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Review Paper Measurement of Pollutant Toxicity To Fish I. Bioassay Methods For Acute Toxicity
Review Paper Measurement of Pollutant Toxicity To Fish I. Bioassay Methods For Acute Toxicity
REVIEW PAPER
J. B. SPRAGUE
Fisheries Research Board of Canada, Biological Station, St. Andrews, New Brunswick, Canada
Abstract-The review describes profitable methods for measuring lethal levels of pollutants
for aquatic organisms. Methods for research in the laboratory are emphasized but the same
principles could be applied in field work. Greater use of standard toxicological methods and
terminology is urged.
For 211 out of 375 toxicity tests reviewed, acute lethal action apparently ceased within 4 days,
although this tabulation may have been biased towards short times by a large number of static
tests.
The incipient LCSO (lethal concentration for 50 per cent of individuals on long exposure) is
recommended as the most useful single criterion of toxicity. If this cannot be estimated, the
Cday LC50 is a useful substitute, and often its equivalent.
A desirable first step in toxicity tests is to estimate median lethal time for each of a series of
concentrations. A toxicity curve should be drawn by plotting median survival times against
concentrations on logarithmic paper. The curve helps to reveal any unusual features of toxicity.
Whenever possible, tests should be prolonged until the toxicity curve becomes parallel to the
time axis, indicating a lethal threshold concentration. The incipient LC50 is then estimated by
selecting an exposure time from the asymptotic part of the toxicity curve; for this exposure
time, observed mortality is plotted against concentration on log-probit paper, and the LC50 is
read from an eye-fitted line. Confidence limits of the LC50 may also be estimated by simplified
methods. These should be given in published work along with a value for slope of the probit
line.
Alternativeapproaches usereciprocal transformations or estimate LCSO’s for a series of expos-
ure times.
A graph is given for estimating partial replacement times of water in tanks of continuous-
flow tests. Rate of flow should give a short partial replacement time, such as a flow equal to
volume in 3-5 hr, and also adequate water for respiration of fish, usually 2 or 3 l/g of fish/day,
or more.
INTRODUCTION
THIS REVIEW is intended for students or scientists entering the field of toxicity tests with
aquatic organisms. It outlines profitable bioassay methods. Later parts (II and III)
will cover associated topics, especially those which relate lethal tests to field problems,
and to “safe” levels of pollutants. This is intended to be a critical review. Favourable
reactions of the reviewer are implied by inclusion of methods and by the amount of
space devoted to them. Direct recommendations are sometimes made, and no doubt
there is room for healthy disagreement on these.
Urgency of water pollution problems is leading us toward official “water quality
criteria” for aquatic life, notably in U.S.A. but also in Europe (U.S. National Tech-
nical Advisory Committee, 1968 ; European Inland Fisheries Advisory Commission,
793
794 J, B SPRAGUE
1965). Information needed for protection of aquatic life has been eloquently described
by TARZWELL(1962a, 1962b, 1966), and toxicity tests provide some of the required
answers. Because of the urgency, such tests should use efficient methods which yield
meaningful results. Effective methods are also appropriate since today's bioasSays
may be costly in automatic equipment or in weeks of laboratory time. It seems only
sensible to analyze expensive experiments by techniques which gather maximal
information.
The need for good methods is further documented in every review of toxic limits for
aquatic life. The investigator looking up a toxicant in the compendium of MCKEE and
WOLFE (1963) should expect a list of "lethal levels" which vary a thousand-fold, and
seldom will he be disappointed. It is time to organize the reasons for such variation.
This requires perceptive research using the most revealing methods.
It is unfortunate that pollution biologists have tended to form a splinter group as
far as toxicology is concerned. Standard techniques of analysis, developed by phar-
macologists and statisticians for testing drugs, have too often been ignored. Successful
adpatations of these methods will be cited frequently.
This review deals primarily with methods for research in the laboratory. However
the same general principles may also be applied to bioassays for management or
control purposes, by industry or government, using aquaria or cages offish in a river.
Similarly most examples are for freshwater fish but there is little difference in basic
approach with marine organisms. We should avoid erecting unnecessary barriers
between the categories above, and between tests with vertebrates and invertebrates,
or lethal and sublethal tests. Examples in this review often cross such lines of distinc-
tion.
The literature contains excellent reviews parallel or complementary to this one.
HERBERT(1965) and EDWARDSand BROWN(1967) discuss the methods used so effect-
ively by the British Water Pollution Research Laboratory. Also highly recommended
are the reviews by ALDERDICE(1967), BURDICK (1967), WARNER (1967), and BEAK
(1958). From Europe, the writing of WtrI-mMANN(1952) remains classic, ROTHSCHEIN
(1964) analyzes bioassay method critically, while BREITIG(1966) discusses standardized
procedures. Among books, that of MARCHETTI(1962) gives a unique major treatment
of aquatic bioassays, JONES (1964) includes much earlier work, while HYNES (1960)
evaluates laboratory tests somewhat skeptically from an ecological viewpoint.
Dealing with bioassays, but not particularly fisheries, the review by GADDUM(1953)
is still a most easily read and informative discussion of methods and is highly recom-
mended. Types of response and analyses are given by BLISS (1957) and BLISS and
CATTELL(1943). FINNEY'Sbook (1964) gives considerable detail of statistical methods.
The present review has benefited greatly from organization and outlook of these and
other works.
MECHANICS OF SETTING UP TESTS
It is encouraging that most investigators now use very similar methods for acute
lethal tests with fish. Furthermore, the methods are advantageous compared to the
variety of approaches which could have been taken, and sometimes were taken in the
first half of the century. Almost without exception, present-day tests incorporate:
(1) a series of test-containers, each with a different but constant concentration of the
toxicant;
Measurement of Pollutant Toxicityto Fish. Part I 795
(2) a group of similar fish, usually ten, in each container;
(3) observations on fish mortality during exposures which last between 1 day and
about 1 week; and
(4) final results expressed as concentration tolerated by the median or "average"
fish.
