Chemical Methods USE in Marine Environmental Monitoring: Commission Manuals

Intergovernmental Oceanographic Commission
Manuals and guides
12
CHEMICAL METHODS FOR USE I N MARINE ENVIRONMENTAL MONITORING
1983 Unesco
TABLE OF CONTENTS
Pages
Preface
..................................................
2.
3.
....................... Determination of Dissolved Oxygen and Oxygen Saturation ....

Potentiometric determination of pH Determination of hydrogen sulphide
1
6
........................
11
16
4.
5.
6.
Determination of dissolved inorganiz phosphate
............ Determination of total phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . Determination of reactive silicate ........................

Direct determination of ammonia with the indophenol blue method ................................................... Determination of nitrite Determination of nitrate
23
23
7.
29
36 40
a.
9.
...................................
...................................
10.
Simultaneous persulphate oxidation of phosphorus and nitrogen containing compounds in water ....................
48
Figure Figurz Figure
1
2
................................................
5
19
................................................
................................................
................................................
43
References
53
This Manual was prepared by the Intergovernmental Oceanographic Commission ( I K ) of Unesco, with the collaboration of a consultant, Mr. Stig R. Carlberg, of the Institute o f Hydrographic Research, National Swedish Board of Fisheries, Gothenburg, at the request of the United Nations Environment Programme (UNEP), for its Co-ordinated Mediterranean Pollution Monitoring and Research Programme (MED POL), whose support in this regard is gratefully acknowledged. The Manual contains descriptions of chemical methods for analysis of parameters of general interest in programmes for chemical oceanography, as well as for marine environmental mcnitoring. It is intended for use by marine science institutes that are or will become involved in such activities, particularly within the Regional Seas Programme of UNEP.
For preparation of practical manuals a supply of good background material is absolutely essential. In this regard, the use of three earlier publications (see Carlberg 1972, FAO 1975 and Grasshoff 1976 in the list of references) is gratefully acknowledged.
The methods presented in this report were produced for the purpose of serving as reference methods for work performed under M D POL. However, since they have much wider applicability than for just one regional area, they are published for the use of interested marine scientists throughout the world.
1 . POTENTIOMFTRIC DFTFRMINATION OF pH
1.1.
SCOPE AND FIELD OF APPLICATION
The determination o f pH d e s c r i b e d h e r e i n i s performed e l e c t r o m e t r i c a l l y , using a g l a s s e l e c t r o d e , w i t h c a l o m e l e l e c t r o d e a s reference. F o r the d e t e r m i n a t i o n o f pH w i t h t h e h i g h e s t p r e c i s i o n a n d accuracy, which represents normal practice i n oceanography, the full d e t a i l s of t h i s methods should be f o l l o w e d . I f the d e m a n d s a r e l e s s stringent (precision and a c c u r a c y of 5 0.1 p H unit o r l e s s ) a simplified p r o c e d u r e , o m i t t i n g the c l o s e d m e a s u r i n g c e l l a n d t h e constant temperature b a t h , may be a p p l i e d . T h e c a l i b r a t i o n p r o c e d u r e is , however, generally applicable.
1.2. PRINCIPLE
pH is defined a s t h e n e g a t i v e l o g a r i t h m o f t h e h y d r o g e n i o n activity (aH+) and t h u s pH -log aH+ is a direct measure o f acidity a c c o r d i n g t o the Brijnsted d e f i n i t i o n of a c i d s a s s u b s t a n c e s t h a t are a b l e to donate hydrogen i o n s , and bases a s s u b s t a n c e s that a r e a b l e t o c o m b i n e with hydrogen i o n s : a c i d s ----base + h y d r o g e n 7----ion.
The principle for the p o t e n t i o m e t r i c d e t e r m i n a t i o n s i s t h a t the potential of the e l e c t r o d e c h a i n c o n s i s t i n g o f a n i n d i c a t o r electrode sensitive t o the h y d r o g e n i o n a c t i v i t y a c c o r d i n g t o t h e Nernst e q u a t i o n - and a pH-independent r e f e r e n c e e l e c t r o d e , both immersed i n t h e test s o l u t i o n , i s measured by m e a n s o f a s u i t a b l e galvanometer (pH meter). T h e m e a s u r e d p o t e n t i a l i s t h e n c o m p a r e d w i t h the electrode c h a i n potential o b t a i n e d w h e n a buffer s o l u t i o n o f k n o w n pH i s used as test s o l u t i o n . T h e a c t u a l pH v a l u e i s found u s i n g the formula for " O p e r a t i o n a l pH" : pH(X) where : pH(X) and pH(S) pH o f test s o l u t i o n and b u f f e r s o l u t i o n respectively E(X) and E(S) = electrode c h a i n p o t e n t i a l i n t e s t s o l u t i o n a n d b u f f e r respectively. R'T t h e Nernst f a c t o r (R' a constant, T the a b s o l u t e temperature).
= =
pH(S)
,%(X)
E(SVT
/R'T
A correctly c a l i b r a t e d pH m e t e r a u t o m a t i c a l l y c o n v e r t s the
measured potential to give direct r e a d i n g i n pH u n i t s .
1.3. REAGENTS
As pH values a r e t e m p e r a t u r e d e p e n d e n t , t h e pH v a l u e s o f the following buffer s o l u t i o n s a r e g i v e n i n T a b l e I. For a proper c a l i b r a t i o n a t l e a s t two o f the s o l u t i o n s a r e r e q u i r e d . T h e c h o i c e of s o l u t i o n s is governed by the pH r a n g e o f t h e s a m p l e s .
- 2 -
1.3.1. Bates 1:l buffer. P o t a s s i u m d i h y d r o g e n phosphate, K H 2 P 0 4 ; 3 . 3 9 g and d i s o d i u m h y d r o g e n p h o s p h a t e , Na2HP04, 3 - 5 3 g are dissolved i n d i s t i l l e d w a t e r f r e e o f c a r b o n a t e s and diluted t o 1 000 ml. P o t a s s i u m dihydrogen p h o s p h a t e , KH2P04; 1.3.2. Bates 1 : 3.5 buffer. 1 . 1 7 9 g and d i s o d i u m h y d r o g e n p h o s p h a t e , Na2HP04, 4 . 3 0 g are dissolved i n d i s t i l l e d w a t e r f r e e o f c a r b o n a t e s and diluted t o 1 000 m l . 1.3.3. Palitzsch borax buffer S o d i u m tetraborate, Na2B407. 10H20, 9 . 5 5 4 g , boric a c i d , H B O , 6 . 2 0 2 g , and s o d i u m c h l o r i d e , N a C 1 , 1.462 g a r e d i s s o l v e d ?n J i s t i l l e d w a t e r free o f c a r b o n a t e s a n d d i l u t e d t o 1 000 m l . 1.3.4. Bates 0.01 M borax buffer. S o d i u m t e t r a b o r a t e , Na2B407. 10H 0 , -___ 3 . 8 1 4 g i s dissolved in distilled w a t e r free o f carbonates a n d diluzed t o 1 000 ml. T h e sodium and potassium s a l t s should be dried before weighing.
