1972.
[11] Durrum instrument Co., Palo Alto, Calif.
R. S. l n g o l s j a n d T. F. Craft I
Sensors for Monitoring

Water Quality
REFERENCE: Ingols, R. S. and Craft, T. F.. "Semors for Monitoring Water Quality,"
Water Quality Parameters, A S T M STP 573, American Society for Testing and Mate-
dais, 1975, pp. 418-423.
ABSTRACT: Scientific principles of operation and general design considerations are

given lot sensors to monitor dissolved oxygen, turbidity, pH, conductivity, temperature,
BOD. and specific ions. The importance of proper sampling procedures to the acquisi-
tion of valid data is emphasized. Solutions to problems encountered with continuous and
semicontinuous monitors in the field and laboratory are detailed.
KEY WORDS: water quality, monitors, environmental tests, automatic control equip-
ment. dissolved gases
T h e technology of water quality m o n i t o r i n g has developed over the last

35 years, b u t very r a p i d strides have been m a d e only in the last d e c a d e .
T h e d e v e l o p m e n t o f solid-state electronics with low electrical c u r r e n t
d e m a n d initiated a g e n e r a t i o n o f field i n s t r u m e n t s c a p a b l e o f e x t e n d e d
o p e r a t i o n in locations r e m o t e from power lines. T h e a n c i e n t a r t o f
m e a s u r i n g water q u a n t i t y has also been improved by m o d e r n electronics.
A t present, there is m u c h activity in the m a r k e t a n d new i m p r o v e d
m o n i t o r i n g devices are c o n t i n u a l l y a p p e a r i n g . As designs evolve we m a y
expect i n s t r u m e n t s of a c c u r a c y a n d reliability an o r d e r o f m a g n i t u d e
b e t t e r t h a n those available today.
W h e r e w a t e r quality m o n i t o r i n g is concerned, effluents from waste
water t r e a t m e n t p l a n t s differ from river water only in the m a g n i t u d e of
values observed. T h e p a r a m e t e r s and principles of o p e r a t i o n are identical
a n d the p r o b l e m s are essentially the s a m e . Therefore, for the m o s t p a r t ,
the following discussion applies to both waste water a n d river water.
D e s i g n Considerations
O n e early c o n s i d e r a t i o n in the design o f any system is the m a n n e r in
Principal research scientist and senior research scientist, respectively, Engineering Experi-
ment Station, Georgia Institute of Technology, Atlanta, Ga. 30332.
418

INGOLS AND CRAFT ON SENSORS 419
which the liquid is brought in contact with the sensor. For instance, if the
sensor is to operate completely submerged it must withstand applied water
pressure and must also function properly when the pressure varies. Where
a partially submerged sensor is used, it may be necessary to pump the
liquid in order to provide a constant level. Different sensors may have
needs for different rates of water flow, but all sensors require a positive,
periodic change of the liquid which is in contact with the sensor face.
Thus, it is an exercise in futility to operate a sensing device which does
not have a properly engineered water flow system.
Other problems may be caused by lack of temperature control or by
interference from electrical equipment in the vicinity. Recent develop-
ments have eliminated some problems but others remain unsolved.
Personal experience indicates that more problems arise from mechanical
failure than from any other cause. As an example, monitoring the quality
of a river which is the discharge of a power impoundment is exceptionally
difficult. It is also impossible to maintain constant flow from a submerged
pump during low night flows when valve settings are disturbed by a 2 to
3-m rise to the daytime river level. This situation was encountered when a
commercial river monitor was first placed in operation on the bank of the
Chattahoochee River near the Atlanta municipal water intake. Water was
supplied to the monitor by a submerged well pump suspended in the river.
All water valves were carefully adjusted and the unit left operating
overnight. The following morning the river had risen 3 m in elevation and
two valve settings were out of balance; the normally dry turbidity sensor
was flooded while the jet to the dissolved oxygen sensor was plugged.
Subsequent experience showed that at sites close enough to the dam to
have no appreciable increase in sediment with the higher flow, flooded
sensors were a frequent problem. Where sediment was picked up by the
turbulence, flow jets became frequently plugged. Most subsequent use of
the monitor was on smaller rivers in flat country where valve settings were
simpler to maintain.
Dissolved Oxygen
Dissolved oxygen (DO) is a parameter of major importance in most
water quality monitoring situations. Polarography was used as early as
1938 for recording DO profiles in a lake. From this start, polarographic
sensors for DO have been developed to a high degree of utility. Oxygen
gas can diffuse readily across a membrane into a carefully controlled
environment for the electrodes of the sensor. A 0.8 to 1.0 V d-c current is
impressed across the electrodes. A compensating thermistor may be
included in the circuitry for increased correlation between signal strength
and DO concentration in spite of varying temperatures. The quantity of
oxygen which diffuses across the membrane depends on a number of
factors including water turbulence at the face of the membrane. Many

devices are commercially available to keep the sensor face clean and to
provide a continuously fresh sample, but mechanical problems generally
develop with jets, pumps, or rotating devices in domestic wastewater flows
unless the sensor is carefully protected by screens. On the other hand, a
vibrating paddle has been observed to provide excellent operating data
without fouling while installed for several weeks in a domestic wastewater
activated sludge aeration tank.
Galvanic electrode pairs are used for measuring DO in the laboratory,
but arc not generally used for on-line monitoring. Batteries can be used
for prolonged periods, but must be replaced eventually to maintain the
proper voltage. The electrodes are generally immersed in large liquid
reservoirs to reduce the frequency of servicing.
Turbidity
Turbidity in water is a property which results from the dispersion of
many different materials either alone or in combination. In general, the
materials are multimolecular aggregates which are greater than 100 nm in
diameter. Some smaller but single molecular dispersions of proteins also
show properties which are typical of turbid suspensions; each may develop
a Tyndal cone when illuminated with a narrow beam of light in the dark.
The particles causing turbidity may have many different colors, though
colors in true solution interfere with the measurement of turbidity.
There are two types of sensors for measuring turbidity. Those which
indicate changes in light transmission and those which measure the
changes in reflected light. Sensors which measure light loss are generally
submerged; problems arise from bacterial and algal slimes on the window
of the light source. Paired photocells with a single light can largely
overcome the variations in light intensity from either voltage variation or
slime buildup. The light path length is fixed and the sensor must be
chosen for the range of turbidity under study. In instruments operating by
light reflectance, both the light and photocell can be placed above the
water surface. Careful control of the water level is necessary to prevent
flooding of the sensor or a change in the light path. Different turbidity
ranges can be measured with a single sensor, but this requires different
electrical signal ranges.
Reference Electrodes
Reference electrodes for pH and specific ion systems in long-term or
monitoring use have been a problem because of crystal formation in the
salt bridge between the calomel half cell and the sample. Solid-state
reference cells apparently can perform well under adverse conditions
including temperature fluctuations and film formation which requires a

strong acid rinse at short intervals, pH electrodes with built-in amplifiers
permit transmission of a signal strong enough to overcome interference
from stray magnetic fields.
Sampling
All sensors require periodic maintenance but some present more
problems than others. Thus, sensors using periodic microliter samples
suffer from severe bacterial slime problems when monitoring waste water.
Also the attainable precision of repetitive delivery of microliter samples is
questionable, and the statistical probability of their being representative of
the whole flow appears low. Of course, all chemical monitoring values for
organic matter, which is done to approximate biologically available food
or BOD, can be meaningful only with enough correlative information.
The direct measurement of BOD within the time-frame required of
monitoring has several pitfalls. The rate of change of DO in an average
unmodified domestic wastewater sample is too slow for accuracy or
significance or both. Measurement of the by-product carbon dioxide in an
infrared spectrophotometer is subject to some of the same limitations. The
time required for obtaining significant correlation with food concentration
or BOD is reduced by the presence of activated sludge as in the treatment
process. A means of accurately measuring the activated sludge concen-
tration is, however, a correlative factor that must be considered for proper
interpretation of the directly obtained BOD values.
The BOD of a mixture of domestic waste water and activated sludge
may be used for monitoring or process control by including the measure-
ment of several parameters. A sensor for DO may be used to note the
extent of change per unit time or the time required per unit change in
DO. We have used the former procedure with limited success. Where a
plant normally uses two compressors a failure of one compressor can
cause an overloaded sludge which will have a prolonged lag period before
responding normally with a discrete change in DO values. Thus, the BOD
appears abnormally low when the activated sludge aeration tanks have so
little DO that an aliquot requires a minute or more to adjust to the
presence of DO in the sample chamber. Otherwise, with normal operating
DO levels in the aeration tank, a rapid drop in DO permits a good
correlation between the oxygen uptake (OU) values and the laboratory-
determined BOD of the raw waste water. Good effluent or normat river
quality would have too low a BOD to obtain values at very short intervals
by such a procedure, but a 1-h OU may possibly give a significant figure.
A commercial BOD apparatus requiring a 1-h observation period and
using gas pressure changes for measuring the OU was demonstrated at the
Atlanta meeting of the Water Pollution Control Federation in October
1972. While lag periods of several minutes totally destroy the usefulness of
1-min OU values, they may cause serious errors with even longer
observation periods unless the lag period is programmed out of the total
OU values.
Measurement of the carbon dioxide product of biological reactions is
possible with an infrared spectrophotometer. Adequate flushing of the
accumulated gas from the liquid system is necessary before an equilibrium
carbon dioxide production rate is possible. Only vague correlation between
BOD and CO2 production with activated sludge have been observed.
The condition as well as the concentration of the activated sludge at the
time of monitoring the mixture is a major concern for interpreting an
OU-BOD value as an indication of the wastewater load. Lag periods from
rapid environmental change are one problem while the relative load from
heterotrophic or autotropbic activity may cause more difficulty in inter-
preting a rapid BOD.
Perhaps the best load measuring device would be a reservoir of 1 m 3
with a 50-1iter/min input. The contents would be maintained at 1.0
mg/liter DO by a variable air supply and a DO monitor. The load would
be equivalent to the necessary air quantity which was monitored elec-
trically.
Monitoring salt concentrations has been done traditionally by measure-
ment of conductivity. These sensors are well-developed and with matching
multi-range, electronic packages can cover a very wide variety of salt
concentrations. Today, it is also possible to monitor for individual ionic
species with an electrode that is selective. Each specific ion electrode must
have its reference electrode and amplifier. The range of concentration and
precision that can be covered by specific electrodes is more limited than
those of simple conductivity devices. Thus, the choice of conductivity or
specific ion electrodes for monitoring raw waste water, an effluent, or a
river will depend upon: (1) the anticipated range of concentration, (2) the
absolute concentration level, and (3) the ease and frequency of available
service. While specific ion electrodes have been widely accepted as a
laboratory tool and advertised for monitoring, no user replies were
received where the electrodes were used for monitoring. A demonstration
of monitoring fluoride ions at a wastewater plant in Atlanta was
reasonably successful on the effluent.
Temperature has been monitored for 70 years or more. Thermocouples
have been the primary choice, because a voltage is produced that is
positively correlated with temperature. Thermistors have been used very
successfully as indicators over a more limited range but have an electrical
signal that is inversely proportional to temperature. They do have the
advantage of a wider range of voltage output for the range of temperature
normal to water monitoring than thermocouples. With a bucking circuit
the thermistor output can be recorded with a positive correlation between
temperature and signal. Recorders which are housed in a variable

temperature environment must be modified to produce a proper record of
the thermocouple signal. This is not necessary for the thermistor signals.
Some water monitoring is done with chemical reactions induced by
added reagents. Thus, chlorine residuals have been monitored in potable
and waste water for many years. Other ions are also monitored in various
process waters. These include phosphates and chromates, but little
information is available to indicate how successfully these work.
Conclusion
A number of commercial instruments are available for monitoring
several parameters of river water and wastewater treatment effluents.
Good instrument design is of prime importance, but experience shows that
careful attention to the details of installation and the frequency of
servicing are highly significant in obtaining valid data.
R. A. O'NeiU A. R. Davis2 H. G. Gross2 and J. Kruus 1
A Remote Sensing Laser

Fluorometer
REFERENCE: O'Neil, R. A., Davis, A. R., Gross, H. G., and Kruus, J., "A Remote
Sen~ing Laser Fluorometer," Water Quality Parameters, A S T M STP 573, American
ABSTRACT: A variety of water quality parameters are accessible to measurement using

the technique of fluorescence spectroscopy. These include such measurements as oil,
chlorophyll, lignin sulfonate, and dye tracing. The availability of reliable lasers has
opened up the possibility of using the fuorescence technique as a water quality analy-
tical tool without being in direct contact with the body of water. For instance, it is
possible to monitor oil on water from such remote locations as a bridge, boat, or
aircraft. A system based on a helium cadmium laser is described and some experimental
results on different materials from various locations are discussed.
KEY WORDS: water quality, fluorometers, lasers, fluorescence, environmental tests,

remote control
In today's context of e n v i r o n m e n t a l m a n a g e m e n t it is often desirable to

have a n i n s t r u m e n t capable of detecting certain specific s u b s t a n c e s at a
distance. The utility of such a n i n s t r u m e n t is greatly increased if it is able
to operate at night, to perform well u n d e r adverse conditions, to m o n i t o r
large areas from a variety of land, ship, a n d a i r b o r n e platforms, or to r u n
u n a t t e n d e d for long periods.
Fluorescence spectra of m a n y substances dissolved and s u s p e n d e d in
n a t u r a l waters are u n i q u e . The fluorescence spectrum of chlorophyll in
water shows a strong peak in the red, whereas oil in water fluoresces with
a blue-green light. I n a d d i t i o n to this u n i q u e n e s s , the integration of the
intensity of the fluorescence radiation in p a r t i c u l a r wavelength intervals
can yield q u a n t i t a t i v e i n f o r m a t i o n on the concentrations. T h e present
work describes a remote sensing laser fluorometer constructed to observe
organic and biological materials in water.
Fluorescence
Fluorescence occurs when a molecule absorbs a photon and sub-
'Water Science Section, Inland Waters Directorate, Environment Canada, Ottawa,

Ontario K1A 0E7, Canada.
424

O'NEII.. ET AL ON A REMOTE SENSING LASER FLUOROMETER 425
sequently emits another photon of less energy. The longer wavelength light
(lower energy photon) is known as the fluorescence radiation. The
optimum wavelength for absorbtion of light by the molecule and the
wavelengths of the subsequent emissions are determined by the molecular
structure.
The spectra obtained by observing the fluorescence of different solutions
are often quite distinct [1]. 2 Figure 1 shows the fluorescence spectrum of
RANGE 60m JULY 18,1973

.J
>-
-r
I.O a.
0
rr
x
~ O.8
E
p-
~. 0.6
I-
o
m 0.4
-3n
< 0.2
0 I-
500 600 700
WAVELENGTH (rim)
FIG. 1--RMeau River water spectrum (Exp 2) obtained with the land based laser
fluorosensor. The spectrum was taken using a filter and a red sensitive photomultiplier tube.
This spectrum is not corrected f o r photomultiplier or filter response functions. The chloro-
phyll at 680 nm is quite prominent as is the very broad peak near 520 nm. The latter
spectral feature is probably due to the fluorescence o f organic material carried by the river
water. A blocking filter, made o f two layers o f Wratten #8 gelatin material and used to
attenuate the laser line, tends to cut out all the light below about 510 nm altering the shape
o f the peaks in the vicinity of 520 nm.
chlorophyll in natural river water. This may be compared to Fig. 2 which

shows a spectrum obtained from an oil-water system. Both spectra were
obtained by exciting the target with a beam of blue light having a
wavelength of 442 nm. The spectrum of chlorophyll [2] in water has a
peak in the deep red at 680 nm, whereas the oil dispersed in water shows
a peak in the blue-green at 520 nm. These spectra were taken with the
remote sensing laser fluorometer which contains filters (see section on the
laser fluorosensor) to render the instrument insensitive to wavelengths less
2 The italic numbers in brackets refer to the list o f references appended to this paper.
RANGE 27m AUGUST I0, 197:5
_1
0
~4
I--
~3
I.-
:D
0
--.2
.2
a.
< I
I- - I
5oo 6oo - Too
WAVELENGTH (nm)
F I G . 2--Spectrum o f Venezuelan crude oil floating on water surface, Exp. 31. This
spectrum was taken with the land based laser fluorosensor using a variable filter and
blue-green sensitive photomultiplier. Laboratory experiments on this oil showed the fluores-
cence spectrum to peak near 480 nm; however, the blocking filter used to attenuate the
reflected laser light also attenuates the peak o f the oil fluorescence. Thus, this spectrum and
the one shown in Fig. 1 do not appear to differ significantly in the region from 520 to 600 nrn.
than 510 nm. Thus, the maximum of the oil fluorescence (below 500 nm)
is not observed. Pure water does not fluoresce.
A general feature of fluoroscence spectra of solutions is the presence of
very broad peaks. The width of these peaks is a handicap in the
identification of specific types of mineral oils. It has been shown [3] that
the fluorescence peaks in the spectrum often shift to the larger wave-
lengths the greater the API density. Even over wide variations of the API
gravity (5 to 40~ the peak emission wavelength does not vary more than
50 nm. In laboratory measurements, the fluorescence spectrum has some
importance as a method of distinguishing oil types. With the device
described in the present work, however, only gross features in the
fluorescence spectrum are used to distinguish oil or chlorophyll from the
background of other fluorescing organic materials in the water.
Another property of fluorescence radiation is a detectable time delay
between the excitation of the molecule and the fluorescence emission.
Many substances have different characteristic decay times; for mineral
oils, however, they are all virtually the same. Fantasia et al [1] found the
lifetime to lie between 9 and 21 ns with most at 10 _ 1 ns. The spread in
the fluorescence lifetimes is insufficient to make use of this property for
identifying different types of oils. The system described in this work is
O'NEIL ET AL ON A REMOTE SENSING LASER FLUOROMETER 427
unable to take advantage of any information about the nature of the

target molecules which could be derived from the fluorescence lifetimes.
Remote Sensing
There are numerous reasons for choosing a remote sensing instrument
over a conventional laboratory instrument when monitoring the general
state of the environment. Perhaps the most obvious is that larger areas
may be observed than by point sampling techniques. This gross overview
sometimes enables one to see the forest in spite of the trees. Another
advantage is that by using noncontacting techniques there are fewer
problems in the construction of instruments and data acquisition systems
to operate in severe environments or unattended for long periods of time.
No sample cells are used so that no special techniques are needed to keep
them clean. Difficulties associated with operating sensors underwater do
not exist with this device.
In the experimental stages of a project there is a great need to make
careful laboratory measurements of selected targets using the remote
sensing instrument. This yields valuable information which can be used
later in the interpretation of field measurements. In the initial stages of
development, too, samples must be taken at the same time as field
measurements are made to establish the "ground truth."
There are two philosophies which may be followed in the interpretation
of remote sensing data. The first (and the ideal) is to expect the sensor to
establish unambiguously the nature of the targets. This involves very
sophisticated equipment, a great deal of work, and a vast amount of data.
The device described in the present work has not progressed to this stage
but is, in some respects, more specific than the approach frequently used
by exploration geophysicists, by whom the sensor is used to detect
anomalies which merit further investigation.
The Department of the Environment decided to develop an airborne
fluorosensor. Initially, it was to be used as a detector of oil spills. Now,
however, there are other potential uses for which the instrument is as well
suited as for its original policing role. The present system is the result of
the part-time activities of two men over a period of two years.
The Laser Fluorosensor

Figure 3 shows a schematic diagram of the airborne laser fluorosensor.
In this system the fluorescence is excited by using a beam of laser light.
Lasers have several distinct advantages over conventional light sources.
The beam has a very high intensity and a divergence of 1 mrad in the
present instrument. Such fine collimation aids in steering the beam and
obtaining a small bright spot on the target.
LOCK- IN J = I PRE-
AMPLIFIER
I
PHOTO--
MULTIPLIER
RECORDER
%;!"
CHOPPER TELESCOPE
LASER b-
Z~ SHOCK-MOUNTED PLATFORM
I
I
I
I
FIG. 3---A schematic diagram of the laser fluorosensor in the airborne configuration. In
this configuration a small mirror is used to direct the chopped laser beam downward onto
the ground. The reflex sight consists of a 45 ~ mirror which may be swung into the light path
so that visual observations may be made. An eyepiece expands the beam of light collected by
the telescope onto the interference filter and photocathode of the photomultiplier tube. The
wavelength interval detected by the photomultiplier is determined by the particular inter-
,terence.filter selected on the filter wheel. A lock-in amplifier detects only the signals from the
photomultiplier which are synchronous with the chopper modulating the incident laser beam.
The present fluorosensor uses a Helium-Cadmium laser. This is a CW

laser 3 with two lines which are useful for exciting fluorescence. A blue line
at 442 nm has provided the excitation for all the field trials of the
fluorosensor. The laser can produce about 25 m W of light in the blue line
whereas an ultraviolet line at 325 nm is about five times less intense. The
ultraviolet line could be useful for exciting the fluorescence of light oils
which do not fluoresce when excited with the blue line. The laser may be
converted to ultraviolet operation by changing the laser cavity reflectors
and beam director which steers the beam onto the target.
As an aid in the detection of the fluorescent signal, the beam is
modulated using a tuning fork chopper. This operates a frequency
of 550 Hz.
The receiver consists of an 8-in. Schmitt Cassegrain telescope which is
focused onto the laser spot on the surface of the water. This gathers the
3CW stands for Continuous Wave. The laser itself is producing light continuously. This is
to be contrasted with a pulsed laser which emits light in short bursts. The laser light in our
system is modulated externally to the laser using a tuning fork chopper.
ambient background light, the reflected laser light, and the fluorescent
light. The light is then focused onto a series of filters. The divergence of
the telescope is approximately the same as the 1 mrad divergence of the
laser beam.
Two sets of filters are used. The first is a high pass filter used to
prevent reflected laser light from reaching the detector. The second is a
narrow band filter which operates in the pass band of the blocking filter.
By changing the narrow band filters, a spectrum of the fluorescence
radiation may be taken. In the aircraft, a single filter is chosen which
admits only light which could come from the fluorescence of the target of
interest. In the ground based system, a variable thickness dielectric
interference filter is used which enables spectra to be taken without the
bulk, weight, or resolution of a dispersion type component.
The filtered beam may be expanded with an eyepiece so that it fills the
photocathode of a photomultiplier tube. In some cases the beam is merely
passed through a field stop to limit the field of view of the photo-
multiplier. The particular photomultiplier selected depends on the ex-
pected fluorescence of the target. For examining oil spills, a blue green
sensitive one is chosen whereas a red sensitive photocathode is necessary
for chlorophyll work.
The signal from the photomultiplier tube is fed into a lock-in amplifier
which also has a reference input from the laser chopper. Provided the
photomultiplier is not saturated, the lock-in amplifier detects the portion
of the returning signal which is in phase with the chopper. In so doing the
signal due to the constant ambient background illumination is rejected.
The output of the lock-in amplifier, then, is a measure of the fluorescence
signal which has been excited by the modulated laser beam.
The overall system weighs 100 kg including all power supplies and
electronics. About 600 watts of 60 Hz 120 V ac are required. The lock-in
amplifier and displays require approximately 0.8 m of rack space. Floor
space required in the present aircraft configuration is a rectangle 1.3 by
0.3 m with at least 0.6 m vertical clearance. The laser can be reoriented to
provide a differently shaped package if necessary.
A land based version has also been built, essentially the same as the
airborne fluorosensor, except there is no beam directing mirror. With a
circular variable filter this device was used to generate the spectra shown
in Figs. 1 and 2.
Field Trials
The airborne system has undergone a series of field trials using a DC-3
as a platform.
The first successful airborne test was over a controlled oil spill in the
Bahamas. Figures 4a, b, c show a portion of a record obtained while
FIG. 4 a, b, and c--Laser fluorometer over Grand Bahama Island, Feb. 1973, altitude o f
500ft. Data and interpretation obtained f r o m flight o f laser fluorosensor over a controlled oil
spill Figure 4b is a continuation o f the same record as Fig. 4a, The aircraft is turning
around over the land at the right hand edge o f Fig. 4a and heads back out to sea and over
the oil in Fig. 4b. A light Louisiana crude oil was spilled in this experiment. The fluoro-
sensor was sensitive to all the induced fiuorescence above the cut-off o f the Wratten #8 filters
at about 510 nm. The land is seen to show a smaller background o f induced fluorescence
than the water. The gradual rise in signal as the aircraft moves o f f the shore is thought to be
due to the lengthening o f the water column seen by the fluorosensor. The fluorescence o f the
water is likely due to the biological materials suspended in the sea water. The water was
clear enough that the low light level television on board the aircraft could see the laser spot
travelling across the ocean floor at depths o f up to 10 m. It is uncertain whether the large
spikes labeled as kelp in the figures arise f r o m a kelp bed near the shore, f r o m the wet
.lbrshore. or f r o m some weathered oil on the rocks. The wave action had piled the oil up into
thick rope-like strands and the fluorosensor, in these figures, shows some evidence o f
detecting this varying thickness. Boat wakes through the oil spill were observed to have
cleared away the oil and is a possible explanation f o r the sharp dip in the trace shown in
Fig. 4b.
Figure 4c shows the record and interpretation o f another pass over the same oil spill, T h i s
record was obtained with a filter with a 10 n m band pass centered at 500 nm. This is the
wavelength interval where the fluorosensor is most sensitive to oil. In this record the thick oil
strands stand out clearly f r o m the thinner sections o f the oil slick. With the narrow band
.filter in the optical path, the rising signal which was interpreted in Figs. 4a and 4b as the
biological material in the water, is not evident.
flying over the land, the water, and the oil spill itself. The wave action
tended to pile the oil up into long thick strips or "ropes." What may be
seen in this diagram is a series of sharp spikes as the laser spot passes
over these thicker sections of the spill. A degree of background fluo-
rescence from the thinner portions of the oil may b e seen between the
spikes. The passage of the laser spot over the oil patches was confirmed
by observation of reflected laser light with a low light level television
system on board the aircraft. Bright flashes were seen to arise from the
thick ropes of the slick and these may have been correlated with spikes on
the record. The interpretation of the remainder of the record is highly
speculative. It is worth noting the step in intensity observed in the
fluorescence as the exciting laser spot moved from the land to the ocean.
The high background fluorescence in the water is probably caused by
chlorophyll and other dissolved organic materials. The sharp spike
dividing the land record from the ocean record could be due to oil on the
beach, but it might result from reflection of the beam by the wet sand on
the foreshore. The intensity of such a reflection could be so high as to be
seen by the photomultiplier tube even through the blocking filters. One
other possible explanation might be fluorescence due to the small organ-
isms living on the wet beach.
The system has also been flown over Rhodamine dye spills in Lake
Ontario. In this case the spill was so small that the spot was only over the
spill for a second at a time. This caused a single sharp spike on the
record.
Pulp plant effluents have also been overflown. Figure 5 shows the
record of a recent flight over the holding ponds of a sulfite process paper
mill. The water in the pond is a deep tea color and has quite high
concentrations of lignin sulfonates. The lignin sulfonates result from the
breaking down of the woody cells in the tree. In addition to this, the pond
contains a number of wetting agents. In the laboratory, these substances
do not cause the water from the ponds to fluoresce much differently from
samples of ordinary river water. In pulp plant effluents, the peak at 520
nm in Fig. 1 is much more intense than in river water. Also, the
chlorophyll peak was not observed in the spectrum of the pulp plant
effluent.
Plans have been made to mount the fluorosensor on a ship from
Canada Centre for Inland Waters in order to monitor the chlorophyll
levels in the waters of Lake Erie. Canadian waters are murky enough that
the laser fluorosensor is only able to analyse the fluorescence in the top
few centimeters of the lake. This is due to both the high extinction
coefficient of the water which varies from 0.3 to 2.0/m [4]. Another
possible use, which has yet to be tested, is the detection of phenolic
residues from petrochemical plants.
Though the present program has emphasized measurements relating to
STATUTE MILES
0 I
ujqlrJ~ iq,, IIr I
••••1• H~vKESBURY
FIG. S---Fluorescence record from a flight over a sulfite process paper mill (Hawkesbury
Mill discharge pools, altitude o f 1000 ft, 16 July 1973). The pools contain large amounts o f
lignin sul.tbnates and wetting agents. Samples o f the water have been tested with a laboratory
.lluorometer and were.found to fluoresce most strongly at 520 nm when excited by light with
a wavelength o f 442 nm. Natural river water also fluoresces at a wavelength o f 520 nm,
though not so strongly as the contents o f these homing ponds. The fluorosensor was operated
with only a Wratten #8 high pass filter, thus the sum o f all the induced fluorescence above a
wavelength o f 510 nm is displayed on the record.
water quality, the same systems could be used on land. Some suggested
users have been looking for oil pipeline leaks and performing airborne
forest health surveys. In such measurements it would be crucial, and quite
difficult, to develop a reliable method of interpretation. Were the system
to have an automatic scanning capability so that the fluorosensor could
display a scene, both interpretation and the navigation would be
simplified.
Although other groups [5,6] have been working on airborne laser

fluorosensors, the Water Science device differs markedly from these
systems because it uses a CW laser. This particular approach may not be
as versatile as a system using a pulsed laser system. The main advantage
of using a CW laser may be found in the simplicity of the signal handling
electronics. The compactness of the fluorosensor package and the low
power consumption of the system have enabled the Water Science device
to be flown before some of the larger pulsed laser fluorosensors.
Conclusion
The present system using a CW laser may be used in applications where
fluorescence is already an established analytical tool but where remote
sensing of the target is desirable.
The remote sensing laser fluorometer is able to detect chlorophyll and
oil. It seems unlikely that a fluorometric device will be able to make
unambiguous identifications of oil types. The chlorophyll concentration
may be related to the chemical oxygen demand or some other gross
indication of water quality. Lignin sulfonates have been shown to fluoresce
in much the same way as naturally occurring waters in the Ottawa area.
This could be used by pulp plants to monitor the contents of holding
ponds before their contents are discharged into the rivers. Investigations
employing the fluorescence of naturally occurring water are likely to make
great use of a remote sensing instrument of the type described in the
present work. I f used in conjunction with stream gaging stations, or
controlling automatic sampling units, a remote sensing laser fluorometer
would provide useful information on the temporal rather than the spacial
characteristics of the water flowing past the sensor.
The fluorosensor may also be employed to make remote reflectance
measurements. In this mode, however, it is limited to a single wavelength.
Such measurements would be more useful if additional exciting wave-
lengths were available.
Possible Future Development
The laser fluorosensor was designed to determine whether it was

possible to make fluorometric measurements remotely. Hence, the
instrument has only the basic components necessary to test the idea. For
detailed or comprehensive surveys the system may need many additions.
The more obvious improvements such as increasing the laser power or the
diameter of the telescope to improve the sensitivity of the instrument are
quite straightforward. The present system is only able to operate in
twilight or darkness because the photomultiplier tends to saturate before
the fluorescence signal is detected. Further study is warranted in this area
so that the fluorosensor's operation may be extended into bright daylight.4

Most of the other improvements are concerned with methods of acquiring
the data and the enhancement of its display to facilitate more certain
interpretation.
A low light level television system has been found to be extremely useful
airborne operations. Primarily, it has shown where the laser spot is
striking the ground. At present, the system has no automatic way of
recording both the fluorescence return signal and a view of the target.
This recording may be done by superposing a measure of the fluorescence
signal on a television display of the laser spot as it moves over the water's
surface. The entire display then could be recorded on videotape. While
interpretative techniques are being developed with a simple fluorosensor,
such a videotape recording would be most useful.
A significant step in the fluorosensor's development would be to
incorporate a beam scanning system so that a scene could be swept out.
This would yield a map of the fluorescence similar to a false color
photograph or to a television display of the target with which it could be
compared directly. The scanning display could be checked for fluorescent
anomalies. In an aircraft a one-dimensional scanning system could be
used so that a swath was swept out as the laser passed over the targets.
An image could be formed of the fluorescent target features as well as
making the navigation easier. The development of a scanning system
could be quite expensive.
A third improvement could be made by exciting the targets simulta-
neously with more than one color of light. The fluorosensor then could be
quite useful for making measurements of reflected light at several
different wavelengths. The additional exciting wavelengths would yield
additional data on the fluorescence which would aid in the interpretation
of returning signals.
The addition of more excitation wavelengths could be usefully combined
with a fourth improvement: a rapid scanning spectrometer. This would
enable the fluorescence spectrum to be taken very rapidly. The process
now is quite time consuming with the circular variable filter and is too
slow to be used in the airborne configuration. Details such as relative
peak heights and peak shapes in the fluorescence spectrum could be quite
important in the identification of returning fluorescence signals from some
targets. Many rapid scanning spectrometers have been developed, some of
which could be used almost without modification on the present laser
fluorosensor.
References
[1] Gross, H. G. and Hyatt, Proceedings, 7th International Symposium on Remote Sensing
of Environment, Willow Run Laboratories, Institute of Science and Technology, The
University of Michigan, Ann Arbor, Mich., 1971, p. 869.
4Daylight operation is possible with pulsed lasers with high peak pulse powers.
[2] Jarret, O., Jr., Mumola, P. B., and Brown, C. A., Jr., paper presented at Remote
Sensing of Water Resources, International Symposium. Canada Centre for Inland
Waters, Burlington, Ontario, Canada, 1973.
[3] Fantasia, J. R., Hard, T. M., and Ingrao, H. C., Report DOT-TSC-USCG-71-7,
National Technical Information Service, Springfield, Va., 1971.
[4] Jerome, L, private communication concerning extinction coefficients observed in the
Great Lakes, 1973.
[5] Kim, H. H., reported in Electro Optical Systems Design, July 1973; and Mumola, P. B.
and Kim, H. H., 1972 IEEE Conference on Engineering in the Ocean Environment,
Newport Rhode Island, 1972.
[6] Measures, R. M. and Bristow, M., Canadian Aeronautics and Space Journal, Vol. 17,
1971.
K . N. B i r c h j
REX, A Computer Controlled Robot

for In Situ Water Quality
Monitoring
REFERENCE. Birch, K. N., "REX, A Computer Controlled Robot for In Situ Water
Quality Monitoring," Water Quality Parameters, A S T M STP 573, American Society for
ABSTRACT: A new, computer oriented system for automatic in situ monitoring of

water quality is proposed which makes full and effective use of modern computer
technology. A concept for in situ computer controlled experimenting is developed as a
reasonable and effective means of automatically observing the wide range of acquatic
parameters needed.
The established capability in the teleprocessing industry is assumed as starting point
and an optimal design is sought for a sensing head which would meet current expression
of requirements for automated water quality monitors while exploiting the advantages of
both hardware and software in a total system context. A working model of such a
sensing head was constructed and operationally tested using an EAI 690 hybrid com-
puter to simulate the central computer and telecommunications facility. This sensing
head consisted of a peristaltic pump, a servo positioned valve, an array of six electrodes
(2 glass pH, 3 silver-silver chloride, and 1 gold micro-electrode) and a thermistor, along
with buffer amplifiers and motor controllers to service these devices. A main feature is
its design as a remote, computer periferal, like a teletype, which understands up to 64
different commands and which returns coded responses. A second feature is its in situ
design making the entire sensor head submersible and operable in depths to 100 m.
A repertoire of programmed experiments was developed for the working model system
to show how, for example, it could monitor the water quality parameters; temperature,
pH, conductivity, dissolved oxygen, chloride ion, total carbon dioxide, total alkalinity,
and a factor called "other ions."
KEY WORDS: water quality, robots, automatic control equipment, monitors, environ-
mental tests, computers
Agencies charged with the responsibility for monitoring the quality and
quantity of water have been anxious to use the technology of automation
t o a i d t h e m in t h e i r c o l l e c t i o n o f b a s i c d a t a a n d t o m a i n t a i n a v i g i l a n c e
over endangered bodies of water. As a result of this need, a generation of
remote water quality monitors have been developed and have found
1 Instrument Development Engineer, Instrumentation R & D Unit, Scientific Support