Given this similar general approach, smaller variations in technique will be com-
pared in this review. Broadly speaking there are two procedures in current use. In the
first, approximate mortality-times are recorded for most individual fish. The time
taken to obtain 50 per cent mortality is estimated for each test-tank. The series of
median lethal times is generally used to estimate an approximate threshold concentra-
tion for lethal effect. This has been used with notable success in British studies of
pollutants, and in studies of lethal temperatures of fish by F. E. J. Fry and associates
in Canada. In the second procedure, customarily used in the U.S.A., mortality is record-
ed only at 1, 2, and 4 days. The concentration lethal to half the fish in each of these
time periods is estimated. The first procedure has more complete observations, and
hence will also provide the answers yielded by the second procedure. The first will
be the standard technique described below, and the second will be discussed separately.
However the two procedures have tended to yield similar estimates of acute toxicity in
recent years when exposure-times of 4 days or longer have become usual.
Many aspects of methodology which have been well discussed by others will be
omitted. These include species, number, size, and condition of fish, kind of dilution
water, temperature and the myriad other conditions of testing. Researchers are urged
to consider the recommendations of DOUDOROFFand his committee (1951) or APHAet al.
(1965), which are clear, sensible, and have stood the test of time. Especially valuable
are the sections on care and handling of fish, experimental concentrations based on
logarithmic bisection, preparation of test animals, diluent, and "other prescribed
conditions". MARCHETTI'S book (1962) is also recommended. ROTHSCHEIN'S(1964)
critical review lists ten common deficiencies of bioassays, with remedies for some.
A few special aspects of test procedure are mentioned below because they are con-
tinuing problems or because of recent progress.
Today the problems may be avoided for continuous-flow tests, by following good
practice such as the suggestions of ALABASTERand ABRA~ (1965). They recommend
that the supply of new test solution should be sufficient to maintain dissolved oxygen
in the test tank. This also keeps toxicant and waste products within desirable limits.
The extreme values which they mention for required amount of replacement solution
are 0.5 and 10 1. per gram of fish per day, the lower values sometimes involving
artificial aeration. Alabaster and Abram chose 10 1/g/day as satisfactory for harlequin
fish, which are small and have relatively high respiratory needs per unit of weight.
Calculations from their data suggest that about 2 or 3 l/g/day, preferably the latter,
would probably be satisfactory in tests with trout. For 3 1/g/day and an oxygen con-
sumption of 0.16 mg/g/hr, used by Alabaster and Abram, this would mean that trout
would use about 1.3 mg of oxygen for each liter of incoming water, so that dissolved
oxygen levels would not be greatly depressed.
When possible, static tests should aim at the same value of 2 or 3 1. of solution per
gram offish, changed daily. This is not too far removed from the preference expressed
in the APHA et al. (1965) method for at least 1 1/g fish. For very small tropical fish such
as the harlequin fish, higher values equivalent to the 10 I/gm of Alabaster and Abram,
might be desirable.
Another useful tool in continuous-flow tests is to estimate the amount of time
required to replace the body of test water. Sometimes the "replacement time" is
published according to the naive calculation: volume of solution divided by flow per
unit time. This would be the time required to fill an empty tank or to empty a full one,
but it certainly is not a reasonable estimate of displacement time of molecules in the
test tank. This may be estimated approximately by using FIG. 1 which is based on
calculations by A. A. HEUSNER (personal communication). It may be compared to
displacement time in lakes, the mathematics of which are discussed by RAINEY(1967).
Many toxicants decrease in concentration with time, apparently by sorption on walls
of the test container, etc. Therefore, when deciding flow rates in holding tanks or test
tanks, even with artificial aeration, I have considered it a reasonable guide to try to
keep the 90 per cent replacement time down to about one-third or one-half of a day,
i.e. about 8-12 hr. This means that flow would equal the test-volume in about 3-5 hr
(FIG. 1).
Such replacement times may be attained by increasing flow or by reducing test
volume, within reasonable limits of each. It was mentioned in the first paragraph of
this section that the volume of solution should be enough for free movement of fish.
The suggested replacement time may be considered to be in general agreement with
the recommendations of ALABASTERand ABRAM (1965). For example, 90 per cent
replacement in 8 hr of 30 1. test-solution containing 100 g of trout, works out to a
replacement of 2 I/g/day.
The forthcoming revision of the APHA et aL (1965) method will recommend a
minimum flow rate which equals the volume of water in the tank in 6 hr (Q. H.
PICKERING,personal communication). This is not much longer than equalling the
volume in 3-5 hr as suggested above. The APHA minimum would mean 90 per cent
replacement of water in about 15 hr, which seems fairly reasonable. However, the
APHA revision will further recommend that flow rates be increased if necessary, until
satisfactory levels of dissolved oxygen are maintained. As a basis for calculation, an
oxygen uptake by fish of 0.2 mg O2/g fish/hr will be suggested, which is in line with the
Measurement of Pollutant Toxicity to Fish. Part I 797
value of Alabaster and Abram. The recommended increased flow could thus corre-
spond with the suggestions in this section, depending on the weight of fish used.
To summarize, continuous-flow tests should allow for both (a) a reasonable rate of
flow of test solution per gram of fish, probably 2 or 3 1/g/day or higher for small fish,
and (b) fairly rapid replacement-times of water, perhaps 90% replacement in some-
thing like 8-12 hr.
IO n
c
,~ 2
E
o
.=
I
-6
no
FIG. 1. Approximate times required for partical replacement of water in tanks, for constant-
flow situations. Example: for a tank containing 30 l., with a flow of 10 I. per hour, there would
be 50 per cent replacement of water in the tank in about 2 hr, 75 per cent in about 4 hr, 90 per
cent in 7 hr, and 95 per cent replacement in 9 hr. Another time-period could replace hours, but
the same time must be used on each axis, and the same unit of capacity must beused for volume
and flow. Based on information supplied by Alfred Heusner.