It i s a l w a y s a d v i s a b l e t o k e e p a s u p p l y o f these s a l t s in a desiccator
c o n t a i n i n g silica g e l ; t h u s , t i m e - c o n s u m i n g drying i n an oven i s avoided. S o m e a u t h o r s r e c o m m e n d r e c r y s t a l l i z a t i o n o f borax b e f o r e use. This s h o u l d be done from w a t e r below 5OOC. T o prevent partial dehydration o f b o r a x , i t s h o u l d be k e p t o v e r a saturated solution o f s u g a r or s o d i u m c h l o r i d e o r s l i g h t l y m o i s t s o d i u m b r o m i d e , i n a closed container. he boric acid c o u l d be k e p t i n t h e s a m e manner. D i s t i l l e d w a t e r f r e e o f c a r b o n a t e s i s prepared by boiling d i s t i l l e d w a t e r f o r a w h i l e a n d , p r e f e r a b l y , passing nitrogen gas t h r o u g h d u r i n g t h e c o l i n g . T h e water should then i m m e d i a t e l y be used f o r the p r e p a r a t i o n o f s o l u t i o n s .
Table I ------Standard p H v a l u e s o f t h e buffer solutions
a~ For P a l i t z s c h buffer o n l y t w o v a l u e s a r e given because a t other t e m p e r a t u r e s t h e v a l u e s a r e u n c e r t a i n , a s found in e.g. ICES i n t e r c a l i b r a t i o n exercises(Gieskes 1969).
- 3 1.4. APPARATUS AND EQUIPMENT
1.4.1. Water b a t h , t h e r m o s t a t e d , w i t h pump for e x t e r n a l c i r c u l a t i o n o f t h e thermosted water. S h o u l d be c a p a b l e o f h o l d i n g a s e l e c t e d temperature t o within + 0.5'.
1.4.2. Measuring cell and h e a t e x c h a n g e r , o f g l a s s , c o n s t r u c t e d a s i n Figure 1. These i t e m s a n d c o n n e c t i n g t u b e s s h o u l d be a s s m a l l a s possible i n order t o r e a c h t h e r m a l e q u i l i b r i u m q u i c k l y , a n d should not r e q u i r e too m u c h s a m p l e w a t e r for w a s h i n g and m e a s u r e m e n t . The e l e c t r o d e s should be k e p t in s e a - s u r f a c e water between measurements. Only d u r i n g l o n g e r s t o r a g e should d i s t i l l e d w a t e r be u s e d . 1.4.3. Electrode (glass e l e c t r o d e a n d c a l o m e l r e f e r e n c e e l e c t r o d e ) any c o m m e r c i a l type o f g o o d q u a l i t y m a y be used. (W. I n g o l d A . G . , Z u r i c h , S w i t z e r l a n d , h a s a g l a s s e l e c t r o d e called L O T 201, w h i c h may be a good choice because i t h a s a f a s t reponse a n d w o r k s properly even a t low temperature). 1.4.4. pH m e t e r of good q u a l i t y . An i n s t r u m e n t w i t h a g a l v a n o m e t e r should be provided w i t h s c a l e e x p a n s i o n f a c i l i t i e s . F o r s h i p b o a r d use a n instrument with d i g i t a l d i s p l a y i s p r e f e r r e d , a s t h e galvanometer needle i s i n f l u e n c e d by t h e m o v e m e n t s o f the s h i p . The pH value must be displayed w i t h at l e a s t t w o d e c i m a l s .
1.5.
SAMPLING PROCEDURE
I t is important t o m i n i m i z e i n t e r a c t i o n b e t w e e n the s a m p l e and t h e carbon dioxide in t h e a i r . H e n c e the s a m e c a r e and p r e c a u t i o n s a s i n oxygen sampling a r e a d v o c a t e d . B e c a u s e o f t h i s s t a t e m e n t O2 a n a l y s e s probably should preceed pH. T h e s a m p l e i s d r a w n v i a a h o s e from t h e w a t e r sampler ( i m m e d i a t e l y a f t e r the o x y g e n s a m p l e ) i n t o a 100 m l glass bottle with a g r o u n d s t o p p e r . T h e s a m p l e s h o u l d be a n a l y s e d w i t h i n one h o u r a f t e r s a m p l i n g .
1.6.
ANALYTICAL PROCEDURE
For t h e exact h a n d l i n g o f the pH m e t e r , t h e i n s t r u c t i o n manual supplied with t h e i n s t r u m e n t s h o u d be c o n s u l t e d .

The p H meter i s s t a n d a r d i z e d w i t h B a t e s 1:l p h o s p h a t e b u f f e r ( 1 . 3 . 1 . ) at t h e chosen c o n s t a n t t e m p e r a t u r e (20 or 25OC). B e c a u s e most electrode pairs a r e not e n t i r e l y l i n e a r o v e r t h e e n t i r e pH s c a l e , the s l o p e (mV/pH) o f t h e e l e c t r o d e s should be c o r r e c t e d . I m m e r s e the electrodes i n B a t e s 0.01 M borax buffer ( 1 . 3 . 4 . ) (rinse well). I f a slope c o r r e c t i o n i s provided o n t h e i n s t r u m e n t , this is used t o get the p r o p e r pH r e a d i n g a c c o r d i n g t o t h e v a l u e s i n T a b l e I. (In t h i s case t h e t e m p e r a t u r e c o m p e n s a t i o n i s set a t t h e m e a s u r i n g temperature and not s h i f t e d a f t e r c a l i b r a t i o n o f t h e instrument). If no s l o p e c o r r e c t i o n i s a v a i l a b l e , t h e t e m p e r a t u r e compensator c a n b e used because i t w o r k s s i m i l a r l y . As a f i n a l check B a t e s 1:3.5 (1.3.2), b u f f e r c a n be r e c o m m e n d e d .
- 4 I n s t e a d o f B a t e s 0.01 M borax buffer ( 1 . 3 . 4 1 , Palitzsch borax buffer (1.3.3) c a n be used. T h e advantage o f this i s that t h e pH v a l u e a t room t e m p e r a t u r e i s c l o s e to t h e maximun! pH value o f sea water. R i n s e t h e m e a s u r i n g c e l l w i t h s e a water several times t o r e m o v e a l l t r a c e s o f buffer s o l u t i o n s . Keep t h e cell filled up w i t h s u r f a c e s e a water.
1.6.2.