Division, Canada Centre for Inland Waters, Burlington, Ontario L7R 4A6, Canada.
437
9
acceptance as useful tools for water quality management as witnessed by

the many automated stations in operation throughout the world. It has
been the practice to employ as many as ten remote stations on a given
water basin, integrating them into a water qualty network. Such integra-
tion almost always involves a computer at a central office with data being
telemetered into this office from the remote stations.
It is the view of the author that, as useful as these present monitoring
networks may be, they fall far short of the performance and capability
that could be realized if the modern digital computer were exploited to its
fullest. Several recent reports, Suffet et al [1],2 Maylath [2], and Klein et
al [3], have commented that more utilization of computers will be
necessary if significant advances are to be made in this field. Un-
fortunately, the basic design of present generation remote monitors does
not lend itself to effective operation with computers. This stems largely
from the isolation of the central computer from the analytical and
measurement processes in the sensors. The "analyzer modules" in each
remote monitor form the first stage of isolation because they not only test
the water, but also interpret the results of these tests with simple
electronic circuits. More isolation is imposed by the data acquisition
subsystem since it is generally restricted to sending only selected sets of
the processed data to the central computer at fixed time intervals.
Consequently, a computer at central office is relegated to simple filing and
summarizing tasks, unable to interact in the primary measurement
processes or assist in their interpretation.
This paper proposes a computer controlled monitoring system using a
relatively simple sensing head capable of performing a repertoire of
analytical experiments, in situ. Some experience gained with the design,
construction, and operation of a prototype sensing head in a simulated
monitoring system is reported to demonstrate the feasibility and exemplify
the concepts involved.
The Basic System Configuration

Figure 1 is a block diagram of a complete water quality monitoring
system based on the in situ, analytical experiment approach. In essence, it
consists of a remote station, called a robot sensing head, and a shore
based minicomputer facility. A full duplex telemetry system communicates
commands from the computer to the sensing head and returns encoded
responses back to the computer. In this configuration the sensing head
becomes a remote terminal to the minicomputer such that the computer
can be located at a convenient office on shore while the sensing head can
be sited in the field. Only one remote station is shown for simplicity,
however, the computer could be time-shared among several other remote
BIRCH ON A COMPUTER CONTROLLED MONITORING SYSTEM 439
Telemetry
41-~Transmitter
Telephone
Computer
Interface
t Telemetry g
~ Telemetry
Receiver
~-~.11 14-t~. e N ~ncode= I Ha
Co
Val o-qr I45-- : ........
Li
FIG. 1--Block diagram of proposed robot experimenter monitoring system.
stations. The detail of the remote station depicts the specific case of a
robot sensing head with a multi-electrode sensing array, as constructed for
this project.
The robot sensing head is the active portion of the remote station. It
includes all the electronic and mechanical hardware to decode and execute
the commands from the computer. It is composed of four types of
functional modules:
(a) System service modules--to handle internal signals or controls
between the active modules and the rest of the system, for example,
A / D converters, D/A converters, power supplies, telemetry
modems, etc.
(b) Motor modules--to effect mechanical action (includes control
circuits) for example, sampling pumps, valves, heaters, etc.
(c) Sensory modules--to sense and measure factors in the water under
test (includes special buffer or driver amplifiers).
(d) Passive modules--to provide other elements that do not interact
with the computer, that is, flow cells, reagent reservoirs, ion
exchange columns, etc.
Using the computer as the master control element is a new idea in
water quality monitoring and an important aspect of the proposed system.
Its primary function, one very easily realized in a computer, is generating
the detailed sequences of instructions for the in situ experiments, and
issuing them with precise timing. These instruction sequences are
arranged into proven, machine language micro-programs which can be
called by an operator program to compose the various experiment
routines. Previously acquired data and inputs from the human operator
can be used to modify the order of events and the timing so as to optimize
information content and accuracy. Where reasonable to do so, confirma-
tion control schemes are employed so that before the computer proceeds
with an instruction sequence, the robot sensing head can be interrogated
to determine if the correct conditions prevail. This implies that the motor
modules in the robot sensing head have feedback transducers. The
computer can also be part of servo-control loops to exercise precise,
programmable control over functions such as pump speed, valve position,
pressure regulation, and water temperature.
The system monitors water quality by conducting experiments with a
sample of water taken into the robot sensing head and then interpreting
the results in terms of useful indices of water quality. Such experiments
may be simple potentiometric electrode measurements from which some
useful factors, such as pH, red-ox potentials, and certain ion activities,
can be deduced directly. However, the experiments can be much more
complex; involving many detailed steps, requiring the addition of reagents
or the execution of sophisticated electrochemical analytical techniques.
Each experiment is defined by a sequence of instructions, called an
experiment routine, which can be executed repeatedly by the computer at
intervals specified by the operator. The measurements made by the robot
sensing head during the course of an experiment are telemetered to the
computer in the raw forms produced by the electrometric sensing devices
(for example, the potential between a specified pair of electrodes or the
collector current of a phototransistor). In the computer, they are
interpreted by fitting them to the best mathematical model of the
experiment (often time variant and very nonlinear). The water quality
parameters are thus assigned values which result in the best agreement
with the observed experiment. Information gained during previous
experiments is available to optimize and supplement later experiments.
The term multivariable is applied to this system to convey the ability to
use all available data in the interpretation of any one particular experi-
ment. This permits exact compensation methods, estimation of confidence
limits, rejection of spurious or questionable observations, and extraction
of more than one water quality parameter from a given experiment.
Outwardly, this proposed system looks very similar to present robot
water quality monitoring networks, employing similar hardware,
(computers, telemetry, in situ sensing devices), and producing similar data
(a time series of parameter values for a given station). The fundamental
difference is that it operates on a batch basis in which a sequence of
analytical experiments are conducted in situ under the direct control of
the computer. The signals derived from the sensing head carry values of
primary, observable quantities, like resistance, potential, or current, not
quasi-processed values of parameters as produced by the analyzer or
sensor modules of conventional monitoring systems. The basic differences

in configuration between this proposed robot experimenter system and the
conventional monitoring system can be seen to be:
(a) shifting interpretation functions from the simple analog circuits
of individual analyzer modules to a digital computer,
(b) integration of the primary sensing elements into a single multi-
purposed test cell, and
(c) substitution of a series of computer controlled analytical experi-
ments, performed at regular intervals, for continuous analysis by
banks of analyzer modules, operating in parallel.
The Robot Sensing Head

The robot sensing head is housed in pressure cases suitable to permit
emplacement in the water body at the desired depth for observation.
Justification for this in situ design comes from the advantages gained from
a stable thermal environment and better sampling handling. Effective
thermal compensation of electrochemical cells demands that one be able
to assume thermal homogenuity throughout the cell. There is also benefit
from relaxed mechanical and electronic design since an operating temper-
ature range from 0 to 30~ could be expected, lying well within the
commercial class .specification. By placing all the water handling and test
apparatus below the surface, at the in situ depth, one can realize a water
sampling system which avoids contamination by contact with the atmos-
sphere, passage through long lengths of pipe, or subjection to violent
pressure changes. The contamination problem is most acute when one
wishes to accurately monitor volatile water components, which are far
from their atmospheric equilibrium conditions. The additional engineering
necessary to achieve this in situ capability is felt to be relatively minor
compared to these benefits and the avoidance of much of the plumbing
and civil engineering costs encountered for installation of each present-day
monitoring station. A depth of 100 m appears to be the point where in situ
design begins to get more involved and where all but a few applications
can be satisfied.
Figure 2 is a simplified block diagram of the complete submersible
sensing head as it was conceived for this project. The modules share a
common interface represented in the figure by the digital command bus
and the analog bus. All inputs to the sensing head are digital codes which
are decoded into module addresses, control functions, and timing signals,
and then distributed to the respective modules through the digital control
bus. All outputs from the sensor head are digital, originating in the
encoder which is essentially an A / D converter fed by a differential
amplifier.
There are 64 different commands. A large subset of these commands is
TELEMETRY LINK TO SHORE BASED COMPUTER I'0
/ (? \
/ \
MODEM I \
/ FULL DUPLEX r \ -I
/ m
\ ~a
/ OUTLINE OF
\ PRESSURE CASE O
c
I im
[~T TELEMETRY ANALOG// J .q

COMMAND TELEMETRY VOLTAGE / DIGITAL
DECODER RECEIVER SOURCE J RANSMITTER t CONVERTER J "13
D,G,TALOOM~N0 ~US DIGITAL C.A~MMAND BUS

[ __IV II
_LI~ 1 1
m
--I
ILuJI__ ~ ABO[~~
NALOG [I . m
POTENTIAL I ~Io I POTENT,ALI IPOTENT,AL INTERNAL I

L_ VALVE ELECTRODEI ITHERMISTER ~|~ I ELECTRODEI ] ELECTRODE CHECK J
BUFFER BRIDGE S~ I BUFFER J I BUFFER
pH GLASS POINTS l
- - t . . . . . . . . . .
TEST CELL
~ _ _
~_j CAP,LLARu
REFERENCE
II
I ~ I CHECKVALVE REAGENT
RESERVOIR
VALVENTAKE FI II-OUTPUT TO WASTE
FIG. 2--Block diagram of submersible sensing head for robot experimenter system.
dedicated to controlling the programmed voltage source, which is

essentially a combination D/A converter and simple signal generator. One
other group of commands selects which, of the many points in the system,
is to be connected to the noninverting input of the encoder. Another
group selects the point for the inverting input. The pump and valve
modules obey commands in the mechanical group (for example, "stop,"
"go forward," "slow," "fast," etc.). The working electrode has three
modes of operation; potentiometric sensing, controlled current, or con-
trolled potential (potentiostat). The mode group of commands allows the
computer to effect the desired mode in the working electrode and, in the
case of the potentiostat mode, to select the reference electrode to provide
the potential feedback.
Figures 3 and 4 show the working prototype apparatus actually built.
The array of six electrodes with their buffer-drive circuitry were mounted
above the pump and the valve mechanisms with their controller circuits.
FIG. 3--Electrode array portion o f robot sensing head.

FIG. 4---Pump and valve mechanism.
The flow cells around each electrode, the valve, and the p u m p were
interconnected by sections of Nylon or Tygon tubing to form the "test
cell" configuration depicted in Fig. 2. The reagent reservoir, shown in
Fig. 2, was a bag (1-in. diameter by 3-in. long) made of supple rubber
and was filled with a standardized 0.1 N hydrochloric acid (HCI) solution.
To simulate a lake environment a special pressure vessel was made. A
short length of pressure casing was sandwiched between these two
bulkheads and secured by six rods. The upper bulkhead held the electrode
array with the flow c e l l arranged so that they would be inside the
pressure vessel. The lower bulkhead held the p u m p and valve mechanism.
T a p water was put inside this vessel and a means for pumping the
hydrostatic pressure up to 150 psig was provided in order to simulate
submergence to 100 m in lake water. The test configuration may be
considered as an "inside-out" configuration of the intended operational
system where all critical aspects of the design are subjected to realistic in
situ conditions.
Separate input and output ports to the test cell were provided so that
known test solutions could be submitted to the robot sensing head without
mixing with the water in pressure vessel. These ports were fitted with
manual valves so that the vessel could be pressurized to its full range
without generating large pressure differences between the water in the test
cell and the tap water surrounding it. A short length of surgical rubber
tubing formed the output section from test cell, thereby providing a
mechanism for equalizing any residual pressure difference due to dif-
ferential volumetric contraction on pressurization, (mostly due to tiny,
trapped air bubbles). Outside the pressure case an enclosed system of
tubing, including a caustic trap for carbon dioxide (COz), was used to
minimize atmospheric contamination of the fairly basic and poorly
buffered test solutions.
The computer, the telemetry link, the programmable voltage source,
and the encoder were simulated on an EAI 690 hybrid computer system.
Thus, a complete working model of the proposed system was realized
without extensive hardware development beyond that which was necessary
to show the feasibility of the peculiar apparatus modules.
A program called ROBOT OPERATOR was written in HOI (Hybrid
Operator Interpreter--a real time, interactive, interpreter language
available on the EAI 690 system). This program was essentially a package
of nested subroutines which could be called in an order specified by an
agenda entered by the operator. Special, machine language subroutines
were written to allow the other subroutines to communicate with the
submersible sensing head in a manner that simulated the code formating
and time delays implied by the proposed configuration. Low order
subroutines, like READ and RUN PUMP, provided control sequences and
calculations that were frequently required. An example of a high order
subroutine is TITRATE, for which a pseudo listing is given in an
appendix to this paper.
The Measurement of Water Quality Parameters
In a typical operation the computer instructs the sensing head to take a

sample by first orienting the valve to accept water from the intake filter
and then pumping the stream past the electrodes and out to waste via the
check valve. While the sensing head is in this "fill" mode, readings of
temperature, pH, conductivity, chloride ion activity, and red-ox potential
can be made.
Temperature is measured by instructing the sensing head to engage the
thermistor bridge module and to transmit the bridge reading to the
computer. Since the reference voltage is used to excite the thermistor

bridge, the "internal check module" is also asked to pass a reading of
reference voltage to the computer. Temperature is computed from a third
order polynomial which had been derived from a regression analysis on
calibration data.
The pH parameter is measured by asking the sensing head to sense the
difference in potential between the glass pH electrode in the test cell and
an identical electrode in the reference chamber. The junction capillary
between the test cell and reference chambers forms the liquid junction
that completes the following cell for potentiometric determination of pH:
glass test liquid 0.1 N glass

electrode water junction HCI electrode
(pHA) (pHR)
The pH value can be calculated from the potential of this cell:
(EpH -- Epz - Ej) 298.16 K

pH = pHA = pHR. +
KpH TA
where
pHR the pH of the reference solution,

=
EpH = the measured potential difference,

Ej = the junction potential that results at the interface of the water
and the reference solution in the junction capillary,
KpH = the sensitivity at 25~ of the pH electrode in volts per pH unit,
Epz = the zero offset of assymmetry potential that is observed if both
the test cell and reference chamber are filled with the reference
solution (approximately 400 #V with electrodes used), and
TA = the absolute temperature in degrees kelvin as determined from
a recent temperature measurement.
The activity of the chloride ion was measured using silver/silver chloride
electrodes in a potentiometric measurement scheme paralleling that for
pH. The computer commands the sensing head to measure the difference
in potential between the chloride electrode in the test cell and the
reference chloride electrode in the reference solution. The value of pC1- is
calculated from the following:
Ect + Ecz + E j 298.16 K

pCI- = pCI-R --
Ko T
where
pCI-R = known from the make up of the reference solution (approxi-
mately 1.098),
ECI the measured potential,
Ecz = the offset of assymetry potential (about 2 mV for the elec-
trodes used),
Ej the same junction potential as appears in the pH computa-
=
tion, and
gCl the sensitivity of the active chloride electrode at 25~
Red-ox potential is measured by commanding the working electrode
into its potentiometric mode and then observing the potential that exists
between this gold electrode and the silver/silver chloride electrode in the
reference chamber. The red-ox potential is computed as follows:
ERED-OX = ERO -- E j -- Eref
where
ERO ---- the measured potential,
Ej = the same junction potential as in the other cells discussed, and
Eref = the standard potential of the silver/silver chloride electrode in
the reference chloride solution.
The scheme for determining conductivity is an adaptation of the four
electrode, d-c method described by Elias and Schiff [4]. The conductivity
cell is created by commanding the sensing head to put the working
electrode into its constant current mode and observing the potential
between the silver/silver chloride electrode and the glass pH electrode,
both of which are in test cell. To measure conductivity with this cell a
programmed current value, I1, is forced to flow from the working
electrode to ground at the ground (or counter) electrode. The only path
for this current is through capillary tubing in the test cell since the other
potential path is effectively interrupted by the pump where the tubing is
pinched closed. Hence, the pair of potential sensing electrodes sees an
additional voltage drop appearing across the water resistance between
them. After reading this difference potential, Ez, the computer next
instructs another current, Iz, to flow in the reverse direction after which it
notes Ez. The computation of conductivity from these data is as follows:
I 1 -- 12
Cond - - Kcell
E 1 - - E2
where Keen is the effective cell constant for this configuration as deter-
mined from calibration with standard potassium chloride (KCI) solutions.
WATER QUALITY PARAMETERS
The residual potential, that normally exists between these two potential
sensing electrodes at zero current, is assumed to remain stable over this
short time interval and therefore cancels out. The conductivity is thus
measured at the in situ temperature and it remains for the computer to
adjust this value to specific conductance (at 25~ by the application of
the best known correction formula for the particular water being
measured.
It was originally planned to measure dissolved oxygen by a chrono-
amperometric experiment using the gold button working electrode.
However, a program to do this could not be perfected in the limited time
that the EAI 690 computer system was available. The lower priority for
this experiment development was based on the knowledge that a Clark
Cell, an acceptable probe for dissolved oxygen, could easily be accom-
modated in the experimenter system concept to obtain this parameter.
One of the more complex experiments which was conducted in this
limited version of the sensing head is an acid titration which can yield
values for total alkalinity and total CO2 (or dissolved inorganic carbon).
These parameters, together with pH and dissolved oxygen, can provide a
means for observing biological productivity (Teal and Kanwisher [5]). A
chloride tag method was devised to demonstrate the feasibility of in situ
titration using a multivariable approach. In this method the concentration
of HC1 added is determined by a direct measurement of the chloride ion
concentration with a selective ion electrode. Since every H § ion introduced
to the test solution will be accompanied by a C1- ion, then the increase in
CI- ion concentration is directly related to the total H § ions added. The
main advantage of the method in in situ titration is that volumetric
measurements are not needed, hence, the apparatus can be relatively
simple. It can be shown that the method is theoretically independent of
any volumetric factors. Apart from this tag method for measuring acid
added, the procedure is very similar to the conventional potentiometric
titration method as would be followed in a laboratory. A series of readings
of pH and pC1 are made, each set of readings being taken after some
small amount of HC1 has been added. The series of pCl readings are
converted to chloride ion concentrations which are then taken to be
equivalent of H § added. A computer program estimates ionic strength and
chloride ion activity coefficients for each point in the experiment. It also
produces a plot of pH versus [CI-] resulting in a curve which is essentially
the same as obtained by the conventional method. From this plot, one can
determine the equivalence points and pick off the concentration of acid
added, which is the alkalinity.
Conductivity also changes with the addition of an acid titrant to water
containing alkalinity, a fact that has led to the suggestion (Park et al [6])
of a conductiometric titration method for alkalinity. Since the working
model could easily make conductivity readings at the same time it made
the pC1 and pH readings, then it was decided to try this conductiometric
method as well. The interpreting program made good use of these
simultaneous conductivity data points.
The titration experiment calls for the addition of small amounts of HCI,
each followed by mixing and the measurement of temperature, pH, pC1,
and conductivity. The process continues until the pH of the test solution is
lowered below a pH of 3. A sequence of pump operations was devised to
pull small amounts of the standard acid reagent into the test cell. With
the pump running "fast forward," a reduced pressure exists in the test
cell which slowly draws acid through the junction capillary. Running the
pump in "fast reverse," pushes a core of mixed test solution into the
junction capillary, both sealing off the test cell and again establishing a
"liquid junction" for the potentiometric measurements. The time period
spent going "fast forward" Was roughly related to the size of the acid
addition. Readings of temperature, pH, pCl, and conductivity were made
using the routines outlined previously. The titration subroutine could
adjust the size of acid additions to give reasonable distribution with pH
and pCl. See the appendix for a pseudo listing of this subroutine.
Figures 5 (top and bottom) show typical plots produced from the
processed data from a "chloride tag" titration of a 0.002 N solution of
sodium carbonate (Na2CO3). Figure 6 (top and bottom) shows the same
solution titrated under simulated conditions of a 100-m depth in lake
water (that is, completely submerged in water in a pressure vessel at 150
psia).
This experiment also provided an opportunity to try various approaches
to computer programs which could extract parametric information from
experiments such as this one. The first step in any of these interpreting
programs was converting the recorded readings of thermistor resistances
and electrode potentials to parametric values using the relationships
presented previously. Since the reference solution was HC1, it was not
possible to assume that the.junction potential, Ej, was negligible as is
commonly done with a conventional reference electrode. Instead, it was
found that the integrated form of the Henderson equation could be used
for estimating Ej to within a few millivolts for most expected cases. The
computation of Ej utilized the measured conductivity of the water plus
estimates for all the significant ionic conductances. Similarly, the con-
version from activity values to concentration values required the estimation
of activity coefficients which could be done using the Debye-Huckle
equation and a computed value of ionic strength. Consequently, the first
stage of the interpreting program required the iterative solution of the
transfer relations and of the estimating equations for ionic strength and
junction potential. The convergence was fast (two passes in general) and
the formulation of the equations in FORTRAN was very straight-forward
despite the complexity.
9oo-
.002 N Na2CO 3 /"
E
800- Feb. 2 1972, Run #5 /
U
In Situ Depth = .3 meters /"
700- In Situ T e m p . = 22~ //
/i
0 /
600- p,
u
j./
c 500- s,I
/,
//
.; 400-
j/l
3oo- //
--O .... %0"- . . . .
2007 ,0- - 0 - - - 0 - ---0--- - ~0~ - - ~ 0 = --- - - - 0 - - - - t
I00-
1 I I
9001 .OO2 .O03
Chloride Concentration
pH
O
O
O
9
8- .002 N Na2CO 3
Feb. 2 1972, Run #5
7-
|
6-
|
5-
4-
O
O
3-
! I
001 .002 .003
FIG. 5--(top) conductivity titration; (bottom) pH titration9

900
.002 N Na2CO 3
800- Feb. 6 1972, Run #6
In Situ depth = 100 meters
In Situ temp. = 25~
8 700-
E
8
u 6oo- /
"E
/
c
,_ 500-
o"
/
/
/
400- /
.6
u 300- /
70 ........... -,~ ............ ~o--
o. --~'- - - -
/
~ 200
I00
I
.001 002 1003
pH
.002 N Na2CO 3
9 Feb. 6 1973, Run #6
8'
7-
6-
5-
4-
|
|
0 9
3-
2 ! I l'
.001 .002 .OO3
Chloride
F I G . 6---(top) conductivity titration at 150 psig; (bottom) p H titration at 150 psig.

The second stage in the interpreting program was linear interpolation

between the points in the data table to determine break points and
equivalence points. This was analogous to that which one would conven-
tionally do on graph paper. This resulted in a value for total alkalinity
which, together with the initial pH value, provided a means for computing
total COz (Park [7]).
Another more sophisticated approach to this second stage is the
application of nonlinear regression analysis. To apply this method one
needs a mathematical relationship between the variables and parameters
of interest. Two models were attempted, one based on the conservation of
alkalinity and the other based on a theoretical calculation of conductivity.
These models allowed the computer to select best values for total CO2,
total alkalinity, plus a quantity called Cx, the concentration of other ions
having an equivalent ionic conductance like sodium.
This process of selecting parameters is iterative wherein guess values for
these parameters are tried in the mathematical model and then "better"
values are computed based on the observed correspondence between this
model and the data table. In this case a modified Gauss-Newton method
(Hartley [8]) was applied which selects the parameters by a least squares
criteria. The degree of correspondence between the theoretical model and
the observations in the data table is expressed as the root mean square
error.
After working with this technique of interpreting the experimental data
it became apparent that this error factor could be a very useful auto-
verification index. When such an index is low, one would have confidence
that the parameter values derived from the experiment were true. How-
ever, when the autoverification index is large, the observer would know to
treat the derived parameter values with caution. Furthermore, a large
index value would suggest that either the robot station was encountering
water having characteristics which invalidated some assumption made in
the theory of the experiment, or the robot station was suffering some
malfunction or drift in calibration. In any event, the h u m a n observer of
the monitor system is alerted so he can take action. It is perhaps not too
far out of line to suggest that the autoverification index is a non-specific
pollution detector and, therefore, a potential tool for the pollution
detection role of monitoring.
Another extension of the regression analysis technique is called auto-
calibration. It is the effect of letting one or more of the calibration factors
for a sensor device become one of the "parameters" selected by the
regression analysis. This should be possible if one takes care to ensure
that the statistical properties of the experiment and the data set will
permit a reasonable estimate for these added factors. In other words, the
experimenter monitoring system should be very capable of calibrating
itself in the course of routine operation and thereby achieve extended

unattended service in the field.
Some typical results of test runs with various prepared solutions and
some river water samples are presented in Table 1. The entries under Qc
and QA are the autoverification indicies for the conductivity and alkalinity
models, respectively. This represents the level of performance reached
after limited programming time and with "first cut" apparatus designs. It
should not be considered typical of the results which should be obtainable
after more development.
TABLE 1--Summary o f in situ titration experiment results.
Alka- Conduc-
linity, CTot, Cx, tivity, QC, QA,
mN b naN mN pH i pCl i /am /am %
Guess values 1.00 1.00 1.00 7.00 4.5

2 naN Na2CO , 1.81 1.01 1.01 10.49 4.4 265. "70 319
2 mN Na2CO3a 1.94 1.54 2.04 9.62 4.3 197. 12 3.1
1 mN Na2CO 3 1.00 0.56 0.63 10.25 4.4 112. 22 2.9
0.5 rn2V Na2CO 3 0.49 0.40 0.50 9.61 4.4 53. 3 4.9
Willamette R. #1 0.37 0.40 0.54 6.76 3.9 54. 9 3.4
(by analysis) 0.38 0.74
Willamette R. #2 0.34 ().51 0.60 6174 410 '53. "7" 217
(by analysis) 0.34 ... 0.85 . . . . . . . . . . . . . . .
aThese data taken at simulated depth of 100 m; all other entries are taken at 0.5 m.
b G r a m equivalents per liter x l000.
Conclusions
The apparatus which constituted the prototype sensing head was a
general success in that it performed satisfactorily in the simulated in situ
environment. The seals and mechanical design worked well enough to
permit operation of the sensing head under the equivalent of 100 m of
water. The capillary junction joining the reference cells to the test cell
produced repeatable stable liquid junction potentials even with very dilute
test solutions. The differential electrode buffer design proved adequate to
reject the significant 60-cycle background which was definitely present in
this laboratory simulation. However, the in situ design was only a first
attempt and several deficiences were recognized. Perhaps the most serious
problem was due to combining reference solution and titrant without
providing direct controls over the flow in the capillary. Flow cell-to-flow
cell connections were prone to minor leaks which led to parasitic electrical
paths of a highly unpredictable nature.
Detailed and elaborated procedures were handled with ease by nested
subroutines, which once devised, could be called by higher level routines
to provide a simple, easy to understand R O B O T O P E R A T O R program.

The idea of performing a series of batch analyses in one test cell was no
problem once good FLUSH and FILL routines were devised. Reagent
consumption by this kind of batch analysis in only 1 ml per titration,
meaning that 1 liter would be enough for a whole month of hourly
titrations. As a result of this work it is felt that the computer-controlled
experiment approach opens the door to many innovative new methods for
observing needed parameters. Novel techniques such as the chloride tag
titration method can overcome the necessity for critical hardware design
which would otherwise be required to directly implement conventional
laboratory analysis.
Bringing the computer into the first stages of the analysis to work with
primary observable quantities has been shown to offer possibilities beyond
the expected ability to just utilize more precise formulations of transfer
functions. A first example was the use of a mu]ti-parameter, iterative
method for adjusting the transfer relations to account for the "not
necessarily minor" factors like ionic strength and changes in "physical
constants" which are, in fact, functions of temperature. Another example
was the use of the computer to interpret data from analytical experiments.
Although the direct programming of conventional graphical interpretation
techniques produced the best results from this working model data, much
promising capability was shown for nonlinear regression techniques. The
concept of autoverification was demonstrated to provide a good index of
the error factor involved in each experiment.
The concept of in situ experimenting has been demonstrated to be an
effective and reasonable means for observing water quality parameters,
including some for which specific sensors are not available. High level
computer languages, such as HOI, proved to be invaluable during the
routine development stages. Similarly, the power of sophisticated numeri-
cal techniques and the convenience afforded by high level languages like
F O R T R A N at the interpretation stages are recognized to be essential.
APPENDIX
The following is a pseudo listing of the titration subroutine used in this working
model. Before it is called, it is assumed that FLUSH and FILL have been executed
such that the test cell is filled with a fresh sample ready to be analyzed.
SUBROUTINE TITRATE
P -- CLOSE (Set valve to circulate
CALL VALVSET (P) (water in closed loop.
DO K ---- 1, 20 (Prepare to take 20 reading sets.
S=+I (Run pump,
CALL PUMPON (S) (slowly forward, while
CALL READT (measuring water temperature and
CALL R E A D E P H (measuring EPH.

S=O (Stop
CALL P U M P O N (S) (pump, and
IF ( E P H . G T . - - . 0 4 0 ) G O T O 2 (Quit if pH under 3.
W=2 (wait
CALL W A I T (W) (2 seconds
CALL R E A D E C L (Measure ECL and
CALL R E A D C O N D (conductivity.
S:+2 (Run p u m p fast forward
CALL P U M P O N (S) t.to draw HC! into test
W:K+5 (cell. Let run for
CALL W A I T (W) ( K + 5 seconds, then
S = --2 (run p u m p full
CALL P U M P ON (S) (speed, reverse
W:5 (for 5 seconds to
CALL W A I T (W) (reset liquid junction.
W R I T E ( - - - - )T, EPH,
ECL, C O N D (File in Data Table.
1 CONTINUE (Make more measurements.
2 RETURN
This program has the temperature and pH measurements made while the p u m p
is slowly circulating the water in the test cell, thereby minimizing self-heating and
the, so-called, alkaline leaching errors encountered with glass electrodes in low
buffered waters.
References
[l] Suffet, J. H., Radziul, J. V., and Goff, D. R., "Continuous Water Quality Measure-
ment, Present Status and Future Trends," Proceedings, 16th National Symposium of the
Analysis Instrument Division of the Instrument Society of America, May 1970.
[2] Maylath, R. E., "Water Quality Surveillance in New York State," unpublished internal
report, New York State, Department of Health, Division of Pure Waters, Water Quality
Surveillance Network, Sept. 1969.
[3] Klein, W. L., Dunsmore, D. A., and Horton, R. K., Environmental Science and
Technology, Vol. 2, No. 10, Oct. 1968, pp. 764-771.
[4] Elias, L. and Schiff, H. I., Journal of Physical Chemistry. Vol. 60, May 1956,
pp. 595-598.
[5] Teal, J. M. and Kanwisher, J., Journal of Marine Research, Vol. 24, 1966, pp. 4-14.
[6] Park, K., Oliphant, M., and Freund, H., Analytical Chemistry, Vol. 35, No. 10, 1963,
pp. 1549-15.50.
[7] Park, P. K., Limnology and Oceanography, Vol. 14, No. 2, 1969, pp. 179-186.
[8] Hartley, H. O., Technometrics, Vol. 3, No. 2, May 1961, pp. 269-280.
G. C. G u n n e r s o n I
Utilization of Data from Continuous

Monitoring Networks
REFERENCE: Gunnerson, C. G., "Utilization of Data from Continuum Monitoring

Networks," Water Quality Parameters. ASTM STP 573, American Society for Testing
ABSTRACT: A large data system is one which is hard to turn off. This inertia is
directly proportional to the amount of money invested in hardware and facilities, to the
number of careers involved, and to the length of record.
Large data systems are concerned with the collection of time series. What eventually
will become a data management problem in a large data system using manual tech-
niques quickly becomes a crisis with automatic equipment. It is accordingly essential in
the design of continuous monitoring networks that the utilization of the data be
adequately considered and optimized.
The arguments for optimization of time-series collection, analysis, and utilization
apply equally to space fields. In either case, the problem is to match the resolution of
the data collection system to that of the response system. For example, the digitizing or
averaging period used for pollution detection should be consistent with the time constant
for pollution control or abatement measures.
The institutional value of monitoring programs may be equally important, but is more
difficult to predict. In any event, the analysis and interpretation of the data must
correspond to the real world use to which it will be put.
KEY WORDS: water quality, monitors, sampling, environmental tests, data processing,
automatic control equipment, pollution, environmental data utilization
T h i s p a p e r is to a l a r g e e x t e n t b a s e d u p o n i n v e s t i g a t i o n s o f o p t i m z i n g
s a m p l i n g i n t e r v a l s w h i c h w e r e p u b l i s h e d six y e a r s a g o [1, 2, 3]. 2 S i n c e
t h e n , t h e r e has b e e n a n e v e r - i n c r e a s i n g n u m b e r o f e n v i r o n m e n t a l m o n i t o r -
ing systems, data storage and retrieval systems, reports, exhortations, and
local, r e g i o n a l , n a t i o n a l , a n d i n t e r n a t i o n a l p r o g r a m s , all g e n e r a t i n g d a t a
w i t h w h i c h we a r e c o n c e r n e d . E v e n so, n o t h i n g h a s h a p p e n e d t o c h a n g e
m y i n t u i t i v e p r e m i s e a n d p u b l i s h e d c o n c l u s i o n t h a t it is p o s s i b l e to collect
t o o m u c h d a t a . I h a v e f o u n d t w o g r o u p s w h o o b j e c t on p h i l o s o p h i c a l
1Director. Marine Exosystems Analysis Program, Environmental Research Laboratories,

National Oceanic and Atmospheric Agency, Boulder, Colo. 80302.
456

GUNNERSON ON UTILIZATION OF DATA ,457
grounds to this assumption. The first group is made up of research

workers whose careers are taken up with the pursuit of either the
techniques, the hardware, or the money to obtain the data they need and
to whom it is apparently inconceivable that one can ever be drowned with
the right kind of data. These people are working with small data systems.
Most of the objections to my thesis come from those who are occupied
with large data systems. A very useful definition, not easily arrived at, of a
large data system is one that is hard to turn off. This inertia is for obvious
reasons directly proportional to the amount of money invested in hardware
and facilities, to the number of careers involved, and to the length of
record already obtained. With that definition it is easy to cite examples of
large data systems.
Large data systems are concerned with the collection of time series; so
are a variety of equipment manufacturers who are making it possible to
collect essentially continuous data on a presently limited, but ever
increasing, number of environmental parameters. What eventually will be
a problem in a large data system using manual techniques quickly
becomes a crisis with automatic equipment.
The automatic measuring devices with which I am familiar are almost
invariably capable of giving me more data than I need, and they usually
do. In estuaries, these have included dissolved oxygen and salinity data
collected at 6-min intervals [1] and velocities determined at 2-min
intervals ]2]. Whether or not the data collection is automated, with a
sufficiently long record the problem becomes one of resolution. In other
words, the question is whether the series from a particular environment
can be digitized at some interval larger than the recording interval without
loss of essential information. If so, future data collection and utilization
procedures for this or similar environments can be designed accordingly,
with consequent savings in data storage and analysis requirements and in
data collection costs as well.
Most of the examples to be presented in the following pages are based
on data from the Federal Water Pollution Control Administration and
from the California Department of Water Resources.
Many ways in which optimum sampling intervals can be determined
have been described in literature. The basic approach that I have used in
studying time series is straightforward. The entire record is examined in
detail by one or more techniques until the maximum understanding or
insight is developed. Next, either the amount of data or the statistical
effort is reduced in a series of successive steps. Sooner or later, this
reduction results in a loss of detail necessary to characterize the particular
environment. This marks the point at which the sampling (or digitizing)
interval is at the optimum.
Data and Analyses