Dosing apparatus
Happily, recent years have seen the advent of a number of mechanical devices for
regulating flow of test water and/or toxicant. Although electric pumps, especially
persistaltic tubing pumps, would seem suitable, most investigators have apparently
concluded that they are too undependable or expensive. Most new devices are home-
made. MARCHETTI (1962) covers the earlier systems and illustrates the best ones so
these will not be repeated. Since then, STARK(1967) has described a desirable-looking
dosing apparatus which operated for 24 weeks within 5 per cent error. It is based on
earlier ones of ABRAM (1960) and GP,ENIZR (1960). All three have the advantage of
"failing safe" because the flow of dilution water controls input of toxicant; if water
slows down, fish are not killed by overdose of toxicant. MOUNT and BRUlqGS (1967)
describe a "proportional diluter" simplified from an earlier serial diluter (Moutqr and
798 J. ]3. SPRAGUE
WARNER, 1965). Both have long-term dependability and fail-safe characteristics, but
successive test concentrations are somewhat dependent on each other in magnitude.*
Other designs are simpler but do not fail safely (BURKEand FERGUSON, 1968), or offer
little advantage over a simple Mariotte bottle (SoLoN et aL, 1968).
Certain specialized pieces of apparatus have been described. CHADWICK and
KnGEMAGI(1968) succeeded in dosing relatively insoluble insecticides by first sorbing
them on a sand column. MOUNT and BRUNGS(1967) inject them as a slurry. A closed
continuous-flow apparatus for small fish is shown by ALABASTERand ABRAM(1965).
Recirculation of test water makes fish swim and would be suitable for static or con-
tinuous-flow bioassays (BETrS et al., 1967). ALOEROICEet aL (1966) describe a simple
tank, suitable for holding or testing, which is self-cleaning and makes fish swim
continuously.
Acclimation and holding
Acclimation of fish to water and general test conditions remains poorly known. "At
least a week" of acclimation is sometimes suggested on little factual basis. The topic
may often be of direct importance for bioassay results. LLOYD(1965) states that trout
transferred from hard to soft water required at least 5 days of acclimation, before their
response to a toxic metal became the same as the reponse of fish held permanently in
soft water. Similarly, Motr~T (1966) mentions that complete acclimation to water
hardness may not have been achieved in his tests with metal at various levels.
Probably more is known about acclimation by fish to temperature than to any other
entity. Even here the knowledge is very incomplete, and has been gained mostly from
responses of fish to lethal temperatures. Judging by this response, change in acclim-
ation state is particularly slow for downward acclimation to low temperatures (BRETT,
1956). However, the rule of thumb sometimes used in such lethal temperature work, of
"at least 1 day per Centigrade degree of change" has no particular scientific basis
and may sometimes contradict the facts (e.g. DOUDOROFF, 1942).
Of much greater interest in toxicity work is metabolic acclimation which could be
different from rate of acclimation as judged by lethal temperatures. The state of
acclimation of metabolism could determine rates of detoxification and other processes
governing survival in a pollutant. On this topic, PEa'ERSOr~and ANDERSON(1969) have
recently concluded that complete acclimation of metabolism of a fish to a temperature
change requires about 2 weeks, regardless of direction of change.
Much more information of this kind is needed on acclimation before tests. Mean-
while the investigator should allow generous time for this process, and state clearly
what regime was followed.
A long-needed set of blood tests, for routinely measuring physiological condition
of fish being held for bioassays, has been provided by HtrNN et al. (1968). They also
suggest desirable holding conditions.
Randomization
Randomization of fish among test tanks is probably neglected in many bioassays.
A formal process of randomization should be used according to FINNEY (1964) and
* N o t e added to proof: This difficulty may be circumvented by using the dosing apparatus for
individual tanks which is described by BRUNGS and MOUNT (1967) A device for continuous treat-
merit of fish in holding chambers. Trans. A m . Fish Soc., 96, 55-57. That device seems equally
as good as the one described by Stark.
Measurement of Pollutant Toxicity to Fish, Part I 799
A variety of terminology has been used in work covered here. I have generally attempted to use one
set of terms and symbols, chosen because of wide usage, early publication, or lack of ambiguity.
Median Effective Concentration (EC50) and Median Lethal Concentration (LC50) will be primary
terms in this review. EC50 may refer to lethal or sublethal responses. The terms correspond to ED50
and LD50, universally used in pharmacology and toxicology, for example insect toxicology (HosKINS,
1960). D represents dose, the amount of drug inside the animal, by injection or ingestion. Fish
toxicology may sometimes be concerned with internal dose, (e.g. pesticide residues), but usually deals
with concentrations in the surrounding water, for which EC50 and LC50 are appropriate.
Use of LC50 (and EC50) in fish toxicology should make the notation more immediately compre-
hensible to workers in other fields of science. LC50 is used in German pollution literature according to
BREmO (1966), is used in Canada ALDERDICE,1967), and generally in Britain where explicit preference
is sometimes stated (BROWNet al., 1967a). In the United States, LC50and EC50 are used to some extent
(U.S. Dept. Interior, 1963; EiSLER,1965; JOHNSON, 1968; STEWARTet aL, 1967b) but the term median
tolerance limit (TLm) has been customary with fish. U.S. readers may wish to substitute TLm for LC50
throughout this review. Unfortunately we cannot speak with grammatical accuracy of lethal "con-
centrations" of water temperature and pH, but the abbreviation will perhaps still be understood. The
wording "median lethal limit" or "median lethal level" could be used if necessary.
The symbol LD50 was first proposed by TREVAN(I 927). He wrote the symbols all on the same line
(not LDso), and major writers in pharmacology follow him (BLISS, 1952; FINNEY, 1964; GADDUM,
1953). Making the 50, or 10, 90, etc., into a subscript is historically incorrect and seems to serve little
purpose beyond complicating the lives of typists and proofreaders.