Measurement
T h e s a m p l e i s brought t h r o u g h t h e heat e x c h a n g e r into the c e l l w i t h t h e aid of a s i p h o n , a r u b b e r tube connected t o the heat e x c h a n g e r . R i n s e t h e c e l l t h r e e to four times w i t h the s a m p l e . F i l l t h e c e l l c o m p l e t e l y a n d let t h e s a m p l e reach constant temperature. (A s m a l l t h e r m o m e t e r s h o u l d extend i n t o the cell a s in F i g u r e I). For h i g h p r e c i s i o n and a c c u r a c y , t h e temperature d i f f e r e n c e between c a l i b r a t i o n a n d m e a s u r e m e n t s h o u l d not exceed 0 . 5 O C . Make a n o t e o f t h e pH and t h e m e a s u r e m e n t t e m p e r a t u r e , and r e m o v e the sample. E x p e r i e n c e h a s s h o w n t h a t n o problem i s encountered when g o i n g from a sample w i t h a high pH t o a sample w i t h a l o w pH (as i s t h e case a t the halocline). H o w e v e r , a f t e r measurement o f a sample w i t h a l o w p H , a d d i t i o n a l r i n s i n g m a y be required.
1.7.
CALCULATION AND EXPRESSION OF RESULTS value) be sample for
T h e measured pH v a l u e i s not t h e true pH v a l u e (in s i t u o f the s a m p l e d water. T h e measured -or apparent- value h a s to c o r r e c t e d for the t e m p e r a t u r e d i f f e r e n c e between measurement c o n d i t i o n s a n d t h o s e a t t h e s a m p l e d e p t h in the water. If the w a s o b t a i n e d from about 500 m or d e e p e r , a further correction t h e d i f f e r e n c e i n h y d r o s t a t i c p r e s s u r e must be a p p l i e d . T h e f o r m u l a f o r c a l c u l a t i o n o f t h e pH i n situ is t h e n :
-------
P H------in situ where : t l t2
PHmeasured
0.0118(t2
t,)
= =
t e m p e r a t u r e __----in Situ measurement t e m p e r a t u r e
T h i s f o r m u l a i s valid f o r work i n areas w i t h water depths n o t e x c e e d i n g 500-1 000 m ( d e p e n d i n g o n the accuracy desired). T h e complete correction formula i s :
i -
i
1 9
Figure
1 . Thennostated pH-cell and Lrat-c>xcharLgPr

7
electrodes ttennome ter heat exchange- (a bout : [ r i m bo sample inlet teflon valve ( 3 w a y - k e y )
;:1
3)
6 ) s n m p l e overflow 7) t:irrnmostaterl hater inlet 8 ) ) t h e n o c t a t e d water outlet (9) to waste
5)
- 6 T h e last t e r m B. Z i s a c o r r e c t i o n f a c t o r for t h e difference in h y d r o s t a t i c p r e s s u r e between the s a m p l i n g depth and the laboratory a t the s e a s u r f a c e . Z i s the s a m p l i n g depth in m e t r e s a n d the 8 - v a l u e i s o b t a i n e d from T a b l e 11.
1.8.
ESTIMATION OF PRECISION AND ACCURACY
It i s o b v i o u s that p r e c i s i o n and accuracy a r e dependent not o n l y on the h a n d l i n g o f the s a m p l e s a n d the quality of buffer s o l u t i o n s . The pH meter and i t s e l e c t r o d e s are important factors. I t i s a b s o l u t e l y e s s e n t i a l t h a t these a r e of good quality and that t h e y are m a i n t a i n e d and used a c c o r d i n g t o instructions given by the m a n u f a c t u r e r . With good q u a l i t y equipment the reproducibility can be a s good a s + 0 . 0 0 2 pH u n i t s and standard deviation between d i f f e r e n t i n s t r u m e n t s may be a s l o w a s kO.005 pH units (Gieskes 1969). For n o r m a l r o u t i n e work a p r e c i s i o n a n d accuracy around 0.02 pH u n i t s m a y be e x p e c t e d with p r o p e r equipment.
2. . -DETERMINATION OF DISSOLVED OXYGEN -- AND OXYGEN - SATURATION
2.1. SC-OPE AND
FIELD OF APPLICATION
The m e t h o d d e s c r i b e d is the c o m m o n Winkler m e t h o d , modified f o r sea w a t e r b y C a r r i t t and C a r p e n t e r . 2.2. PRINCIPLE T h e method i s . b a s e d on the f o l l o w i n g principle : Manganous i o n s are p r e c i p i t a t e d i n a l k a l i n e m e d i u m , forming manganous h y d r o x i d e . T h i s i s o x i d i z e d b y the dissolved o x y g e n : 2 M r 1 ( 0 H ) ~+ 02- 2MnO(OHI2.The m a n g a n e s e hydroxide is dissolved with a c i d and r e d u c e d by i o d t d e i o n s : MnO(OH)2 + 4 H O + + 31---+Mn2+ + I '
7H20. T h e I - i o n s produced
are d e t e r m i n e d by t i t r a t i o n with thiosulphate i o n s :
2s2023
I3
.S40E-
31-
- 7 2.3. REAGENTS
S t o r e in a plastic bottle. (MnS04.4H20, 670 g , or MnCL 2 in equivalent a m o u n t may be u s e d . 1. 2.3.2. S o d i u m iodide, N a I . 600 g i s d i s s o l v e d by w a r m i n g i n t h e s m a l l e s t possible volume o f d i s t i l l e d w a t e r . d r o x i d e , N a O H . 320 g i s dissolved i n the s m a l l e s t 2.3.3. Sodium h y_-----possible v o l u m e of d i s t i l l e d water. G r e a t care s h o u l d be e x e r c i s e d i n d o i n g t h i s a s the h y d r o x i d e p r o d u c e s c o n s i d e r a b l e heat w h e n it d i s s o l v e s . A d d the h y d r o x i d e p e l l e t s , portion by p o r t i o n , i n t o the w a t e r , s t i r r i n g c o n t i n u o u s l y . 2.3.4. S o d i u m azide 9 NaN3. 1 0 g i s dissolved i n t h e s m a l l e s t possible v o l u m e of distilled w a t e r . ( T h i s r e a g e n t i s used i n o r d e r to destroy n i t r i t e in s t a g n a n t waters). Alkaline iodide r e a g --ent ________________---
_____________
________
____________
2.3.5.
T h e s e three s o d i u m r e a g e n t s ( 2 . 3 . 2 . - 2.3.4) a r e m i x e d together and diluted t o one l i t r e w i t h distilled w a t e r . S t o r e i n a plastic bottle. These r e a g e n t s a r e v e r y concentrated and g e n e r a l l y will not b e c o m e clear u n t i l t h e y a r e d i l u t e d to the full v o l u m e . If c r y s t a l s a r e f o r m e d , t h e c l e a r l i q u i d i s decanted i n t o a n o t h e r vessel.
2.3.6.
Sulphuric a c i d , H2S04 ( 1
1)
solution about 9 moles/liter.