Continuous data from tidal estuaries have been selected for analysis.
Although different record lengths have been analyzed for special studies,
most of the work has been based on a 1-month period. This is a
reasonable period in descriptive analyses for pollution surveillance work.
Also, it is about as long as water quality monitoring equipment will
provide an essentially continuous record without interruptions due to
breakdown, power failure, vandalism, or acts of God [4].
Tidal estuaries provide excellent environments for developing methods
for optimizing sampling intervals. There are known semidiurnal, diurnal,
fortnightly, and annual periodigities which can be used as benchmarks. I
have examined data from the Potomac River estuary, Raritan Bay, and
San Francisco Bay with consistent results.
Additional records based on grab-sampling from inland streams have
also been analyzed in order to relate the distance downstream from major
sources of pollution to the optimum sampling interval.
Ranges, Means, and Quartiles
In pollution surveillance, the simplest, and often the most revealing,

statistic is the range. It is the extreme values that cause trouble. Figure 1
shows the variation of extreme, mean, and quartile values of dissolved
oxygen and specific conductance for 31-day periods in late summer and in
spring in the Potomac River in Washington, D.C. Sampling intervals of
from 6 min to 2 weeks were examined. For intervals of 1 day or more,
only noontime data were used. This choice was purely arbitrary;
comparable results would have been obtained by using data collected at
midnight or any other time.
Inspection of Fig. 1 reveals that the slopes of curves representing mean
and quartile values are almost fiat between the 0.1 h and 24th h. Because
these dissolved oxygen and conductivity values were essentially constant
throughout the range of sampling intervals, daily sampling would have
been sufficient to determine them.
Extreme values were more affected by the sampling interval. Even here,
in three of the four cases shown, maxima and minima were well
established by a 12-h sampling interval. The remaining example, dissolved
oxygen during 27 Aug. to 25 Sept. 1963, showed significant increases in
range as the interval was reduced to about 1 h.
The statistics given in Fig. 1 are consistent in showing that, with the
single exception noted, little change resulted from sampling at intervals of
less than about 12 h. However, this method of analysis is insensitive to
much of the information contained in the original data.
GUNNERSON ON UTILIZATION OF DATA 459
SPECIFIC CONDUCTANCEAUG 2?-SEPT ZSr196.= DISSOLVEDOXYGEN ~UG 2T-SEPT 25rl963

I I I -- I I /
450 .~_AXI~UM
MAXIMUM 8
3 dQUARTILE " ~ ' ~

40o - --
MEAN 7
SCdQUARTILE ~ - - )
350 -I~QUARTILE - "= .. ~. .. MEAN
' ___'2__"
300 . . . . . .
5
_ M'N2My__M
.............. MINIMUM ~ _ _ - - ~
250 . . . . .
I I I
OI ~ io TOO ,coo 4O I L =0 I00 I000
SPECIFIC CONOUCTANCEAPRtL I-MAY I, 1964 OIS$OLVEO OXYGEN APRIL I-MAY 1,1964

220 MAXIMUM ------ __MAXIMUM
2'0 ~ F ~ U A R TILE ~"~, ~ ..... "~
~90 ....
9 MEAN
~,,o -J -- . . . . . . 22 ~ -
'~ 17o ..... I~QUARTiLE
~ ISO . . . . !u QUARTIL,.E. . . .
.,,o . . . . . . . .. . . J ./ . '
J 9
/ T I I I I
Is~ i *o ioo iooo o.i i ~o ioo r
SAMPLING INTERVAL, HOURS SAMPLING INTERVAL, HOURS
Potomoc River at Washington, D.C
FIG, l--Variation of mean, quartile, and extreme values of specific conductance and
dissolved oxygen for selected sampling intervals during 31-day record.
Frequency Distributions
Plotting frequency distrubutions on normal probability paper is one of
the easiest and most revealing exercises in evaluating environmental
pollution data. In most cases, normal paper is sufficient, although
occasionally an extreme-value distribution, particularly Gumbel's [5], will
be useful. Normal paper provides an adequate basis for studying sampling
intervals in polluted water. Data from Refs 6 and 7 on coliform bacteria
and from Ref 8 on iron and manganese were used.
Bacteriological results are notoriously variable, and little faith is placed
in any single observation. These data thus provide an extreme case for
evaluating sampling requirements. Figure 2 shows frequency distributions
of coliform bacteria at four water pollution surveillance stations with a
wide range of pollution conditions, which are summarized in Table 1.
Figure 2 compares frequency distributions based on weekly or monthly
sampling, where the latter is the first value reported for a given month [6,
7]. At New Orleans, the two sets of data are in excellent agreement
between the 10 and 90 percentiles.
460 W A T E R QUALITY P A R A M E T E R S
, , i w . , i + v i ,A,
9 ,tee 9
Lo" I i + i i i i , ~ f , ~ 1 i
BIG S I O U X R I V E R AT M I S S O U R t R I V E R AT
SIOUX F A L L S . S. DAKOTA KANSAS C I T Y , MO.
WATER Y E A R 1963 WATER Y E A R 1 9 6 3
io o 9 %.
! ~.A .e*
[ A& **t"*
b ...,.-
..'g 9
r ..-%
_i 9 9 A
0 =~ t
0
L
I
.i . . . +. . . I. . . . .I. I * I ,
" " J-
bl
, -T-. i J I i , + , , , , + , + , , , I 84 i t + i i I
m M I S S O U R I R I V E R AT M I S S I S S I P P I R I V E R AT
S t L O U I S , MO. / NEW ORLEANS. LA.
WATER Y E A R 1 9 6 2 . / " ~ WATER YEAR 1963 J e
&* 9 9
0
9 9
i e9
9 WEEKLY SAMPLES
9 ,~, , ' . . . . . . . . .
~5 SO SO 4O SO SO 70 eo es 90 9n 9S
PERCENT SAMPLES WITH CONCENTRATIONS LESS THAN INDICATED VALUES
F I G . 2--Frequency distribution of coliform bacteria in suoCace water.
TABLE l--Bacteriological pollution at selected water pollution surveillance system stations.
Mean Coliform Bacteria

per 100 M1 Source of
Station (Fig. 2) Pollution
Mississippi River at New Orleans, La. 1 500 Diffuse

Missouri River at St. Louis, Mo. 10 0 0 0 Diffuse
Missouri River at Kansas City, Kans. 28 0 0 0 Diffuse
Big Sioux River at Sioux Falls, S. Dak. .500 0 0 0 Dominant
GUNNERSON ON UTILIZATION OF DATA 46"/
At St. Louis, the agreement is excellent for the mean and higher values.
Breaks in the curves indicate a bimodal log-normal distribution. The
higher coliform levels are largely due to municipal and industrial dis-
charges from the Kansas City metropolitan area; the lower levels are due
to runoff.
At Kansas City, coliform levels show the effects of discharges from St.
Joseph, approximately 70 miles upstream. Although mean concentrations
from the weekly and monthly data are practically identical, both higher
and lower percentile values from the two sets of data show significant
differences.
At Sioux Falls, the sampling station is located a few miles downstream
from the sewage treatment plant. It is obvious that the stream cannot be
characterized by monthly samples.
In summary, Fig. 2 shows that with a diffuse source or sources of
pollution, monthly coliform sampling is adequate for establishing annual
statistics and thus for determining longer term trends. If there is a need to
evaluate conditions during a particular season, more frequent sampling
will be required. As a single dominant source of pollution is approached,
a shorter sampling interval is required. The need for a shorter interval is
accompanied by a concurrent requirement for closer distance spacing so
that at, say, 20 miles from the outfall, cross-sectional sampling is
generally necessary.
A similar conclusion may be drawn from data published in Ref 8. This
sampling program provides for analyses, at 10-day intervals, of combined
daily samples so that Variations in water quality are minimized. Figure 3
shows the frequency distribution of concentrations of iron in the Ohio
River and its tributary, the Mahoning. Similar results were obtained for
manganese. The Mahoning shows the effects of industrial discharges from
the Youngstown, Ohio, area. Figure 3 shows that although the differences
between the weekly and monthly data are never large, these differences
become more apparent as the source of pollution is approached and
indicate the need for more sampling in the source area.
Comparisons of Departures from Continuous Record

In another approach to optimizing sampling intervals, Carter et al [9]
examined three recording frequencies in the operation of the network of
U.S. Geological Survey stream gages. Output from these gages is punched
onto paper tape at 15-min intervals. Daily stream flows computed from
the 15-min data were accepted as base values. Flows were also computed
from 30 and 60-min data, and their departures from the base values
determined. The standard deviations of the departures, expressed as a
percentage of the base or 15-min values, were determined for a total of 59
gaging stations in five widely dispersed study areas.
2.8 I t I I l I I I I I I 0.4 I I I i i i I I I ,.i. i
MAHONING RIVER AT I OHIO RIVER AT NEWELL,W, VA,
LOWELLVILLE, OHIO O.2J- (mile 46.4) 9
m
2,6 =o
A
s
/
2,4 0 I- L_=:~ - ~ ~ 9162149 99 i i r-
m .<
0.6 I I I l
I I I I I i i
9 IO-DAY S A M P L I N G I N T E R V A L OHIO RIVER AT RAVENSWOOD, W. V A . -o
2,2
z 9 MONTHLY SAMPLING INTERVAL (mile 220.9)
0 0.4
o 9
-
I,,~ z:
2.0 o m
I-
z 0.2 9149 m
t=J 20
u 1.8 if)
z
o 0 I: ,L I- :~ d.~.~."L"mV,~'~dr ~I
~.)
0.4 0.4 I I i 1 i i l I I I I
I OHIO RIVER AT FLORENCE, INDIANA
.,A,o "&
/ (mile531.71
0.2 =~
ee~lk 0'2I
j 9149149176
L ~,.I.. 9149176 41 I I I I I I l 0 I- ~ I= . = ~ - i - - ~ . ' l i ' ' ' i " a L i ' d l b ' ~ ' ' ' i ~ 9 9 & " 9 Mi. i
5 I0 2 0 30 40 50 60 70 80 90 95 98 2 5 I0 2 0 SO 4 0 SO 6 0 7 0 80 90 95 98
PERCENT OF SAMPLES WITH CONCENTRATIONS ~,INDICATED VALUE
FIG. 3--Frequency distribution o f iron in M ahoni ng and Ohio Rivers, Oct. 1957 to Sept. 1958.
Figure 4 shows two sets of results reported by Carter et al [9]. Their

limiting standard was an arbitrary 1 percent maximum allowable standard
deviation from the base value. On this basis, they showed that 60-min
intervals were satisfactory for east coast drainage areas over 100 square
miles in area, while 15-min intervals were required for areas of less than
40 square miles. In contrast, 15-min values were found necessary for
Kansas stream gages with drainage areas of less than 300 square miles,
and 30-min data were required for areas from 300 to at least 5000 square
miles.
51 T - I wi I I I I I I I I I i I
4 84 9
i - - wz 3 , % ATLANTIC
0-"~w" z x o SEABOARD
==
~ 'i ;-o.- ~ o~
z>o
w- o I J I I I ~I L l I I i I I i
_- ;..- .A.SAS -
zO 2 _ " ~ . . . ~ -. -
~- _
OQ O "~ "~ 9
I--
g
0 I I I II I I I I I I I I I
IO 20 40 60 I00 200 400 tO00 2 0 0 0 4 0 0 0 I0r
STREAM FLOW, CFS
FIG. 4----Comparisons of average daily flow, computations from 30-rain (open circles) and
60-min (black circles) data with computations for 15-rain data.
Spectral Analysis
Spectral analysis provides one of the most powerful tools for determin-
ing optimum sampling intervals. An introduction to the mathematics of
spectral analysis is presented in Appendix A. A bibliography for further
reading is given in Appendix B.
Power spectra were computed for 31-day lengths in the Potomac River
[11]. Typical results are shown in Figs. 5 and 6 for sampling intervals of
from 0.1 to 6.0 h. Results of the power spectrum calculations based on 27
Aug. to 26 Sept. 1963, data are shown in Fig. 5. The sampling interval
was varied from 0.1 to 6.0 h, while the computation was held constant at
100 lags.
WATER Q U A L I T Y P A R A M E T E R S
&=O.l h r i
OA
0,01
r
"~=I hr 9
O.I
v
E Ol
A=2hr
OJO
.01
/~.: 4 h r
.oo i
00010"0~ ~ •. 6h, NOTE:ALLTo

oIoCOMPUTATO
I NSL-AGS
0.0001
o.oooo, I / 1 I I I I I I I I I
.(X 0.02 0,0~4 0.06 0.06 0.10 O, 12 O. 14 0.16 0.18 0.20 0.22 0.24
14d
!
24hr 12hr
JCYCLESPERHOUR
FIG. 5---Estimates o f spectral density---dissolved oxygen in Patomac River at Washington,
D.C., 27Aug. to 26 Sept. 1963.
Spectral density peaks at 14 days and 12 h for dissolved oxygen show

the effects of lunar fortnightly and semidiurnal tides. There is a vertical
oxygen gradient in the river so that variations in depth are reflected in the
oxygen concentration. In the case of the semidurnal tide, concentrations
are also affected by mass water movements (advective changes). The 24-h
peak is due to daily variations in photosynthesis. The most significant
io,ooo~.~ I 1 I I I I I I I I I"
t,O00
I,OOO
A=O. l hr
I 0 0 L AGS
10~3
I A " 0.2 h r ~ "
&=O.5 hr
I OO LAG S
IO0
I,OO0
0 A" Ihr
I OOO
=o
E
,o ~,o~"~'AGs '
I 0 0 LAGS =
1,000
I0 ~." 0.2 hr
1 I. L I I I I I I I I I
o.o, 0.02 Q~ o.o~ Qo1 o.,o o.,z o.,4 o.,6 o.,8 o.zo o.zz a2,~
CYCLES PER HOUR
7d 24h t2h
FIG. 6---Estimates of spectral density--specific conductance in Potomac River at Waah-
ington, D.C., 27Aug. to 26 Sept. 1963.
result of this series of analyses is that, with a 100-lag computation, the 0.1
0.2, 0.5, and 1-h data failed to reveal one or more of the frequencies at
which significant events occured. On the other hand, use of 4 or 6-h data
frequently gave erratic or unstable results because the number of data
points was too small for the 100-lag computation. The 2-h sampling
interval most clearly reveals the nature of the variance in the record.
Similar results (not shown) were obtained with a 3-h period.
Comparable results for specific conductance are shown in Fig. 6. Here
the only peak in the spectral density was found at 12 h; the driving force
behind this peak is presumably semidiurnal tide. The mechanism that
couples the tide to the specific conductance or salinity is not clear because
ocean salts are believed to remain well downstream from the sampling
station. One possibility is that the salinity variations are caused by releases
to the river, during ebb tide, of water from tidal flats, or other backwater
areas subject to evaporation or to discharges of wastes.
The bottom part of Fig. 6 shows the results of an increased statistical
eftbrt. Computations were carried out to 300 and 621 lags. This figure
shows that even these massive computational efforts, which required 21/2
and 5 min. of time, respectively, on an IBM 7090, were less rewarding
than the 100-lag computations on 2-h data. In order to obtain comparable
resolution from 12 or 6-min data, 1000 or 2000-lag computations would
have been needed.
It was thus determined that for conditions prevailing from 27 Aug. to
26 Sept. 1963, a sampling interval of 2 or 3 h was sufficient for resolving
the frequencies of the dominant factors that affected salinity and dissolved
oxygen in the Potomac River.
The adequacy of a 2-h sampling interval was confirmed by analyses of
data collected at from 1/2 to 2 h during the period 1 April to 1 May 1964.
Also, the statistical effort was varied from 20 to 100 lags. With 2-h
interval data, a 40 to 60-lag computation was sufficient to describe the
dominant variations in the dissolved oxygen and specific conductance.
The Potomac River data were obtained from one point in the stream.
Similar water quality data from the Raritan Bay were obtained for
surface, mid-depth, and bottom waters [11]. Raritan Bay occasionally has
a three-layer circulation system, presumed to be due to density flows
which carry high sediment loads during peak runoff. Spectral analyses
showed that a 2-h sampling interval was the optimum here also.
Analyses of San Francisco Bay data gave comparable results. Estimates
of spectral density, coherence, and phase were computed for stage,
salinity, and currents. Record lengths of 31 days, 4 days, and 4 h were
examined with 60, 60, and 30-lag computations, respectively, using
B I M E D Program BMDO2T [10].
Figure 7 shows the effects, on monthly records, of varying the sampling
interval from 1 to 6 h for surface currents. Large variability in 4 and 6-h
I~ I I I I I J I I I I I I I I I I I I [ I I I lid
OI --
I0
OI
~ IC-- --
,oh2 A. / -
OI --
001 --
L I [ i ] I i t I I I I I i I i I I [ I I I I L I
O07d 02 04 06 08 I0 12 14 16 [ 18 20 22 24
FREQUENCY, CYCLES PER HOUR
14d 24h
]
12h
I
83h
I
6h
]
5h PERIOD
FIG. 7--Estimates of spectral density for surface currents, 1 Aug. to 1 Sept. 1963
(Z~ indicates sampling interval).
spectra shows that the analyses were becoming computationally unstable.

Spectral peaks corresponded to currents associated with fortnightly,
diurnal, and semidiurnal tides and to the 5, 6, and 8-h periods between
successive tidal stages. It should be remembered that each tidal stage or
half cycle requires a full cycle of current velocities; a tidal period of 12 h
is associated with a velocity period of 6 h. The actual values of the peaks
in Fig. 7 reflect flood or ebb dominance during the particular tidal
periods. In addition, the 4 and 6-h data show effects of aliasing. Here,
energy associated with the 6-h current period is folded so as to appear at
12h.
Figure 8 shows the results obtained from surface current data collected
at intervals of from 8 to 30 rain. There is some resolution of spectral
--7~, r- I I I I I I I I I I I I I I I I I I I I I
o? ' / ~ SURFACECURRENTS
oo1 ~
001 ~ --
~- OOI -'~
IC --
OI =30m
OOt -- 1 /~/~fl -
OOOl -- ~N V~ W~
I J K I L I J [ I I I I ] ] I I i J ] ] I J I I
DO I i02 04 06 08 I0 12 14 16 18 20 2 2 24
12II
24 865 PERIOD.hours
FREQUENCY. CYCLES P E R HOUR
F I C . 8--Estimates of spectral density for surface and bottom currents, 14-18 Aug. 1963
( Z X indicates sampling interval).
peaks between S and 24 h. More importantly, the general impression is

that there are no appreciable amounts of variance in the record at periods
of less than 5 h.
Figure 9, which is based on 4-h records of 2-min velocity data during
flood or ebb tides, supports the prior impression that high-frequency
variations are not a significant factor. This figure also shows the results of
linear detrending of the data. Although the 4-h records during ebb or
flood tides were deliberately chosen to enhance this computation, the
results showed no particular advantage in detrending. Similar results were
obtained when the detrending option was applied to 4 and 31-day record
lengths.
Figure 10 shows the effect of varying the sampling interval upon
estimates of coherence between stage and surface current for a 31-day
record. The results are typical of cross-spectra computations for other
parameters and record lengths. Resolution, computational stability, and
identifications of significant frequencies are in accord with those for the
individual power spectra. Here again, a 2-h sampling interval appears to
provide the optimum amount of information.
Other Approaches to Digitizing Continuous Records

Regression analyses are often simplified by sequential selection from
large bodies of data. In stream quality studies, the results are consistent
with those for the frequency distributions shown in Figs. 2 and 3. For
example, a detailed analysis was made of inverse relationships between
streamflow and water quality in the Columbia River Basin. This was
supported by similar analyses of hydrologic data from stream stations
throughout the continental United States [11].
The study showed that streamflow and quality in the Columbia River
Basin are predictably related. Maximum concentrations accompany
minimum flows. Each reach of river has a unique signature which defines
the local dependence of mineral quality upon flow. The basic form of flow
quality relationships is a continuous annual cycle reflecting variations in
rates at which m i n e r a l s a r e weathered or leached from rocks or soils and
in streamflow rates. These cyclic variations are most pronounced in upper
reaches of streams where they may be described by elliptical doughnuts on
logarithmic plots of concentration versus flow, an example of which is
shown on Fig. 11. The pattern becomes more complex as tributaries from
different elevations and snowmeit periods combine. Rising (spring) flows
flush accumulated salts from the basin While falling (summer) flows
remove only newly leached salts. It was found that identical results were
obtained from either weekly data or monthly data (using the first sample
of the month) from the Columbia River Basin. The same was true for data
from the rest of the United States in spite of even greater variations in
HIGHER HIGH WATER TO LOWER LOWER LOW WATER TO LOWER

LOW WATER HIGH WATER
I I I I 1 I I I I I I [ I I I I I I I I I I I I I I '] [
001
2,1963 A.U.GUST 2, 1963
001
OI •AUGUST SURFACE CURRENTS ~ SURFACE CURRENTS
o
u
o Ol \ SEPTEMBER I, 1963 SEPTEMBER I, 1963
SURFACE CURRENTS
O.O01
oo, \, I -
OOOl BOTTOM CURRENTS BOTTGIM CURRENTS
I I I I I I I I I I I I I I I I I I I I I I 1 I I I I I
0 2 4 6 8 I0 12 14 2 4 6 8 I0 12 [4
FREQUENCY, CYCLES PER H O U R
FIG. 9---Estimates of spectral density for surface and bottom currents during 4-h periods
qf ebb or flood tides (dashed lines indicate detrended data).
i.O
' ' ' ' ' Y~' ' ' IA' A
(15
0.0
0.5
w
o~
0.0
t~ O.5
z
m 0.0
o 0.5
~ A=4hr
0.0
0.5
0.0
rjv [j
,I I
, ~.6.r
I
I
+ 360*
+180 =
J j A.,h,
O*
+360 ~
+180'
0o
+360' "~q
LU §
03
+180 ~ -- /j. ~ ~
A. .A
0 Vv., Ifvv V
-180* --
+360 ~ --
i
+,8o.-A . ~^-^~ . _ / k a . r ,~ ~.6,r
0 a
_180 *j/~/le" I ]- W
I - I L I I I I I I I
O0 02 0 ~, 06 08~ I0 .12 14 16 I 18 20 .22 ,24
FREQUENCY, CYCLES PER HOUR
l 1 I I
24d 24h 12h 63h 6h 5h PERIOD
FIG. lO---Coherence and phase between stage and surface currents, 1 Aug. to 1 Sept.
1963 (A indicates sampling interval).
hydrologic factors which resulted in such other interrelationships of

streamflow and quality as shown on Fig. 12.
A simple and direct method, sort of a plumber's proof, was used for
demonstrating the validity of a 2-h sampling interval in San Francisco Bay
just described. Total inflows and outflows over a single tidal cycle of 25 h
SOOL" I I I I $ I I I I r I I I I _J
/ TOTAL /
~-- DISSOLVED -1
/ ~SOLIDS /
- ~d~c ~-t~ HARDNESS ALKALINITY -
It~,,w"e~
~ . 1 . ~ . ,~I - - ~ OCT-DEC AN
~
z 10 ~ SULFATE _
I lo 20 40 60 I00 20 40 60 I00 20 40 60 I00 200

FLOW, THOUSANDS OF cfs
FIG. 1 l - - M i n e r a l quality and stream f l o w in Snake River at Wawawai, Oct. 1959 to
Sept. 1962.
were computed. The summation of flows determined for even-numbered

hours was compared with that for the odd-numbered hours. In either
case, appropriate adjustments were made for the 25th h. The computation
involved a considerable amount of manual plotting, interpolating, veri-
fication, and planimetering of the data and was not amendable to
computer processing.
Tidal flows computed at 2-h intervals from the velocity cross sections for
the period 0630 August 28 to 0730 August 29 are as follows:
Total Total Net
Outflow Inflow Outflow
(Ebb) (Flood) (Ebb)
(Acre-Feet) (Acre-Feet) (Acre-Feet)
Even-hour data 162 400 139 000 23 400

Odd-hour data 161 600 126 200 35 000
The agreement is considered reasonable, although the computed net
FIG. 12--Concentrations of mineral constituents and stream flows: A--Chamberlin Creek,

Alaska; B--Mississippi River at Vicksburg, Miss.; C--Gunnison River at Grand Junction,
Colo.; D---Ohio River at Cairo, IlL; E--Canadian River near Canadian, Tex.; F and
G--Salt River at Shepherdsville, Ky.
outflows, whose determination was the reason for the whole measurement
program, varied significantly. The net outflow values are about twice the
net inflows to the delta [12]. Some of the difference may be due to the
emptying of the delta from the lunar fortnightly tide. Figure 13, on which
the hourly flows are plotted, suggests that most of the difference is an
artifact due to the selection of the particular 25-h period, since both the
even and odd-hour data conform to a single smooth curve.
C~
Z o 200,000
0
(.~
I.d
O0
o 100"000'
..2
n- tL
LO
(1.
0
I--
bJ
bJ
LI..
, n - I 0 0 , OOC
m
rn IM
0 - 200,00C
J - E v e n - numbered hours
& - O d d - numbered hours
-300,00C , .... I , , , , , I .... , I ..... I , , , , , I
06 12 18 30'01 06 12
August 28, 196 August 29
FIG. 13---Tidal-period variations in outflows from Sacramento and San Joaquin Rivers.
Note that even-hour data and odd-hour data conform to the same curve so that either will
provide necessary information and that it is not necessary to compute both.
Discussions and Conclusions
All of the preceding examples relate to descriptive models. Optimizing

sampling or digitizing of time series can be based on spectral analyses,
probability distributions, or regression analyses, depending on the type
and amount of data. In general, spectral analyses appear to be most
useful for this.
In estuaries where the greatest proportion of the variance is associated
with tide~w~th periods of 1/2, 1, and 14 days, spectral analyses show that
a 2-h interval is optimum. This is not to say that information does
not exist at higher frequencies. In studies of turbulence, for example,
D.W. Pritchard 3 has used velocity observations taken at 1/20th s over
12-min record lengths. However, the turbulence structure of a stream or
estuary need be determined only once for a given set of hydrographic
conditions and is thus outside the scope of this paper. The available
evidence indicates that for engineering purposes in water resources
development or pollution control, the variances found at the shorter
3 D. W. Pritehard, personal communication.

periods are not large enough to affect operations.

The ebb and flow of the tides in an estuary not only provide for dilution
but cause waters (including pollution slugs) to pass a particular point
many times. Pollution surveillance programs in this sort of environment
must be designed accordingly. When an intermittent waste discharge must
be fully characterized, continuous sampling at the outfall itself is re-
quired. This is supported by evidence from inland streams where studies
of bacteriological and trace element pollution show conclusively that, as a
dominant source of pollution is approached, the sampling interval must be
shortened. With increasing distance downstream, mixing and stabilization
processes and dilution 'from tributaries result in increasing homogeneity,
which can be described by less frequent sampling. This is precisely the
conclusion that was reached for tidal estuaries. It follows that each
environment has peculiar data collection requirements.
There are other hazards in developing descriptive models from time
series analysis, particularly those of nonsense correlations. Pritchard [13]
has followed Kinsman in describing the dangers of manipulating time
series to conform to established hypotheses. An extract of Pritchard's
paper is presented in Appendix C; the point is that no matter how elegant
the arithmetic may be, findings based on statistical analyses must conform
to the real world.
An instance where continuously collected environmental data were being
used to control a waste discharge is that of the Enrico Fermi Atomic
Power Plant near Detroit, Mich. Gill and Bierly [14] have described
details of the operation of instrumentation and associated computer
equipment used to ensure that concentrations of radioactivity downwind
from the stack will remain below the maximum permissible concentration.
Wind velocity and direction are monitored and running averages are
computed over periods of 3, 6, 12, or 24 min:
The CAUTION signal (is actuated) whenever average wind speed drops
below 7 mph and remains below 10 mph and the standard deviation of
wind direction drops to 5 ~ or less and remains at 8 ~ or less... The 5TOP
signal (is actuated) whenever wind speed drops below 4 mph and remains
below 7 mph...Averaging times of 12 or 24 minutes appear better than
shorter averaging times with some preferences for the 24-minute period.
One cannot help wondering whether, if it were possible to average over 36

or 48-rain periods, one of these might be preferred.
In the study of water pollution surveillance in estuarial waters, the
significance of the foregoing results derived from air pollution practice is
that individual power plants are inherently rapidly responsive to changes
in either power demands or environmental factors. In contrast, wastewater
treatment facilities or reservoirs operated for low-flow augmentation
typically require hours or days in order to respond to changes in
environmental conditions downstream from outfalls. The 2-h period for

sampling in the P o t o m a c River estuary, Raritan Bay, and upper San
Francisco Bay is tllus an o p t i m u m tbr descriptive models, but it is almost
certainly too short tot predictive models used to control waste treatment
plant or low-flow augmentation operations.
Continuous monitoring data are useful when they are collected from an
outfall and immediately fed back into process control operations or used
as a sort of fire alarm to warn downstream water treatment plant
operators. Enforcement actions and remedial programs have much longer
time constants and cannot be expected to respond to short-term variations
in continuous monitoring data.
In brief, optimizing data collection means maximizing data utilization.
Unfortunately for us in planning data systems, we are not exactly sure of
what we do in data analysis. In a 1963 symposium, John Tukey developed
this theme in a paper whose conclusion was announced in the title, " T h e
Inevitable Collision Between Computation and Data Analysis" [15].
As a goal, we can look at the startling observation made by Kneese: " I n
the R u h r area of Germany, the only area where basin-wide effluent
changes are assessed (as an integral part of regional water quality
management), the quantity and quality of effluent are sampled once or
twice a year" [16]. This is surely a m a x i m u m in utilization of data.
APPENDIX A
The Arithmetic of Power Spectrum Analysis 4

Appendix A is based on Ref 17 which is a primer of the mathematical basis for
and application of spectral analysis. Power spectrum analysis is one method for
time series analysis. The term is frequently shortened to spectral analysis. Spectral
analysis provides measures of the variance of a record. Variance is expressed as
the mean square or, following communications usage, the power of the units being
measured; hence, "power spectrum analysis."
In recent years, the analysis of sequential data has been greatly facilitated by
the use of power spectrum analysis. The application of this tool to geophysical
problems was first described by Tukey in 1949 [18]. Originally developed for
analyzing noise in communications systems, spectral analysis has since been ap-
plied to a variety of problems in meteorology, oceanography, and engineering
where the basic data were arranged in time series. The most obvious bonefit of this
application has been, paraphrasing Munk [19], the condensation of "...miles of
wiggly curves in the time domain to a few simple traces in the frequency domain."
One feature of tl~ method (the number of lags) will be referred to repeatedly.
Lags are defined explicitly. Their importance lies in the fact that total computer
time is a linear function of the record length (number of data points) multiplied by
the number of lags. The reason for using larger numbers of lags is to improve the
4Appendices A and B are reproduced essentially in their entirety from an earlier publi-
cation [1].
resolution of the calculation. This means that the frequencies (or periods) of cyclic
events in the record can be more precisely defined and separated. It is possible in
this way to measure and compare the effects of both the semidiurnal tide and the
solar day on dissolved oxygen.
The dependence of computer time on the record length and the number of lags
needed to unscramble the record suggests that when more data are collected, more
work is needed to examine them. In this respect, power spectrum analysis is
similar to simple averaging. It takes more effort to determine the average of many
numbers than it takes for a few numbers.
A time series is a record of repeated observations made at a particular location.
Each observation is a momentary summation of the effects of everything that is
happening to the particular parameter. These effects may be caused by cyclic or
random phenomena. A trend throughout a given record length may be real or
apparent; for example, a short segment of a sine wave will appear as a trend.
Power spectrum analysis identifies the frequencies at which different factors
cause the record to vary. The analysis also provides an estimate of the variance
that derives from each of these factors.
Figure 14 shows some simple spectra (more precisely, estimates of spectral
density) from several types of curves. Where the original curve is made up of more
than one factor, as in Cases V and VI, the power spectrum clearly reveals the
nature of the components.
Four steps in the computation of individual power spectra are as follows:
1. The mean and the square of the mean of the record are determined.
2. The autocorrelation function of the record is formed. This is the basic
operation in spectral analysis, and it is described in detail later.
3. The Fourier cosine transform for each autocorrelation is computed. This
defines the difference between spectral analysis and standard Fourier analysis
because in the latter, the Fourier transform is applied to the raw record
rather than to the autocorrelation function. In spectral analysis, the Fourier
transform serves to smooth out some of the fluctuations present in the auto-
correlation function.
4. A second weighting operation provides the estimate of spectral density.
The Autocorrelation Function
Wastler's description is essentially as follows: the autocorrelation function is

obtained by first multiplying each number in the record by another number in the
record. From the mean of the sum of these products is subtracted the square of
the arithmetic mean of the entire series. In determining the autocorrelation at lag
0, the record is multiplied by itself so that the result is the variance as defined in
statistics. The autocorrelations at Lag 0, Lag 1, Lag 2, and Lag 3 are computed as
shown in Table 2.
The entire operation may be expressed mathematically as
Cr : (n - - r) -1 ~ , x t x t + r - - [n -1 xt] 2 (1)
1 I
in which Cr = autocorrelation at lag r, xt = record value at t, t = O, 1, 2 ...n =

sequential index of values, r = O, 1, 2 . . . . m = lag number, and m = total
number of lags.
4'/8 WATER QUALITY PARAMETERS
RECORD POWER
S PECTRUM
Case I
I
Cose Tr
I I
Case rrr
t~
Case ]~
.. II
Case ~Z
(-- T r + T r r )
C a s e ~rr
( =Tn + ]~Z:)
TIME FREOUENCY
FIG. 14--Spectral arithmetic.
In the simple case of a pure sine wave, the computation is physically analogous
to looking at a white picket fence through one of a series of vertical gratings. Both
the fence and the gratings are constructed so that the bar width always equals the
slot width. The pickets are a square-wave approximation of the sine wave. The
resolution of detail with which the fence may be seen is a function of the spacing
between grates. Similarly, the effective resolution (R) of the sine wave is a function
of the number of lags (m) used for computing the autocorrelation function and of
the sampling interval (AT), where R ---- 1/2mAT.
If the spacing of the grates is too large, some of the pickets will not be seen.
The total amount of light seen by the viewer will be less than the amount reflected
T A B L E 2--Typical computation of autocorrelation function, a
C o - Autocorrelation Ca - Autocorrelation 6"2 - Autocorrelation

at Lag 0 at Lag 1 at Lag 2
1,30 x 1.30 = 1.69 1.30 x 2.57 = 3,34 1.30 x 3.79 = 4.93

2.57 x 2.57 = 6.60 2.57 x 3.79 = 9.74 2.57 x 1.49 = 3.83
3,79 x 3.79 = 14.36 3.79 x 1.49 = 5.65 3.79 x 2.30 = 8.72
1,49 x 1.49 = 2.22 1.49 x 2.30 = 3.43 1.49 x 4.73 = 7.05
2,30 x 2,30 = 5.29 2.30 x 4.73 = 10.88 2.30 x ......
4.73 x 4.73 = 22.37 4.73 x ...... 4.73 x ......
3.10 x 3.10 = 9.61 3.1() x 1'.~16 = z1153 3.1() x 3.16 = 9.~)

1.46 x 1.46 ---- 2.13 1.46 x 3.16 = 4.61 1.46 x 3.30 = 4.82
3.16 x 3.16 = 9.99 3.16 x 3.30 = 10.43 3.16 x 1.41 = 4.46
3.30 x 3.30 = 10.89 3.30 x 1.41 = 4.65 3.30 x 2.35 = 7.76
1.41 x 1.41 = 1.99 1.41 x 2.35 = 3.31 1.41 ......
2.35 x 2.35 = 5.82 2.35 ...... 2.35 ......
Sum = 1046.9 Sum = 804.67 Sum = 764,62
1046.9 804.67 764.62

-- 7.22 -- 5.588 -- 5.347
145 --5.90 144 --5.90 143 --5.90
Co = 1.32 Ca = --0.312 Cz = --0.553
a Number of observations = 145; m e a n of observed values = 5.90.
by t h e fence. W h e n t h e g r a t i n g w i t h t h e o p t i m u m s p a c i n g is selected, t h e f e n c e
c a n be d e s c r i b e d precisely. T h i s also h a p p e n s w h e n t h e o p t i m u m n u m b e r o f l a g s is
a p p l i e d to t h e s i n e w a v e r e c o r d .
A s t h e g r a t e s p a c i n g c o n t i n u e s to b e d e c r e a s e d , all o f t h e p i c k e t s c a n still b e
s e e n , b u t s o m e o f t h e l i g h t is a g a i n lost to t h e viewer. Soon, only h a l f t h e r e f l e c t e d
light is a v a i l a b l e a n d e v e n t u a l l y , t h e g i s u a l r e s o l u t i o n o f t h e f e n c e is d e s t r o y e d
entirely b e c a u s e t h e g r a t i n g d i m e n s i o n s a p p r o a c h t h o s e o f l i g h t waves. H e r e ,
i n t e r f e r e n c e a n d d i f f r a c t i o n p a t t e r n s a r e set u p . S o m e t h i n g c o m p a r a b l e , a l t h o u g h
n o t strictly a n a l o g o u s , h a p p e n s w h e n t h e a u t o c o r r e l a t i o n is c o m p u t e d to t o o m a n y
l a g s for t h e p a r t i c u l a r r e c o r d l e n g t h . T h e c o m p u t a t i o n b e c o m e s u n s t a b l e .
C o m p u t a t i o n a l i n s t a b i l i t y is s h o w n in Figs. 5 a n d 6 o f t h e t e x t w h e r e 1 0 0 - l a g
c o m p u t a t i o n s w e r e a p p l i e d to d a t a collected at a 6 - h i n t e r v a l (A). A c o m m o n rule
o f t h u m b is t h a t t h e n u m b e r o f l a g s s h o u l d n o t e x c e e d 10 p e r c e n t o f t h e n u m b e r
o f d a t a p o i n t s . T h e r e a r e e x c e p t i o n s , h o w e v e r . T h e 2 - h d a t a were in all c a s e s
r e a s o n a b l y w e l l - d e f i n e d w i t h 1 0 0 - l a g c o m p u t a t i o n s . T h e n u m b e r o f p o i n t s h e r e is
(31 d a y s t i m e s 12 p o i n t s p e r day) e q u a l to 372 p o i n t s , a n d t h e n u m b e r o f lags is
27 p e r c e n t o f t h a t n u m b e r .
A s m a l l e r n u m b e r o f l a g s will o f t e n s u f f i c e . It w a s n o t e d in t h e t e x t t h a t 6 0 - l a g
c o m p u t a t i o n s were a d e q u a t e to resolve 3 1 - d a y r e c o r d l e n g t h s in tidal e s t u a r i e s .
Sixty lags a r e 16 p e r c e n t o f t h e n u m b e r o f p o i n t s .
F i g u r e 15 i l l u s t r a t e s t h e a u t o c o r r e l a t i o n f u n c t i o n s o f t w o p u r e s i n e w a v e s a n d o f
t h e i r s u m . A l t h o u g h t h e p a r t i c u l a r e x a m p l e is m o r e s i m p l e t h a n t h o s e f o u n d in
480 WATERQUALITYPARAMETERS
RECORD A U TOC ORRELATION
5
Case I 2 ~ i
5
Case2 ~
0 TI" 211" 311" 0 2 4 6 8 LAGS

0 "l'r 2"11PHASE
FIG. 1S---Autocorrelation functions of simple sine waves.
nature, it shows how the number of lags (in this case, eight) relates to the
description of the record by means of an autocorrelation function.
It can be seen intuitively that any variance due to a secular trend in the record
will be measured at lag O. This is because a secular variation has an infinite period
(zero frequency). This variance will, of course, be added to that due to harmonic
motion.
Fourier Transform and Smoothed Spectrum

The Fourier cosine transform is calculated as
m-I
Vr = (k/m) [Co + Cm cos rn + 2 ~ Cq cos (qrn/m)] (2)
q-I
in which Vr = Fourier cosine transform of the autocorrelation at lag r, q ---- lag

number having values between 1 and m - - l , and k = constant,
k= 1 forr= 1,2...m--I]
k IA f o r r 0 (3)
r m,
and the other letters have the definitions previously given.