Time and concentration are inseparably linked in tests with aquatic organisms. Hence LC50's
should be qualified by an indication of exposure-time, such as 24-hour LC50, 4-day LC50, etc.
Another important parameter is the concentration at which acute toxicity ceases, given several
suitable names: incipient lethal level (FRY, 1947); ulitimate median tolerance limit (DouDoROFF, 1945;
DotrDOgOrF et al., 1951); lethal threshold concentration (LLOYDand JORDAN,1963); asymptotic LC50
(BALL, 1967a); kritische schwelle (DENZER, 1959); Schwellenwerte (WUHRMANNand WOKER, 1950;
NEHRING, 1962); Todlichkeitsgrenze (LmPOLT and WEBER, 1958). For the English language, incipient
lethal level has the advantage of being part of a general scheme of classifying environmental factors
800 J . B . SPRAGU~
and may be defined as "that level of the environmental entity beyond which 50 per cent of the popula-
tion cannot live for an indefinite time" (modified slightly from FRY, 1947). The word "ultimate" in
the second term was used earlier (FRY, BRETT and CLAWSON, 1942) not to describe the length of
exposure-time, but the highest degree of resistance attainable by acclimation to the lethal condition.
Perhaps it might be reserved for acclimation effects, which hopefully will become more widely
studied in toxicity tests. Lethal threshold concentration seems a good term although some investigators
still feel that threshold effects should not deal with 50 per cent mortality but with the most sensitive
individual in a population. That concept has no precise mathematical meaning as discussed further
on in the review. According to BUERKLE(1967), most modern investigators would agree that a thres-
hold is "that stimulus intensity at which a positive response can be expected in 50 per cent of the
presentations." Nor should the threshold for acute toxicity be misinterpreted as a "safe" concen-
tration. It is merely a convenient and reproducible reference point: that concentration which would
kill the average fish on long exposure.
To avoid such misunderstandings, the term incipient LC50 is proposed to describe threshold values
as defined in the previous paragraph. The term will be used in this review, and also lethal threshold
concentration.
Another set of terms is used for response time in fixed concentrations, usually Median Lethal Time
(LT50), and Median Effective Time (ET50). Other words such as median survival time, and resistance
time are also used. The symbols follow the established pattern, are useful with fish (ALDERDIeE,1967),
and are standard for time-series in pharmacology (LITCHFIELD, 1949). Unfortunately confusion is
likely between this notation and the abbreviation TL50 which is to replace TLm (P. Doudoroff,
personal communication) in the 13th edition of"Standard Methods" (APHA et al., 1965,12th edition).
To avoid misinterpretation, LC50 will be used in preference to TL50, and LT50 will be written in
words when required, as in the section immediately following.
T h e r e a s o n f o r u s i n g a g r o u p o f fish in e a c h t e s t - t a n k , i n s t e a d o f o n e fish, is t h a t
i n d i v i d u a l s v a r y in resistance. I n this section, v a r i o u s m e t h o d s o f e s t i m a t i n g s u r v i v a l -
t i m e o f t h e " a v e r a g e " o r " t y p i c a l " fish will be d e s c r i b e d . O b t a i n i n g t h e s e m e d i a n
l e t h a l t i m e s is t h e first step in a n a l y z i n g results o f t o x i c i t y tests. T h e y c a n be u s e d to
construct median survival curves and estimate incipient LC50's.
,
60
ILl:?/•
i;i.+ ....
. . . . . . . . . .
R
i •IF!?
201- /•^
./
o---~ /
// /
/• ,,g
/
/ • I w
joL d/
o
t
J"
o-~ /
• __~.. /
. ~ ./
f
o/--i.86
'
I
t_____J_ .; ~ _ J u_i ~J~__ __ t , _.L~L_LU +'. . . . ~. , ,
20 50 tO0 200 500 1,000 5,000
Resistance Time, minutes
FIG. 2. An example of mortality in individual test-tanks plotted against time on logarithmic-
probability paper. Results are for trout tested at 10 levels of oxygenindicated in the body of the
graph. Similar results could be obtained with a toxicant. The lines on the left are for more
severely lethal conditions. Exposure was for 5000 rain, equal to 3.5 days. Modified from
SHEPPARD (1955).
Plotting results on log-probit paper also makes it apparent whether there are two or
more different modes o f toxic action. Changes in slope or grouping of lines provide the
clues. In FIG. 2, the slopes change for the less toxic concentrations at the right, but
Shepard interpreted this merely as increasing variability.
Much more complicated and unusual "split probit" lines are demonstrated in FIG. 3.
From these and similar results, TYLER (1965) concluded that high temperature had
three separate mechanisms of death which operated sequentially, the fastest apparently
being heat shock. Split probit lines of the same pattern are used by entomologists as
the classical indication that insecticide-resistance is developing in a population. The
802 J.B. SPRAGUE
two components of the log-probit line represent normal and resistant individuals
(HosKINS, 1960). HARRIS (1968) has shown how two such super-imposed normal
distributions may be separated into individual components. Application of this
technique could be very useful in evaluating modes of toxicity.
Estimating median response-time
The commonest method is to plot the results on logarithmic-probability paper and
fit straight lines by eye as described above and shown in FIG. 2. Median survival times
may then be read directly from the graph, where the fitted lines cut 50 per cent mortality
(LITCHFIELD,1949). The 95 per cent confidence limits may also be estimated easily by
Litchfield's method, and this should be done especially if the median lethal times are
to be published without supporting data. This is further discussed in the section on
estimating error. Again, more exact mathematical methods could be used if desired
(BLISS, 1937).
Non-lethal effects may also be analyzed by the very same methods; BRETT (1967)
used log-probit methods to estimate median fatigue time of fish swimming at various
speeds and temperatures.