Concentrated a c i d s m i x e d w i t h t h e s a m e v o l u m e o f distilled w a t e r . The a c i d i s slowly poured i n t o t h e w a t e r u n d e r c o n s t a n t m i x i n g and cooling. The reason for u s i n g s u l p h u r i c a c i d instead o f the c o m m o n l y recommended concentrated phosphoric acid i s because d u r i n g f i e l d work there i s a great r i s k o f C o n t a m i n a t i n g s a m p l e s for the p h o s p h a t e a n a l y s i s when phosphoric a c i d i s used. 0-.5H20, 0.02 mo-les/liter. Sodium thiosulphate , Na2S2 3.7 2-3 __ 25 g is dissolved in distilled water. 50 ml isobutylalcohol is added and the solution is diluted to 5 litres.
~
______
2 . 3 . 8 . ' S t a r c h solution. 1 g s o l u b l e s t a r c h i s dissolved in 100 m l

distilled w a t e r by w a r m i n g u n t i l t h e s o l u t i o n b e c o m e s c l e a r . T h e s o l u t i o n may be p r e s e r v e d by t h e a d d i t i o n o f 0 . l g s a l i c y l i c acid (HOCOC6H4COOH). 2 . 3 . 9 . 'ihyodene indicator ( C a m p b e l l W i l l i a m s & C O , 14 S t . N e o t s R o a d , A b b o t s l e y , H u n t s , England). T h i s i n d i c a t o r i s better than t h e s t a r c h s o l u t i o n , because the s a m p l e r e m a i n s c l e a r d u r i n g t h e whole t i t r a t i o n and t h e end point is e a s i l y d e t e c t e d . W h e n using t h e s t a r c h s o l u t i o n the s a m p l e mostly becomes t u r b i d . T h i s i n d i c a t o r a l s o can be used a s a s o l u t i o n , prepared a c c o r d i n g to t h e i n s t r u c t i o n s on t h e c o n t a i n e r
_______________
__ _______________
- 8 2.4.
APPARATUS AND EQUIPMENT
2 . 4 . 1 . Calibrated b u r e t t e , f o r i n s t a n c e a 12 m l Derona burette w i t h a n a u t o m a t i c z e r o a d j u s t e r , o r a motor-driven piston burette.
2 . 4 . 2 . Automatic s y r i n g e p i p e t t e s , p i s t o n pipette or s i m i l a r . As t h e r e a g e n t s have t o be a d d e d s w i f t l y , common t y p e s o f pipettes w i t h o r without s u c t i o n d e v i c e s s h o u l d be avoided. 2 . 4 . 3 . W i n k l e r b o t t l e s , v o l u m e about 100-130 m l , carefully c a l i b r a t e d . T h e s e m a y be s u b s t i t u t e d by common 50 ml g l a s s bottles w i t h g r o u n d s t o p p e r s , i f t h e s e s t o p p e r s a r e long enough t o extend t h r o u g h t h e bottle n e c k a n d i n t o t h e body of t h e bottle, thus e l i m i n a t i n g the r i s k o f t r a p p i n g a i r bubbles in t h e bottle.
2.5.
SAMPLING AND PRE-TREATMENT
T h e s a m p l e should i f possible be drawn from water s a m p l e r s o f plastic or plastic-coated metal. T h i s should be done a s soon a s possible a f t e r the h y d r o g r a p h i c cast i s brought o n board (sampling). T h e o x y g e n s a m p l e s h o u l d be t h e first sample d r a w n from t h e water s a m p l e r . For t h i s p u r p o s e a r u b b e r t u b i n g is fitted to t h e outlet o f t h e s a m p l e r . A g l a s s t u b e a b o u t 10 cm l o n g is connected t o the t u b i n g . T h e g l a s s t u b e i s i n s e r t e d t o t h e bottom O f the Winkler bottle a n d at l e a s t one v o l u m e of s a m p l e w a t e r i s allowed to f l o w through t h e bottle before f i l l i n g with t h e s a m p l e . The w a t e r should f l o w at a m o d e r a t e s p e e d , a v o i d i n g a i r bubbles. (Air bubbles trapped in the r u b b e r t u b i n g a r e r e m o v e d by squeezing). The t u b e i s then carefully removed from the bottle w h i c h i s filled t o overflowing. With a u t o m a t i c s y r i n g e p i p e t t e s t h e r e a g e n t s manganous sulphate ( 2 . 3 . 1 ) a n d a l k a l i n e - i o d i d e ( 2 . 3 . 5 ) a r e a d d e d simultaneously. T h e syringes s h o u l d h a v e l o n g p l a s t i c t i p s , e x t e n d i n g deep i n t o the bottle. 1 ml of e a c h reagent is a d d e d t o t h e s a m p l e v o l u m e 100-130 ml. ( T o the s m a l l e r bottle 0.5 ml o f e a c h i s added). T h e w i n k l e r bottle is careful-ly closed w i t h the s t o p p e r a v o i d i n g t h e trapping o f a i r b u b b l e s , a n d the bottle is v i g o r o u s l y shaken w i t h a s n a p p i n g motion o f t h e wrist.
2.6.
ANALYTICAL PROCEDURE
2.6.1.
---A n a l y --sis
W h e n the p r e c i p i t a t e h a s s e t t l e d to at l e a s t about half w a y d o w n t h e b o t t l e , 1 ml s u l p h u r i c a c i d (2.3.6) i s added with a n a u t o m a t i c s y r i n g e . The bottle i s closed a g a i n , a v o i d i n g a i r bubbles, a n d s h a k e n until the p r e c i p i t a t e i s dissolved. N o w the bottle should be stored i n a dark p l a c e and t i t r a t e d after a maximum t i m e o f o n e h o u r . If the a n a l y s i s h a s to be d e l a y e d , the bottles should be stored w i t h the o x y g e n precipitated. T h e a c i d should by n c meanste added i f the bottles have t o be stored for a l o n g time. I f d i s s o l v e d , t h e i o d i n e will e v a p o r a t e , passing the glass s t o p p e r . If the s a m p l e s h a v e t o be stored for s e v e r a l d a y s , or until the ship a r r i v e s at t h e home p o r t , the W i n k l e r bottles should be stored in g l a s s or plastic j a r s filled w i t h s a m p l e water. T h e bottles s h o u l d be c o m p l e t e l y s u b m e r g e d , a n d the j a r s c l o s e d ( i t is a good i d e a t o s t o r e the bottles u p s i d e - d o w n in the jars).
- 9 The dissolved s a m p l e i s q u a n t i t a t i v e l y t r a n s f e r r e d i n t o a wide-necked 300 m l E r l e n m e y e r f l a s k a n d titrated w i t h s t a n d a r d i z e d thiosulphate solution ( 2 . 3 . 7 . ) , u n t i l a very pale s t r a w c o l o u r remains. The titration must be c a r r i e d out a t a n e v e n speed. Three drops o f starch s o l u t i o n (2.3.8.) freshly prepared (or Thyodene indicator ( 2 . 3 . 9 ) are a d d e d a n d the t i t r a t i o n i s continued u n t i l t h e s o l u t i o n i s c o l o u r l e s s . If the t h i o s u l p h a t e i s added too s l o w l y at the e q u i v a l e n t p o i n t , t h e r e w i l l a l w a y s be a s l i g h t c o l o u r in the s a m p l e due to o x i d a t i o n .