The smoothed estimate of spectral density is obtained from a final weighting

function, expressed mathematically as
U0 = 0.54V0 + 0.46V 1 (4)
Ur = 0.23Vr-I + 0.54Vr + 0.23Vr+l (5)
for r : 1,2,3 .... rn - - 1, and
U m = 0.46 Vm-l + O.S4Vm (6)
in which Uo, Ur, Urn, the power spectrum estimates corresponding to the
=
respective lags, and the remaining symbols have the meanings previously assigned.
Sample calculations of the smoothed spectrum are presented by Wastler [17].
A liasing
The designs of both the sampling interval and the subsequent statistical or
spectral analysis require consideration of aliasing. Aliasing is defined graphically
in Fig. 16 and results from the high-frequency events that add variance to the
record but that are not "seen" by the particular sampling interval. Figure 16
illustrates how the variance from this type of event is folded into the record and
reappears at a lower frequency. Where the period of the high-frequency event is
known, the period of the aliased record can readily be determined analytically.
Inspection of Fig. 16 reveals that any cyclic event that occurs at a period less
than twice the sampling interval will result in aliasing. Where the period equals
twice the sampling interval, the event will never be seen. (The same is true for any
event whose period (P) is related to the sampling interval (/) by P = 21/n, in
which n is a positive integer. In the design of sampling programs, those cases in
Legend: N TRUE RECORD

~--- ALIASED RECORD
9 SAMPLING TIME
/"X A.--'7'X--.../X
"--V---"k.,/ ",J"'--V--'"k.J V " - -
TIME
FIG. 16--Typical effects of observing periodic events at intervals of more than one-half the
period.
which n > 1 are not normally considered.) Only when the period exceeds twice the
sampling interval can the event be measured. As the period of the cyclic event
approaches a value of twice the sampling interval, the record length necessary to
describe the event increases. The corresponding frequency, f~, which limits the
events seen by a particular sampling frequency, fs., is known as the Nyquist
frequency, f~ = 1/2 fs.
General
The interrelationships between sampling interval, record length, number of lags,
degrees of freedom, and confidence limits are examined briefly by Wastler [17].
Definitive treatments are presented in several of the works listed in Appendix B.
Appendix B also includes works that describe the calculation and interpretation
of cross spectra. These are basically a comparison of pairs of individual power
spectra, which produce the equivalent of correlation coefficients between time-
series measuring different events (for example, tidal stage and salinity). In addi-
tion to providing a measure of the correlation or coherence between the two
events, the phase relationship is established and other criteria are determined.
APPENDIX B
Bibliography on Spectral Analysis and Related Studies
Barber, N. F., Experimental Correlograms and Fourier Transforms, Pergamon
Press, Inc., New York, N.Y., 1961.
This is an excellent introduction, useful to the engineer or physical scientist, which
clearly shows the physical significance of Fourier transforms, correlograms, and
power spectra. The underlying theory and a wide variety of mechanical, optical,
and electrical analogs are reviewed.
Barber, N. F. and Tucker, M. J., "Wind Waves," The Sea, Vol. I, edited by M.
N. Hill, Interscience Publishers, Inc., New York, N.Y., 1962, pp. 664-699.
Presents example of need for and results from spectral analysis.
Blackman, R. B. and Tukey, J. W., The Measurement of Power Spectra, Dover
Publications, Inc., New York, N.Y., 1958.
This is the standard reference for the theory and application of spectral analysis.
Cartwright, D. E., "Analysis and Statistics," The Sea, Vol. I, edited by M. N.
Hill, Interscience Publishers, Inc., New York, N.Y., 1962, pp. 567-589.
Summarizes statistical formulation of sea waves and introduces concepts of
spectral analysis.
BMD--Biomedical Computer Programs, edited by W. J. Dixon, Health Sciences
Computing Facility, Dept. of Preventive Medicine and Public Health School of
Medicine, Univ. of California, Los Angeles, Calif., 1 Jan. 1%4.
Includes digital computer programs for individual power spectra, cross spectra,
and other time-series analyses.
Dronkers, J. J., Tidal Computations, Interscience Publishers, Inc., New York,
N.Y., 1964.
A basis and complete treatment of tidal hydrodynamics, harmonic analysis, and
computational methods.
Holloway, J. L., Jr., "Smoothing and Filtering of Time Series and Space Fields,"
Advances in Geophysics, Academic Press, New York, N.Y., Vol. 4, 1958, pp.
351-389.
Reviews and presents basis for selection from a variety of methods for smoothing
and filtering serial data.
Kinsman, B., Wind Waves, Their Generation and Propagation on the Ocean
Surface, Prentiss-Hall, Englewood Cliffs, N.J., 1964.
An outstanding description of the theory and application of spectral analysis.
Munk, W. H., Snodgrass, F. E., and Tucker, M. J., "Spectra of Low-Frequency
Ocean Waves," Bulletin of the Scripps Institute of Oceanography, Univ. of
California, Los Angeles, Calif., Vol. 7, No. 4, 1959, pp. 283-362.
Includes what the senior author identifies as a cookbook of numerical recipes.
Panofsky, H. A. and Brier, G. W., Some Applications of Statistics to Meteor-
ology. Pennsylvania State Univ. Press, University Park, Pa., 1958.
Chapter VI is an excellent introduction to the subject.
Time Series Analysis, edited by M. Rosenblatt, Proceedings of the symposium at
Brown University, Providence, R. I., 11-14 June 1962, John Wiley & Sons, Inc.,
New York, N.Y., 1963.
Includes papers on a broad range of topics, including statistical theory, spectral
analysis, and applications to geophysical, engineering, economic, and biological
problems.
Snodgrass, F. E., Munk, W. H., and Miller, G. R., "Long-Period Waves over
California's Continental Borderland--I: Background Spectra," Journal of
Marine Research, Vol. 20, 1962, pp. 3-30.
Presents an example of results derived uniquely from spectral analysis of ocean
waves.
Southworth, R. W., "Autocorrelation and Spectral Analysis," Mathematical
Methods for Digital Computers, edited by A. Ralston and H. S. Wilt', John
Wiley & Sons, Inc., New York, N.Y., 1960, pp. 213-220.
Presents digital computer program for determining autocorrelation function.
Stommel, H., "Varieties of Oceanographic Experience," Science, Vol 139, 1963,
pp. 572-576.
Presents magnitudes and interrelationships of time and space characteristics for
both sea level and ocean currents.
APPENDIX C
Spurious Conclusions from Valid Data
Most of us engaged in environmental data analysis have a particular collection
of horrors by which we humble ourselves when we are inclined to stretch data
beyond the breaking point. D. W. Pritchard has provided one of the most
instructive papers in this regard [13], and this appendix is drawn largely from his
work. A major hazard lies in the ability to correlate almost any two sets of
numbers, such as monthly means, which show cyclic variations.
Pritchard reviews an earlier work by Kinsman who had...
analyzed a paper in which the author attempted to show that the number of ice-
bergs counted by the Ice Patrol in the North Atlantic in any given year was related
to the monthly mean sea-surface temperature anomalously obtained from measure-
ments at the end of a pier at Key West, Fla. In order to show the relatively high
probability of obtaining apparently significant correlations between finite series
when, in fact, any physical connection is nonsense, as long as some choice for
manipulation of one set is allowed, Kinsman counted the number of commas per
page in the issue of the journal in which the original paper on icebergs was pub-
lished. Kinsman correlated the number of icebergs in a given year to the number of
commas per page in the subject journal, but left himself the option of proceeding
either forward or backward in the page count, and of selecting which page he
would start his comparison with. He found that when the computed the number of
icebergs per year based on the number of commas per page in the journal, starting
with the last page of the article he was analyzing and proceeding in page sequence
toward the front of the paper, he obtained a correlation of 0.95 with the observed
iceberg count for the years 1942 through 1951. The comparison is shown graphically
in Figure 17 taken from Kinsman's paper. He then proceeded to use the relation-
ship thus obtained, together with the number of commas per page, running back-
wards, in the article just preceding the one he had analyzed, to "predict" the ice-
berg count for succeeding years. As shown in Figure 1, the prediction for the three
years 1952, 1953, and 1954 is quite good. Thereafter, as would be expected, the
prediction failed completely.
Evidently Kinsman's selection of data was fortuitous; however, this example does
serve as a vivid warning about the way environmental data are often used.
1200 I ! I I I I I I I I I
: 7- I c e Patrol counts of Icebergs

1000
o.-~,.o Iceberg counts estimated from
commas in T e l l u s
800
I/I
0
Correlation 1942-1951: ---- 0.95
IiI
I.} "l P'--
ll,l
I0
z
6OO
I
/
/
A
\
-IA
-
4OO
2OO
Ol l ! t I I I I I I ~ n I
42 43 44 45 46 47 48 49 50 51 52 53 54
YEARS
FIG. 1T--CorreLation o f number of icebergs in a given year to iceberg counts estimated
f r o m commas in Tel/us.
T h e r e are o t h e r t r a p s . O c c a m ' s R a z o r a s s u r e s us o f a simplicity in n a t u r e w h i c h

m a k e s o u r scientific w o r k easier a n d , to m e at least, m o r e satisfying. This r e q u i r e s
only a b e l i e f t h a t relations b e t w e e n p h e n o m e n a are simple, a b e l i e f w h i c h
P r i t c h a r d d e m o l i s h e s by p o i n t i n g out t h a t one m a n ' s simplicity m a y b e a n o t h e r
man's utter confusion. The heliocentric universe of Copernicus simplifies an

astronomer's calculations, but it would make life miserable for a navigator unless
he stayed with the Ptolemaic geocentric universe.
Both of the preceding aspects were brought to life for me in an analysis of the
effects of winds upon sea level to quantify the frequently reported tendency of
surface currents in the Bosporus to reverse under the influence of sustained
southerly winds. It seemed that all I had to do was to compute cross-spectral phase
relationships between the southerly component of winds and the sea surface slope.
The computation showed clearly that no such phase relationship existed. It was
not until we had manually plotted the raw data that we discovered that the ability
of southerly winds to reverse the flow depended on the amount of outflow from the
Black Sea and was thus strongly dependent on the annual cycle of runoff from the
Danube River basin [20]. One can still learn from plotting points.
The conclusion from all this is the same as Pritchard's, that too much environ-
mental data has . . . . "been collected under programs developed without adequate
consideration of how the results will be used. The time has come when we should
severely limit the amount of effort being expended on the general collection of
environmental data for which we have only vaguely or partially conceived the use."
References
[1] Gunnerson, C. G., Journal, Sanitary Engineering Division, Proceedings of the American
Society of Civil Engineers, Vol. 92 No. SA2, 1966, pp. 103-125.
[2] Gunnerson, C. G., Water Pollution Research. Vol. 3, No. 2, 1967, pp. 491-504.
[3] Gunnerson, C. G. in Proceedings, IBM Scientific Computing Symposium on Water and
Air Resources Management, 23-25 Oct. 1967, IBM Data Processing Division, White
Plains, N.Y. 1967, pp. 115-140.
[4] "Continuous Water Pollution Surveillance Monitoring, Potomac River at Washington,
D.C., July 19, 1963-December 31, 1964," Water Quality Activities, Division of Pollu-
tion Surveillance, F.W.P.C.A., Cincinnati, 1965.
[5] Gumbel, E. J., "Statistical Theory of Extreme Values and Some Practical Appli-
cations," No. 33, Applied Mathematics Series, National Bureau of Standards, Washing-
ton, D.C., 1954.
[6] "Water Pollution Surveillance System Annual Compilation of Data, October 1, 1961-
September 30, 1962," Publication No. 663, U.S. Public Health Service, Washington,
D.C., 1962.
[7] "U.S. Public Health Service Water Pollution Surveillance System Annual Compilation
of Data, October 1, 1962-September 30, 1963," Publication No. 663, Vols. 7 and 8,
U.S. Public Health Service, Washington, D.C., 1963.
[8] "Quality of Surface Waters of the United States, 1958-Parts 1-4: North Atlantic Slope
Basins to St. Lawrence River Basin," Water Supply Paper 1571, Geological Survey,
U.S. Department of the Interior, Washington, D.C., 1958.
[9] Carter, R. W., Anderson, W. L., Isherwood, W. L., Rolfe, K. W., Showen, C. R.,
and Smith, W., "Automation of Stream Flow Records," Circular 474, Geological
Survey, U.S. Department of the Interior, Washington, D.C., 1958.
[10] "BMD-Biomedical Computer Programs," W. J. Dixon, Ed., Health Sciences Com-
puting Facility, Department of Preventive Medicine and Public Health School of Med-
icine, University of California, Los Angeles, Jan. 1964.
[1l] Gunnerson, C. G., Journal, Sanitary Engineering Division, Proceedings of the American
Society of Civil Engineers, Vol. 93, No. 6, 1967, pp. 1-16.
[12] "Hydrologic Data, 1%3, Vol II, Northeastern California," State of California, Depart-
ment of Water Resources, Sacramento, 1965.
[13] Pritchard, D. W. in Proceedings, Symposium on Environmental Measurements, July
1964, U.S. Public Health Service, Publication No. 999-WP-15, 1964, p. 235.
[14] Gill, G. C. and Bierly, E. W., Journal of Applied Meteorology, Vol. 2, No. 4, 1963,
pp. 431-439.
[15] Tukey, J. W. in Proceedings, IBM Scientific Computing Symposium on Statistics,

21-23 Oct. 1963, IBM Data Processing Division, White Plains, N.Y., 1965, pp.
141-152.
[16] Kneese, A. V., The Economics of Regional Water Quality Management, The John
Hopkins Press, Baltimore, Md., 1964.
[17] Wastler, T. A., "Application of Spectral Analysis to Stream and Estuary Field Sur-
veys--I. Individual Power Spectra," Publication No. 999-WP-7, U.S. Public Health
Service, Washington, D.C., 1963. This reference has been superseded by "Spectral
Analysis Applications in Water Pollution Control," U.S. Federal Water Pollution Con-
trol Administration, 1969.
[18] Tukey, J. W. in Symposium on Application of Autocorrelation Analysis to Physical
Problems, Woods Hole, Mass., Office of Naval Research, 1947, pp. 47-67.
[19] Munk, W. H. in The Sea, M. N. Hill, Ed., Interscience Publishers, Inc., New York,
1962, pp. 647-663.
[20] Gunnerson, C. G., and Ozturgut, E., "The Bosporus," The Black Sea, D. A. Ross,
Ed., American Association of Petroleum Geologists, Tulsa, Okla., 1973.
73
@
03
c
B . -.~
o~
0 9
0
m .
0 0
0.
W. A. A d a m s ? B. F. Scott? and N. B. Snow 2
Environmental Impact of
Experimental Oil Spills in the
Canadian Arctic
REFERENCE: Adams, W. A., Scott, B, F., and Snow, N. B., "Environmental Impact
of Experimental Oil Spills in the Canadian Arctic," Water Quality Parameters, A S T M
STP 573, American Society for Testing and Materials, 1975, pp. 489-513.
ABSTRACT: In August 1973, oil spills (using tx/,o different crude oils) were carried out
near lnuvik, in the Mackenzie River Delta. One oil was from the Canadian Subarctic at
Norman Wells, and the other was from the Pembina oil field in Alberta. Each oil was
poured into a separate typical productive Delta lake that had been partitioned to
provide a spill and a control area. Bioassays of water and sediment were regularly
determined, as was the dissolved oxygen content, for each spill. Samples were taken of
the weathered bulk oil. For the spill involving Norman Wells crude, incident solar
radiation and temperature were continuously monitored at various depths below the oil
and compared to the control area. Results obtained during the first two weeks of the
spill will be discussed.
KEY WORDS: water quality, oil spills, sediments, crude oil, environmental tests, cold
weather tests
The problem of the accidental introduction of crude and refined

p e t r o l e u m p r o d u c t s i n t o o u r e n v i r o n m e n t is a very real o n e , a n d f u r t h e r -
m o r e , is o n e t h a t is on t h e i n c r e a s e . It is a p r o b l e m t h a t is as old as m a n ' s
e f f o r t s to e x t r a c t fossil fuels f r o m t h e e a r t h ' s crust, a n d it has always
r e p r e s e n t e d a c o n s i d e r a b l e h a z a r d to e c o s y s t e m s . Now t h a t o n e o f t h e
f r o n t i e r s o f oil e x p l o r a t i o n activity is t h e A r c t i c , t h e p r o b l e m is likely to
b e c o m e a c u t e , since t h i s e n v i r o n m e n t a d d s n e w d i m e n s i o n s . T h e r i g o r o u s
c l i m a t e i n c r e a s e s t h e l o n g e v i t y o f d e l e t e r i o u s effects, d e c e l e r a t e s n a t u r a l
b i o d e g r a d a t i o n p r o c e s s e s , a n d h i n d e r s r e m e d i a l m e a s u r e s by i m p o s i n g
severe c o n s t r a i n t s on m e n a n d m a c h i n e r y .
N o r e l i a b l e e s t i m a t e o f t h e m a g n i t u d e o f this p r o b l e m ( a c t u a l or
a n t i c i p a t e d ) a r e a v a i l a b l e for t h e C a n a d i a n A r c t i c . It is possible, h o w e v e r ,
1Research scientists, Water Science. Inland Waters Directorate, Environment Canada,

Ottawa, Ontario K1A 0E7, Canada.
2Research scientist, Freshwater Institute, Environment Canada, Winnipeg, Manitoba.
489
9
to obtain some idea of what can be expected from an Alaskan case his-
tory. It has been reported [1] 3 that approximately 0.03 percent of all the
oil produced and handled in Cook Inlet is accidentally spilled. If this
figure is reduced to 0.01 percent and applied to the anticipated 1975 to
1980 Alaskan North Slope oil production, a spillage rate of 0.38 liters/s
(33 600 liters/day) may be expected. It should be noted that this is a
minimum figure.
In addition to the handling and transportation of crude oil, the
stockpiling of a variety of fuels and hydrocarbon additives in conjunction
with exploration and transportation activities constitute another hazard.
Spills from these sources (including three major ones in 1972 alone, with
which one of the authors was involved) represent the current hazard to
Canadian Arctic ecosystems.
For an appreciation of the magnitude and nature (but not effectiveness)
of cleanup and contingency plans currently envisaged for the Arctic, see
Kay et al [2]. This field is beyond the scope of this paper. Most major oil
spills to date involving aquatic ecosystems have occurred in the marine
environment, and a perusal of any of the currently available bibliographies
[3] reveals the sparsity of data concerning the effects of oil on freshwater
habitats. Data concerning the nature of oil toxicity to aquatic biota is
likewise scant. Many fractions of crude oil, however, are known or
suspected to have varying levels of toxicity to living organisms in the long
or short terms. The aromatic hydrocarbons represent the most dangerous
fraction: low boiling point components (benzene, toluene, xylene, and
their derivatives, etc.) are acute toxins for all living systems. In addition,
hydrocarbons are known to be involved in the reception of chemical
stimuli by aquatic organisms. Such stimuli are important for purposes of
food and mate locations; escape from predators, habitat selection, and
homing ability. The presence of oil may affect all of these activities by
blocking receptor sites or mimicking natural stimuli [4].
Another area where data are lacking is the effect of oil on the physics
and chemistry of natural fresh water and its corollary: the effect of a
natural body of water on the oil itself. This test spill was an opportunity
to apply to an Arctic situation a continuous in situ water quality
monitoring system which had been developed for winter oil pollution tests
at Shirley's Bay near Ottawa i_n 1972 thru 1973 [5, 6, 7]. Automatic water
quality monitoring in England has been recently reviewed by Briggs [8].
Systems for use in the marine environment are available commercially, for
example, Inter-Ocean Systems and others. The Inland Waters Directorate,
Water Quality Branch, has automatic monitors in use in Southern
Canada, usually associated with water treatment facilities or power dams.
The harsh environment and the great distances in the Arctic make
ADAMS ET AL ON EXPERIMENTAL OIL SPILLS 491
frequent visits for water quality sampling extremely difficult and

expensive. Furthermore, in many situations, because of short-term
fluctuations in waterflow or mixing processes, it is not sufficient to take
only weekly or even daily measurements to characterize a body of water.
This was a definite factor in the shallow Mackenzie Delta lake used for
these monitored test spills. Wind-induced mixing processes and fluctuat-
ing water levels produced significant short-term fluctuations that would
have been difficult to observe by conventional sampling techniques, and
would have obscured the effects of the oil on the parameters studied.
Special consideration was given to provide a system able to withstand an
Arctic winter and the spring flood of the Mackenzie River, since there is
only scanty information available on water quality parameters during these
periods. Such water quality monitoring stations may be required to
operate year round to detect possible breaks in Arctic pipelines at critical
river crossings. In general, they will provide the baseline data on the
variations of water quality parameters throughout the Arctic that cannot
be obtained by conventional field party techniques because of the costs
involved and the difficulties presented by the Arctic climate.
The present paper represents an attempt to elucidate the effect of crude
oil on selected physical, chemical, and biotic components of a low Arctic
lake by means of an in situ study. The ultimate goal is to be able to
extrapolate the results to similar ecosystems, to predict the nature and
magnitude of harmful effects, and the rate of recovery of such systems.
Study Area
The lakes selected for experimental oil spill work are located in the
southern Mackenzie Delta, midway between Inuvik and Aklavik, to the
west of the Main Channel (Lat. 68 ~ 19'N, Long. 134 ~ 34' W/O). They are
small, flood-plain lakes (areas of 1 to 4 ha, depth = 2 to 3 m) in a region
which represents an extension of boreal forest into tundra biome.
The Mackenzie Delta itself is a bewildering 13 x 103 km 2 complexity of
sinuous channels and myriads of lakes. For a general discussion of the
area, see Mackay [9]. The channels, during the open-water season, carry
large amounts of suspended sediment (0.5 to 1.5 g/liter), and annually
deposit 160 million megatons in the vicinity of the Delta. Consequently,
the channels represent an impoverished habitat for zoobenthos (0 to 400
organisms/mZ). The smaller floodplain lakes, however, represent "oases"
of productivity (1000 to 20 000 organisms/mZ). The reason is that after
the vernal inundation of many of the lakes, the smaller ones with isolated
drainage areas sediment the Mackenzie muds very quickly and the
increased water transparency, coupled with their shallow depth (2 to 3 m),
allows the development of a substantial microphytic flora. This, in turn,
provides food and shelter to aquatic animals. Nutrients appear to be
4,92 WATER QUALITY PARAMETERS
supplied to the Delta drainage in excess quantities [10]. Most of the larger
lakes remain silty as a result of wind action (resuspension) or channel
connections. These generally support smaller standing crops of plants and
animals. The experimental oil spill lakes are representative of the general
area.
Although the Delta itself does not include marine habitats, as a result
of the enormous Mackenzie River discharge (up to 28 000 m3/s), it is at
its northern extremity an estuarine zone. As such, it is an example of
what is usually considered to be a very productive ecosystem. This feature,
together with the virtual absence of tidal action and its location in a
continuous permafrost zone, places it high on the list of sensitive areas.
Moreover, it is extremely important in terms of the wildfowl and fur
bearer populations which are as much an integral part of this ecosystem
as are indigenous fish and invertebrate faunas. The Delta thus occupies a
key position with respect to the socio-economics of the settlements in the
area.
Methods
The two lakes chosen for the 1973 oil spills (named 30 and 8), which
flank another lake (named 4) that was used in a previous oil spill
experiment in 1972, had areas partitioned off by polyethylene sheeting. To
ensure as complete a seal as possible, the sheeting was weighted and
buried in the sediment to a depth of approximately half a meter. The
partition was supported by wooden posts and extended half a meter above
the lake surface. Sampling stations were set up in the littoral zone on
either side of this structure. An anemometer was installed on the shore of
Lake 8, to obtain local wind direction and speed. The air temperatures
provided by the Inuvik Aeradio office were found to be within a degree of
those obtained at the experimental site. Weather data are presented in
Fig. 1.
Zoobenthos and bottom sediment samples from these experimental
lakes were obtained using a modified Ekman-type grab [11]. Water
samples were taken using standard limnological gear and all samples were
processed as soon as possible after their collection. Dissolved oxygen
samples were titrated by the Winkler method within an hour. Most other
samples were treated to allow their storage, and subsequent shipment for
final processing in Yellowknife, Winnipeg, or Ottawa.
For a detailed consideration of the sampling and analytical procedures
see Brunskill et al [10].
Twenty physical and chemical parameters were measured, but only data
for those which we have complete runs to date are included in this paper.
They include temperature, conductivity, dissolved oxygen, pH, major ions~
seston, and macronutrients.
FIG. l--Weather data for spill site, August 1973.
A block diagram of the automatic monitor system and a sketch of the

equipment shelter are given in Figs. 2 and 3. The basic component of the
system consists of the two cassette tape data loggers (Datal Systems,
Canton, Mass.) which each accept 16 analog inputs. Their operation is
indicated in Fig. 2. The sensors were chosen to monitor the physical
changes in the water quality brought about by the oil that would have
importance to both biological activity and ice dynamics. Light intensities
were measured using UDT-500 photodiode/operational amplifier units
(United Detector Technology, Santa Monica, Calif.), combined with
Schott filters (450 nm and 680 nm with 20 nm bandwidths). These were
fitted into water-tight black-anodized aluminum housings which were built
from a similar design to that discussed by Tyler and Smith [12] (see Fig.
4). 4 These light units were placed at three levels from the surface to the
bottom on equipment towers which were self-supported on tripods (see
Fig. 5), one in the control and one in the partitioned-off part of Lake 8.
No attempt was made to obtain absolute light intensities. Results in the
oil-covered section are being compared to those of the control section. The
frequencies monitored are those of photosynthetic importance. Temper-
ature was monitored using (Yellow Springs Instruments) Y.S.I. 44033
thermistors installed in Belden multiple conductor rubber cables (Belden
4The contribution of A. R. Davis, Water Science, Department of the Environment,
Ottawa, to the design of the light sensors and his help with the field study is gratefully
acknowledged.
m
0
c
t-
-<
-u
m
m
~J
co
FIG. 2 - - B l o c k diagram o f data logger system.

FIG. 4---Underwater irradiance collector.
No. 8418). Accuracy is better than _+0.05~ while sensitivity is _-_~.01~

The conductivity probes were similar to those used at Shirley's Bay [5]
with a bridge frequency of 10 kHz. The thermistor cable was attached to
the equipment tower using cable ties (Fig. 5). The capacity of the tape for
an hourly record of all sensors is five months. Readings can be taken
more frequently, if desired, but there is then a shorter remote monitoring
period; for example, for five days following the oil spill, full sets of
readings were taken each minute, 24 h a day. The tape cassettes are
UNDERWATER
LIGHT
SENS(]R
ALUMINUMPLATE9"x9"x3/8" THK:K
9 " A.
SURF~,CETHERMISTOnF I " O A T ~ ~' REFERENCELIGHTSENSOR
L I
CONDUCTIVITYCELL THERMISTORCHAIN
i* c4A ALUMINUMTOBE,I/8" W 18"SECTtON~ t NYLONpIN TyPECONNECTORS
NYLONPINTyPECONNECTORS
CABLESTO ~ fCONDUCTNITY CELL
SHORE CASLE TOSHORENSTALLATON
SC/~WED
~,.ATE
....
IN
V
TO/~J.UMiNUM
eocM
L I
FIG. 5--Underwater equipment support tower.
transferred to Ottawa where a Hewlett-Packard 9830 computer-calculator

(8K-16 bit-word memory) is used with a Datel Systems (LPS-16R) reader
to convert taped coded information into plotted, printed, or stored results.
Of special interest are the environmental precautions we have taken to
protect this equipment from weather extremes, both high and low, in the
Arctic. Figure 3 shows the installation of the electronics and batteries in
well-insulated boxes themselves placed in a well-insulated shelter (Kemso
Canada, Winnipeg, Man.). This shelter was heated by the cooling fins of
a propane thermoelectric generator. A radio-telephone and automatic

35-ram camera with a 250-frame back were placed in the shelter as
shown in Fig. 3. Data could be read from LED panel displays on the data
loggers for immediate parameter evaluation and checking of the system.
This was of great help since one of the data loggers used to record light
levels failed because of an over-voltage problem. Five days of data were
recorded manually.
A gas chromatographic analysis of the oil is described in this volume
[6]. Samples for these analyses were collected in nitrogen gas purged
bottles and stored in darkness at low temperature ~-1~
Experimental Oil Spills

On 5 Sept. 1972 an experimental spill was carried out on Lake 4. Two
barrels (360 liters) of Norman Wells crude were pumped into this small
Delta lake. It dispersed in the prevailing wind (SE) and the bulk of the oil
came to rest, within a matter of hours, amongst the macrophytic
vegetation at the NW lake margin. Here it was protected from wind action
and formed a slick about 2-cm deep.
Within a few days, following several wind changes, the oil slick had
affected the whole shoreline and its presence was still largely confined to
the lake margin.
The initial effect of the spill upon the lake biota was felt by the littoral
benthos, the deeper water benthos showed no effects initially and even to
date (October 1973) remains relatively unchanged in diversity and abun-
dance as compared to pre-spill levels.
The oil film itself gathered large numbers of zoobenthic invertebrates
derived largely from the littoral area. The littoral benthos reflected this
observation and suffered a considerable reduction in numbers of organ-
isms during the same time. After one year's regular monitoring of the
littoral benthos, a distinct trend has emerged (Fig. 6). It can be seen that
following the spill the littoral benthos were reduced to one third of their
pre-spill abundance, followed by a period of recolonization, when the
standing crop increased to two-thirds of the pre-spill value. During 1973,
fluctuations resulting from adult insect emergence occurred, but by the
end of the open-water season, numbers of littoral benthic organisms had
stabilized to one-third of the pre-spill abundance level and there had been
a change in species composition.
Two of the ten major taxa (Ephemeroptera and Coleoptera) have
disappeared altogether. The abundance of all taxa is decreased although
gastropods (predominantly Valvata sincera helicoidea) still comprise one-
quarter of the samples. Oligochaetes now represent a much larger and
significantly increased proportion of the samples over pre-spill values.
A basic weakness of this experiment was the failure to obtain a
300-
200-
I00-
470 16-12-75
0
IO0- 180 25-9-73
'~
139(I) 17-8-73
0 I001 169 (6) 15-7-73
w
Q. 0--
136 (0 I-7-73
1 Uq
z 22 27-5-73
0
I001 25-11-72
m
I00- 19-8-T2
0
I00 - 177 (I) 12-8"72
30~-
200- 5"8"72
100 -
0
I 1 I ! I I f l r t t l ] I I l
%, % % %/%~%#^'eo %'eo^ ~,~%,'% % . %
FIG. 6--Changes in composition of littoral benthos of Lake 4.
satisfactory control lake which necessitated our falling back on one year's
data for the same lake (Lake 4) prior to the spill, with which to compare
post-spill changes. Drawing upon the experience of this first experiment,
it was decided to carry out two more spills on adjacent lakes which would
be partitioned to obtain experimental and control areas within the same
lake. It was also decided to concentrate upon the littoral benthos since
this is the biotic component immediately affected and expand the chem-
ical parameters studied and also include changes in the composition of the
spilled oil with time.
Both of the lakes chosen for the experimental spills differ from Lake 4
with respect to their flora and fauna (and also from each other) but
bathymetrically and in terms of physical and chemical regimes they are all
fairly similar.
At 14:48 mst, on 6 Aug. 1973, 60 liters of Pembina crude oil was
poured onto the experimental area of Lake 30. This oil was the closest
approximation to Prudhoe Bay crude which could be obtained in the
absence of the latter oil itself. From the air, the oil was seen to spread
evenly and fairly slowly, presenting a "jagged" leading edge with no
evidence of a slick of faster moving components as was apparent in the
Lake 8 spill. At ground level, the separation of slick and bulk components
did become apparent as a surface reticulation (Fig. 7a). The difference in
the appearance of the two types of crude oil is easily seen by comparing
Fig. 7a, Pembina, to Fig. 7b, Norman Wells. Two-thirds of the shoreline
of the partitioned area had been affected by the oil within the space of 1 h
and the whole lake margin was affected within 2 h of the spill.
Within 24 h, the thin, even oil film had piled up to form a bulk oil
slick amongst the emergent macrophytic vegetation (predominantly
Menyanthes sp.). Six weeks after the spill there was little obvious evidence
of the oil apart from a "tide-line" along the polyethylene sheeting, and
some pockets amongst decomposing marginal vegetation.
Under the spill conditions, this oil exhibited an initial rapid evaporation
which decreased steadily. This is to be expected as a large proportion of
the crude oil is composed of components having low vapor pressures. The
proportion which had evaporated was determined by analysis of the
fractions collected from a modified Hempel distillation [13]. Chromato-
grams of the original Pembina crude oil and two of the Hempel
distillation cuts (#7 and #10) are shown in Fig. 8, and the results of the
modified Hempel distillation are shown in Table 1, After 2 h, 21 percent
by volume of the bulk oil had evaporated and this increased to 26 percent
after 4 h. Evaporative losses after 16 h were 30 percent a n d b y 36 h, 39
percent of the oil had evaporated. Within 48 h of the spill, 45 percent of
the oil had been lost, Very little evaporation occurred after this time.
Immediately following the spill and to date (October 1973), there has
been no significant difference between control and experimental area
zoobenthos samples. The bulk slick was sampled for the organisms it
contained using a hand-skimmer, 0.2-m wide, fitted with a 400-~m mesh
screen.
The results are shown in Table 2. Organisms collected in this way
represent components of the littoral benthos of the lake as well as aerial
insects. Dipteran adults and larvae predominate in most samples. Skim
FIG. 7--Spreading o f oil as observed close to water surface, (a) Pembina crude oil on
Lake 30, (b) Norman Wells crude oil on Lake 8.
o
h)
m
2o
s
C
t'-
-<
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2o
m
m
2o
FIG. 8 - - C h r o m a t o g r a m s o.f P e m b i n a crude oil (top) a n d H e m p e l distillation fractions, (a) Fraction 10. (b) Fraction 7.
TABLE 1--Modified Hempel distillation, Pembina crude oil
Cut, ~ Volume (ml) Volume, % Sum, % Comments
50 6.6 2.2 2.2