Median survival times have occasionally been estimated from arithmetic plots
(HERBERT and DOWNING, 1955). Smooth sigmoid curves are drawn and medians read
90:
0
0
70
50t
o// 27. 5
./.
30 ]
f
/
26.5 J
I
i t t zI t , l L2_a~_,[ ~ J ~2_~t_L~l_ -_
I0 50 I00 500 ~000 5000
Time of Observat[o n, rain
Fio. 3. Split probit response of a fish to certain lethal temperatures. This unusual response
indicates two modes of lethal action at 27.5° and 29.5°C, with a less distinct effect at 30.5°C. The
format of the graph is the same as Fie. 2, Modified from TYLER(1965).
from the graph. Use o f log-probit paper seems preferable: firstly, differences in slope
of lines are more easily detected; and secondly, confidence limits may be placed on the
median lethal time.
Median survival time m a y also be calculated by various arithmetic procedures.
Measurement of Pollutant Toxicity to Fish. Part I 803
The simplest is to adopt the response-time of the median fish and this is often adequate
(DouDOROFF et al., 1966). However, this does not fully use information provided by
response times of the other fish and provides no estimate of error. Geometric mean
survival time has often been used. It is calculated as the antilog of the mean of
logarithms of individual survival times (e.g. BROWN et al., 1967a). This would be the
same value as the median for regular lognormal data. Such purely arithmetic pro-
cedures should be confirmed by simple graphs (FINNEY, 1964; GADDUM, 1953). For
irregular or truncated data, "graphical analysis is essential for the derivation of
unbiased statistical constants" (Buss, 1937).
The harmonic mean survival times has been effectively used by ABRAM (1964, 1967).
It was about equivalent to the median for some toxicants, and ALDERDICEand BRETT
(1957) found the same thing for pulp mill waste. However, SSEPARD (1955) found that
harmonic means were consistently and significantly shorter than observed times to
50 per cent mortality. The harmonic mean may be calculated as the reciprocal of the
arithmetic mean of reciprocals of individual response times. Or it may be estimated
by plotting survival time on a reciprocal scale against per cent mortality on an arith-
metic scale. Such a graph fitted toxicity data for Malachite green as well as a log-probit
plot or better (ABRAM, 1967). Unfortunately, reciprocal of exposure times does not
always yield a linear plot with percentage response. For example, logarithm of time
is more suitable for cyanide poisoning (HERBERTand MERI<ENS, 1952). For the moment,
use of harmonics may be considered as a possibly useful alternative technique.
"If only one quantity may serve to characterize toxicity of a substance, we consider the threshold
of concentration C, as the most appropriate quantity." WUnRM~ (1952).
The next step in analysis is to plot all the median lethal times and look for a lethal
threshold concentration. Rough plotting should be done as tests proceed so that
suitable concentrations may be used and exposures carried on long enough to define
the threshold adequately.
Each median survival-time may henceforth be regarded as the response to a given
concentration. Details of log-probit analysis used in estimating this value are no
longer of great concern. The important thing is interpretation of a series of such time-
concentration measurements. This has usually been done by drawing toxicity curves
(WurmMANlq, 1952; BUgDICK, 1967; MATIDA, 1960) with semi-logarithmic, logarithmic
or reciprocal transformations, all of which seem to have merit.
804 J.B. SPRAOUE
NH 3
=
c I000
I i I [ i i i ii i i i ~ 1 [
lO I00
Concentration of Poison , p.p.m.
F=G.4. Examplesof toxicitycurves for trout. The lethal thresholdconcentrationof ammonia
has obviouslybeen defined.This is not clear for zinc; longer experimentsmight have shown
additional mortality.From HERBERT(1961).
200
=
o
I00 Copper
=
Zinc
:'z
o
o
50
ti ° o
.i I
o
i.=
20
i=
2 , i [ r illl k I t ~ l l t f i i__.i t II
2O 50 I00 300 I000 5000
Concentration of Metal t .~.O / I
FIG. 5. Examples of straight-line toxicity curves for salmon. The lethal thresholds are indicated
by sharp breaks in the lines. F o r each median survival time, 95Yo confidence limits are indicated
From SPP.AGtm(1964).
described using Ostwald's formula of concentration, time, their threshold values, and
two constants (WUHRMA~% 1952; GADDtrM, 1953; MATIDA, 1960). If the lethal
threshold concentration and minimum survival time are estimated and included in the
formula, the hyperbolic function is expressed linearly (WUrmMA~,q,~and WAKER, 1950).
A simple trial-and-error method of doing this is described by BtmDICK (1957). He
shows that transformation of the toxicity curve to a straight line indicates that the
lethal threshold concentration has been estimated correctly. Burdick's method of
806 J.B. SPRAGUE
estimating the threshold seems to have been little used, since proposed, but could be a
useful tool and is worthy of further trials.
Further mathematical consideration of toxicity curves is given by MATIDA (1960)
with applications to thresholds, body size, and modifying factors. HEY and HE',"(1960)
show efficient calculation of thresholds using the same formula for a rectangular
hyperbola mentioned in the previous paragraph.
Irregular curves appear for some toxicants. Tests with suspensions of inert solids
failed to show any well-defined relation between concentration and survival time
(HERBERT and MERKENS, 1961). BALL(1967C) reports an anomalous S-shaped toxicity
curve for cadmium, in which all concentrations from 0.01 to 1.0 rag/1 killed fish in
about 6 days, although a threshold appeared at a somewhat lower concentration.
ALDEgDICE and WORTmNOTON (1959) show a "bizarre distribution of resistance
times" for DDT-in-oil and emulsifier. Maximum toxicity was at intermediate con-
centration, and fish survived longer in higher concentrations! (The authors credited
this to unstable emulsions.) One such peculiar case has been given an adequate
chemical explanation. DOOOOROFF(1956) found distinct "primary" and "secondary"
mortality in tests with mixtures of cyanide and nickelous sulfate. He proved that this
resulted from a great change in cyanide toxicity with a small change in pH from
respiratory carbon dioxide.