2.6.2.
Standardization o f the t h i o s u l p _____________ hate solution. ___-______________________
The exact titre o f t h i s s o l u t i o n c a n be d e t e r m i n e d by t i t r a t i o n a g a i n s iodate i o n s in a c i d s o l u t i o n :
IO; + 51- + 6 H O+
21- + 9 H 2 0
T h u s 1 mole i o d a t e is e q u i v a l e n t t o 6 m o l e s t h i o s u l p h a t e . Analytical g r a d e potassium i o d a t e (KIO molecular weight 2 1 4 . 0 4 ) , i s dried at 18OOC to c o n s t a n t w e i g h t ? E x a c t l y 1.1891 g KIO i s dissolved in d i s t i l l e d w a t e r a n d diluted t o 1 000 rnl. 5 m? o f this 0.00555 moles/litre s o l u t i o n i s added t o a g l a s s stoppered Erlenmeyer f l a s k (250 m l ) c o n t a i n i n g 40 m l d i s t i l l e d w a t e r , 1 ml o f the sulphuric a c i d ( 2 . 3 . 6 ) s o l u t i o n , 1 ml m a n g a n o u s a n d 1 m l sodium reagent. After five m i n u t e s t h e solution i s t i t r a t e d with the thiosulphate s o l u t i o n a s described for the s a m p l e s . T h e r e a g e n t blank i s obtained by o m i t t i n g the a d d i t i o n of i o d a t e a n d t h e n repeating the procedure. The a d d i t i o n of s t a r c h s h o u l d g i v e n o , or o n l y a f a i n t , blue c o l o u r .
M
a y
b
the c o n c e n t r a t i o n o f the t h i o s u l p h a t e s o l u t i o n , moles/litre
rnl KIO
solution
the c o n c e n t r a t i o n o f the KIO
s o l u t i o n , moles/litre
ml thiosulphate s o l u t i o n c o n s u m e d , c o r r e c t e d f o r blank
2.7.
1 0
o f Results
C a l c u l a t i o n and R e p o r t i n g __________-________-____
___c-__-___
T o c a l c u l a t e t h e c o n c e n t r a t i o n o f dissolved oxygen , use t h e f o r m u l a below. a v o l u m e o f t h e t h i o s u l p h a t e consumed i n m l c o n c e n t r a t i o n o f t h e t h i o s u l p h a t e s o l u t i o n , moles/litre
M
V
v o l u m e of t h e W i n k l e r b o t t l e i n ml
then
O2
ml/l
a.M.22393
4 (V
f o : .
2)
_--___M.22393 as
the c a l c u l a t i o n s i t m a y be p r a c t i c a l t o calculate the term a c o n s t a n t f a c t o r ( w h i c h i s close to 112 for a s o l u t i o n 4 t h a t c o n t a i n s 0,02 moles/litre
If t h e s m a l l s a m p l e b o t t l e (50-6Onl)is used the v o l u m e o f e a c h a d d e d r e a g e n t is 0 . 5 m l . I n t h i s c a s e the ( V - 2 ) in t h e formula i s r e p l a c e d by ( V - 1 ) .

At a n y g i v e n t e m p e r a t u r e a n d salinity a w a t e r is s a t u r a t e d w i t h o x y g e n w h e n it h a s d i s s o l v e d a c e r t a i n , specified quantity. T h i s q u a n t i t y i s referred t o a s t h e s a t u r a t i o n c o n c e n t r a t i o n o r 100 percent s a t u r a t i o n . T h u s , a n y o x y g e n concentration f o u n d by a n a l y s i s c a n be expressed a s a p e r c e n t a g e o f the s a t u r a t i o n value i n s t e a d o f ml/1. T h e s a t u r a t i o n c o n c e n t r a t i o n o f t h e water i s obtained f r o m a s t a n d a r d t a b l e w h e n t h e t e m p e r a t u r e and s a l i n i t y o f the s a m p l e a r e known.
T h e main s o u r c e f o r s y s t e m a t i c errors in a n a l y s i s o f d i s s o l v e d o x y g e n i s i n t h e s a m p l i n g . I f t h i s is l z f t out o f the d i s c u s s i o n t h e i n d i v i d u a l r e p r o d u c i b i l i t y + can be 0.02 ml/litre for o x y g e n c o n t e n t s b e l o w 2 m l / l i t r e a n d -0.04 ml/litre a t higher c o n c e n t r a t i o n s . T n t e r c a l i b r a t i o n e x e r c i s e s between five different l a b o r a t o r i e s , e a c h u s i n g i t s o w n m o d i f i c a t i o n of t h e m e t h o d , d e m o n s t r a t e d a s t a n d a r d devia-tion o f + 0.03 ml/litre (Grasshoff 1976).
2.9.
Remarks -_-____
T h e c o n c e n t r a t i o n o f s o d i u m i o d i d e has t o be at l e a s t 600 g/l in- t h e r e a g e n t , a c c o r d i n g to C a r r i t t a n d Carpenter. Potassium i o d i d e m a y a s well be u s e d , but m a y ' b e more difficult to dissolve i n t h e c o n c e n t r a t i o n used. P o t a s s i u m hydrogen i o d a t e , KH(I0 may a s well be used i n e q u i v a l e n t c o n c e n t r a t i o n a s s t a n d a r d 2iZl.e substance. I t i s v e r y i m p o r t a n t t h a t the entire s a m p l e volume i s t i t r a t e d . D u e t o t h e l o s s e s o f i o d i n e , it i s impossible to t a k e out a repres e n t a t i v e a l i q u o t for t h e t i t r a t i o n a s described i n some procedures.
3.
3.1.
11
DETERMINATION OF HYDROGEPJ SUL,PHIDE SCOPE AND FIELD QE APPI.ICATION
S u l p h i d e is found in a n o x i c w a t e r s , w h e r e it i s formed by microbiological reduction o f s u l p h a t e i o n s . T h e method d e s c r i b e d h e r e , depending on the formation o f m e t h y l e n e blue from d i m e t h y l p - p h e n y lene d i a m i n e , i s a s i m p l e a p p l i c a t i o n o f a w e l l e s t a b l i s h e d colorimetric method for s u l p h i d e . T h e method i s a s n e a r l y s e n s i t i v e a s i s theoretically p o s s i b l e , and i s f o r d i r e c t d e t e r m i n a t i o n applicable t o concentrations up t o a b o u t 100 moles/liter. At h i g h e r c o n c e n t r a t i o n s of hydrogen s u l p h i d e t h e s a m p l e has t o be d i l u t e d with o x y g e n - f r e e distilled water prior t o t h e a n a l y s i s .
3.2.