75 7.6 2.5 4.7
100 14.1 4.7 9.4
125 18.8 6.3 15.7
150 16.2 5.4 21.1 no pentane
175 13.4 4.5 25.6 no hexane
200 12.7 4.2 29.8 no heptane
225 13.3 4.4 34.2 no nonane
250 14.2 4.7 38.9 liRle decane
275 19.4 6.5 45.4 no undecane
TABLE 2--Percentage composition and numbers of invertebrates collectedfrom

oil-slick (0.2 m2), Lake 30.
~ Composition of Taxa
Time Chiro- Total

After nomi- Culici- Other Gerri- Cole- Odo- Pelecy- Gastro- Col-
Spill (h) dae dae Diptera dae optera nata poda poda Other leeted
1 19.1 28.6 38.0 14.3 . . . . . . . . . 21

4 3313 iii 66.7 . . . . . . . . . 6
8 . . . . . . . . . . . . . . . 0
10 ... 100.0 ... . . . . . . . . . 2
12 100.0 ... . . . . . . 1
13 76.3 ... 5.1 68 "3.4-- ... 8.4 59
14 42.9 ... 57.1 . . . . . . . . . 7
16 100.0 ... . . . . . . . . . 9
18 57.1 28.6 14.3 . . . . . . 7
20 14.3 28.6 42.9 ...... 14.2 7
24 100.0 ... 1
36 40.0 ... 13.3 i;.3 zi.? ;5 15
42 . . . . . . . . . ...... 100.0 1
72 . . . . . . . . . ...... 100.0 1
96 . . . . . . . . . . . . . . . . . . 0
samples taken from the control area at the same intervals are not included
as t h e y c o n s i s t e d o f s i n g l e e x a m p l e s o f l a r v a l o r a d u l t i n s e c t s , o r m o r e
u s u a l l y n o n e a t all. T h e oil slick t h e r e f o r e h a s a n e n t r a p m e n t effect, w h i c h
in t h e c a s e o f t h i s spill w a s o f s h o r t d u r a t i o n , ( a p p r o x i m a t e l y t h r e e d a y s )
p r o b a b l y as a r e s u l t o f t h e s m a l l a m o u n t o f oil s p i l l e d a n d its r a p i d
a g g r e g a t i o n in a t h i n m a r g i n a l b a n d .
B u l k a n d slick oil s a m p l e s w e r e t a k e n a t t h e s a m e t i m e t h a t c o l l e c t i o n s
o f o r g a n i s m s c o n t a i n e d in t h e oil w e r e m a d e .
Most of the physical and chemical parameters being monitored showed
no significant differences between control and experimental areas. (This
was also true for Lake 8). They showed the normal open-water patterns
throughout the season. However, Fig. 9 shows that the ratio of the average
temperature indicated by the six thermistors placed in each side of the
partitions underwent a significant variation after the spill. This was an
increase of temperature of less that 1~ on the oil-covered side of the
lake. Some of the results obtained from the light sensors are presented
graphically in Fig. 10. A spike occurs in both the green (450 nm) and red
(680 nm) detectors at the time that the slick passed over the sensors. The
average variation of this ratio from unity before the spill is partly due to
the lack of a perfect match between control side and oil spill side of the
lake. A ratio of unity, however, would not imply that each side was
receiving equal light intensity because of instrumental effects. Photo-
graphy from a helicopter using both color 16-mm movie film and 35-mm
still photography has provided results which can be used to correlate the
movement and thickness of the slick with time.S The thickness of a slick
may be estimated from the appearance of the oil film on the water as has
been described for marine oil spills [14]. The applicability of this method
may depend on the nature of the spreading oil. Infrared photography
taken from a camera-equipped airplane, by the Glaciology Division,
Environment Canada, provided results showing a striking correlation with
the sediment loads of the lakes and the nearby river channel. Lake 8 is
connected to a larger lake containing a higher suspended sediment load
and appeared lighter than either Lake 4 or Lake 30.
The vanadium content of the aged oil has been measured by neutron
activation analysis (NAA) as a function of time. An increase of approx-
imately 20 percent is observed after two days. Other trace elements have
also been studied by NAA, but the results are not yet available. The
increase in concentration of vanadium with time is less than that expected
based on the gas chromatographic results, that is, less than would be
expected from evaporation effects [13]. These results would suggest that
there is a loss mechanism for vanadium, perhaps solubility in the water or
evaporation in association with organometallic complexes.
The ranges of the parameters for which we have complete analyses to
date are presented in Table 3. Two other parameters also show some
variation between control and experimental areas. These are the levels of
total dissolved nitrogen (TDN) and seston. The variations between TDN
levels of control and experimental areas of Lake 30 are not as consistent
as for Lake 8 values, but at several sampling times show a significant
increase in the oil-affected area (Table 4). Similarly, increases in seston
levels are also apparent in the experimental area (Table 5).
s A print of this 16-mm film is available on loan for research purposes only from Water
Science, Department of the Environment, 562 Booth St., Ottawa, Ontario Canada, or from
Fisheries Research Board, Fresh Water Institute, Winnipeg, Manitoba, Canada.
- 109
~-+#. I.OB
- 107
§ §
106
+
~J
+ - tO5 Cl
1500 4" 1.04

+ ..J
4" § § 4.:~: -J
- i.o3 ~" 0
ffl ~>
§ + § 9 4"+ 9
- - 1.02 I0"
_j 1400 + 4" U~
0 i.Oi W
0 m
§ § I.OO ~
z
0 4- + ,.J ~>
o t3DO- + -4" o.g9 t'-
Z
+ ++ +
+++ + 4- I- 0
0.98 z
o.. 4- o z
u
4- + + 0.97 m
4- §
~ 12.oo -4- n, x
+ ++ ++ 9
O . N ~J
> + m
D- 2D
+
I 1.00
rrl
O_ z
--4
t-
+ ox
~.00 ,-- 9 AV TEMP O
- o.91 r-
+ RATIO
i'-
~.o_~ ~ I ,hi L,,, I I I ,,, 1 I ,I r-
o.oo i .oo 2.00 3.00 4,00 5.00 600 T~ 0.~ 9,
TIME IN DAYS ( S P I L L + 0 . 6 3 , AUG. 7 , 1 9 7 3 )
01
F I G . 9---Temperature results, Lake 8. 0
01
0
m
:IJ
s
c
i'-
--4
.<
D
:D
m
m
FIG. l O ~ U n d e r w a t e r i r r a d i a n c e results, L a k e 8.
TABLE 3--Range of physical and chemical parameters, Lake~ 30 and 8

~27 May 1973 to 2 Oct. 1973).
Parameter Lake 30 Lake 8
Temperature (~ 1.0 - 22.0 1.0 - 21.3

Conductivity (~mho/cm) 151 -240 150 -305
pH 7.2 - 8.0 7.25- 8.75
Dissolved Oz (mg/liter) 6.75- 12.56 5.6 - 13.6
Alkalinity (M/m 3) 1.15- 2.58 1.02- 2.88
TDP (mM/m 3) < 0.05- 0.83 < 0.05- 0.80
Si (mM/m 3) 7.6 - 36.7 13.6 - 66.0
S04 z- (mM/m3) a 106 -224 212 -362
C1- (mM/m3) a 169 -359 155 -418
PP (mM/m 3) 0.12- 0.77 0.19- 0.73
a Data to August 28 only.
TABLE 4--Total dissolved nitrogen (mMm -3), Lakes 30 and 8.
Lake 30 Lake 8
Station 1 Station 2 Station 1 Station 2

Date (with oil) (without oil) (with oil) (without oil)
27.5.73 . . . . . . 18.1 18.4

15.6.73 21.3 20.7 23.8 24.7
16.7.73 19.9 24.6 24.7 18.2
6.8.73 23.0 30.1 . . . . . .
7.8.73 23.3 24.0 12.0 19.4
17.8.73 28.1 20.2 19.5 15.7

21.8.73 20.0 20.7 17.4 13.1
28.8.73 22.5 15.6 17.4 14.0
4.9.73 22.3 30.0 21.4 16.8
11.9.73 24.4 28.9 16.8 18.3
18.9.73 32.2 27.8 25.0 19.9
23.9.73 30.4 27.1 19.7 14.8
2.10.73 26.7 39.4 24.2 24.1
NOTE--Above horizontal line are pre-spill measurements.
T h e L a k e 8 spill w a s c a r r i e d o u t a t 1500 r e s t o n 7 A u g . 1973. A t t h i s

t i m e , 180 liters o f N o r m a n W e l l s c r u d e oil w e r e p u m p e d o n t o t h e s u r f a c e
o f t h e e x p e r i m e n t a l a r e a . T h e oil s p r e a d in t h e d i r e c t i o n o f t h e p r e v a i l i n g
N W w i n d very r a p i d l y a n d h a d r e a c h e d t h e s h o r e 7 m i n l a t e r . W i t h i n 30
m i n , h a l f o f t h e s h o r e l i n e w a s a f f e c t e d . T h e s h o r e l i n e o f L a k e 8 is q u i t e
d i f f e r e n t f r o m t h a t o f e i t h e r L a k e 4 or 30. I t h a s n o e m e r g e n t m a c r o p h y t i c
v e g e t a t i o n a n d is i m p o v e r i s h e d in t h i s r e s p e c t . T h i s is p r o b a b l y b e c a u s e o f
f l u c t u a t i n g w a t e r levels r e s u l t i n g f r o m t h e c o n n e c t i o n o f t h i s l a k e w i t h
a m u c h l a r g e r o n e . T h e s p r e a d i n g o f t h i s oil w a s s i m i l a r t o t h a t o b s e r v e d
TABLE 5---Seston values (rag~l), Lakes 30 and 8.

Lake 30 Lake 8
Station 1 Station 2 Station 1 Station 2
Date (with oil) (without oil) (with oil) (without oil)
15.6.73 5.6 6.4 15.9 15.6
16.7.73 5.0 7.1 11.8 5.7
6.8.73 2.9 4.0 . . . . . .
7.8.73 2.3 1.0 12.3 6.4
17.8.73 5.7 3.6 2.7 2.2
21.8.73 2.9 1.1 6.8 6.0
28.8.73 8.8 2.3 10.3 3.2
NOTE--Values above horizontal line are pre-spill measurements.
on Lake 4 in the preceding year, but quite different from the Lake 30
Pembina crude (see Fig. 7).
From the moment of contact with the water, a rapidly moving slick
preceded the brown sludge of heavier components in their advancement
towards the shore. A distinct sheen, distinguishable from the bulk oil was
still discernible days later. Also, by this time the bulk oil had affected the
whole experimental area margin and was evident against the polyethylene
partition. Within the next two months, following a 1-m drop in lake level,
the bulk oil was largely stranded along the mud and moss of the
shoreline.
The initial evaporation of the Norman Wells crude oil was greater than
that of the Pembina crude used 24 h earlier. Thirty minutes after the
spill, 27 percent by volume of the bulk oil had evaporated. After 4 h, 32
percent of the oil had evaporated (chromatogram shown in Fig. 11) and
this increased to 37 percent within 24 h. Samples collected 48 h after the
spill showed evaporative losses of approximately 49 percent by volume,
and after 62 h, 52 percent of the oil was calculated to have been lost in
this way. The oil continued to evaporate at a slower rate and there was no
evidence for dodecane in the bulk oil samples after 120 h. All samples
collected after this time contained some tridecane.
Biological and chemical samples were taken concurrently with Lake 30
samples, and, in general, results showed the same trends as for the
previous spill. Data for organisms collected in the oil slick are presented
in Table 6. As with control area of Lake 30, very few organisms were
taken in surface skims. The difference in the composition of these samples
compared to those of Lake 30 reflect differences in the zoobenthic fauna
of these lakes. It can be seen that in these samples from Lake 8, corixids
( C a l l i c o r i x a sp.) are as numerous as dipteran adults and larvae (pre-
dominantly chironomid midges). Adult beetles are also in evidence to a
~>
-C n-Cl4 0
n-Cl2 13
~>
n ~ C15
n-CH E
0')
n-el6 n-Crf ~Cl8 n-Ci9
m
--4
r-
0
Z
m
x
I I I I I I I I I ...... I I [ f "13
m
.-m
FIG. l l--Chromatogram of Normal Wells crude oil 2 h after spill, using FID detector and SCOT columns (O V-I). E
m
z
.-I
r-"
0
r"
co
"1o
F
r-
r~
01
0
r
TABLE 6--Percentage composition and numbers of invertebrates collectedfrom

oil-slick (0.2 mZ), Lake 8.
Time Chiro- Tri-
After nomi- Other Cole- chop- Ephem- Corixi- Pelecy- Gastro- Total
Spill (h) dae Diptera optera tera eroptera dae poda poda Others Number
2 66.7 ... 16.6 16.7 6
4 . . . . . . . . . . . . 0
5 . . . . . . . . . . . . 0
7
9 solo .. . .. . .. . ... . . . . . . o5
11 . . . . . . . . . . . . 0
13 ,.. 0
14 : g.o ...... i2.; 8
17 ... 0
19 i617 .. i 2s.o 16.7 8.3 33.3 ... 12
24 6.8 .. 22.7 68.2 ......... 44
31 50.0 .. 25.0 25.0
42 1~i0 .. .. .. .. .. . . . . . . . . 4
4
48 50.0 ......... 2
59 33.3 .. 67.7
92 14.3 ... 14.3 5711 .. . .. . .. . .. . .. . . . . 37
10 days 93.8 ... ... 6.2 . . . . . . . . . . . . 16
14 days 100.0 ...
3 weeks 11.1 44.5 333 iii iih .. . ... . .. . ... . . . . .
39
4 weeks . . . . . . . . . . . . 0
g r e a t e r extent t h a n L a k e 30. The longer d u r a t i o n of the e n t r a p m e n t effect

of the L a k e 8 slick p r o b a b l y results from the larger a m o u n t of oil spilled
c o u p l e d with the lack of m a r g i n a l vegetation which a p p e a r s to " h o l d "
most of the bulk oil.
Increases in T D N a n d seston levels in the e x p e r i m e n t a l a r e a are even
m o r e a p p a r e n t for this spill t h a n for t h a t on L a k e 30 (Tables 4 a n d 5).
Discussion and Condusions

In the m a r i n e e n v i r o n m e n t , littoral o r g a n i s m s are the g r o u p most
adversely affected by oil [15,16,17] a n d this a p p e a r s to be the case in
lacustrine ecosystems as i n d i c a t e d by o u r results. T h e r e is, in a d d i t i o n ,
one c o m p o n e n t of the latter ecosystems which is likely to b e the first to be
adversely affected by oil. This is the pleuston: those a n i m a l s associated
with the surface film. It includes such f a m i l i a r forms as the water-striders,
p o n d - s k a t e r s , a n d water b o a t m e n , several species o f d i p t e r a n larvae, a n d
m a n y beetle larvae and adults. I n all our e x p e r i m e n t a l spills these forms
are i m m e d i a t e l y affected, some p e r h a p s seriously as they have failed to
recolonize after one year. O u r results indicate t h a t water-striders a n d
p o n d - s k a t e r s d i s a p p e a r e d i m m e d i a t e l y after the oil spills. Large n u m b e r s
o f Gerris sp. were observed actually being t r a p p e d in L a k e 30 oil an h o u r
after the spill. T h e y h a d been " p e n n e d " into an unaffected e m b a y m e n t .
Also at this time adult Gyrinus sp. were found dead in the oil slick in
large numbers. This indicates that Pembina crude is more toxic to these
organisms than Norman Wells crude, as gyrinid and dytiscid beetles
survived for several hours after total immersion in the latter oil on Lake 4.
Only one species of beetle remained in the littoral benthos of Lake 4
following the spill, whereas dytiscids disappeared completely. The surviv-
ing species is Haliplus immaculicollis, and its success is probably the
result of the mode of respiration of the larvae, which still occur in deeper
water benthic samples. The larva has a closed system and respires
cutaneously. It is therefore independent of the surface film for gaseous
exchange. Dytiscid adults and larvae both require contact with the
atmosphere via the surface film for respiration purposes and so both life
stages are likely to be affected by the presence of oil.
The way in which oil actually brings about the death of aquatic
organisms may take two forms. There may be a specific toxicity due to
components of the oil (physiological effects), or vital processes may be
physically impeded, for example, respiratory surfaces or feeding mech-
anisms become clogged (physical effects). It would seem that actual
contact with the oil is necessary as evidenced by the oil slick samples.
Furthermore, the zoobenthos of bottom samples in our experimental lakes
is largely unaffected by the oil. This is not true when extensive littoral
areas become affected by oil, and distinct changes in littoral benthos
become apparent.
Certain zoobenthic organisms, notably oligochaetes and other species
characteristic of oxygen-depleted habitats, are reported to be tolerant to
the presence of oil in fluviatile ecosystems [18]. Our data indicate that this
may also be true for lacustrine systems.
The increases in TDN and seston values in the oiled areas of both Lakes
30 and 8 may indicate a eutrophying aspect of oil pollution. Increases in
this nutrient (TDN) brought about by oil may cause algal blooms which
are reflected in increased seston levels. The largest increase in this latter
parameter in both Lake 30 and 8 was apparent on 28 Aug. when a
blue-green algal bloom was also evident. This was determined from
periphyton artificial substrates. Those placed in the experimental areas
were covered by an enormous growth of the algae, whereas the substrates
placed in the control areas were virtually devoid of this growth. The
presence of oil is known to enhance algal growth of some species whereas
it will depress thgt of others [19].
The major effects of the oil spills appear to be an initial heavy mortality
of certain components of the ecosystem with concommittent degradation
and impoverishment of the littoral areas in terms of zoobenthos. A
secondary effect may be eutrophication, which, if it proceeds, will result in
increased productivity of probably undesirable species, a decrease in
habitats and ecological niches, followed by a decrease in diversity of the
flora and fauna. The change may be an undesirable one. The effects
reported here are probably not in the realm of catastrophes, but they are
nonetheless real and it should be remembered that very small amounts of
oil were spilled. The amounts of oil used in these experiments are
insignificant compared to actual amounts which would result from even a
minor accidental spill of the kind with which we are familiar. The physical
effects observed in the spill of Lake 8 indicate that solar radiation
absorption by the water in the spill area is affected by even a very thin oil
slick. Underwater light intensity effects were observed from such a thin
slick during this test coupled with an observable increase in the average
water temperature. During winter test oil spills by the Oil Pollution
Research Group, Inland Waters Directorate, Ottawa, similar effects were
noticed even though the duration of daily incident solar radiation in
Ottawa, November to January, is much less than in the Arctic summer
[13. 7]. Such observations give some support to scenarios by Ramseier [20],
and Campbell and Martin [21], in which it is suggested that an oil spill in
the Arctic Ocean could bring about sufficient lowering of the albedo to
prevent the formation of the following winter's ice cover. This could bring
about significant changes in global weather patterns.
Different types of crude oils have different physical characteristics, such
as pour point, viscosity, percentage of classes of components, trace metal
content, etc. The behavior of these two dissimilar crude oils, when spilled
on the water and allowed to interact with the environment also varied.
Differences in manner of spreading and the toxicity of these oils to various
organisms were noted. Another important difference was the amount of
the weathered oil remaining when the evaporative processes had dimin-
ished. Although the Pembina crude oil was exposed for a longer period of
time, it did not volatilize to the same extent as the other crude oil.
Therefore, when considering the effects of spills and estimating possible
environmental repercussions, some consideration must be given to the oil
properties themselves.
The data presented in this paper are largely concerned with the
short-term effects of oil pollution. The research is on-going and long-term
effects will be monitored. The main value of this approach is that it will
eventually be possible to predict the rates of recovery of systems subjected
to this kind of disturbance.
Acknowledgments
We wish to gratefully acknowledge the assistance of the following people
who devoted a great deal of personal time to the execution of these
experiments: R. Bernhardt, C. Bosworth, B. Bowen, R. M. Chatterjee,
G. Crum, V. Fraser, W. Ferguson, C. Johnston, C. Metcalfe, J. Robillard,
P. Stewart, M. van Det, and G. Walker.
W e also t h a n k P o l a r C o n t i n e n t a l S h e l f P r o j e c t ( E . M . R . ) a n d t h e I n u v i k
R e s e a r c h L a b o r a t o r y s t a f f ( I . A . N . D . ) for t h e i r g e n e r o u s s u p p o r t a n d t h e
G l a c i o l o g y D i v i s i o n , E n v i r o n m e n t C a n a d a for p r o v i d i n g a e r i a l i n f r a r e d
i m a g e r y o f t h e e x p e r i m e n t a l site. I m p e r i a l O i l L i m i t e d p r o v i d e d t h e oils
u s e d in e a c h e x p e r i m e n t a n d t h e i r h e l p in this r e g a r d is a p p r e c i a t e d .
M e t e o r o l o g i c a l d a t a were p r o v i d e d by t h e I n u v i k A e r a d i o Office, N . W . T . ,
( D . O . T . ) . H e m p e l d i s t i l l a t i o n s were p e r f o r m e d by t h e F u e l s R e s e a r c h
Centre (E.M.R.).
References
[1] Glaeser, J. L., Proceedings, Joint Conference on the Prevention and Control of Oil
Spills, Washington, D. C., 1973, p. 479.
[2] Kay, G. B., Humphreys, V., and Peterson, E. V., Oil Week, Vol. 24, 1973, p. 15.
[3] Moulder, D. A. and Varley, A., A Bibliography on Marine and Estuarine Oil Pollution,
Marine Biological Association of the United Kingdom, Plymouth, England, 1971.
[4] Blumer, M. in Oil on the Sea, D. P. Hoult, Ed., Plenum Press, New York, 1969,
p. 114.
[5] Adams, W. A., "Continuous Water Quality Monitoring Associated with Experimental
Oil Spills" (M.S.), Technical Bulletin, Inland Waters Directorate, Department of the
Environment, Ottawa.
[6] Scott, B. F., in this volume; also in "Report on the Activities of the Oil Pollution
Research Group," Inland Waters Directorate, Proceedings, "Workshop on Oil and the
Canadian Environment," University of Toronto, 1973, p. 28.
[7] Scott, B. F., Adams, W. A., Chatterjee, R. M., and Chert, E. C., "Behaviour of Oil
Under Canadian Climatic Conditions: II, Oil on Ice" (M.S.), Technical Bulletin, Inland
Waters Directorate, Department of the Environment, Ottawa.
[8] Briggs, R., "Monitoring of Water Quality," Report of the Water Pollution Research
Laboratory, Department of the Environment, Stevenage, England, 1973, p. 103.
[9] Mackay, J. R., "The Mackenzie Delta Area, N. W. T.," Memoir 8, Geographical
Branch, Canada, Department of Mines and Technical Surveys, Ottawa, 1963.
[10] Brunskill, G. J., Rosenberg, D. M., Snow, N. B., Vascotto, G. L., and Wagemann, R.,
"Ecological Studies of Aquatic Systems in the Mackenzie-Porcupine Drainage in Rela-
tion to Proposed Pipeline and Highway Developments," Report of Environmental-Social
Committee, Northern Pipelines, Department of the Environment, 1973.
[ll] Burton, W. and Flannagan, J. F., Journal, Fisheries Research Board of Canada, Vol.
30, 1973, p. 287.
[12] Tyler, J. E. and Smith, R. C., Measurements of Spectral Irradiance Underwater,
Gordon and Breach Science Publishers, New York, 1970, p. 19.
[13] Scott, B. F. and Chatterjee, R. M., "Behaviour of Oil Under Canadian Climatic Condi-
tions: I, Oil on Water Under Ice Forming Conditions" (M.S.), Technical Bulletin,
Inland Waters Directorate, Department of the Environment, Ottawa.
[14] "Report on the Oil Blowout in the Santa Barbara Channel," County of Santa Barbara,
General Research Corp., Petroleum Engineering Office, 1970, p. 15.
[15] Carthy, J. D. and Atther, D., "Field Studies (Supplement 2)," Field Studies Council
Ltd., London, 1968.
[16] Torrey Canyon, J. E. Smith, Ed., Pollution and Marine Life, Cambridge University
Press, Cambridge, England, 1968.
[17] Woddin, S. A., Nyblade, C. F., and Chia, F. S., Marine Pollution Bulletin, Vol. 3,
1972, p. 139.
[18] McCauley, R. N., Limnology and Oceanography, Vol. 11, 1966, p. 475.
[191 Hutchinson, T. C., Kauss, P., and Griffiths, M., Proceedings, Water Pollution
Research of Canada, 1972, p. 52.
[20] Ramseier, R. O., Proceedings, International Symposium on Identification and Measure-
ment of Environmental Pollutants, Ottawa, 1971, p. 271.
[21] Campbell, W. J. and Martin, S., Science, Vol. 181, 1973, p. 56.
B . F. S c o t t I
Investigation of the Weathering of a

Selected Crude Oil in a Cold
Environment
REFERENCE: Scott, B. F., "Investigation of the Weathering of a Selected Crude O11 in a

Cold Environment," Water Quality Parameters, ASTM STP 573, American Society
for Testing and Materials, 1975, pp. 514-525.
ABSTRACT: In large ponds, two test oil spills using crude oil were made near Ottawa
during the winter of 1972 through 1973. The first spill was carried out on water under
ice-torming conditions, and the second was carried out on ice. While these spills were
allowed to age naturally, the weather parameters of precipitation, wind speed, tempera-
ture. and light were monitored. The results of the monitoring were then correlated with
the composition of the residues of the spills. In addition, the effects of precipitation and
wind were correlated with the behavior and movement of the oil in the ponds.
KEY WORDS: water quality, environmental tests, residues, cold weather tests, oil
spills, crude oil
The occurence a n d effects of oil spills in warm e n v i r o n m e n t s are well

d o c u m e n t e d . The n u m b e r of spills in cold e n v i r o n m e n t s has been fewer,
a n d the overall effects of the oil on the e n v i r o n m e n t are not well k n o w n .
With the increased oil exploration in the North, it is imperative that we
u n d e r s t a n d the influence of oil on the ice-water systems that exist in such
areas a n d the weathering of the oil u n d e r these conditions.
As water-ice systems are c o m m o n to all regions of C a n a d a from the cold
Arctic regions to b a l m i e r regions in British C o l u m b i a and s o u t h e r n
regions in O n t a r i o , such a study has n a t i o n a l implications. T h e work
presented here concerns two different oil-water-ice systems: oil on water
u n d e r ice-forming conditions a n d oil on ice. Instead of quickly cleaning
up the oil after the spills were made, we allowed the oil to r e m a i n in the
spill area for ten m o n t h s . T h u s , the oil experienced extreme cold
conditions as well as very hot weather. D u r i n g this time a n u m b e r of
p a r a m e t e r s were m o n i t o r e d , especially in the first m o n t h . The m a j o r
aspects investigated were the physico-chemical changes associated with the
1Research scientist, Water Science Subdivision, Water Quality Research Division, Inland
Waters Directorate, Department of the Environment, Ottawa, Ontario K1A 0E7, Canada.
514

SCOTT ON WEATHERING OF CRUDE OIL IN A COLD ENVIRONMENT 515
weathering of the oil and the influence of the oil on its immediate
environment, such as the ice surface below it, as a function of the weather
conditions.
Study Area Preparation

The study area was 32 km northwest of Ottawa where four large ponds
(15.6 by 7.2 m) were constructed. Each pond was doubly lined with
polyethylene sheets and extra sheeting was placed between the ponds so
that all four ponds could, if required, be flooded to yield one large pond
and also to prevent oil from fouling the area around the ponds.
An anemometer was positioned near the ponds to record local wind
direction and speed. Temperatures at the site were checked against those
recorded at the Ottawa weather station and at the Central Experimental
Farm, and found to agree to within I~ Atmospheric conditions were
recorded at the site and, during site visits, the behavior of the oil on the
ponds was noted.
Analytical Procedures
Oil samples were collected in small glass-stoppered bottles, placed
under nitrogen and stored in the dark and cold, either at the site or in the
laboratories. Before analysis, the cold samples were allowed to warm
slightly, shaken, and small aliquots were removed with care taken to
exclude water; the original sample being returned to the refrigerator for
possible further analysis. Chromatographic (GLC) and spectroscopic
techniques were used to analyze the oil.
For the GLC analysis, a Perkin-Elmer model 900 gas chromatograph
was used in conjunction with a flame ionization detector. All chro-
matograms were obtained using temperature programming; for well-aged
samples, an initial temperature of 60~ was used, but for reasonably
unaged samples, an initial temperature of 35~ was maintained for 3 min.
The temperature increase was 4~ per minute and the maximum tempera-
ture was maintained for 32 min. The maximum temperature depended on
the type of column used. Only support-coated open tubular (SCOT)
columns, 13 m in length were utilized. Before each working column, a
1.8-m empty precolumn was employed to retard the introduction of
heavier components of the oil onto the columns. Two types of columns
were used, one with Dexsil 300 and the other with OV-1 as the liquid
phase. The Dexsil columns were used at higher temperatures (305~ and
this permitted analysis of the higher boiling components but with a loss in
the resolution of components with high vapor pressures. The OV-1
columns permitted a maximum temperature of 210~ to be used.
Norman Wells crude oil, provided by Imperial Oil Limited, was used
for the two spills. An abridged Hempel distillation (maximum temperature
275~ was carried out for us by Fuels Research Division of the Depart-
ment of Energy, Mines, and Resources. A chromatogram of the original
oil and of Fractions #5 and #8 are shown in Fig. 1. The chromatograms of
each fraction of the Hempel distillation were scrutinized for the presence
of the N-alkane constituents and then compared with those of the
weathered oil samples.
Spectroscopic techniques were also applied to the weathered oil
samples. Used in this aspect of the work were a Perkin-Elmer model 457
grating infrared spectrophotometer, a Perkin-Elmer model MPF 11
fluorescence spectrophotometer, and a Beckman DBG recording ultra-
violet and visible spectrophotometer and occasionally a Cary 14 spectro-
photometer. For the infrared analysis small samples were dissolved in
either chloroform or carbon disulfide and the spectra were run in cavity
cells. Hexane was the solvent for the ultraviolet and fluorescence spectra
Sample size was kept to a minimum so that the Beer-Lambert law was
followed and so that quenching did not occur in the fluorescence.

The first spill occurred on 20 Nov. 1972. At that time an approximately
3.6 by 3.6-m block of ice was removed from the center of one pond with
an additional area removed accidentally from one side. Ninety liters of
crude oil were pumped onto the open water surface. This amount of oil
was sufficient to provide a depth of 4 m m of oil.
Weather conditions are shown graphically in Fig. 2. Throughout
November, the temperatures remained near freezing but turned much
colder during the first few days of December. A few days of milder
weather followed, but colder weather returned. Wind speeds were
relatively constant (2.0 m/s), but its direction often changed. Pre-
cipitation, mainly in the form of snow, was minimal until 2 December
when snow completely covered the oil to a depth of 200 m m . The oil did
not reappear until 6 December, during a freezing rain storm. Only about
12 h of sunlight were recorded between 20 November and 2 December and
about 8 h occurred between 2 December and 8 December, at which time
the test was suspended.
Within 24 h after the spill, approximately 27 percent by weight of the
oil had evaporated and by 93 h, about 37 percent had been lost. By 28
November, an estimated 42 percent of the oil had evaporated. The
chromatograms of the exposed oil altered very little after this time. In-
deed, a sample taken on 17 January corresponded closely to that taken on
28 November. Chromatograms obtained by using the Dexsil columns
indicated that no alterations of the heavier ends had occurred. In Fig. 3
are the chromatograms of samples taken on 30 November.
0
0
--I
-.-I
0
z
m
~-C 7
-.-I
"1"
m
=0
n-CI5 n-CI4 n-Cl5 n-Ci 6 n-Cl7 n-Cl8
z
0
I I I I I I I I I "71
C~
n-Cil c
m
0m
I1-CI2 I'-"
n-C 7
0
0
r'-
n-C 8 m
n -C 9 z
<
0
z
m
z
l l i I I I I I i --4
FIG. 1--Chromatograms of Norman Wells crude oil and of Fractions #5 and #8 of Hempel distillations.
-M
FIG. 2 - - W e a t h e r conditions f o r spill o f oil on water under ice f o r m i n g conditions.
No correlation with weathering could be found using the spectroscopic

techniques.
During the period of observation of the spill, there was precipitation in
the form of snow which was then melted by the oil, passed down through
the oil because of its greater density, and then formed additional ice on
the ice surface under the oil. This was most noticeable in the triangular
section of the exposed water surface where ice was removed just prior to
the spill, shown in illustration I of Fig. 4. The increased ice height around
this area allowed the oil to run off this section towards the center of the
pond. Prevailing winds then caused the oil to be concentrated in the
northwest section. It was from this area, later in the winter, that oil was
seen oozing from cracks in the ice, which were created by pressures on
the ice surface. The movement of the oil about the pond can be seen in
Fig. 4.
Our second spill occurred 9 Jan. 1973. For this spill, 300 liters of
Norman Wells crude oil were poured onto an ice surface of another pond.
A thermistor was situated to monitor, on an hourly basis, the temperature
of the oil. Only the behavior of the oil during the first month will be
presented in any detail. Figure 5 shows the temperature of the oil and the
ambient air. The temperature of the oil is generally higher than that of
the air. The next illustration (Fig. 6) presents a weather summary for the
month of January. Figure 7 shows the location of the oil in the pond.
Originally, the oil was bounded on all sides by snow, and the snow
extended out to the sides of the pond. Its average height was 90 m m . As
(3
O
.--4
.-.4
O
n-CI7 n_Ct8
z
m
n-el 5 n-Cl6 n-Ci9
.--4
-1-
n_CL2 CI3 CI4
m
2o
z
O
O
71
C)
m
c
~C o
i
ITI
L I I I I I I I I I I l I I I 0
r
n-Cpl
n.Cl z "-cm .-C m
n-C~ n_Cl 7 0
fl_ClI ~C ta II-C19 I--
o
m
z
<
0
z
I I I I I I I I I I I I I I I I I
m
z
FIG.
3---Chromatograms o f weathered Norman Wells crude oil samples taken on 30 November. (Top} using Dexsil 300 SCOT columns and ( b o t t o m ) --t
using OV-I liquid phase SCOT columns.
cO
FIG. 4---Movement o f oil on water ice surface. I = Nov. 20; I I = Nov. 21, 22 and 23:
I11 = Nov. 24: I V = Nov. 25; V = Nov. 26 and 27; V I = Nov. 28; V I I = Nov. 29, 30,
Dec. I and 2: V I I I ----- Dec. 3, 4, and 5; I X = Dec. 6, 7, and 8; V I I I = Dec. 9.
indicated from the last figure, the oil penetrated the snow on the pond
and finally covered the entire ice surface. During January, after the spill,
there was about 200 mm of snow. This snow was blown off or sublimed
from the adjacent ponds, but on this pond the oil caused the snow to
melt, so that water passed to the ice surface beneath the oil and then
added onto the ice layer. This action caused the ice level and also the oil
level to increase. In February, the oil eventually overflowed this pond and
some of the oil went into an adjacent pond where another experiment was
underway. After this time, daily samples were still collected and analyzed,
but this was only oil exposed on the surface. One extremely important
point is that some oil was always visible after any amount of precipitation.
Initially very little oil evaporated, in part because of the low ambient tem-
peratures at the time. Chromatograms of oil artifically aged in a cold room
JANUARY
--~--OIL TEMP C~
+30 ..... AIR TEMP C ~
+20
+10 '-~:00 JAN. I

~ o
-10
-20
-30
25=00 JAN. 51
\
TIME (days)
FIG. S---Temperature of ambient air and oil during January.
(-1S~ with no incident radiation and very little air movement over
the oil showed only a small portion of the oil (22 percent by weight) had
volatilized in 23 days. Initially, there was little change in the composition
of the spilled oil, though the oil was affecting the environment. Stakes
driven into the ice to a depth of 40 mm for a spreading experiment, fell
over within the first week. After seven days, 32 percent of the oil had
evaporated and by the end of the month of January, 42 percent of the oil
had been lost. After that time, very little more oil evaporated. Chro-
matograms of the weathered oil taken on 10 January and 17 April are
shown in Fig. 8. Comparison of these two chromatograms with that of the
original oil (Fig. 1) illustrates typical evaporative losses due to weathering
observed for oils. The chromatogram of the sample of 17 April represents
the nature of the oil present in the pond shortly after spring break-up.
During break-up, the oil which had been held fast in the ice on this
pond mixed with the exposed oil. Chromatograms of samples taken at this
time showed mainly the components of the oil exposed for several months,
however, traces of the components of oil not weathered as long also
appeared in the chromatogram. At break-up, the ice in the oiled ponds
disappeared at least one week before the ice in the adjacent control pond.
During the summer months, a noticeable difference in the appearance
of the oiled and control ponds was observed. This difference prompted a
biological assessment. From this assessment it was shown that the
diversity of the zooplankton, diatoms, and other phytoplankton was much
FIG. 6--Weather conditions .for spill o f oil on ice (first month).
greater in the control pond than in the oiled pond. The diversity of the
periphyton was about equal in both ponds. Although there were a number
of species of diatoms and phytoplankton common to both types of ponds
(Tables 1 and 2), very few abundant species of zooplankton were common
to both control and oiled ponds. Bacteria were found to be in higher
concentrations in the oiled ponds.
Summary
These studies indicate that at low temperatures, near or below freezing,
components of an exposed oil whose vapor pressures are greater than
undecane can evaporate from the oil spill while other components will
J 0 0 d
JAN. 9 JAN. II JAN. 12 dAN.14
JAN. 16
D
JAN. 18 JAN. 19
63
JAN.20
L3
JAN. 22 dAN. 24 dAN. 25 d AN. 28
FIG. 7--Location qf oil on pond.
TABLE 1--Phytoplankton and periphyton.
Number of phytoplankton species found in control pond--28.