Sometimes the relation is regular but no threshold is apparent. The toxicity curve
continued with no threshold during 15 days for high pH (JORDAN and LLOYD, 1964)
and during 12 weeks for detergent (HERBERTet al., 1957).
These examples of unusual toxicity curves by no means invalidate the usefulness of
logarithmic plotting. On the contrary, they show the value of constructing such a
survival curve, so that unusual toxicity relationships will be uncovered. Such relations
could easily be obscured by incomplete toxicity curves obtained by arbitrarily restrict-
ing length of exposure or frequency of inspection times. Weak or erroneous inter-
pretations could result. The value of obtaining complete toxicity curves is particularly
well illustrated in the work of BROWNet al. (1967a); complete curves allowed them to
interpret and demonstrate a complicated reversal of temperature effects on toxicity of
phenol. DOUDOROFFet al. (1966) used the toxicity curve to demonstrate that mole-
cular hydrocyanic acid is the cause of acute toxicity, not total cyanide. BALL(1967a)
showed that full curves were necessary to evaluate ammonia. One-day tests indicated
trout were more sensitive than coarse fish, but actually the coarse fish were merely
slow to react. Completion of toxicity curves, requiring 3- to 5-day tests, showed that
all fish were equally sensitive in terms of incipient LC50.
An additional minor irregularity has been pointed out by ROTHSCnEtN(1964). Some
pollutants which occur naturally in trace amounts (e.g. copper) are necessary for life.
Below an optimum concentration, the toxicity curve would double back to shorter
times. However, this would only occur if the test-water were deficient in the necessary
element. Furthermore the irregularity would not exist for most man-made pollutants.
Two approaches f o r construction of toxicity curves such as those in FIGS. 4 and 5
may be used: firstly, median lethal times may be estimated for a series of concentra-
tions, as described above; secondly, median lethal concentrations may be estimated
for a series of inspection-times (see section on U.S. standard method). If the response
is reasonably regular and inspections of mortality are equally frequent, either method
should give essentially the same curve.
Measurement of Pollutant Toxicityto Fish. Part I 807
Semi-log toxicity curves
These are relatively uncommon in fish toxicity work. However, the logarithm of
survival time plotted against an arithmetic scale of temperature is customary in lethal
temperature work with fish, and yields linear or nearly linear relations (FRY, 1947).
Curves with reciprocal survival time
The reciprocal of time was used by early investigators of fish toxicology (see JONES,
1964).
Recently, the usefulness of this transformation has been demonstrated for several
toxicants by ABRAM (1964, 1967). Since infinity appears as one extreme value on
reciprocal graph paper, suitable choice of time units can cause infinite time to be
physically very close to useful finite times--Abram shows times from 1 week to 10
weeks in such proximity. Extrapolations can be made to infinite exposure-time.
Abram extrapolates to (a) ~ kill at infinite time for various constant concentrations,
and (b) concentration lethal to half the fish at infinite time. The extrapolated values
from (a) could also be used to estimate (b).
The technique would seem very useful. Obviously no fish would live for an infinite
time. Also, the linear relation would often be disturbed by secondary or tertiary toxic
effects at long exposures. However, this is essentially a technique for extrapolating to
the threshold of a given kind of toxic action, say primary. Unfortunately, this re-
viewer's experience has been that .not all data are amenable to analysis on reciprocal
paper. However, Abram showed relatively straight plots of log concentration vs.
reciprocal time for data on phenol, sodium sulphide, potassium cyanide, and 3
pesticides. Undoubtedly the technique would be applicable to other pollutants.
Results could easily be plotted on reciprocal paper on a trial basis according to
Abram's methods. Such trials would seem desirable as an additional way of estimating
reasonable threshold concentrations.
ALDERDICE and BRETT (1957) concluded that reciprocals were the best transform-
ation for both survival time and concentration in tests with salmon and pulp mill
effluent. Their procedure for mathematically estimating the threshold concentration
would no doubt frighten most biologists, but a line fitted to their data by eye would
almost certainly give much the same threshold for infinite survival. They obtained
confidence limits from their caclulations.
Toxicity curves using the U.S. standard method
The "routine bioassay method" given in "Standard Methods" (APHA et al., 1965)
is more or less standardin the U.S.A. and will be called hereafter the "U.S. standard
method". It is discussed separately since it has a somewhat different approach. It is
described by DOUDOROFFand his committee (1951) and almost identically by APHA
et al. (1965). These descriptions are based on a more extensive outline, now out of
print, by HART, DOUDOROFF,and GREENBANK(1945), in turn derived from research
methods of DOUDOROFF(1942, 1945).
The method is well-known, and yields median tolerance limits (TLM's or LC50's),
customarily for 1, 2, and 4 days. For each fixed time, percentage mortalities are
plotted against test concentrations. Concentration lethal to half the fish is estimated
by interpolation. This is very much the same as standard quantal bioassays in phar-
macology, which plot percentage response against dose. -Hence, standard techniques
of analysis may be easily adapted.
808 J.B. SPRAGU~
It is clear that the U.S. standard method was intended to be a valid tool for pollution
control, yet simple enough for industrial laboratories unfamiliar with bioassays and
fish. The committee succeeded admirably in its purpose, judging by the wide use of the
method. However it is unfortunate that many biologists, particularly in the U.S.A.,
have been content to use for research, the method described by DOUDOROFFet al., as
"the simplest routine procedure". Only in recent years have researchers started to
follow some of the low-key suggestions for obtaining increased information. For
example, following the description of the routine method for calculating LCS0, the
A P H A et al. 0965) suggest: "Other widely accepted and often more satisfactory pro-
cedures include graphical methods which involve fitting a smooth, sigmoid c u r v e . . .
or fitting a straight line to data plotted on logarithmic probability paper, as well as the
more refined methods o f probits, logits, or angles." Some recent research papers have
followed suggestions of the committee on three topics which will be described below:
(1) placing confidence limits on the LCS0; (2) longer exposure times: and (3) more
frequent inspection of mortality.