PRINCIPLE
The method is based o n the f o l l o w i n g principle : the a c i d i f i e d s a m p l e i s a l l o w e d to react w i t h d i m e t h y l p-phenylene d i a m i n e , w i t h ferric ions a s catalyst. A c o m p l e x o x i d a t i o n a n d s u b s t i t u t i o n t a k e s p l a c e , r e s u l t i n g in t h e q u a n t i t a t i v z i n c o r p o r a t i o n o f a n y s u l p h i d e sulphur present into a h e t e r o c y c l i c d y e called m e t h y l e n e blue. T h e absorption o f light by the s a m p l e i s m e a s u r e d before or a f t e r dilution in 1 , 5 or 10 cm c e l l .
3.3. 3.3. 1 .
REAGENTS
___________________ N , N - d i m e t h y l p-phenylene _____ di _a _m _ i_ n_ e _d _ i_ h_ yd _r _ o _ c_ h_ lo _r ide _ _ _ _ _ vL _ ~ ~ ~ .
(CH NC6Hq.NH72HC1( 1 , 4 ) . 1 g i s d i s s o l v e d i n 500ml 6M hydrochloric. . 3) 2 This acid may be prepared by diluting concentrated HCI (37 perce-t, 1.19) with an equal amount of distilled water.
3.3.2. _______________L_El_. F e r r i c chloride a 7 FeCl . 8 g is dissolved in hydrochloric acid (6 moles/liter) (prepared a s a a o v e ) to 500 m l . 3-3.3.
__ O x y g_______________________ e n - free di st i 1 led water
A s u i t a b l e v o l u m e o f d i s t i l l e d w a t e r i s boiled f o r 3 0 m i n u t e s Nitrogen g a s may be bubbled t h r o u g h t h e w a t e r d u r i n g t h e b o i l i n g . As the water c o o l s down t o room t e m p e r a t u r e , n i t r o g e n g a s must be continually bubbled t h r o u g h t h e w a t e r . T h i s i s c o n t i n u e d a s l o n g a s water i s needed for t h e c a l i b r a t i o n .
3.3.4.
S u l p h i d e stock s o l u t i o n . _______________________
( A b o u t 3 . 1 2 pmoles/mlS2-). . A s o l u t i o n o f s o d i u m s u l p h i d e i s prepared w i t h oxygen-free distilled w a t e r . C r y s t a l s of Na2S.9H 0 2 p.a. a r e q u i c k l y washed w i t h distilled water by s q u i r t i n g from a w a s h i n g bottle. The c r y s t a l s a r e dried w i t h f i l t e r p a p e r and p l a c e d in a p r e - w e i g h e d , g l a s s s t o p p e r e d , w e i g h i n g g l a s s . 0.750 g i s w e i g h e d on a n a n a l y t i c a l balance a n d dissolved i n o x y g e n - f r e e d i s t i l l e d w a t e r (added with aid o f a s i p h o n ) t o 1 000 r n l in a volumetric flask.
3.3.5.
12
--_ S u l p ___-______h i d e w o r k i n g______-__ s o l u t i o n (about 0,156 !Jmoles/ml S 7
S i p h o n a s u i t a b l e v o l u m e o f oxygen-free distilled water i n t o a 5 0 0 ml v o l u m e t r i c f l a s k . I n t o t h i s i s pipetted 25 ml of the sulphide s t o c k s o l u t i o n a n d o x y g e n - f r e e distilled water i s added t o the m a r k . F o r the g r e a t e s t a c c u r a c y , t h e concentration i s determined by t i t r a t i o n a s described below. T h e solution i s stable f o r only 15-30 minutes.
T h e s a m e s o l u t i o n a s f o r the determination o f dissolved o x y g e n . 5 g Na S 0 .5H20 p.a. i s dissolved in 1 000 ml distilled w a t e r . Add 5 mf ?sJbutyl a l c o h o l before diluting t o the full volume o f t h e v o l u m e t r i c flask.
3.3.7.
Potassium i o d a t e s o l u t i o n ( 0 . 0 0 5 5 5 moles/litre)
W e i g h o u t a c c u r a t e l y 1.1891 g KIO analytical g r a d e (molecular w e i g h t 2 1 4 . 0 4 ) , dried a t 18OOC f o r one h o a r . D i s s o l v e i n distilled w a t e r a n d d i l u t e t o 1 000 m l . T h i s s o l u t i o n is t h e same a s used f o r t h e o x y g e n d e t e r m i n a t i o n s a n d i t i s very s t a b l e .
T h e s a m e s o l u t i o n a s f o r t h e dissolved o x y g e n determination. O n e v o l u m e o f a c i d i s c a r e f u l l y mixed w i t h one v o l u m e o f distilled water under constant cooling and mixing.
3.3.9.
Starch solution
1 g per 100ml distilled water or Thyodene indicator as for the dissolved o x y g e n d e t e r m i n a t i o n .
3.3.10.
Potassium iodide K I , p.a. c r y s t a l s .
3.4.
3 . 4 . 1 . Winkler b o t t l e s , volume calibrated o r volumetric flasks of 100rnl volume. 3 . 4 . 2 . S p e c t r o p h o t o m e t e r o f filterphotometer w i t h filter at or c l o s e t o 670 nm. I c m a n d 5cm o r 10cm c e l l s . 3.4.3. Automatic s y r i n g e pipettes.
3.5.
SAMPLING
13
The s a m p l e s must be o b t a i n e d with plastic w a t e r s a m p l e r s . If these are not a v a i l a b l e , m e t a l l i c o n e s may be used provided t h a t the inner s u r f a c e s are p l a s t i c - c o a t e d .
3.6.
ANALYTICAL PROCEDURE Calibration ----------_ S t a n d a r d i z a t i o n o f the thiosul-phate _solution. P r o c e e d a s described i n s e c t i o n 2.6.2. a b o v e .
3.6. 1. 3.6. 1 . 1 .
3.6.1.2.
S t a n d a r d i z a t i o n o f th.e s u l p h i d e working s o l u t i o n .
T h i s is carried out w i t h i n m i n u t e s a f t e r p r e p a r a t i o n o f the working solution ( 3 . 3 . 5 ) and a t the s a m e time a s the p r e p a r a t i o n of t h e photometric s t a n d a r d s a m p l e s (3.6.1.3). T a k e six E r l e n m e y e r f l a s k s with ground g l a s s s t o p p e r s a n d add t o each about 1 0 ml distilled w a t e r a n d 1-2 g potassium i o d i d e (3.3.10). P i p e t t e i n t o e a c h f l a s k 10.00 m l i o d a t e s o l u t i o n (3.3.7). A d d 1.0 ml sulphuric a c i d (3.3.8) i n t o e a c h flask. I n t o t h r e e o f the flasks pipette 5 0 ml o f the s u l p h i d e w o r k i n g s o l u t i o n a n d t o t h e other three a d d about 50 m l d i s t i l l e d w a t e r . S e t a l l flasks a s i d e i n a cool p l a c e whilst t h e c o l o r i m e t r i c s t a n d a r d i z a t i o n i s b e g u n t h e n titrate the c o n t e n t s of the f l a s k s w i t h t h i o s u l p h a t e u s i n g the starch indicator (3.3.9).