Number of phytoplankton species found in oiled pond--10.
Number of species common to both ponds--9.
Number of periphyton species found in control pond--13.
Number of peripbyton species found in oiled pond--14.
Number common to both ponds--6.
TABLE 2--Number of diatom species present in ponds.
Control Pond Pond 2 Pond 3 Pond 4
24 7 6 5
Number of diatom species present in control pond and one other
pond--9.
Number of species present in control pond only--lS.
Number of species present in oiled pond only--3.
,E
o"
,E
i
c
,.E_
,E__
i
r
evaporate to a lesser degree; that the oil will influence its immediate
environment; and that there will be movement of the oil, once spilled,
perhaps long distances away from the original spill area. Also, as noted
from this work, the oil will aid in the melting of ice surfaces, and if in
sufficient quantity, may not disappear under subsequent snow falls.
Acknowledgments
The author wishes to thank the Department of Communications for
allowing us to use their site for these experiments, Imperial Oil Limited
for providing the oil, R. Draper for performing the Hempel distillation,
and M. Dickman, D. Shindler, and L. Krelina as well as the Museum of
Natural Sciences for their help in the biological assessment. W.A. Adams
is thanked for help during the spills, as are E. Nagy and D.B. Carlisle for
helpful discussions.
J. M a r s a l e k j
Sampling Techniques in Urban

Runoff Quality Studies
REFERENCE: Marsalek, J., "Sampling Techniques in Urban Runoff Quality Studies,"

Water Q uafity Parameters, A S T M STP 573, American Society for Testing and Mate-
dais, 1975, pp. 526-542.
ABSTRACT: Composite as well as sequential sampling techniques are examined with

regard to their applicability in urban runoff studies. While inexpensive composite
sampling is adequate for the determination of the total pollutant yield from a watershed,
the study of variation in pollutant concentration requires sequential sampling.
A rational approach to the development of a sequential sampling program is pro-
posed, weighing the cost of data acquisition against the detail and significance of the
information obtained. The design of such a program is based on the adoption of an
empirical model for the variation of runoff quality and on the techniques of prediction
analysis.
Practical aspects of sampling installations are also considered. Such considerations
include the depth of sample withdrawal in a nonhomogeneous media, the capability of a
sampler to collect solids, sample cross-contamination, and synchronization between flow
records and collected samples.
KEY WORDS: water quality, water pollution, sampling, waste water, urban areas,
environmental tests
Historically, the interest in u r b a n r u n o f f has been limited to the volume

a n d rate of discharge, b u t in the last 15 years increasing emphasis has
been placed on the r u n o f f water quality. This change in the attitude has
been required by new priorities in u r b a n water m a n a g e m e n t : r e d u c t i o n in
water pollution, conservation of water resources, a n d e n h a n c e m e n t of
urban environment.
Lately c o m p u t e r models have been developed for the s i m u l a t i o n of the
q u a n t i t y and quality of u r b a n runoff. The availability of these models does
not eliminate completely the ,need for field studies of u r b a n runoff,
because all the existing models have to be calibrated by c o m p a r i n g the
model predictions with a limited n u m b e r of field m e a s u r e m e n t s of r u n o f f
q u a n t i t y a n d quality. The model p a r a m e t e r s can then be adjusted, a n d
1Research scientist, Hydraulics Division, Canada Centre for Inland Waters, Burlington,
Ontario L7R 4A6, Canada.
526

MARSALEK ON URBAN RUNOFF QUALITY STUDIES 527
the calibrated model is used for predicting the drainage system behavior
for low-frequency storms, or for computing the response of the system to
proposed changes in land use.
In a typical field study of urban runoff, storm water flow rates and
water quality are monitored simultaneously at one or more stations in the
sewer system. Though some water quality parameters can be monitored
continuously, in most eases owing to either technical or economical
considerations, the water quality is monitored by discrete samples which
are analyzed later in the laboratory.
The selection of a s~mpling technique depends on the objectives of a
particular runoff quality study. The objectives are usually to estimate the
total pollutant yield from a runoff event, or to estimate the pollutant
concentration variation during the event or both.
Various sampling techniques applicable in urban runoff studies will be
discussed and mention made of some practical aspects and the limitations
of sampling installations.
Composite Sampling Techniques to Determine the Total Pollutant

Yield from a Runoff Event
Composite sampling involves lumping together all samples collected
during a runoff event. The composite sample then represents an average
pollutant concentration for the event. The main advantage of the com-
posite technique is the low cost of quality analysis, since only one sample
per runoff event has to be analyzed.
In practice, the following sampling techniques are used:
(a) simple composition (nonweighted),
(b) flow-weighted composition, and
(c) pollutant mass-flow-weighted composition.
The first two techniques are rather ~ommon and will be applied to some
typical runoff quality data (adopted from Ref 1) 2 to demonstrate the
application of these techniques and to obtain some indication of the
accuracy of techniques.
Simple Composite Sample (Nonweighted)

In this sampling technique, constant aliquots of storm water are
withdrawn at regular time intervals and collected in one container to
constitute the composite sample. The technique is rather inexpensive and
the least sophisticated samplers and flow recorders may be used.
The simple composition is not acceptable for urban runoff studies,
because it significantly underestimates the total pollutant yields. The
r e a s o n for this u n d e r e s t i m a t i o n is t h a t t h e h i g h c o n c e n t r a t i o n s a n d flow

r a t e s in t h e initial r u n o f f p e r i o d a r e a s s i g n e d t h e s a m e s t a t i s t i c a l w e i g h t in
t h e s a m p l e c o m p o s i t i o n as t h e low c o n c e n t r a t i o n s a n d flow r a t e s in t h e
l a t t e r p a r t o f t h e r u n o f f . T h e d a t a in T a b l e 1 i n d i c a t e t h a t this u n d e r -
e s t i m a t i o n c o u l d b e in t h e o r d e r o f 30 p e r c e n t o f t h e a c t u a l v a l u e . T h e
e r r o r is little a f f e c t e d by t h e f r e q u e n c y o f a l i q u o t w i t h d r a w a l s .
Flow- Weighted Composite Sample

The flow-weighted sample composition requires more sophisticated and
m o r e e x p e n s i v e e q u i p m e n t t h a n t h e s i m p l e c o m p o s i t i o n . T h e flow-
w e i g h t e d c o m p o s i t i o n is d o n e e i t h e r a u t o m a t i c a l l y o r m a n u a l l y . T w o
a u t o m a t i c t e c h n i q u e s are a v a i l a b l e . I n t h e first one, a c o n s t a n t f r a c t i o n o f
TABLE l--Application of compoMte sampling techniques to runoff quality data (runoff

quality data after Ref 1.)
Flow Total Suspended Settleable
Sample Time Rate COD BOD Solids Solids Solids
No. (rain) (mUs) (rag/liter) (rag/liter) (rag/liter) (rag/liter) (rag/liter)
1 0 0.00 430 13 14 600 9 600 6 756

2 5 4.14 400 15 12 560 11 200 7 640
3 10 4,73 280 11 6 638 6 050 3 330
4 15 3.65 170 16 5 830 5 520 2 660
5 20 3.01 310 1S 10 002 9 020 6 528
6 25 2.73 300 15 10 632 10 010 6 906
7 30 3.65 370 5 10 242 9 170 5 702
8 35 0.97 240 8 8 676 8 150 6 662
9 40 0.79 230 13 7 196 5 560 2 913
10 45 0.64 210 15 6 092 S 900 2 332
11 50 0.50 210 16 4 898 4 620 2 530
12 55 0.38 230 4 4 598 3 920 3 616
13 60 0.29 150 15 3 908 3 140 2 792
14 65 0,30 135 16 3 403 2 650 1 904
15 70 0.32 120 17 2 898 2 160 1 016
16 75 0.28 130 15 2 604 2 040 1 026
17 80 0.24 140 14 2 310 1 920 1 036
18 85 0.23 130 14 1 990 1 470 698
19 90 0.23 120 14 1 670 1 020 360
20 95 0.26 89 13 1 562 1 090 442
21 100 0.30 58 12 1 454 1 160 524
Actual total yield (kg) 2 377 97 72 202 63 348 41 326
Simple composite sampling (5 min
interval), total pollutant yields
in percent of actual yields 76% 113% 69% 67% 66%
Flow-weighted composite sampling,
total yields in percent of actual
yields. Sampling intervals: (a) 100% 108% 100% 100% 100%
(a) 5 rain (b) 10 min (b) 106% 99% 101% 97% 95%
(c) 20 min (c) 108% 121% 115% 103% 109%
the flow is collected continuously. In the second automatic technique,

samples of constant volume are withdrawn at irregular time intervals,
based on a preselected constant quantity of storm water passing through
the metering station.
The former automatic technique is rather impractical in urban runoff
studies because of the large flows involved. The latter technique requires
the installation of a flow meter with a flow integrator to activate the
sampler.
The manual flow-weighted sample composition is achieved by propor-
tioning the volume of individual sequential grab samples relative to the
flow which passed between the collection of two consecutive samples. The
proportioning is done after the runoff event using the flow record.
The manual flow-weighted sample composition was applied to the
runoff quality data in Table 1. The results show that the method gives
good estimates of the total pollutant yield provided the samples are
withdrawn at a rate of one sample every 5 to 10 rain. As the sampling
interval increases, the error in the calculated yield to pollutant increases
too, because the variation in pollutant concentration between the sampling
events is neglected. At closer time intervals, the variation in concentration
becomes negligible. The results in Table 1 show that for average sampling
intervals of 5 to 10 min, the error in the calculated yields is less than 10
percent.
In automatic flow weighting, the sampling interval varies. The interval
could be as short as 1 to 2 rain during the peak flows. The setting for the
flow volume passing between the two consecutive samples should be done
in such a way that the average sampling interval during the runoff event is
not greater than 10 min. This requires some knowledge of the volume and
duration of runoff in the studied area..
Pollutant Mass-Flow-Weighted Composite Sample

This composition technique requires continuous approximate monitor-
ing of the quality parameter under study, or of a parameter which can be
related to the one being studied. In the latter instance, for example, the
monitoring of suspended solids was approximated by monitoring the
turbidity [2].
The output from the quality probe, pollutant concentration, is
multiplied by the flow rate to yield the pollutant mass flow. The mass flow
is then integrated and samples are collected at preselected constant
increments of the cumulative pollutant mass flow. The laboratory analysis
of the composite sample is believed to give a more accurate and reliable
result than the direct processing of the quality probe output.
The pollutant mass-flow-weighted sample composition is the best
composition technique, because it takes into account not only the flow
rate variation but the pollutant concentration variation as well. The

application of the techniques in urban runoff studies is rather limited
owing to the difficulties with continuous monitoring of water quality
parameters.
Sampling Techniques to Determine the Pollutant Concentration Variation

To study and obtain information on the variation in pollutant con-
centration, sequential grab sampling is necessary. The sample collection
has to be done automatically, because in the case of manual grab
sampling, it is practically impossible to dispatch the field personnel in
time to start sampling at the very beginning of a runoff event.
A practical objective in studies of pollutant concentration variation is to
obtain an acceptable accuracy at minimal costs. This objective may be
met only by applying experimental design techniques. For urban runoff
quality studies, the main problem in designing an effective sampling
program is to determine the least number of samples required to
accurately delineate the variation in concentration. Careful consideration
of the number of samples is warranted because the cost of detailed
analyses of storm water samples usually exceeds $100 per sample. Signif-
icant savings may be realized by the application of the experimental
design to runoff quality studies.
The application of experimental design techniques, in this case of the
prediction analysis, requires the adoption of an empirical model of the
studied phenomena. Such an empirical model based either on the
experience from other studies or on the results of preliminary observations
is used to guide the data collection. In the initial stages of a study, a
crude model is sufficient.
Empirical Models j-br Urban Runoff Quali~

There have been several attempts to establish an empirical model for
the quality of urban runoff water [3-5]. The runoff quality is described in
these models either by the rate at which the pollutant is washed out from
the drainage system, or it may be described directly by the variation of the
pollutant concentration during the runoff event.
The former description of runoff quality may be expressed as follows [4]
P = Po exp ( - k Vt) (1)
where
Po ---- the amount of pollutant on the surface of the drainage area and
in sewers at the beginning of a runoff event,
P -- the amount of pollutant remaining at time t,
t ---- real time (t ---- 0 at the beginning of runoff),
lit = the cumulative runoff at time t, and

k = a constant.
The pollutant concentration in runoff water can then be determined by
dividing the mass of pollutant washed out by the runoff volume corre-
sponding to the same time period
P/I -- e l 2
Car(t,, t2) -- Vt, -- Vt, (2)
where subscripts t 1, t 2 refer to real time, and Cav is the pollutant concen-
tration averaged over the time interval (t2 -- tl). The latter, direct de-
scription of runoff quality has a form C = C(t). The results of some
studies [1.6, 7] suggest the feasibility of assuming the exponential decay of
the pollutant concentration during the runoff event as expressed by the
following equation
C = Co exp ( - k z t ) (3)
where
C -----the time varying pollutant concentration,
Co = the pollutant concentration at the beginning of runoff, and
k2 = a constant.
Other expressions similar to Eqs 1 and 3 have been proposed [3,5], but
quite often these empirical relationships apply only to the urban areas for
which they were originally proposed. The question of the differences in
urban runoff quality observations for different areas requires considera-
tion. A possible explanation of the variation may be found by examining
the sources of pollution in urban runoff.
Generally, three sources may be considered:
1. the contamination of rain water,
2. wash-outs from the urban area surface (including erosion), and
3. deposits inside the sewer system (in catchbasins and sewers).
The first source is usually regarded as not of major significance. The
pollutants from the other two sources are of a similar character and occur
with similar variation with time. During the initial period of the runoff,
the pollutional load on the surface and in the sewer system itself is
dislodged and transported through the sewers. Since the quantity of the
available pollutants is gradually reduced during the runoff, the pollutant
concentrations in the runoff decrease as the storm progresses. A possible
exception to this rule would be the soil erosion which could increase with
time, depending on the rainfall intensity variation, and could have an
unlimited source of supply. Concentrations may also decline in the latter
part of the runoff as the transporting capacity of the storm water flow
declines. Consequently, some solids will settle out in sewers without

reaching the outfall. These solids will be dislodged and transported again
during the next increase in the runoff flow. Thus, the general tendency for
a gradual decrease in pollutant concentrations with time, at a point just
downstream from a supply source, seems plausible.
For the field studies, however, the observations are made only at one
point within a complex system. Consequently, the time required for
pollutants to arrive at this point from various parts of the system plays
some role. This time of travel and the nonuniform distribution of the
pollutional load over the area is the reason for the non-universal nature of
the observations. In fact, the observations represent the integrated effect
of many factors most of which are unique to the area under study. It is
little wonder, therefore, that oversimplified models are not universal.
Example of an Experimental Design for Sequential Sampling in an Urban

.Runoff Quality Study
The experimental design technique is demonstrated in this section on

the urban runoff data adopted from Ref 1. For a simple runoff event, that
is, with a single peak hydrograph, the pollutant concentration variation is
assumed in the form of Eq 3. For this illustration, the chemical oxygen
demand (COD) was selected. The problem is to find the number of storm
water samples to be analyzed to determine the parameters Co and kz with
a prescribed uncertainty (say 0.05) provided that the measurements of
concentrations are done with an uncertainty Kc. The estimated uncer-
tainty of Co and k2 can be predicted by the prediction analysis and would
approximate the uncertainty determined by the least squares analysis of
the actual data.
The uncertainty is defined here as the estimated value of the standard
deviation. When the uncertainty is expressed in percent it will be referred
to as the accuracy.
The detailed development of the prediction analysis equations for an
exponential decay function is presented in Ref 8; only the modified final
expressions are presented below.
Case / - - T h e pollutant concentration is measured with a constant
uncertainty (_+AC); the uncertainty in time recording is negligible.
The uncertainties for Co, K2 can be expressed as follows [8]
Kc(Zb -- Za) 1/2

Oco = N1/2
x ( 2(Za2 + Za + 0 . 5 ) - (Zb2 + ZbD1

+ 0.5)exp [-2(Zb - Za)]} )1/2 (4)
Ok, : Kck''(Zb -- Za)l/z ( 2 { 1 - - exp [--2(Zb

D1 -- Za)]} ) (5)
Kc2k, '(Zb - Za)~

Oco,K2 :
Co'N
2{(za + 0.5) - (zb + 0.5) exp [ - 2 ( z b - za)] }~ (6)

x
D1 !
where
tl = the real time when the first m e a s u r e m e n t was taken,
tn = the real time of the last measurement,
Kc = the uncertainty of the concentration measurements,
N = the n u m b e r of measurements
kz : an estimate of the magnitude rof kz (this estimate m a y be crude),
C o ' = an estimate for Co,
D1 = exp ( - 2 z a ) ( 0 . 2 5 - (2ZaZb - Zb' - Za' - 0.5)
x exp [ - 2 ( Z b - Za)] + 0.25 exp [ - 4 ( z b - za)]},
tn --t 1 )
Za = kz' t~ 2N , and
Zb = k z ' ( tn q- tn -- tl )
2N "
Case / / - - T h e pollutant concentrations are measured with a constant

fractional uncertainty (_+pC); the uncertainty in time recording is
negligible.
The prediction equations for this case will read [8]
KcCo' ( --12ZaZb ) 1/z

OCo N1/z 4 d- '(Zb : Za) 2 (7)
Kck2' 121/2
(8)
Ok,.- N1/z Zb -- Za
:6(z~ + zb)
oc~ k, -- (9)
(zb - za) 2
The notation is the same as in Case I.

Equations 4 through 9 were applied to data from Ref 1. For this

particular runoff event the COD data is listed in Table 2 and plotted in
Fig. 1 together with a least squares estimate for the COD variation during
the runoff. After estimating Co, k2, Eqs 4 through 9 were evaluated for a
2-h runoff event and the number of samples varying from 2 to 50. The
results of these calculations are plotted in Fig. 2 in the following form
Oco = AzK~, = f ( N ) (10)
o,~. = A 2Kc = f(N) (11)
2O 0
?
O COD CONCENTRATIONS
400 0
LEAST SQUARES APPROXIMATION FOR COD

z~ VARIATION DESCRIBED BY EQ (3)
O 200 eK2t
COD = (COD) o
0
O 2'0 4'0 60 8'0 1()0 120
t = TIME SINCE THE START OF RUNOFF (rain)
FIG. 1--Application o f an empirical model to runoff quality data. (Basic data: COD con-
centrations and flow rates [1].)
TABLE 2 - - R u n o f f flow rate and COD variation during a runoff event [1].
Time (min) 0 10 20 30 40 50 60
Flow rate (m3/s) 0.00 2.17 1.06 0.64 0.36 0.28 0.22
COD concentrations
(mg/liter) ... 400 259 216 140 184 119
Time (min) 70 80 90 100 110 120

Flow rate (m3/s) 0.16 0.12 0.13 0.10 0.09 0.09
COD concentrations
(mg/liter) 108 140 129 65 86 54
A = EXPERIMENTAL ACCURACY
6K c"
C = COST OF N OBSERVATIONS
5% 5K c- .10- s z*
o_
E
4K c- .08' -4 4
z
O O
3K c- z .06.! 93 r
8
b ,,=,
zo
o
2K c-
z~ O~e CAsE I o
==
1Kc- .02. -/~"---~.~-~
~pv ~ MINIMAL C O S T
O
o ,~ 2b 3b 4'0 ~o o.o' lb 2b 3b
N= NUMBER OF EXPERIMETS N = NUMBER OF EXPERIMENTS
FIG. 2--Application of prediction analysis and least-cost approach.
A brief examination of Eqs 4 through 9 and results plotted in Fig. 2

indicates that both uncertainties Oco.Ok~ increase with the increasing error
in the pollutant concentration measurements and the increasing range of
the independent variable (time in this case). Both uncertainties decrease
with the increasing number of experimental observations.
For both Cases I and II, the uncertainties in Co, the initial pollutant
concentrations, are rather small. For the number of observations N = 8
the uncertainties in Co are smaller than those for the observations.
The uncertainties in k2, the rate of decay, are considerable. If only the
more typical Case II is considered and the uncertainty of the concentra-
tion measurements is, for instance, 0.10, then to estimate k2 with a 0.05
uncertainty would require 17 experiments.
In some cases, the uncertainty of experimental observations can be
reduced by using more sophisticated and more costly equipment. If cost
curves can be developed for these different levels of experimental un-
certainties, a minimal cost approach can be used for the selection of the
number of experiments and the experimental uncertainty. An example of
this approach is shown in Fig. 2 for hypothetical data. In this case, the
cost curves were assumed for three levels of experimental uncertainty,
0.05, 0.075, and 0.10. If the desired uncertainty in k2 determination is
0.05, then the cheapest alternative is to perform eight observations with a
0.075 uncertainty. Since most of the water quality analytical techniques
are standardized, and consequently there is no freedom in selecting the
experimental uncertainty, the minimal cost approach would rarely apply.
The technique demonstrated in this section can be used for any
functional relationship. The technique offers a good indication of the

number of samples which have to be analyzed to obtain acceptably
accurate data.
Practical Aspects of Sampling Installations in Urban Runoff Studies

The discussion in the preceding section assumed that the collected
samples are truly representative for the storm water quality. To achieve
this sample representativeness, systematic errors in the sampling technique
should be avoided. The main sources of systematic errors in the sampling
in urban runoff studies are as follows:
1. the depth of sample withdrawal in a nonhomogeneous media,

2. capability of the sampler to collect solids,
3. sample cross-contamination, and
4. lack of synchronization between the flow recording and the sampling
The Depth of Sample Withdrawal in a Nonhomogeneous Media

The sample withdrawal depth is important for those flows where the
solids concentration distribution along a vertical is nonuniform. The
concentration distribution depends on the flow characteristics (shear
velocity) and the characteristics of solid particles (fall velocity). Such a
relationship is plotted in Fig. 3 [9]. As an example, a shear velocity of 0.4
m/s was assumed which corresponds to a 4-ft concrete pipe flowing nearly
full on a 1 percent slope, and the concentration distributions were
identified by the particle sizes. An acceptably uniform concentration
distribution is obtained in this case only for sand-like particles smaller
than 0.12 mm. Larger particles which still may contain a significant
portion of the pollutional load have nonuniform concentration distribu-
tions. Thus, the measured concentrations of solids and solids-bound
pollutants will be affected by the depth at which the sample was
withdrawn.
The theoretical relationship between the solids concentration distribu-
tion and the depth of flow was confirmed by field measurements. Several
samples were collected in a straight reach of 4-ft sanitary sewer at
different levels above the pipe invert. The samples were analyzed for
suspended solids and five-day biochemical oxygen demand. The results are
shown in Fig. 4 and indicate a sharp gradient in the solids and BOD
concentrations in the vertical plane.
Since it is practically impossible to account for the sewage density
stratification in a sampling operation by means of hardware, a partial
remedy may be achieved by destroying the stratification by means of
mixing and turbulence. As a stopgap, it is recommended to locate the
M A R S A L E K ON U R B A N R U N O F F Q U A L I T Y STUDIES 537
Om "LO
~ 0.8
~ 0.6
+
/~ \" \o \" /=t /
~, I 0.4
rr 0.2
"qa= 0.05 O.2 0.4 0.6 0.8 1,0
C.. D
Ca
RELATIVE CONCENTRATION
LEGEND
RELATIVE ELEVATION ABOVE THE CHANNEL BOTTOM
)l (=ELEVATION ABOVE THE BOTTOM/ DEPTH OF FLOW)
C VOLUMETRIC CONCENTRATION OF SUSPENDED SOLIDS

MEASURED AT ELEVATION "11
lqa REFERENCE RELATIVE ELEVATION
Ca VOLUMETRIC CONCENTRATION OF SUSPENDED SOLIDS

MEASURED AT ELEVATION
V~ SHEAR VELOCITY
W FALL VELOCITY OF SEDIMENT PARTICLES
d SIZE OF SAND-LIKE PARTICLES CORRESPONDING TO

THE m VALUES FOR V~=.4m (THIS VELOCITY
CORRESPONDS TO A 4ft CONCRETE PIPE FLOWING
NEARLY FULL ON A l~ SLOPE)
FIG. 3--Theoretical solids concentration distribution with depth [9].

1.0- FI = RELATIVE ELEVATION ABOVE THE

SEWER BOTTOM ( E L ABOVE
B O I T O M / DEPTH OF FLOW)
.8- C = CONCENTRATION OF SUSPENDED

SOLIDS ( r a g / I ) MEASURED AT
ELEVATION I1
.B. Cr = CONCENTRATION OF SUSPENDED
SOLIDS MEASURED AT Fir
I .4. f i r = REFERENCE RELATIVE ELEVATION

(= .04)
it
BOD r = BIOCHEMICAL OXYGEN DEMAND
(5 DAY) AT f i r
q r = .04
Z/HZ/I'HH/Z/~
0
9 C/cr o eOD/BODr
FIG. 4--Distribution of BOD and suspended solids concentration with depth. The result
represents an average of four sets o f measurements in a sanitary sewer; depth of flow
h = 0.61 m.
sampler intake at a cross-section where the flow is very turbulent, close to

the lateral inflows or vertical drops.
Capability of the Sampler to Collect Solids

The capability of a sampling installation to collect solids is particularly
important and should be evaluated in those studies of urban runoff in
which the solids and solids-bound pollutants represent a significant
portion of the total pollutant yield.
According to Ref 5, most of the solids which eventually end up in the
runoff contain various pollutants in significant concentrations. Some data
from Ref 5 are shown in Table 3 and indicate the pollutant concentrations
found in the street surface contaminants of various sizes. Thus an
installation with a limited capability to collect solids may lead to an
erroneous estimate of not only solids yields, but of other pollutant yields
as well. The data in Table 3 should be used only as a general guide,
because they apply to the dry street surface contaminants of which specific
gravity is not known. The particle size might be reduced by attrition
during the transport to the sewer inlet and through the sewer pipe. Also
the collection and transport of particles having a smaller specific gravity
than sand is more easily achieved.
The efficiency of the solids collection by a sampler is affected by the
orientation of the intake and by the velocities in the intake nozzle and
line.
The orientation of the sampler intake with regard to the flow direction
has a definite effect on the concentration of solids in the collected samples.
TABLE 3--Fraction of pollutant associated with each particle size range for street su(face
contaminants (% by weight) [5].
Particle Size ~m)
>2000 840-2000 246-840 104-246 43-104 <43
Total solids 24.4 7.6 24.6 27.8 9.7 5.9

Volatile solids 11.0 17.4 12.0 16.1 17.9 25.6
BOD5 7.4 20.1 15.7 15.2 17.3 24.3
COD 2.4 4.5 13.0 12.4 45.0 22.7
Kjeldahl Nitrogen 9.9 11.6 20.0 20.2 19.6 18.7
Nitrates 8.6 6.5 7.9 16.7 28.4 31.9
Phosphates 0.0 0.9 6.9 6.4 29.6 56.2
Total heavy metals 16.3 17.5 14.9 23.5 27.8
Total pesticides 0.0 16.0 26.5 25.8 31.7
This effect was studied in the laboratory [10] using sand particles. Some
data from Ref 10 is replotted in Fig. 5. The data show that unless the
intake is oriented axially, pointing upstream, the solids concentration in
the sample is always smaller than that in the sampled flow. In the studied
case (sand, 0.15 mm), the bottom intake underestimated the sample
concentrations constantly by about 15 percent over the whole range of
intake velocities. A laterally oriented side intake underestimated the
10
' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' 1
~7
SAND,d= 045ram
Z
_o 2
z S A N D , ~ ~
9
1
.9 B ~..-.....~ --
.8 B O T ~
u_ .7
.6
.5 ~ '- "*'--'SAND, d= 0.15ram
.4 SIDE (90~ 5 FPS
.3
.~[ I I I I I I I ,T
.1 .2
INTAKEVELOCITY
FLOW VELOCITY
FIG. 5---A = effect q f sampling velocity on representativeness of suspended solids [8,10].
B : e~'ect of lateral orientation of sample intake [8,10].
sample concentration by 15 to SS percent depending on the ratio of the

intake to flow velocity.
The axial intake pointing upstream gives solids concentrations which
depend on the ratio of the sampling (intake) velocity to the flow velocity.
This dependency was significant only for large particles (sand, 0.45 mm),
it practically disappeared in the case of small particles (sand 0.06 ram).
In field studies of urban runoff, the sampler intake is typically
suspended from the top or oriented axially but pointing downstream.
These intake arrangements are usually necessary to prevent the clogging
of the intake. Solids concentrations in the samples collected in these
installations will be underestimated according to the previous discussion.
Another factor which affects the solids collection by a sampler is the
intake line velocity. The intake line velocity differs from the sampling
velocity if the line diameter differs from the nozzle diameter. While it is
desirable to keep the line diameter reasonably large, say 3/8 in., to avoid
clogging, a velocity sufficient to transport even the largest collected
particles should be maintained. This means that the line velocity should
be higher than the particle fall velocity and the lift should be minimal.
If the grab sample is withdrawn directly from the sewer and deposited into
a bottle, the initial part of the sample will have a smaller solids concentra-
tion than the rest, because the particles have a relative negative velocity in
relation to the ambient fluid. Only for small lifts is the initial portion of
the sample with its underrated concentrations insignificant in comparison
to the total sample volume.
To minimize the error caused by the deviation of the intake velocity
from the flow velocity, typical flow velocities during the peak runoff flow
should be considered for the selection of the intake velocity. The feasibility
of using commercial samplers with constant sampling velocity which varies
only with the lift should be checked from this point of view.
It should be stressed that the prior discussion applies mainly to the
sand-like particles (same shape and specific gravity). For lighter particles,
the density effects described are less pronounced.
Sample Cross-Contamination
Cross-contamination of samples is another source of error in sampling.
It can occur because of deficiencies in the sampler plumbing, or because
of residue in the intake line. The former possibility is uncommon and the
latter source of error can easily be eliminated by an air-purge preceding
the sample withdrawal. Such an air-purge does not however eliminate the
possibility of the bacteriological cross-contamination in those samplers
where all the samples have to pass through the same intake line and flow
distributor. Therefore, some commercial samplers have separate intake
lines for each individual sample.
Time Synchronization Between the Sample Withdrawal and Flow

Recording
Poor time synchronization between the sample withdrawal and flow
recording can cause appreciable errors in the calculation of the total
pollutant yields, because wrong pollutant concentrations will be assigned
to the measured flow rates. The proper synchronization is best achieved by
simultaneous recording of the runoff flow rates and sampling cycles on the
same chart or tape.
The first sampling cycle should start as closely to the beginning of the
runoff as possible. The sampling cycle can be triggered by the first
impulse from the precipitation sensor, or better, by the rise of the storm
water level in the sewer by a preselected increment.
Conclusions
The sampling programs in urban runoff quality studies have to be
designed according to the objectives of these studies. If only the total
pollutant yields are of interest, an inexpensive flow-proportioned compos-
ite sampling technique is recommended. The average sampling interval
should not be greater than 10 min. When the time variation of pollutant
concentration is o f interest, it is recommended that an empirical model be
established for the concentration variation. This model can then be used
in the experimental design of the data collection program for a runoff
quality study. The use of such an empirical model expressing the pollutant
concentration variation as an exponential decay function has been demon-
strated on runoff quality data.
Great care has to be devoted to avoid systematic errors in the sampling
technique and thus to ensure the true representativeness of samples. The
first step in this direction is to locate the sampler intake at a cross-section
where the sampled medium is rather homogeneous. The capability of the
sampling apparatus to collect solids should be evaluated, mainly with
regard to the intake orientation and the intake nozzle and line velocities.
Good time synchronization between the precipitation, runoff, and storm
water quality monitoring must he ensured, best by recording all these data
on the same chart or tape.
Typical sampling installations with intake nozzles oriented laterally,
pointing downwards or downstream, will collect samples generally leading
to an underestimate of the total pollutant yields. This is especially true for
those pollutants which are associated with large heavy particles.
References
[l] "Combined Sewer Overflow Abatement Alternatives, Washington, D.C.," Water Pollu-
tion Control Research Series, Environmental Protection Agency, Report 11024 EXF
08/70, Aug. 1970.
[2] Nedved, T. K., Fochtman, E. G., Langdon, W. M., and Sullivan, F. O., Journal o f the
Water Pollution Control Federation, Vol. 44, No. 5, May, 1972, pp. 820-828.
[3] "Storm Water Management Model," Water Pollution Control Research Series, Environ-
mental Protection Agency, Report 11024 DOC 07/71, Part 1, July 1971.
[4] "Urban Runoff Characteristics," Water Pollution Control Research Series, Environ-
mental Protection Agency, Report 11024 DQU 10/70, Oct. 1970.
[5] "Water Pollution Aspects of Street Surface Contaminants," Water Pollution Control
Research Series, Environmental Protection Agency, Report EPA-R2-72-081, Nov. 1972.
[6] "Stream Pollution and Abatement from Combined Sewer Overflows, Bucyrus, Ohio,"
Water Pollution Control Research Series, Environmental Protection Agency, Report
11024 FKN 11/69, Nov. 1969.
[7] "'An Assessment of Automatic Sewer Flow Samples," Water Pollution Control Research
Series, Environmental Protection Agency, Report EPA-R2-73-261. June 1973.
[8] Wolberg, J. R., Prediction Analysis, Van Nostrand Publishing Co.. Princeton, N.J.,
1967.
[q] Krishnappan, B. G., "A Probabilistic Method of Determining the Distribution of
Suspended Sediment in an Open Channel Flow," PhD thesis, Queen's University,
Kingston, 1972.
IlO] "LaboratGry Investigation of Suspended Sediment Samplers," Inter-Agency Committee
on Water Resources, Report No. S, 1941.
F. C. T a n ~ a n d G. J. P e a r s o n j
Stable Carbon Isotope Ratios as