Criticism as a research method eentres mainly on using data in its quantal form (reacted or not
reacted at an arbitrary time) instead of utilizing the graded time-response which is available. Each
animal supplies more information if its survival time is measured, rather than being classified as
positive or negative (BLISSand CATTEL,1943). If most of the animals die during the test, "neglect of
the information conveyed by the detailed time measurements may seriously reduce the precision. . . . "
(FINNEY, 1964). GADDUM(1953) estimates that "theoretically it may be expected that about half the
information will be lost, so that twice as many observations will be needed for any given degree of
accuracy." An even higher estimate is "ten times the number of replicates to obtain the same con-
fidence limits that would be reached through the use of the logarithmic mean time of the death time
of all fish" (BURDICK,1960).
This criticism would seem to be negated if estimates of LC50, by the regular U.S. standard method,
were made more frequently during a test. The resulting toxicity curve should look the same as one
constructed from median lethal times. They should be equally valid if based on the same observations
of mortality at successive times. Since the same data on fish mortality could be used either way, my
own preference is to estimate time to 50 per cent mortality in each concentration; this shows in a
simple way what happens in each test tank (FIG. 2) and is easy to understand if complications arise
(FIG. 3).
LC50'S based on short exposure times, often 24 hr, were frequently published in
research articles some years ago, although the U.S. standard method recommends
continuation for at least 48 hr. DOUDOROFFet aL (1951) seem to have had research
work in mind when they stated: "Median tolerance limits for longer exposure periods
(for example 8 days, etc.) thus can be determined. The concentration at which half of
the animals are killed eventually, while the rest . . . survive indefinitely ( . . . the
ultimate median tolerance limit) sometimes can be established." Obviously this
describes a lethal threshold. Happily, most researchers in recent years have carried
on their tests for at least 48 hr and usually 96 hr, and for certain purposes to 7-12 days
(PICKERINGand VIGOR, 1965), or even 30 days (PICKERINGe t al., 1962; JENSEN and
GAUFIN, 1966).
Confidence limits have customarily been omitted for LC50's estimated by this
method. However this has been done recently, by American workers CAmNS and
SCHEIER (1963) and PICKERINGand VIGOR (1965). The topic is an important one, and
is discussed in a separate section.
Differences between 1-, 2-, and 4-day LC50's, or lack of difference, can indicate
whether or not a threshold has been reached. This may be formalized by statistical
Measurement of Pollutant Toxicity to Fish. Part I 809
o
u 5
o- 2 0
vo
5O
x 80
(_)
a. 95
99
99.8 I I t I I Itll I t I
2 4 6 8 I0 20 40
Concentrotion of Fluoride, p.p.m. F
FIG. 6. Estimating the median lethal concentration. In this case the incipient LC50 is estimated
since the exposure time was long. Percentage response of trout is plotted on the vertical
probit scale. The median lethal concentration is 8.5 mg/l, and its confidence limits could be
estimated as described in the text. The 5 per cent response is also shown. F r o m HERBERT and
SmmBEN (1964).
routine U.S. standard method except that it uses probits, following standard phar-
macological techniques. Analysis of results by the rapid graphic methods of LITCH-
FIELDand WILCOXON(1949) is recommended. (These improvements are also suggested
by A P H A et al. (1965) as steps beyond the routine method.)To carry out the Litchfield-
Wilcoxon procedure, actual percentage mortality in each test tank at the selected time
beyond the lethal threshold, is plotted on log-probit paper as in FIG. 6. A line is fitted
to the points by eye. Its goodness of fit is estimated by a rapid chi-square test. In
814 J.B. SPRAGU~
doubtful cases, several lines may be drawn and the best chosen by minimum chi-
square value. The incipient LC50 is then read from the graph. Its confidence limits may
be estimated as described in the section on errors.
If desired, the more formal and time-consuming mathematical procedures of
FINNEY (1962) may be used to estimate the incipient LC50. The line is fitted by suc-
cessive approximations based on maximum likelihood methods, since the well-known
method of least squares is unfortunately not valid for these quantal log-probit
relations. The extra computations would seldom seem warranted, since it has been
shown (LITCHFIELDand WILCOXON,1953) that log-probit lines fitted by eye are often
highly accurate. EISENBERG(1952) finds graphical estimates of ED50 are "in very good
agreement" with the formal mathematical estimates. Even FINNEY (1964) gives
limited approval to eye-fitted lines for moderately good data. GADDUM(1953) states
bluntly that "there is no evidence against the view that the calculation of successive
approximations is a pure waste of time."
As mentioned in previous sections, sometimes a lethal threshold may not be found,
even after weeks of testing. If not, some arbitrary time period could be substituted, and
a 4-day LC50, one-week LC50, etc., could be estimated in the same way as described
above.
The incipient LC50 must be estimated by using the actual observed percentage
mortalities from each test tank. Derived values for a selected exposure time should not
be taken from fitted lines such as those in FIG. 2. Only by using original observations
to construct graphs such as FIO. 6 can confidence limits be calculated and comparisons
of statistically significant differences be made.
Alternative methods of estimating thresholds
Other techniques have been developed and may sometimes be preferable or could be
used for confirmation. As mentioned before, the logarithmic toxicity curve may be
treated as a hyperbola, and its asymptote with the time axis estimated mathematically
(WUHRMANN,1952; MATIDA,1960; HEY and HEY, 1960) or graphically (BuRDICK,
1957). In the section on curves with reciprocal survival time various techniques of
ABRAM (1964, 1967) and ALDERDICEand BRETT(1957) were described, for estimating
the infinite LC50.