A B M
mean of the t i t r a t i o n s o f t h r e e s o l u t i o n s with n o added s u l p h i d e , i n m l . m e a n of the t i t r a t i o n s o f the t h r e e s o l u t i o n s c o n t a i n i n g sulphide, in ml.
concentration o f the t h i o s u l p h a t e s o l u t i o n , moles/litre
(the individual t i t r a t i o n s c o n s t i t u t i n g a t r i p l i c a t e s h o u l d agree t o within 0.05 ml).
3.6.1.3.
14
P h o t o m e t r i c s t a n d a r d samples.
From t h e w o r k i n g s o l u t i o n (3.3.5) the following standard series is prepared.
Too 100 ml v o l u m e t r i c f l a s k s or volume calibrated Winkler bottles the f o l l o w i n g v o l u m e s o f w o r k i n g solution are a d d e d by means o f a pipette or burette ( T a b l e 1 1 1 ) :
T h e s e c o n c e n t r a t i o n s c o r r e s p o n d to a s u l p h i d e working s o l u t i o n w i t h precisely 0 , 1 5 6 p m o l e s / m l . Thus t h e y have t o be c o r r e c t e d a c c o r d i n g t o the t r u e v a l u e found b y titration. I f W i n k l e r bottles are used the concentration values g i v e n a b o v e have to be r e c a l c u l a t e d accordingly for the calibration g r a p h described below. With the a i d of a s i p h o n the bottles a r e filled up with o x y g e n - f r e e d i s t i l l e d w a t e r t o the 100 ml mark, o r in c a s e of c a l i b r a t e d Winkler b o t t l e s , to the neck. I n the l a t t e r c a s e a c o r r e c t i o n for the v o l u m e has to be made f o r each bottle. As s o o n a s a bottle i s f i l l e d , 1 m l o f each r e a g e n t i s a d d e d w i t h a n a u t o m a t i c s y r i n g e p i p e t t e a n d the c o n t e n t s of the bottle a r e mixed well. After 60 m i n u t e s , the s a m p l e s are measured a g a i n s t the blank, a t 6 7 0 nm i n 1 cm and/or 5 cm c e l l s , a s suitable. F r o m the r e s u l t s a c a l i b r a t i o n g r a p h for each o f the cell lengths i s prepared on m i l l i m e t e r paper. T h e g r a p h should b e a straight l i n e and g o through origin.
If higher c o n c e n t r a t i o n s a r e analysed, the graph will s t a r t d e v i a t i n g f r o m the s t r a i g h t l i n e at 40-50 p m o l e s / l i t e r . The e.xact point for the s t a r t o f the d e v i a t i o n depends on the quality o f the a m i n e s o l u t i o n . H o w e v e r , i f t h i s i s taken i n t o account when c a l c u l a t i n g the a n a l y t i c a l r e s u l t s , the graph a n d thus the working r a n g e o f the method can be extended UP to about 100 pmoles/liter.
3.6.2.
15
---Analxsis _--
An ordinary W i n k l e r b o t t l e i s filled w i t h t h e s a m p l e i n e x a c t 1 the s a m e m a n n e r as d e s c r i b e d f o r the d i s s o l v e d o x y g e n determination (2.5.). The reagents d i a m i n e and ferric ( 3 . 3 . 1 . and 3 . 3 . 2 ) c h l o r i d e are i m m e d i a t e l y added w i t h pipettes ( p r e f e r a b l y a u t o m a t i c s y r i n g e pipettes) t o the s a m p l e . Let the p i p e t t e t i p s e x t e n d d e e p i n t o t h e s a m p l e bottle. The s t o p p e r i s n o w i n s e r t e d a v o i d i n g a i r b u b b l e s . The blue c o l o u r s t a r t s d e v e l o p i n g i n a f e w m i n u t e s a n d t h e s a m p l e is r e a d y for measurement in a p h o t o m e t e r a f t e r 30 m i n u t e s . H o w e v e r , i f t h e s a m p l e contains high c o n c e n t r a t i o n s o f h y d r o g e n s u l p h i d e , one hour must be a l l o w e d for f u l l c o l o u r d e v e l o p m e n t . T h e c o l o u r intensity m a y be regarded a s c o n s t a n t f o r a t l e a s t 24 h o u r s . The colour i n t e n s i t y o f t h e s a m p l e i s m e a s u r e d a g a k n s t distilled w a t e r (or, i f n e c e s s a r y , c o m p e n s a t e d f o r by r e a g e n t b l a n k s , a s described i n the n o t e below) a t 670 nm u s i n g 1 o r 5 cm c e l l s a s required. I f t h e sample c o n t a i n s higher c o n c e n t r a t i o n s o f h y d r o g e n s u l p h i d e t h a n 100 p m o l e s / l i t e r , it h a s t o be d i l u t e d prior t o t h e analysis. T h i s is done by p i p e t t i n g a s u i t a b l e v o l u m e o f t h e s a m p l e i n t o a m e a s u r i n g flask o r W i n k l e r bottle c o n t a i n i n g s o m e o x y g e n - f r e e distilled w a t e r . The pipette t i p should e x t e n d b e l o w t h e s u r f a c e of t h e water. Then m o r e o f t h e d i l u t i n g w a t e r i s a d d e d by m e a n s o f a siphon u p to the c a l i b r a t e d v o l u m e of t h e f l a s k o r bottle. T h e reagents a r e added a n d t h e s a m p l e i s then t h o r o u g h l y mixed. A c c o u n t has t o be t a k e n of t h e d i l u t i o n f a c t o r w h e n c a l c u l a t i n g the r e s u l t of the analysis. According to S t r i c k l a n d a n d P a r s o n s (1968) it may be n e c e s s a r y to c o m p e n s a t e the a b s o r p t i o n v a l u e o f e a c h s a m p l e for t h e a b s o r p t i o n v a l u e of a reagent b l a n k . T h i s l a t t e r v a l u e i s o b t a i n e d by a d d i n g reagents t o filtered s u r f a c e w a t e r a n d m e a s u r i n g t h i s a g a i n s t t h e same filtered water c o n t a i n i n g no r e a g e n t s . T h e a b s o r p t i o n v a l u e should not e x c e e d 0.5 i n a 10 c m c e l l and s h o u l d preferably be l e s s than 0.25. H o w e v e r , t h e blank v a l u e s a r e u s u a l l y n e g l i g i b l e , e v e n if a discoloured a m i n e s o l u t i o n i s used.
Note : It is difficult to prepare a standard solution of sulphide with a high degree of accuracy. Therefore a systematic error may be included in the analysis. Using the method described, this error will be below 2%.
3.7.
CALCULATION AND REPORTING OF RESULTS
For the routine analysis a calibration factor is calculated. Select from the calibration graph a corrected absorbance value (e.g. 0,500)and find its corresponding concentration figure (e.g. 20,2 pmoles/litre) and calculate the factor F for the cell length that was used for the measurement :
20,2 0,500
which gives F
40,4
Then the concentration of the sample, inmnoles/litre, is obtained by multiplying its abosrbance with the factor F.