Water Quality Indicators
REFERENCE: Tan, F. C. and Pearson, G. J., "Stable Carbon Isotope Ratios as Water
Quality Indicators," Water Quality Parameters, ASTM STP 573, American Society for
ABSTRACT: Stable carbon isotope ratios (C13/C 12) of man-made organic substances
such as petrochemicals and pulp mill effluents are generally different from those of the
"natural" organic matter in marine environments. By measuring the C*VC t2 ratios of
background organic matter in the marine environment and the pollution sources, an
estimate of the degree of man-made pollution can be obtained.
Principles of the Ct3/Ct2 technique, analytical methods, and an example of its use are
discussed together with the limits of applicability of the approach.
KEY WORDS: water quality, water pollution, carbon isotopes, environmental tests
The concentrations of total organic carbon (TOC) and its dissolved and
particulate constituents (DOC and POC) are important in water quality
sampling programs. However, these indicate only the amount of carbon
present in the system at the time of sampling, and if increases should
occur, it is much more beneficial to distinguish between the effects of
man-made and natural sources of the elevated carbon levels.
Considerable progress has been made towards an understanding of the
variation of stable carbon isotope ratios (Ca3/C 12) in nature during the
past 20 years. It is now known that various carbon reservoirs have discrete
ranges of values of the Ca3/C a2 isotopic ratio. This parameter has been
used to investigate various geological and chemical problems with some
success, but as yet has received little attention in water quality programs.
Organic effluents such as those from petrochemical plants, pulp mills,
and domestic sewage treatment plants generally have C~3/C z2 values which
are different from that of the natural organic carbon in fresh water or
marine environments. Therefore, it should be possible to estimate the
x Research scientist and scientific officer, respectively, Chemica! Oceanography Division,
Atlantic Oceanographic Laboratory, Bedford Institute of Oceanography, Dartmouth, Nova
Scotia B2Y 4A2, Canada.
543

contribution from pollutant sources to the organic carbon present in a

water body by using the characteristic CIa/C lz values. In this paper, we
outline the principles, methods, applicability, and limitations of the
Cla/C lz method in water quality studies.
Carbon Isotope Ratios of Various Carbon Reservoirs

The C13/C 12 ratios in various reservoirs differ due to the fractionation
effects of certain biological, geological, and chemical processes. Carbonate
equilibria and photosynthetic conversion of inorganic carbon into organic
are the major processes of carbon isotope fractionation in aquatic systems.
Generally, organic matter is enriched in C x2 with respect to inorganic
carbon. Figure 1 shows the ranges of C13/C 12 values associated with
various natural and m a n - m a d e organic carbon reservoirs [1-5]. z The
Ca3/CZZ ratios are expressed as dC 13 which is defined by
(C'3/C'2)sample - (C'3/C'2)standard x 1000

6C~3(~176176
= (C'VC'~)smd~d
where the standard is the universally used PDB 3 Cretaceous belemnite,

and 6C 13 is in units of parts per thousand.
It is immediately apparent that marine and land plants have very
different 6C 13 values. Pollutants such as effluents from petrochemical
plants and pulp mills can, in some instances, be distinguished from
natural marine DOC, POC, and sedimentary organic carbon, while
certain crude oils exhibit a range of tic la values somewhat different from
that of the background organic constituents.
It should be realized that the ranges of 6C 13 values in Fig. 1 represent a
combination of available data, and that specific samples would have a
much narrower range of values. For example, marine sediments from a
specific area would have a narrower range of 6C aa values than that shown,
and a particular crude oil would have a single, measurable value of 6C ~3.
Principles of 6C~3 Technique

The carbon pool of an aquatic system such as a marginal marine bay
which is receiving m a n - m a d e organic pollutants may be said to consist of
two components: natural and pollutant. Since man-made organic pollutants
usually show the most negative 6C a3 while the marine organic matter the
least negative, the mixture of the two would result in an intermediate value
3PDB is a carbonate standard prepared from a belemnite rostrum collected from the
Cretaceous Peedee formation of South Carolina.
TAN AND PEARSON ON STABLE CARBON ISOTOPE RATIOS 545
Land Plants
~ceanlc
HCO~
Marine Plants. Organics in
Marine Sediments
Domestic Sewage~
coi~ p6c-o ~-~oc
Petrochemical & N a t u r a l Gas
Marine
,DOC
Marine
POC
i
Crude Oil
i ii
Pulp M i l l Effluent
2 0 "4 "8 "12 "16 "20 "24 -28 "32 "36 "40 "44 "48
8c1:3%0
FIG. 1----~C t3 of various carbon reservoirs.
depending upon the dC 13, the concentration, and the volumes of natural
and pollutant sources.
The mathematical expression for the resulting 6C 13 may be derived as
follows, where
Cn = concentration of ~aatural DOC in receiving waters,
Va = volume of natural component,
dn = dC 13 of natural DOC,
Cp = concentration of pollution-derived DOC in receiving waters,
Vp = volume of pollutant component,
6p = dC 13 of pollutant DOC, and
do = dC a3 of resultant DOC at a particular sampling location.
Considering material balance for a two-component system of natural
and pollutant carbon sources
- CnVndn "[- C p V p d p
60 = (1)
CnVn + cpvp
Let
c.v. cpvp
Fn = CnV,, + CpVp and Fp = CnVn + CpVp
then
do = Fll ti. + Fp tip (2)
and
Fll + Fp = ] (3)
Combining Eqs 2 and 3
till - - ~0
Fp -- (4)
dn - dp
Therefore, Fp is a measure of the fraction of the total DOC which is

derived from pollutant sources. It is expressed only in terms of ti values
which are measurable quantities. In the case of a number of pollutant
outfalls on the same water body, tip must be deduced by a weighted
average of the individual dC13s considering DOC concentration and flow
rate.
From the form of the expression for Fp it can be seen that the
sensitivity of the method increases with the difference between tJn and tip.
If tin is equal to tip no useful information can be gained. The preceding
derivation will hold for particulate as well as dissolved organic matter, and
simultaneous studies of both parameters should yield very reliable values
of Fp. The inorganic carbon component or total dissolved CO2 may be
used to yield qualitative information.
Analytical Techniques
The basic technique for measuring C 13 of organic samples is to quanti-
tatively convert the organic carbon to CO2 gas which is analyzed on an
isotope ratio mass spectrometer. The principle and techniques of this
machine have been described by Craig [6].
The methods for converting POC and DOC samples to CO2 are
discussed in detail by Calder [7]. Craig [1], and Williams and Gordon [5].
Briefly, in the case of POC the sample is filtered through a 0.5-W'a glass
fiber or silver filter. The POC retained by the filter is oxidized to CO2 at
900~ in the presence of CuO and purified 02. The evolved gases are
cycled over heated MnOz and CuO to convert any CO to CO2 and to
remove oxides of sulfur or nitrogen. The COz yield is measured in a
manometer and the gas sample saved for mass spectrometric analysis.
This method is capable of a tiC la reproducibility of 0.30/00.
In the case of DOC, the pH of the filtrate is adjusted to less than 1 with
100 percent phosphoric acid, and the inorganic carbon is removed by
flushing out the evolved COo with pure nitrogen gas. Potassium persulfate
is added to the filtrate which is then autoclaved at 120~ for 1 h. After
removing water vapor by using dry ice traps, the volume of the evolved
CO2 is measured and the gas is saved for 6C 13 analysis.
Application
An example of the application of the 6(213 technique is given by Calder
and Parker [2] in a study of the Houston Ship Channel in Texas. This
waterway receives effluent principally from petrochemical plants, and to a
much smaller extent from domestic sewage and a paper mill. A m a p of
the area showing sampling locations and 6C 13 values is shown in Fig. 2.
7 .~
2
::i
- 31-2
7~
10
::2
-27-1 / Galveston
3 - 31.5 11 -27"5 ~ Bay
4 - 29-3 12 -28.2
5 -30"! 13 -28.2 16
6 -26"9 14 -32-0
7 ---- 15
8 -48"8 16 -24.8
17 -24-9
FIG. 2--603 qf Houston Ship Channel Tex. [2].
In order to estimate the extent to which man-introduced organics are

contributing to the DOC, the authors estimated 6n as --20o/oo based on
measurements in the Gulf of Mexico, 6p was taken as - 3 9 % 0 which is
the measured value at the effluent outfall of a petrochemical plant, and 6o
as the average of all observations in the channel. Obviously, this will give
an estimate of the average effect of man-introduced organics over the
entire sampling area.
Substituting into the expression for Fp, and assuming an analytical
uncertainty of _+0.30/00 for 6C a3
6n - 60 --20 - (--30.5)
= 0.55 +_ 0.05
Fp-- 6n - 6p -20- (-39)
Therefore, more than 50 percent of the DOC in the channel is probably

derived from petrochemical effluent.
This example could also be used to examine the limit of applicability of
the method. Assume a total analytical uncertainty for 6C 13 in DOC of
-+0.30/oo (mass spectrometer precision 0.05~ Then the lowest meaning-
fully measurable value of 60 would be -20.60/00, that is, twice the
analytical precision removed from the value of the natural background.
Then
-20 - (-20.6)
Fp -- -20- (-39) ----0"03
Therefore, a minimum of 3 percent effect due to effluents could be

detected under these conditions. Similarly, if the major input were due to
pulp mill effluent and 6p were, for instance --270/00, the minimum de-
tectable effect would be 9 percent due to pollutant sources.
Selection o f B a c k g r o u n d dC 13 V a l u e s
Since meaningful estimates of the effect of man-made organics on the

aqueous environment depend upon the background 6C 13 value of the
system studied, it is essential to have reliable values of 6n. It would be
extremely difficult, if not impossible, to obtain a universal background
6C 13 value. One could use an average open ocean value which would
presumably be free of significant effect by man-made organics. Williams
and Gordon [5] have shown that the 6C x3 values of DOC from the
southeast Pacific have a range of values between - 2 1 . 2 and -24.40/00 which
is constant with depth and time. Possibly a mean value of these could be
used for 6 , , but this is a very arbitrary procedure and perhaps not valid
until more data are available.
A more meaningful value of 6n can be obtained by utilizing a clean
area comparable to the one being studied for effect of organic pollutants.
The shape, size, physical oceanographic characteristics, and surrounding
terrain would have to be similar for the two areas in order to obtain a
reliable 6, from the nonpolluted one.
If a suitable nonpolluted reference area is not available, a comparative
method within a particular waterway could be used. This is fairly simple
for a system which is not tidal since values of tin can be obtained from
sampling locations upstream of the area of organic outfalls. For tidal
environments, an arbitrary starting value for 6n would have to be
established at a particular time by measuring present 6C 13 levels. This
range can then be used to assess quantitatively the m a n - m a d e organic

additions to the system in subsequent years.
Limitations and Applicability of the Method

Several factors control the applicability of the method to environmental
problems. As mentioned previously, the sensitivity increases with increas-
ing difference between the 6C la values of the D O C of the natural and
pollutant water. Since marine D O C has t~Cla values which are relatively
constant and C la rich in comparison with most organic effluents, the
method is particularly useful in coastal environments.
The stability of P O C and D O C in the aqueous environment is also
important for the method to be successful. Recent studies by Williams
and G o r d o n [5] and Calder [7] have shown that these are relatively stable,
and for the case of continuous input into a waterway will be u n i m p o r t a n t
for the validity of the method. Also, it is necessary that the m a n - m a d e
and natural components be well-mixed at any one sampling location in
order to give consistent results. This is especially important for investi-
gating streaming and tidal effects upon the distribution of organic
pollutants.
For open ocean work, the b a c k g r o u n d 6C la is in the range of - 2 0 to
-25~ This means that the 6C la for the raajority of crude oils will be
more negative than the b a c k g r o u n d range, and therefore readily de-
tectable by this method.
A disadvantage of the method is that no information is obtained
regarding the chemical structure or toxic properties of the organic
pollutants. It can best be utilized in conjunction with other techniques
such as gas-liquid c h r o m a t o g r a p h y , CHN ar~alysis, or high resolution
organic mass spectrometry.
References
[1] Craig, H., Geochimica Et Cosmochimica Acta, Vol. 3, 1953, p. 53.
[2] Calder, J. A. and Parker, P. L., Environmental Science and Technology, Vol. 2, 1968,
p. 535.
[3] Reimers, R. S., MS thesis, University of Texas at Austin, 1968.
[4] Silverman, S. in Isotopic and Cosmic Chemistry, H. Craig, S. L. Miller, and G. J.
Wasserburg, Eds., North-Holland Publishing Co., 1964.
[5] Williams, P. M. and Gordon, L. I., Deep Sea Research, Vol. 17, 1970, p. 19.
[6] Craig, H., Geochimica Et Cosmochimica Acta, Vol. 12, 1957, p. 133.
[7] Calder, J. A., PhD thesis, University of Texas at Austin, 1969.
J. M. Bewers2 L D. Macaulay2 Bj#rn Sundby, I
and D. E. Buckley 2
Data Are for Looking At or

Quality Control Through
Interpretation
REFERENCE: Bewers, J. M., Macaulay, I. D., Sundby, Bjcrn, and Buckley, D. E.,
"Data Are for Looking At or Quldlty Control Through Interpretation," Water Quality
Parameters, ASTM STP 573, American Society for Testing and Materials, 1975, PI~
550 -565.
ABSTRACT: A logical interpretive procedure for the examination ot environmental data

is described and its use illustrated. The procedure is used to eliminate errors, detect
redundancies, detect and validate anomalies, examine water quality data sets, and
improve analytical and sampling techniques. It is a first step towards thorough data
analysis and should ensure that the data, which are subsequently used for geochemical
or environmental purposes, are valid.
KEY WORDS: water quality, environmental tests, data processing, interpretation,

sampling, analysis (mathematics)
A number of Canadian programs (the largest of which is the National

Water Quality Network) exist for the collection of water quality data. As a
result a veritable mine of environmental information is now available in
data banks such as "'NAQUADAT." It is apparent, however, that much
of the potential value of this information has been negated by the
omission of preliminary data inspection and review procedures. 3 This
paper discusses the design of a simple data interpretation system and
demonstrates its value when applied to contemporary NAQUADAT data.
: Head, water geochemist, and NRCC postdoctoral fellow, respectively, Inorganic Chemis-
try Section, Chemical Oceanography Division, Atlantic Oceanographic Laboratory, Bedford
Institute of Oceanography, Dartmouth, Nova Scotia, Canada.
z Head, Environmental Marine Geology, Atlantic Geoscience Centre, Geological Survey of
Canada, Dartmouth, Nova Scotia, Canada.
3NAQUADAT data listings distributed to users contain the conditional statement "All data
appearing in this report have not been verified and must be considered provisional and subject to
revision."
550
9
BEWERS ET AL ON QUALITY CONTROL 551
Water quality objectives include:

(a) the enhancement of water quality,
(b) the management and protection of water bodies,
(c) the protection of aquatic ecosystems from the effects of anthro-
pogenic activities, and
(d) the assessment of the probable impacts of proposed major develop-
ments on water quality and water uses.
These objectives place some constraints upon the quality and type of data
which are required. It is obvious that water quality criteria cannot be
based solely upon the suitability of water for human or industrial
consumption. One can argue about what constitutes high or low "water
quality" but there are no a priori reasons to exclude any parameter or
substance from such an assessment. Thus, enhancement of quality implies
that one should, within practical limitations, make and use all possible
measurements of the chemical composition and physical state of a water
body. The other objectives require the inclusion of sampling stations both
within natural aquatic environments and at critical locations close to
municipal and industrial discharges.
Storage of data from these programs is necessary because of the large
quantity involved and because much of the interpretation depends upon
repeated analyses over long time periods. The use of a computer enables
more reliable and economical manipulation of the data. These con-
siderations justify the use of a large central file, such as NAQUADAT, as
a repository for all the data from a water quality program. They do not,
however, obviate the necessity for some evaluation and interpretation of
the data before they are permanently stored. Such interpretation includes
error detection and quality control functions which facilitate corrective
feedback to the analytical and sampling operations. It is also possible to
use the interpretation to provide a certain degree of guidance to the
overall direction of such programs.
Data Interpretation and Corrective Feedback

One would like to avoid the situation depicted in Fig. 1 where corrective
feedback is confined to the sampling and analytical stages of the system.
A better approach is shown in Fig. 2 in which information is channeled
back to the analytical and sampling operations at two stages of data
interpretation. First, raw analytical data are inspected before they are
entered into the file. Errors detected at this stage can be used to provide
immediate or short-term corrective feedback to the field and laboratory
operations. Second, the master data file is periodically surveyed to detect
more fundamental faults in all stages of the water quality system. The
information derived from the latter process provides some long-term
Data ~l
~[ Data
File
FIG. l--Limited feedback system.
Data~ Data Data

file
Y
I ,.riodi*T |
< 1
Information
FIG. 2--System with correctivefeedback.

interpretatio~
l
guidance with regard to the extent and type of activity in which the
network is engaged.
It is this process of interpretation and corrective feedback with which
we are concerned. A set of logical interpretive procedures is described and
the application of each stage is demonstrated with a selection of
NAQUADAT data shown in Tables 1 to 5.
Input Data Inspection

Let us consider the situation in which semi-raw analytical and field data
in recognizable units are submitted for entry into the master file. How can
we evaluate these data to provide as much corrective feedback as possible
to the sample collection and the analytical work and, at the same time,
avoid the inclusion in the master file of dubious numbers? A method 4
currently in use for this purpose compares each measurement of a
particular parameter with two ranges, during the input stage. If the entry
value lies outside the wider range it is assigned the indicator "'impossible
value" and then excluded from the file. If the entry value lies inside this
4Peters, R. H. and Demayo, A., paper presented at the Workshop Seminar on Computer
Storing and Processing of Hydrological Data, 19-20 Oct. 1971, Laval University, Ste. Foy,
Quebec.
TABLE 1--Water quality data from S. W. Miramichi,

Blackville. N.B.
29306L 30304L
Copper Extractable, Zinc Extractable,
mg/liter mg/liter
0.025 .. 9
L.005 a ...
L.OO5 010i9
oi& s 004
0.04 0.065
0.05 0.02
O.13 0.03
0.05 0.010
0.005 0.007
0.005 0.003
0.005 0.010
0.005 0.02
0.005 0.011
0.005 0.009
0.005 0.015
0.005 0.02
0.005 0.016
0.008 0.02
0.18 0.03
0.006 0.000
0.000 0.000
0.002 0.000
O.10 0.010
0.002 0.000
0.000 0.000
0.000 0.000
0.07 0.03
0.08 .. 9
Detection limit = 10 pg/liter Detection limit = 10 # g / l i t e r
a Less than 0.005 mg/liter.
r a n g e b u t o u t s i d e a n a r r o w e r r a n g e it is assigned the label "'unusual

v a l u e " a n d r e m a i n s within the file. W h i l e such a process is a d e q u a t e for
p a r a m e t e r s such as dissolved oxygen a n d p H , it is far from a d e q u a t e for
m o s t o t h e r species a n d p a r a m e t e r s . T h e limits set on each range have to
be e s t a b l i s h e d in the light o f the e x t r e m e m a t r i c e s which m a y exist at
water quality n e t w o r k stations. Thus, for e x a m p l e , we m i g h t well set
c h l o r i d e limits for i n l a n d waters of 0.1 to 150 p p m for " u n u s u a l " a n d
0.001 to 1000 p p m for " i m p o s s i b l e , " b u t these would b e invalid for
e s t u a r i n e a n d c o a s t a l w a t e r q u a l i t y stations where u s u a l should be 1.0 to
19 400 p p m a n d i m p o s s i b l e to 0.1 to 20 000 p p m . T h u s , one t e n d s to use
the wider limits for b o t h fresh a n d saline water stations t h e r e b y d e f e a t i n g
the object o f the excerise.
TABLE 2--Water quality data from Harrys River, Black Duck, Nfld.
82301L 48302L
Lead Extractable, Cadmium Extractable,
mg/liter rag/liter
0.6 ..,
L.00 02L ...
0.01 02L ...
0.00 02L
L.00 02L 0.001 01L
0.00 02L L.001
0.00 02L L.001
0.00 02L L.001
0.00 02L L.001
L.00 02L L.001
82301L, detection limit = 50 ~g/liter 48302L, detection limit =- 1 ~g/liter
82302L, detection limit = 1 /~g/liter 48301L, detection limit ----10/~g/liter
It is obviously desirable to be more selective and realistic. A flow

diagram for a system of checks on the input data is shown in Fig. 3.
Using the Water Quality Dictionary and a library of independent geo-
chemical compliations as references, the system inspects the incoming
data. In cases where errors are detected or suspected, the routine either
removes or modifies the data or requests validation by resampling and
analysis. It can be demonstrated that the checks incorporated in this
routine are not, in general, being applied to N A Q U A D A T data. The
copper and zinc values in Table 1 include both zeros and values below the
detection limit of the methods. These data are of little value. The high
lead concentration of 0.6 mg/liter in Table 2 and the low sodium datum
of 800 mg/liter in Table 3 should have been identified and referred for
verification.
Data File Interpretation

Let us assume that the numbers pass our simple input inspection
routine and are entered into the master file. Are the data then worth
owning? As J. R. Macdonald of the National Research Council Numerical
Advisory Board recently observed, the answer to this question is usually
"No" throughout the whole spectrum of research. He concludes that
much better data analysis is required before a high proportion of
published data can be properly used for more than qualitative purposes.
In the case of water quality data there are several ways in which
we can improve its usefulness. One such method is depicted diagrammat-
ically in Figs. 4 to 6. This routine is divided into three parts, each of
which sequentially carries out numerical, analytical, and geochemical
TABLE 3
WATER DUALITY DATA REQUEST 0002 PAGE 23
STATION 02PE01CD00O2 LATITUDE 460 20M SIS LONGITUDE 62D ISM 16S
COLVILLE ~AY+ APPROX. 125 YDS. S~ OF BEACON ON B R E A K W A T E R ~ SOURISI PRINCE EDWARD ISLAND
SAMPLE }0ISIL 10AOIL 10501L 10451L I0551L 0BA01L 06201L 06301L

DATE TIME ALKALINITY RESIDUE RESIDUE RESIDUE RESIDUE OXYGEN BIEARBONT~ CARBONATE
PHENOL NONFILTR~ FIXED FILTERABLE FIXED CONSUMED (CALCD,} (CALCD,)
AST PHTHALEIN NONFILTR. FILTERABLE
CACQ3 02 HCD3 CO3
D M Y H M MG/L MG/L MG/L MG/L MG/L MG/L MG/L MG/L
20 6 72 11 00 O.0 I41 O
20 6 72 II Ol 0.0 IAl o
23 7 ~2 I i DO o.o z4o o
23 T 72 It Ol 0,0 146 O
22 8 ?2 II O0 0.0 127 0
22 8 72 II 01 0.0 tZB 0
31 l 0 72 11 40 5~ 02L
31 ]0 72 II S0 3.2 02L
IB I 73 14 O0 24~ W
IR I 73 14 10 3.2 m
IS 2 73 13 30 I~
IS 2 73 13 40 0,2 m
~0
WATER DUALITY DATA REQUEST 0003 PAGE 23
m
STATION 02PE01CD0002 LATITUDE 46D 20M SiS LONGITUDE 62D 15M IAS -I
COLVILLE DAYI APPROX. 125 YDS. SoE. OF REACDN ON B R E A K W A T E R I SOURIS, PRINCE EOWARD I S L A N D
r"
SAMPLE |0602L 20IOIL 1210AL IlIO3L 19103L IAI02L I7203L 091ObL
DATE TIME HARDNESS CALCIUM MAGNESIUM SODIUM POTASSIUM SILICA CHLORIDE FLUORIDF 0
TOTAL DISSOLVED DISSOLVED DISSOLVED DISSOLVED REACTIVE DISSOLVED DISSULVED z
AST (CALCDo|
CACO3 CA MG NA K SI02 CL F O
O M Y H M MG/L MG/L MG/L MG/L MG/L MG/L MG/L MG/L c
20 6 72 |I O0 4741.3 250. 03L 1000. O?L 8550~ OAL 500~ OAL 0~ 16200, 2.1 04L
20 6 72 II 01 5072.0 300~ 03L 1050, 02L 8600. 0AL SOD, 0AL 0.1 16200. 2~ 04L r-
23 7 72 11 00 5377.7 3A0~ 03L 1100. 02L 8600~ 04L 305. 04L 0+2 16400+ 2+~ 04L
23 7 72 II 0I 5377*7 340. 03L I100+ 02L 8600, 0AL 310o 04L 0=3 16400~ 3.0 0AL ..<
22 A 72 II O0 5171~ 340. 03L lOS0+ 02L 800.0 OAL 295. 04L 0ol 1~600= 2.~ 04L
22 B F2 l] OI 4941.0 330. 03L tO00* 02L 8300. 04L 305. OAL 0.3
0
15BO0. 3,0 04L
3| l 0 72 II A0 1025+ 02L 0.6 0
31 l0 72 I1 50 I000~ 02L 0~ Z
IE 1 73 14 O0 1000~ O2L 1~ --I
18 I 73 14 I0 IOS*O O2L 0.8
I% 2 73 13 30 BSO~ 02L I,0 0
IS 2 73 13 40 1050, 02L 0~ r-"
O1
Salinity ( %o ) 29.1 28.4 2.6 26.7 28.0 82.3 U1
01
ol
TABLE 4 01
o)
WATE~ Q U A L I T Y DATA REQUEbT 0001 ~AG~ 2J
STATION 02PE01CD0007 LATITUDE AbD 20M 515 LONGITIJDE 620 ISM 16S
COLVILLE IJAY+ APPROX, 12S YDS. S,E, UF BEACON ON ~ R E A K ~ A T ~ R + SOUR|St PRINCE EDWARD ISLAND 3>
SAMPLe 020615 O7061L 02OAIL 00202L 10301L 02073L 0?0ILL 10101L
-i
DATE TIME TEMP. TEMP* SPECIFIC TOTAL PH TURBIDITY COLOUR ALKALINITY ITI
CONO. DISSOLVED TOTAL ARPARENT TOTAL
AST SOLIDS
(CALCD. I JACKSON CACO3 s
D M Y H M 0EGeC, DEG,C. UMHO/CM MG/L PH U N I T S TURB, UNTS 9 REL, UNITS MG/L C
20 0 72 II 00 13 21 44100 28692 ~.8 0.2 3>
20 6 72 II 01 9 21 44200 28922 6.9 0,5 r-
LS, 116
23 7 72 tl O0 |7 22 44200 2896A 7.3 0,3 LS, I20 .-I
23 7 72 II Ol I& 21 44400 ~RqoA 7+3 I,? LS+ I?0 -<
22 8 72 11 00 18 22 38700 20601 7*q 1+7 5, 10%
-o
22 8 72 It 01 16 22 39800 27915 7,R 1,0 10% 3>
31 10 72 II 40 8 4SOR0 AIS 3252 7.0 OIS "n
31 10 72 II AS 8
31 10 72 II 50 8 ASAIO 41S 3163 7,8 0IS 3>
18 1 7] 14 00 0 21 O1200 3023 7.7 O|S
18 1 73 IA I0 0 21 A3600 2275 7,7 OIS

rn
1% 2 73 13 30 0 32700 2427 8,0 OIS
.-I
15 2 73 13 40 0 All00 3264 7,7 DIS rn
~o
6o
WATER QUALITY DATA REOUEST 0 0 0 S PAGE 23
STATION 02PE01CD0002 LATITUDE A6D 20M 5 I S LONGITUDE 62D 15M I6S
COLVILLE DAY+ APPRUX= 125 YDS, S.E= OF BEACON ON OREAKWATER+ SDURIS+ PRINCE EDWARD ISLAND
SAMPLE 26302L 26302P 26|02L 26102P 25304L 2530AR 2S104L ~5104#
DATF TIME IRON IRON IRUN 1NON MANGANESE MANGANESE MANGANESE MANGANESt.
EXTRBLEo EXT~BLE= DISSOLVED DISSOLVED EXTRBLE, EXTRRLE, DISSOLVED I)ISSt)LV~D
AST FE+2~FE§ FE+2~FE§ FE§247 FE+2~FE+3
FF FE FE FE MN MN MN MN
O M Y H M MG/L MG/L MG/L MOIL MOIL MG/L MG/L MG/L
20 6 72 I1 00 0~ O.Ol L.00 0SP
20 6 72 11 Ol 0,09 0o01 L,00 05P
23 ? 72 II 00 0.02 L,0I
23 7 72 11 01 0.02 L.01
22 8 72 i~ 00 0,09,, L.01 ,,, L+O0 OSP L+O0 05L
22 B 72 II 01 0,07 L*OI L.00 05P L.00 0SL
31 I0 72 II 40 L,O0 OSL LeO0 OSL
3I 10 72 It 50 L,00 0SL L.00 05L
lS I 73 IA 00 0,01 05 o L,00 0SL
18 1 73 IA I0 O*OI 05P L+00 05L
15 2 ~3 13 30 O,OI O~P L+00 OSL
IS 2 73 13 A0 L,00 05P L.00 05L
dl 0.001 dl O.O01 dl 0.001 dl o.ool dl o.o01

TABLE 5
WATER QUALITY DATA REQUEST 0006 PAGb 23
STATION 02REOICO0002 LATITUDE AbD 20M BIS LONGITUDE 62D ISM 16S
COLVILLE BAY, APPROX. 125 YDS* S*E. OF BEACON ON B R E A K N A T E R * SOUR1S, PRINCE EDNARD ISLAND
SAMPLE 29306L 29306P 29106L 29106R 30304L 30304P 30104L 30104P
DATE TIME COPPER COPPER COPPER COPPER ZINC ZINC ZINC ZINC
EXTRBLE* EXTRBLE* DISSOLVED DISSOLVED EXTRBLE. EXTRBLE. DISSOLVED DISSULVED
AST
CU CU CU CU ZN ZN ZN ZN
O M Y H M MG/L MG/L MOlL MG/L MG/U HG/L MG/L MGIL
20 6 72 II 00 L*002 0SP L*O02 OSP

2O 6 72 11 01 L.DO2 OSP L*O02 OSP
23 7 72 ii O0 L*O02 05P 0,016 05P
23 7 72 II OI L*O02 O~P 0.021 OBP
22 B 72 II O0 L*OO2 05P L*O02 05P
22 S 72 II OI L.O02 OSP 0*021 OSP

31 I0 72 II AO L.O02 05P 0.01 OSR
31 I0 72 11 50 L*O02 OSP 0.008 OSP
18 I 73 14 O0 0.018 05P 0.016 OSP m
18 I 73 IA IO O*09b OSR 0.175 OSP
m
IS 2 73 13 30 0.007 OSP 0,012 OSP
15 2 73 13 40 0.023 OSP 0.023 OSP m
~0
dl o.ool d ! O.OOl O~
WATER QUALITY DATA REQUEST 0007 PAGE 23 m
-4
STATION 02PEOICDO002 LATITUDE 46D 20M 51S LONGITUDE 67D 15M 16S
COLVILLE BAY, APPROX* 125 YOS* S*E. OF BEACON ON B R E A K t A T E R * SOURIS. PRINCE EDMARO'|SLANO r-'
SAMPLE BZ30]L B2301P 82101L B21OIP 80301L 80101L AR302L 13301L 0
DATE TIME LEAD LEAD LEAD LEAD MERCURY MERCURY CADMIUM ALUMINUM Z
EXTRBLE. EXTRBLE. DISSOLVED DISSOLVED EXTHBLE. D|SSOLVEO EXTRBLE* EXTRULF*
AS? 0
PR PB PB PB HG HG CD AL c::
D N V H q MG/L MGIL MG/L MG/L MG/L MG/L MG/L MG/L
20 b 72 11 00 L.OO 02P O*O01R OiP LEO01 02P r-