Another method used to calculate LC50's is that of moving averages, in conjunction
with angular transformations. Because of special interest in the error calculation, all
aspects of this method are discussed together for convenience in the section on con-
fidence limits.
Mortality in controls
Substantial mortality in control tests, merging with the mortality at some concen-
trations of toxicant, sometimes interferes with interpretation of experiments, especially
long ones. For example, this occurred in tests lasting a few weeks against lobster
larvae, which are difficult to rear in the laboratory without some mortality (SPRAGUE
and MCLEESE, 1968). It is possible to consider the threshold concentration as that
which allows survival time equal to the control. However this is not deemed entirely
satisfactory although it would be similar to medical studies, in which equal survival in
control and experimental groups is taken to indicate absence of chronic illness.
A correction can be made by the simple method of TATTERSFIELDand MORRIS
(1924). The observed percentage mortality at a given test concentration is corrected by
Measurement of Pollutant Toxicity to Fish. Part I 815
deducting the percentage dead in the control; the result is multiplied by 100, then
divided by the quantity (100 minus the percentage dead in the control). This yields a
corrected percentage mortality at the given concentration.
However, even that procedure does not solve the main problem, which is the
possibility of interaction of stress from the experimental condition with whatever
stress causes mortality in the controls. In view of the additive effects now shown for a
number of diverse toxicants, such interaction of stresses should be considered a prob-
ability rather than merely a possibility. (Joint toxicity will be discussed in Part II).
Working with fish, it would seem such preferable, and usually quite possible, to
create laboratory conditions which permit indefinitely long survival of the animals.
ESTIMATING ERROR
For the sake of completeness, pertinent references and procedures have been
gathered in this section, although this involves some repetition.
Confidence limits of median response
For median lethal times, confidence limits are often omitted. However, they can be
very useful to the investigator when fitting a line by eye to a set of median survival
times (e.g. FIo. 5). Also the reader could more easily evaluate results to his own
satisfaction. The rapid graphic methods of LITCHFIELD(1949) are suitable for estimat-
ing confidence limits of median survival times and have been used (SHEPARD,1955;
SPRAGUE and RAMSAY, 1965) as have the more formal methods of BLISS (1937) by
BROWN et al. (1967a). Litchfield also provides simple methods for testing whether
two median survival times are significantly different, and whether slopes of two
probit lines are different. These could sometimes be useful in evaluating results.
For median lethal concentrations, confidence limits are even more valuable (BALL,
1967a). The standard and preferred (BLISS, 1956; FINNEY, 1964; GADDUM, 1953)
methods are based on logarithmic-probability transformation. The easiest and most
rapid of these is the graphical procedure of LITCHFIELDand WILCOXON(1949). It has
been used for 96-hr LC50's (CAIRNSand SCHEIER,1963), and incipient LC50's (BALL,
1967a; BROWN et al., 1967a). The more time-consuming formal method of probit
analyses (FINNEY, 1952) has also been used (BROWN et al., 1967b). However, the
Litchfield-Wilcoxon method compares well with the formal mathematical procedure,
and if anything is somewhat more conservative in that it tends to give larger values for
confidence limits (EISENBERG,1952).
A case has been made (BALL, 1967a) for publishing the parameter termed "slope
function" or " S " by Litchfield and Wilcoxon. It is the factor by which a dose must be
multiplied or divided to produce a standard deviation change in response. Knowing
LC50 and S, a probit line may be reconstructed. The point seems a good one, and
publication not only of incipient LCS0 and its confidence limits, but also S, should be
done inasmuch as it may have value for future investigators.
Confidence limits based on reciprocal transformations have been calculated by
ALDERDICEand BRETT(1957) for an infinite LC50.
The only other method which seems to have been used in fish toxicity studies is
based on the angle transformation instead of probits, and a moving average to
estimate the LC50 instead of a graph (HARRIS, 1959; THATCHERand SANTNER,1966).
It was conclusively shown that LC50's for zinc, petrochemicals and detergent were
816 J.B. SPRAGUE
almost identical for this moving average method and the U.S. standard method
(PICKEmNG and Vie,OR, 1965; PICKERII,rG and HENDERSON, 1966b; PICKERING, 1966).
Nevertheless it would seem prudent to plot the original unsmoothed data on a graph
and examine them for any interesting irregularities, as a supplement to the arithmetic
estimate.
Estimating confidence limits by the angle transformation instead o f probits could
have some disadvantages. Equal numbers of animals must be used at each concen-
tration, and concentrations must be separated by a constant interval, usually logar-
ithmic (HARRIS, 1959). Malfunction of apparatus, which breaks the series of concen-
trations, prevents use of the method, as encountered by PICKEmNG (1966).
The angular transformation is considered reasonable for bioassays only if extreme
responses of zero and one hundred per cent do not occur (BLISS, 1956; FINNEY, 1964),
but these are frequent when using the U.S. standard method, as pointed out by
PICKERING and HENDERSON (1966b). Probably they would also be frequent in cal-
culations of incipient LC50's. GADDUM (1953) recommends that the angular trans-
formation should only be used when responses are within the limits of 20 to 8 0 ~ .
Serious misinterpretations of estimates of error are possible by any of the above
methods. The confidence limits are based only on internal evidence from one experi-
ment. They are merely indicators of what might be expected if the very same stock of
animals were immediately retested under identical conditions! They do not show the
differences that might be expected in any repetition of an experiment (FINNEY, 1952);
those would be considerably larger (LITCHFIELDand WmCOXON, 1953). For example,
at different times of year, the same stock of fish in the same laboratory may give LC50's
which differ by a factor of 2.5 (BRowN, 1968). Variation with time, place, and species
is well known to most biologists. DOUDOROFF et al. (1951) make it clear that such
variation played an important part in arriving at a suitable compromise between
precision and simplicity of methods for their routine bioassay procedure.
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