20 6 ?2 11 Ol ODD0 02P 0,0004 OlP L.Q01 OEP .-I
23 ? ?2 It 00 0.00 OEP 0.00107 Ol~ LEO0| 02P .<
23 ? ?2 !1 OI 0.01 02P 0.00072 DiP L*001 02P
22 8 72 I1 O0 0.00 02P 0,002 OlP L*O0| 02P C3
22 8 72 II OI 0.00 O2P 0.0054 OIP LEO01 OEP 0
31 I 0 72 II A0 0.00 02P OeOOI 02P z
31 lO 72 I1 SO 0*00 02P L*001 02P -I
18 / 73 IA O0 0.00 OEP L*0OI 02P -11
IB 1 73 IA I0 O.AO L*O01 OEP
0
IS 2 73 13 30 0.00 02P O.OOl 02P i-"
I5 2 73 13 40 0,00 OEP L*O0| 02P
ol
dl 0.001 dl 0 . 0 0 0 0 5 di 0 . 0 0 1
"'Less than I
ql data
inpu_..~.~t________.t I detecti_onlimitJ
'F
] Should
I eturn data Are there
zero values? yes••
/
"Less than
detection limit"
~ b e assigned?/
Water Quality t
Dictionary
f Are there ~
ralues which are
below d e t e c t i o n S /
/
" ~~impr~
Has
methodology~
/ lJ"
yes
T update
Independent
a
Are there h
bnormally
d o t a ,
validated by
Geochemical
Data
CompilationsI
--- . . . . . . . ,ou .... piing~
values? / ~ analysis? /
Accept data t
FIG. 3--Input data inspection routine.
checks on the stored data. This simple interpretation system has the
ability of reducing the errors and of providing further corrective feedback
to the laboratory and field operations.
Software and Input Checks
The first step in the interpretation routine (Fig. 4) consists of two

checks for simple numerical errors which arise because of software or
transcription faults:
Round-off Errors--In m a n y cases such as those of iron and m a n g a n e s e

in T a b l e 4 a n d lead in T a b l e 5 the round-offs are a n order of m a g n i t u d e
File t
File o u t p u t
Software i
Ct heef f edcatt a
Central file -- - - - - anyArevaanl uother e

malous
es?
yes Does d a t e t a l l y
with unalytlcal
returns?/
there
FIG. 4---Interpretation Routine A--software and input checks.
higher than the respective detection limits. The software therefore needs to
be corrected.
Anomalous Values--It is essential that anomalies in the data be

detected so that they can be substantiated or corrected. The method of
doing this is similar to that proposed by Peters and Demayo 4 in which
probable and extreme ranges of concentration are placed into a control
file for each sample matrix (atmospheric precipitation, rivers/lakes,
estuarine and marine waters for a range of salinities) analyzed within the
water quality network. In this way the low sodium concentration in Table
3 would have been checked against the analytical return and corrected to
8000 mg/liter. The potassium data in Table 3 also contains anomalies
%
ontly below detec
tion limits?
Garycontoina more.~"-- ,~ J[ sensitive
~ sensitive m o t h - . /
Employ more
onolyt- J
ica, technique J
[
~n yes
I sensitive
Develop more I
onalyt.J
J icol technique I
~ i v u muthJ
r" > I DiscontinueI

analysis
iS there
ity?
high variabil- / f ~ ~
~yus no I precision
imp.... I
FIG. S--InterpretationRoutineB--analytical checks.
which require investigation. The anomalous lead concentration of 0.6

mg/liter in Table 2 would also have been questioned and validation
requested.
Analytical Checks
In the second step of the interpretation, which is shown in Fig. 5, the
routine is devoted solely to corrective feedback with respect to analytical
procedures.
Comparison of Detection Limits with Experimental Data--If the file
values are predominantly below the detection limit for a substance, there
is little purpose in continuing the analysis unless the station constitutes a
spill detector or some other industrial monitor. The cadmium levels in
Is Discontinue I I Increase
parameter analysls. sampling
redundant? frequency.
v;e-~l"co-m-p:r : ~ w i ht ~
geochemical c o r n - / niques re s p o n s l b l e ~
pilations? /
ATTENTION!
Investigatel [
~ / body be char- ~ y e s J Reduce samp-
low! / j ~ - . - - - - - ) . - ~ acterized or rood-~ ling frequency.
9 ~ elled ?
I Continue as
before. ~ [c .......... ,ysls I
9 further ~ yes . J for new parameters.|
eters re- ~ Reduce frequency I
q uired~ /
LII~sting p..... ,....
I of analysis f . . . . . |
J g frequency.
FIG. 6---Interpretation Routine C--geochemical checks.
Tables 2 and 5, as well as throughout NAQUADAT, are predominantly

below the detection limit of the method. Therefore, a decision needs to be
made as to whether the analyses should be discontinued or whether
cadmium is of sufficient importance to justify development of a method
having at least 20 times better sensitivity. There certainly is little point in
the continued use of the present method for baseline applications when
one realizes that environmental concentrations of cadmium in fresh water

are typically less than 0.1 /ag/liter.
Variability---A qualitative judgment regarding variability is relatively
easy to make where the NAQUADAT data is obtained at a reasonably
high frequency. It is, however, virtually impossible to make a quantitative
judgment because of the absence of information on analytical precision.
Every effort must be made to include estimates of analytical precision in
the Water Quality Dictionary in order to determine real environmental
variability. If the variability in the data is essentially derived from poor
analytical precision, the latter has to be improved. On the other hand, if
there is significant variability which cannot be ascribed to experimental
imprecision, the frequency of sampling should be increased to investigate
further its amplitude and periodicity.
Geochemical Checks
The third step in the interpretation routine (Fig. 6) consists of a series
of checks on the geochemical validity of the data. This final routine would
be essential in any interpretation of a perfect file in which routine
numerical and analytical checks had been made.
Redundancy---There is a need, for reasons of economy, to detect
superfluous analyses in the water quality system. Examples of such
redundancies may be found in Table 3 where six conservative properties
(calcium, magnesium, sodium, potassium, chlorine, fluorine) of a saline
water body are being measured in addition to conductivity. All of these
analyses are redundant since they can be simply computed from the
conductivity value given in Table 4. For each analysis of a conservative
parameter on the sample collected on 22 Aug. 1972, we have calculated
an approximate salinity and included these in Table 3. Some appreciation
of the magnitude of the analytical uncertainties can be obtained from an
intercomparison of these calculated salinity values. We would recommend
that for water bodies having salinities in excess of 5 parts per thousand
determination of conservative properties be discontinued immediately.
Geochemical~Environmental Anomalies--The input inspection proce-
dure (Fig. 3) should ensure that a geochemically anomalous datum is only
included in the file if it is validated. However, we need to identify real
anomalies so that we can investigate them further. It is essential that
further action be taken if the anomalies represent inherently hazardous
conditions. This stage is analogous to the red light on a control panel
which warns of malfunctions. The method of detection is relatively simple.
There exist a number of geochemical compilations of ground, river, lake,
and seawater analyses. A good example is Livingstone's review, s Although
SLivingstone. D. A.. "Chemical Composition of Rivers and Lakes," U.S. Geological
Survey Professional Paper 440-G, 1963.
some of the trace metal concentrations in this document are now suspect
in the light of improvements in methodology made since the early 1960's,
the data given for most substances are quite realistic. Incorporating such
compilations into a geochemical reference file would allow file values to be
examined and compared with typical environmental concentrations. If file
values are found which are anomalous, the sampling and analytical
techniques must first be closely examined for faults. If the anomaly still
appears to be real, further investigation will obviously be required,
especially if the substance concerned is hazardous at elevated concentra-
tions. In the event that the anomaly is attributed to analytical or sampling
errors, the erroneous file data should be erased. A good example of a case
in which such anomalies can be assigned to analytical error are the
fluoride results in Table 3. The analytical method that was used is only
valid for media of low ionic strength. Since fluoride is a conservative
property of sea water, the true concentration at 30 parts per thousand
salinity should be about 1.15 nag/liter or half the listed values. This
discrepancy should have been detected much earlier. If the real value were
as high as given, the coastal waters of Prince Edward Island would be
unique.
Similarly, the mercury data in Table 5 are very suspect. In examining
the analytical and sampling procedures it comes to one's immediate
attention that the alphanumeric code on the method, " L " implies
laboratory analysis without sample preservation at the sampling site.
There is an abundance of evidence that mercury analyses carried out on
samples which are not meticulously collected and preserved are invalid. 6
Low Variability--In cases where little temporal variability is encoun-
tered for a parameter it constitutes sound sense to reduce its measurement
frequency. If there is sufficient interest in the parameter, attempts could
subsequently be made to develop a mathematical model for it. The savings
in time and effort should be used to expand the range of substances which
are being analyzed. Badly needed trace element analyses are often
neglected in favor of continuing the analysis of low variability major
elements.
Sediment Data
Data interpretation and corrective feedback should also be carried out
for chemical analyses of sediments. There are several factors, however,
which make these interpretations and evaluations more difficult. The
variability in mineral composition and texture of sediments make the
selection of "possible" or "usual" ranges impractical. There are a
bewildering number of techniques and methods for the analyses of silicate
in sediments, each of which may produce significantly different results. The
5Coyne. R. V. and Collins, J. A., AnalyticalChemist~. Vol. 44, 1972, pp. 1093-1096.
one advantage that the reviewer of sediment data may have over the
reviewer of water chemistry data is that of being able to refer to
well-established international standards (U.S. Geological Survey Standard
Rocks), provided that the analyst has reported his results in comparison
with analyses of these standards.
Table 6 may be used to illustrate several problems of reporting
analytical data for the chemical analyses of marine sediments. The four
TABLE 6--Analysis of sediment samples (ppm).
Sample Mg K Ca Na Cu Pb Zn Cd Hg
A 5 800 22 350 3 300 13 000 `5.0 31.0 <2.0 <1.0 6.1

B 13 600 27 850 7 400 24 000 17.0 31.0 <2.0 <1.0 2.6
C 9 000 19 850 $ 500 22 000 9.0 31.0 <2.0 <1.0 0.9
D 3 000 24 850 1 300 1,5 000 `5.0 31.0 <2.0 <1.0 0.5
samples were collected in a small bay in Prince Edward Island on the

same day from adjacent sample sites in approximately the same depth of
water. The analyses will presumably be incorporated in NAQUADAT.
The practice of reporting data to a greater number of significant figures
than is warranted by the analytical method is illustrated by the major
element analyses.
The variability in the reported method major element composition is
remarkable considering that the texture and mineralogy of the sediments
in this area should be quite constant. The apparent chemical variability is
illustrated by comparing the four magnesium results, which vary by more
than 400 percent. Similarly, the ratio of potassium to magnesium varies
from 2.05 to 8.38. This variability, if real, would indicate drastic
differences in mineralogy or texture or both. The apparent discrepancies
between analytical results and anticipated constancy of the sedimentolog-
ical environment could perhaps be resolved if the precision and accuracy
of the chemical data were known from replicate analyses and comparison
with analyses of standard silicates.
The minor element data illustrate several additional problems. Both
copper and lead results appear to be anomalously high. It is remarkable
that lead should be so constant at 31.0 ppm while copper values vary by
more than 360 percent. The reported mercury result of 6.1 ppm is clearly
anomalous and could represent gross contamination or a highly polluted
environment. This result demands verification by repeated sampling and
analysis. The consistent reporting of zinc and cadmium results below the
detection limit provides no useful information. In the report there were
208 analyses for zinc and cadmium, none of which were above the
detection limit.
Conclusions
There is a danger that persons receiving NAQUADAT data will accept
the data at face value. This could be quite embarrassing to the Water
Quality Division and Department of the Environment. NAQUADAT data
must be carefully evaluated before they are released to potential users.
The suggested procedure of data inspection is a first step towards
thorough data analysis. It should ensure that the data which are subse-
quently used for geochemical or environmental purposes are valid and
realistic. It is apparent that better feedback to the analytical and field
operations in the water quality program could be provided, and there is
an urgent need for more information on the precision of the analytical
methods. These suggested improvements would allow for greater diversity
in the range of parameters and elimination of redundancies in the entire
operation. Greater attention must be given to anomalies in the
NAQUADAT data. The ultimate goal is to improve data quality and to
optimize the costs of environmental monitoring programs.
J. A . W i n t e r j a n d H. A . C l e m e n t s I
Interlaboratory Study of the Cold

Vapor Technique for Total Mercury
in Water
REFERENCE: Winter, J. A. and Clements, H. A., "Intedaboratory Study of the Cold

Vapor Technique for Total Mercury in Water," Water Quality Parameters, ASTM STP
573, American Society for Testing and Materials, 1975, pp. 566-580.
ABSTRACT" The American Society for Testing and Materials (ASTM) and the U.S.
Environmental Protection Agency (EPA) conducted a joint study of the cold vapor
technique |'c~r total mercury in water, before formal acceptance of the method by each
organization. The method employs an aeid-permanganate-persulfate oxidation step with
heat.
Following Youden's design, samples were prepared in pairs as similar yet different
concentrates in sealed gla~s ampuls. Analysts added an aliquot of each to distilled water
and to a natural water of choice. Single analyses were made on each sample and
rccoveries comnared. Results from 90 analysts showed a positive bias of 53 to 99 percent
and a relative deviation of b7 to 79 percent at the m i n i m u m detectable level of 0.2
tag/liter. At the 0.5 tag/liter level, the bias was still positive but reduced to 18 to 32
percent whereas the relative deviation held at 55 to 80 percent. At the 3 to 10/ag/liter
Icvcl. the bias was small (--7 to + 4 percent) and the relative deviation was reduced to a
30 to 40 percent level. Natural waters exerted little effect on the precision or accuracy of
the method.
KEY WORDS: water quality, mercury (metal). environmental testing, statistical analy-
sis, water pollution
The U.S. Environmental Protection Agency (EPA) gathers water quality

data to determine compliance with water quality standards, to provide
information for planning of water resources development, to determine the
effectiveness of pollution abatement procedures, and to assist in research
activities. In a large measure, the success of the pollution control program
rests upon the reliability of the information provided by the data collection
activities.
To ensure the reliability of physical, chemical, biological, and microbio-
logical data, the Methods Development and Quality Assurance Research
1Method Development and Quality Assurance Research Laboratory, U.S. Environmental
Protection Agency, National Environmental Research Center, Cincinnati, Ohio 45268.
566

WINTER AND CLEMENTS ON TOTAL MERCURY IN WATER 567
Laboratory (MDQARD, formerly the Analytical Quality Control Laborato-

ry, was established in Cincinnati, Ohio, as part of the National Environ-
mental Research Center (NERC-Cincinnati). The quality assurance pro-
gram of this laboratory was designed to assure the reliability, and when
necessary, the legal defensibility of all water quality information collected
by EPA.
In MDQARL, the Quality Assurance and Laboratory Evaluation Branch
is responsible for conducting all interlaboratory method studies for water
and waste-water analyses in EPA.
Before this study, the chemists in EPA proposed a method of analysis
for total mercury (organic and inorganic) in water. 2 The method includes
an acid-permanganate persulfate digestion at 95~ for 2 h followed by
reduction and measurement by the cold vapor technique. The same basic
method was being considered also by Subcommittee D19.05 of Committee
D-19 on Water, American Society for Testing and Materials (ASTM).
Because chemists in EPA also play an important roll in the task groups
of ASTM Committee D-19, it was logical to propose a joint ASTM-EPA
study for total mercury in water. This report describes the joint study that
was conducted and its conclusions which include a statement of precision
and accuracy for the ASTM-EPA method.
Experimental
Test Design
Youden's nonreplicate technique '3t x and y samples ~ was used in a
test design as follows:
1. Each sample was prepared as a stable concentrate in a sealed glass
ampul.
2. Using Youden's x-y sample technique, similar yet different sample
concentrates were prepared in pairs.
3. When an aliquot of a concentrate was diluted to volume, mercury
was present at levels found in natural waters.
4. Four levels of mercury were tested to cover the normal levels found
in surface waters.
5. Each of the x-y sampte pair was added independently to a distilled
water and natural water of choice and the samples were analyzed.
The natural waters were analyzed with and without increments and
the added levels determined by difference. Recoveries from distilled
and natural waters were compared. Precision, accuracy, bias, and
interferences were determined.
2 Kopp, J. F., Longbottom, M. C., and Lobring, L. B., Journal o f the American Water
Works Association. Vol. 64, No. 1, 1972, pp. 20-25.
3Youden, W. J., Statistical Techniques for Collaborative Tests, Association of Official
Analytical Chemists, Inc., Washington, D.C., 1967.
Details o f the S t u d y
Eight water sample concentrates were prepared for this study by

dissolving weighed amounts of reagent-grade chemicals in ultrapure water
(distilled water polished by passage through a Millipore 4 cartridge Super
Q4 system) to produce accurately-known concentrations of organic and
inorganic mercury. All eight samples contained the same ratio of in-
organic to organic mercury, 40:60, as mercuric chloride and methyl
mercury chloride, respectively.
The distilled water--natural water spike technique was used in this
study. Each analyst was instructed to dilute a separate 5.0-ml aliquot of
each concentrate to 1 liter with distilled water and a river, lake, or
estuarine water of their choice. For each sample, a result was obtained
for: (1) distilled water plus increment, (2) the natural water, and (3) the
natural water plus increment. Recovery of the increment from natural
water was determined by difference.
The concentrates were preserved with 0.15 percent redistilled nitric
acid, and were checked for stability by repeated analyses over a period of
three months. These analyses established that the solutions were stable
and verified the concentration of each constituent. Further confirmation of
the true values was obtained from determination by an independent
referee laboratory. The true values are shown in Table 1. When diluted to
volume according to the instructions, the samples contained the con-
centrations of total mercury in ~g/liter.
Since dilution of the concentrates renders the acid ineffective as a
TABLE 1--True valuesfor total mercury, a

Mercury Ratio
Concentration Inorganic/Organic
Ampul t~g/liter Mercury
1 0.21 40160
2 0.27 40160
3 0.51 40160
4 0.60 40160
5 3.4 40160
6 4.1 40160
7 8.8 40/60
8 9.6 40/60
a The concentrations given are the actual levels calculated and

added. These are not based on analyses, the latter being used only
for verification.
4Mention of trade names or commercial products does not constitute endorsement by the
EPA or ASTM.
preservative, the analyst was instructed to add 1.5 ml of redistilled nitric

acid/liter during sample preparation.
Study Logistics and Methodology

The mercury study was announced in an invitational memorandum sent
to each EPA regional Analytical Quality Control (AQC) Coordinator
(regional representative for quality control) in September 1972. Further,
the study was announced in EPA's AQC Newsletter which is distributed to
about 7000 laboratories and technical offices of governmental and private
agencies throughout the United States and Canada.
Seventy-six laboratories from EPA, Canada, other federal, state, and
local agencies, universities, and the private sector took part. After a
cut-off date of October 23 for responses, samples were packed and
shipped on 30 Oct. 1972. Each laboratory was sent one set of eight sample
concentrates, instructions for sample preparation, a copy of the full
method writeup, and duplicate report sheets. The final date for reporting
data was 20 Dec. 1972. All data returned to MDQARL by that date are
included in this report.
The method developed by ASTM and EPA and evaluated in this study
includes an acid-permanganate-persulfate oxidation procedure and heat,
before reduction, to ensure conversion of all organic mercury compounds
to ionic form before measurement. Kopp et al ~ described the method.
In the present study, mercury was measured by atomic absorption spectro-
photometers, ultraviolet/visible spectrophotometers, and meters such as
the Coleman MAS-S0 specificially designed for measurement of mercury.
Results
Screening for Nonuseable Data

Because the mercury study was done at low to fractional microgram/
liter levels, some of the data received were orders of magnitude away from
the true values. It was necessary to screen out these completely-extraneous
values before beginning any data evaluations. If this was not done first,
the standard deviation values calculated from these data would be
excessively large and would misleadingly indicate large deviations for the
method. Further, the rejection of outliers using the t test, which includes
the standard deviation in its calculation, would be unable to identify
outliers because of the overwhelming effect of the excessively large
standard deviation values in the denominator of the t test.
With the need to reject the meaningless values before any data
evaluation we chose to screen out values that exceeded four standard
deviations. With normal distribution, this mechanism yields only six
chances out of 100 000 that a valid data point is being rejected. This
probability is sufficiently small to allow use of this screening device before
full data treatment.
Rejection o f Outliers
To determine the accuracy of the analytical method, it was necessary to

remove those extreme values that had only a small chance of validity and
that would significantly change the reported precision and accuracy
values. These values were probably caused by gross instrumental,
chemical, or human error and were rejected by applying the two-tail t test
to all values at a 99 percent probability level; that is, with a 99 to 1
assurance that the data rejected were invalid and should be rejected.
A greater spread of data round the true value causes rejection of fewer
outliers because a larger standard deviation in the denominator of the t
test reduces the calculated t value and fewer extreme values are rejected as
outliers. With better accuracy and precision, the t test is more powerful
and more outliers are rejected. However, the data rejected in either case
are true outliers and laboratories reporting such values should carefully
review procedures for the cause of inaccuracy.
Statistical S u m m a r i e s
Statistical summaries are given in Tables 2 thru 5. Each sample is

discussed in turn, with the data displayed by type of water sample and
method of analysis. A statistical evaluation is provided for each concentra-
tion with each set of sample-test conditions by type of water sample.
All statistical parameters: mean, range, accuracy, standard deviation,
and relative deviation are based on retained data, that is, the data
remaining after rejection of outliers.
TABLE 2--Statistical summary of recovery of total mercuryfrom distilled and

natural waters.
Sample t Sample 2
Distilled NaturalWater Distilled NaturalWater
Statistic Water by Difference Water by Difference
True value,/ag/liter 0.21 0.21 0.27 0.27
Mean recovery,/ag/liter 0.418 0.349 0.450 0.414
Accuracy as %
relative error (bias) 98.9 66.4 66.5 53,3
Standard deviation, ~g/liter 0.279 0.276 0.325 0.279
Relative deviation, % 66.9 78.9 72.2 67.5
Range, ~g/liter 1.60 1.27 1.80 1.20
TABLE 3--Statistical summary of recovery o f total mercury from distilled and

natural waters.
Sample 3 Sample 4
Distilled Natural Water Distilled Natural Water

True value, tag/liter 0.51 0,51 0.60 0.60

Mean recovery, tag/liter 0.653 0.674 0.744 0.709
Accuracy as %
relative error (bias) 28.1 32.1 24.0 18.1
Standard deviation, tag/liter 0.376 0.541 0.466 0.390
Range, tag/liter 2.26 3.99 2.97 2.30
TABLE 4---Statistical summary of recovery of total mercury from distilled and

natural waters.
Sample 5 Sample 6

True Value, ~g/liter 3.4 3.4 4.1 4.1

Accuracy as %
relative error (bias) 0.03 0.34 3.85 --7.11
Range. t~g/liter 11.1 12.4 10.4 6.3
TABLE S---Statistical summary of recovery of total mercury.from distilled and

natural waters.
Sample 7 Sample 8

True value, ~g/liter 8.8 8.8 9.6 9.6

Accuracy as %
relative error (bias) --3.7 --0.4 --2.3 --5.2
Range, tag/liter 19.4 27.5 24.4 25.8
572 WATER Q U A L I T Y P A R A M E T E R S
Two-Sample Charts
To effectively describe the recovery values obtained for each parameter,
a series of two-sample charts, Figs. 1 to 8, were prepared as suggested by
Youden. 3 Results were plotted by parameter and by water sample type.
Two results for each set of similar samples (1 and 2), (3 and 4), (5 and 6),
and (7 and 8) were the coordinates on the x and y axes that determined a
single point for each analyst. The distribution of points (analysts)
characterized the method-results for that sample. However, the plotting of
the statistically acceptable data for each sample pair resulted in severe
crowding of data points < to the true values, because of the large number
of values greater than the true values. To expand the presentation of the
data ~<to the true value, plot scales were selected to place the true value at
least one-third of the distance from the origin. This general rule proved
acceptable and was used on all data plots. With its use, data points were
distributed evenly over each plot to show a truly representative picture of
method performance. Extreme values which characterize a small segment
of the data were represented as > values in the extreme upper right
quadrant.
If random error was primarily responsible for the deviation of results
from the true values, then the data from the sample pair would be equally
distributed among the four quadrants ( + + ) ( - - + ) (----) ( + - - ) . How-
~IO~A~ ~ E ~ P E R LITFJ~ XXXX ( > 0 . 6 )

O.G i XXX
OISTILLEO WATER
0.5 X ~OKxX
x
X x
W
0.4 x
x x x
0.3 x ~x x
X
XX
0.2 X X X
O.1 X
X
(3.0 .~_E i
0.0 O.i 0.2 0.3 0.4 0.5 ).G
FIG. 1--Two-sample chart for total mercury, Samples 1 and 2.

WINTER AND CLEMENT$ ON TOTAL MERCURY IN WATER 573
MICR[]3~A~4S M E R O J R Y P E R LITER xxxxx (,0.6)

0.6 m i ,
J D(xxx
NATURAL WAT~:~
X
0.5 X X X x
X
~nJ x
X
0.4 X X
x x x
x
x
0.3 x x
x
7
x
~x x
x
x
x
0.2 x x ~ x
x
x x x
x
0.1 x
x
0.0 SAIW=UEI
0.0 0.I 0.2 0.3 0.4 0.5 O.G
FIG. 2--Two-sample chart for total mercury, Samples 1 and 2.
MIL~AMS IvE'I:;~L-L~Y PER LITER XXXXX (,I,2)

1.2 , ~ I I !
OISTILLEO WATER X
X
i-O X X
xXX
X
x
0.8 X X X
x
>x xX
O,G
xXx~x xx ~ x
x XX
0.4 X
X x
X
0.2 X
X X x x
0.0 X I /
),0 0,2 0.4 0,6 0,8 :1.',0 1,2
FIG. 3---Two-sample chart for total mercury, Samples 3 and 4.

MIC3~[Z3RAM5 M E R [ ~ Y PER LITER xxxx ('13)

.t.2 , XXX
NATURAL WATER
X
x
1.0 X X X
X x
X X
bJ
0-8 x x
x
~x x
0.6
xX
X Xx IN
XxX
0.4 X X X
XX
X X
X
0.2
0.%~0 SAMPLE 3
0.2 0.4 0.6 0.8 l.O 1.2
FIG. 4---Two-sample chart for total mercury. Samples 3 and 4.
MZCI~Q(3~AMS ~ Y PER LITER x (,8,0)

8.0
OISTILLEO VC&T E R
x
6.0 x
W x
Ld x x
x
x x x xX
x
X
4.0
xX~ X
2.0 X
X
x
x
x
0.0 x SAW~ 5
0.0 2.0 41.0 6.0 8.0
FIG. S---Two-sample chart for total mercury, Samples 5 and 6.

MI~Ab~3 bERELRY ~ LITER X (>8.0)

8.0
NATURAL WATER
G-O
CD x
W X x
x
X x x X
x x X
4.0 X X"~
XX
X
x
x x
2.0
x
x
x x
0.0 x SAMPLE 5
04 2.0 ,4.0 6.0 1.0
F I G . 6--Two-sample chart for total mercury, Samples 5 and 6.
MICROGRAMS ~ Y PER LITER X (:'20)

2 0 .C i v i
OISTILLEO WATER
J-g 9C
bJ X
X X
Xxx x
iR .( xx x
xxXxx
XX x
..,;~ x x
x~ ,~•
8.0 X ~)4 X
xx
4.0 Xx
x
0.0 x SAMPLE 7
I
0.0 41.0 B.O IR.O I-6.0 20,0
F I G . 7--Two-sample chart tbr total mercury. Samples 7 and 8.

MI~_~U_~A~ ~ PE~ LITER xx (='20)

20.Q
NA'I"I~ WATER
X
.1.6.Q
W x
X
X
X x
.I.2 9Q >K yx
x XX x X X
X
8.0 X
X
XX
4.0 Xx
X x
x
X
0.0 x ~q~=l_E 7
I I I
0.0 4.0 8.0 s 0 s 20.0
FIG. 8---Two-sample chart for total mercury, Samples 7 and 8.
ever, this did not occur. Systematic error influenced the data plots most,
and the distribution of values was not random but on a 45 ~ slope line in
the ( - - - - ) and ( + + ) quadrants, since an analyst obtaining a high or low
value on one sample also got a high or low value on the similar sample.
This distribution of values on a 45 ~ slope forms an elliptical pattern. D a t a
points far removed from the elliptical cluster indicate a large systematic
error for one analyst compared with the other analysts. As random error
decreases, data points move closer to the 45 ~ slope. Extreme values or
outliers suggest a procedure or instrument out of control. General scatter
of data points away from the 45 ~ line indicates poor precision in the
method. When all data are spread far out along the 45 ~ line, a large
systematic error is indicated for the method. When precision is satisfactory
but a significant bias or interference occurs, the data grouping is low
( - - - - ) or high ( + + ) .
Single Laboratory Precision

In Tables 2 thru 5, the precision measured as standard deviation
represents the deviation expected among values received from a number of
laboratories. This between-laboratory error is important. It represents the
broad error that operates in any mass of data collected from a number of
laboratories.
However, a n o t h e r m e a s u r e o f precision i m p o r t a n t to i n d i v i d u a l l a b o r a -
tories a n d analysts is the m e a s u r e m e n t of how well an i n d i v i d u a l a n a l y s t
can e x p e c t to p e r f o r m in his own l a b o r a t o r y . This within laboratory
pe~brmance is m e a s u r e d here as the sr value. It is used in T a b l e 6 to
c a l c u l a t e the single a n a l y s t 9 5 % Confidence Interval (95% C . I . ) for each
set o f s a m p l e s in distilled a n d n a t u r a l waters. It was defined by Y o u d e n 3
as
(Di - D) 2
sr = 2(n- 1)
where
(n - 1) = degrees o f f r e e d o m ,
D! = difference between values for s a m p l e pair, X j and Yi, a n d
D : X - Y o f a series o f X a n d Y values.
T h e f o r m u l a m e a s u r e s precision w i t h o u t d u p l i c a t i o n a n d hopefully
avoids s o m e o f t h e well-intentioned d a t a a d j u s t m e n t t h a t can occur in any
l a b o r a t o r y d o i n g r e p l i c a t e analyses.
TABLE 6---Single analyst precision valuesfor mercury analyses.
95% C.I.
Sample Water Type Level/~g/liter (X _+ 1.96s)
1 and 2 distilled 0.21-0.27 0.42 • 0.38

natural 0.21-0.27 0.38 _ 0.32
3 and 4 distilled 0.51-0.60 0.69 + 0.72
natural 0.51-0.60 0.71 • 0.32
5 and 6 distilled 3.4 -4.1 3.77 • 1.55
natural 3.4 -4.1 3.51 • 0.72
7 and 8 distilled 8.8 -9.6 8.92 • 1.74
natural 8.8 -9.6 9.01 • 2.74
Precision Statements
L i n e a r regression analyses were c o m p l e t e d on t h e statistical s u m m a r y
d a t a for m e r c u r y analyses in distilled a n d n a t u r a l waters. T h e results o f
these analyses a r e shown in Figs. 9 a n d 10. T h e precision s t a t e m e n t s for
t o t a l m e r c u r y analyses in distilled a n d n a t u r a l waters in the 0.2 to 10
/~g/liter were c a l c u l a t e d as follows: in distilled water, s t a n d a r d devia-
tion --- 0.1828 + 0.3071 X - ' m e a n ) ; and in n a t u r a l water, s t a n d a r d
deviation = 0.1071 + 0 . 3 8 5 5 X (mean).
Y AXIS
The precision of this method for total

mercury in distilled water samples
within the range of concentration of
0.2 - I0 ug/liter may be expressed as
follows:
4.
s = 0.1828 + 0.307L~
I ~ 3. .........."
~i "" +
~ 2.
~d : ...~"
~i ~"
' 1.
#~....""
-:~ ........ :; ........ :; ....... :'; ........ ~ ......... ~: ....... -2: ....... ,':........ ;: ..... :--:x,,
X Recovery of Mercury, ug/llter
~-i
FIG 9--Precision statement f o r analysis o f total mercury in distilled water.
Y AXIS
The precision of this method for total

mercury in ~ samples
~thin the range of concent rat i on of
0,2 - i0 ug/liter may be expressed as
follow:
s = 0.1071 + 0 . 3 8 5 ~
+..r
..~176176
3.
oe00~l~ee
...**"
J .~
'$ 2.
..,...'*'"
o~
.~
..~ §
,~ .....""
, + ~
i 4e.~.'*
._A . . . . . . . . . A. . . . . . . . . A. . . . . . . . . A. . . . . . . . . x L A A
-4 -3 -2 -1 2. ~.. 6. 8. X AXIS
R e c o v e r y os MercurT, u g / l i t e ~
FIG lO--Precisioa statement /br analysis o f total mercury in natural water.

Discussion
The low levels of 0.21 to 0.27 /ag/liter total mercury tested here
approach the minimum detectable level. The calculated 53 to 98 percent
positive bias and a 67 to 79 percent relative deviation are shown
graphically in Figs. 1 and 2. Since the measured level is such a small
increase above background, any variation in measurement of peak heights
will produce fairly large deviations. It is also notable that the water type
(distilled versus natural) had relatively little effect on the method. The
positive bias is expected because the analyst reporting 0.2 /ag as a
minimum would report 0, 0.2, or >0.2 values. Hence, positive measure-
ments are automatically 0.2 or more and just as automatically produce a
positive bias in the method.
At 0.51 to 0.60 /ag/liter levels of total mercury, somewhat above the
minimum detectable level, the positive bias in the method is reduced to 18
to 32 percent. This improved bias is evident in Figs. 3 and 4. However,
the relative deviation of the data is still at 55 to 80 percent. As with
Ampuls 1 and 2, the water type had no particular effect on the results.
At the 3.4 to 4.1 /ag/liter level of total mercury, the bias of the method
is sharply reduced and is variable, ranging from - 7 to + 3 . 9 percent.
Likewise at this level, the relative deviation is reduced, ranging from 29 to
44 percent. Water type produced no apparent effect on the data. The
improved precision and bias, which are obvious in Figs. 5 and 6, sharply
contrast the data distribution in Figs. 1 to 4.
At the 8 to 10/ag/liter levels of total mercury, the bias of the method is
small and negative, 0.4 to 5 percent, with a relative deviation of 31 to 42
percent. Figures 7 and 8 sfiow data distributions similar to those at the 3
to 4/ag/liter levels, but with a somewhat greater deviation in natural water
than in distilled water.
Conclusions
An interlaboratory study was conducted with 90 analysts in 76 labora-
tories. A series of eight samples in pairs at similar yet different concen-
trations in the 0.2 to 10 gg/liter range were added to distilled water and
natural water of choice by each laboratory. Samples were analyzed using
the EPA-ASTM method for total mercury. Precision and accuracy state-
ments, two sample data charts, and single laboratory precision measures
were determined on each sample for distilled and natural waters.
The most significant factor in analyses in the 0.2 to 10 /ag/liter range is
the concentration of mercury itself. At the lowest levels of 0.2 /ag/liter
which is also taken as the detection limit, the data show a large positive
bias of 53 to 99 percent. Similarly, the relative deviations range from 66 to
78 percent of the mean values reported.
As expected, accuracy and precision improve and remain improved as
concentrations increase. Further, as the level of mercury increases, we

begin to see natural water samples having a slightly greater standard
deviation.
The single analyst precision measures (st) are expected to be somewhat
smaller than the standard deviations generated for the total data mass. In
this study, the Sr values were smaller, relative to concentrations, with sr
values about one half of the total standard deviation at <1 #g/liter levels
of mercury. As concentrations increased to mercury levels of 3 to 9
/~g/liter, the sr values improved--one half to one fourth of the total
precision both in natural and distilled waters.
The precis!on of the A S T M / E P A method of analysis for total mercury
in natural waters is linear within the range tested as follows
standard deviation ---- 0.1071 + 0.3855 X (mean)
Acknowledgments
The authors wish to thank Tom Bennett, Jr., Mercury Task Group
Chairman of ASTM Committee D19.05, and Elmo Julian and John Kopp
of the Methods Development and Quality Assurance Research Laboratory,
NERC-Cincinnati, USEPA for their cooperation and assistance in this
study.

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WATER QUALITY PARAMETERS

ASTM SPECIAL TECHNICAL PUBLICATION 573

List price $29.50

qSTAMERICAN SOCIETY FOR TESTING AND MATERIALS

Printed in Baltimore. Md.

Second Printing, 1979

Microorganic Matter in Water, STP 448 (1969) $9.25, 04-448000-16

Manual on Water, STP 442 (1969) $16.50, 04-442000-16

Asbestos Fibers in Lake Superior--R. w. DUarIAM AND T. PANG 5

On 19 to 21 November 1973, the Symposium on Water Quality Para-

Copyright9 1975 by ASTM International www.astm.org

Acknowledgment is due to the session chairmen of the symposium for

Asbestos Fibers in Lake Superior

REFERENCE: Durham, R. W. and Pang, T., "Asbestos Fibers in Lake Superior,"

Asbestos is a commercial product derived from several minerals of the

Copyright9 1975by ASTMInternational www.astm.org

similar in size and appearance to a standard sample of chrysotile and

ultrasonically in 1 ml of distilled water without further treatment because

FIG. 1--Lake Superior showing locations of sample stations.

FIG. 2--Chrysotile fibers in water sample from near Isle Royale.

TABLE 1--Asbestos fiber concentrations in Lake Superior.

103a 203 ,50 to 15 87.3

aCruise 103, 15-28 June.

FIG. 3---Cummingtonite fibers in water sample near Silver Bay.

tiffed this fiber as cummingtonite by comparing its electron diffraction

filtered. It is unlikely, however, that a standard water filtration system

REFERENCE: Armstrong, F. A. J., "Spectrophotometrie Determination of Sulfide in

KEY WORDS. water quality, spectrophotometry, sulfide minerals, environmental tests

Hydrogen sulfide is highly objectionable a n d is toxic to m a n a n d to fish.

Copyright9 1975by ASTMInternational www.astm.org

TABLE l--Typical concentration rangefor determination of sulfide.

In the course of some unrelated investigations it was noticed that

R e a g e n t s - - S o d i u m sulfide approximately 0.1 M. Crystals of the hydrate

Results and Discussion

FIG. l~Absorption spectrum of sodium aulfide.

much weaker maximum at 189 nm. If the absorption of solutions is

FIG. 2--Change o f absorbance o f sulfide solution (10-4 M S 2-) with pH.

FIG. 3----Change of absorbance o f sulfide solution (I0 -4 M Na2S) with time.

photometer and pumping solutions through a flow cuvette with a peri-

Difficulty with this determination may be expected if much alkali must

FIG. 4--A bsorption spectra of natural waters.

nm can be appreciable, and of course must not change significantly when

TABLE 2--Comparison of methods for sulfide in water.

1.37 1.37 0.00

REFERENCE: Barica, J., "Electrochemical Detection of NH,*-NH3 Systems in Water,"

KEY WORDS: water quality, nitrogen inorganic compounds, ammonia, environmental

Total a m m o n i a = ionized N H { + unionized N H 3

Research scientist, Department of the Environment, Freshwater Institute, Winnipeg R3T

LC 50 [113 The concentration of free unionized ammonia depends on the

1. the ammonium ion (NH4§ using a univalent (monovalent) cation

In this paper a summary of the aforementioned approaches is pre-

Determination of Ionized NH4 + with a Univalent Cation Electrode

The univalent cation electrode (Corning monovalent cation electrode

than 3 mg/liter NH4+-N the method of standard known addition can be

A calculation using the ionization constants [2] shows that ammonium

Determination of Total A m m o n i a with an A m m o n i a Probe

Direct Determination of Free Unionized A m m o n i a

Determination of Total Mercury

REFERENCE: Nishi, Sueo, and Horimoto, Yoshiyuki, "Determination of Total Mer-

ABSTRACT: A rapid and simple preconcentration technique for the determination of

Copyright9 1975 by ASTM International www.astm.org

MV 253, equipped with a 300-mm long, 30-mm internal diameter optical

Preparation of GoM Trap

Results and Discussion