3.8.
I 6
A t a c o n c e n t r a t i o n level o f 25 p m o l e s / l i t e r , the precision ~ d be n e x p r e s s e d a s +0.3%lnu;;;F;er o f r e p l i c a t e s a m p l e s .
3.9.
REMARK
I t s e e m s i m p o s s i b l e to o b t a i n a diamine that is not more or l e s s d i s c o l o u r e d . H o w e v e r , t h i s d o e s not seem t o affect the r e s u l t s . O c c a s i o n a l t e s t s u s i n g a o n e - y e a r old brownish diamine s o l u t i o n produced r e s u l t s w h i c h differed only slightly from t h e r e s u l t s o b t a i n e d w i t h a f r e s h l y prepared s o l u t i o n .
DETERMINATION OF DISSOLVED INORGANIC PHOSPHATE
4.1. SCOPE AND FIELD OF APPLICATION

T h i s i s a m o d i f i c a t i o n o f t h e w e l l known S i n g l e solution method by M u r p h y a n d Riley (1962). T h e u s e o f t w o different solutions m a k e s the reagents more stable.
N o e r r o r from s a l i n i t y w i l l o c c u r i n the d e t e r m i n a t i o n s a s t h i s d e v i a t i o n is l e s s t h a n 1 percent. No i n t e r f e r e n c e s from c o p p e r , i r o n a n d s i l i c a t e o c c u r a t c o n c e n t r a t i o n s s e v e r a l t i m e s higher than t h o s e i n s e a water. Arsenate r e a c t s g i v i n g a similar c o l o u r , but i s regarded a s e x i s t i n g i n m i n i m a l a m o u n t s i n s e a water. Recent results by J o h n s o n ( 1 9 7 1 ) i n d i c a t e , h o w e v e r , that t h e concentration o f arsenate i s higher t h a n w a s believed. I n periods o f high primary production - a n d hence l o w c o n c e n t r a t i o n s o f p h o s p h a t e a r s e n a t e a n d phosphate l e v e l s a r e s a i d t o be e q u a l a n d s o m e t i m e s arsenate may be dominating?
T h e d e t e r m i n a t i o n , on a n unfiltered s a m p l e , gives t h e concent r a t i o n o f d i s s o l v e d i n o r g a n i c phosphate ions i n t r u e s o l u t i o n and probably a l s o i n c l u d e s a s m a l l f r a c t i o n o f these i o n s w h i c h are a b s o r b e d i n t o p a r t i c l e s and a r e s u b s e q u e n t l y dissolved by the acid i n the mixed r e a g e n t . T h e l a t t e r f r a c t i o n i s not included i f the a n a l y s i s i s performed o n a filtered s a m p l e .
4.2. PRINCIPLE
T h e p h o s p h a t e in water i s allowed t o react w i t h ammonium m o l y b d a t e , f o r m i n g a c o m p l e x h e t e r o p o l y acid. T h i s acid i s reduced by a s c o r b i c a c i d t o a blue-coloured c o m p l e x , the light a b s o r p t i o n of which i s m e a s u r e d in a p h o t o m e t e r . Normally, t h i s reduction i s s l o w , b u t by a d d i n g a c a t a l y s t - i n this case antimonyl tartrate t h e r e d u c t i o n proceeds s w i f t l y .
1 7
This i s a modification o f the well k n o w n s i n g l e s o l u t i o n method b y Murphy and Riley (1962). T h e use o f t w o different s o l u t i o n s m a k e s the reagents more stable. According t o e x p e r i e n c e from i n t e r c a l i b r a tions it i s necessary to measure t h e s a m p l e s o f s e a water a g a i n s t water from t h e same depths i n order t o c o m p e n s a t e f o r the v a r y i n g turbidity. This turbidity i s s o m e w h a t by the acid in the m o l y b d a t e r e a g e n t . The turbidity blank s h o u l d f o r t h i s r e a s o n be o f t h e s a m e acidity as t h e sample. Therefore it i s convenient t o use t w o r e a g e n t solutions - o n e containing a l l c o m p o n e n t s except t h e a s c o r b i c a c i d and a second which i s the a s c o r b i c acid s o l u t i o n .
4.3. 4.3. 1
REAGENTS
__-___________L_-------L_______________ Sulphuric acid about moles/litre
4 75
253 m l o f concentrated s u l p h u r i c acid ( d e n s i t y 1.84) i s , under cooling and continuous m i x i n g , carefully poured i n t o a s u i t a b l e volume (600 m l ) of distilled water. After cooling t h e v o l u m e i s adjusted to 1 000 ml with distilled water.
Dissolve 9.0 g o f the salt (NH4!6M~ 024.4H 0 i n distilled water and dilute to 100 ml. A n e w s o l u t i o n zhould 6 e made i f a n y precipitation occurs.
4 . 3 -3.
_________________ Potassium a n t i m o n x________________ l t a r t r a t e s o l u t i o-L_---------_----_ n 0.1 moles/litre
Dissolve 3.25 g of t h e s a l t K(SbO)C4H406 i n distilled water and dilute to 100 ml. T h e s o l u t i o n should be renewed i f a precipitation occurs. 4 . 3 - 4 - Acid
- molybdate reagent __________ ___-_------
200 m l of the sulphuric a c i d ( 4 . 3 . 1 ) i s mixed under continuous stirring with 45 ml o f t h e molybdate s o l u t i o n (4.3.2). Finally, 5 r n l of the tartrate s o l u t i o n ( 4 . 3 . 3 ) i s a d d e d . I f s t o r e d cold i n a d a r k bottle the reagent i s stable for s e v e r a l m o n t h s .
7 . 0 g of ascorbic a c i d , C H 0 i s dissolved i n distilled water and diluted to 100 ml. If s g o F e 8 c o l d i n a dark b o t t l e , t h e reagent i s stable f o r some weeks. F o r preservation, 0.05 g of EDTA ( e t h y l e n e diamine t e t r a a c e t i c acid) and 1 m l formic a c i d , H C O O H , may be a d d e d .
4.3.6.
1 8
Phosphate stock solution
P o t a s s i u m d i h y d r o g e n p h o s p h a t e i s dried o v e r concentrated s u l p h u r i c a c i d o r a t 100C. 0.3403 g o f KH2P04 p.a. is dissolved i n distilled w a t e r a n d d i l u t e d t o 1 000 ml in a v o l u m e t r i c f l a s k . S o m e d r o p s o f chloroform a r e a d d e d for preservation. This s o l u t i o n c o n t a i n s 2.5 p m o l e s / m l PO4-P. The solution is s t a b l e for many m o n t h s i f kept i n a r e f r i g e r a t o r .
10.0 ml o f the s t o c k s o l u t i o n i s diluted with distilled water t o 1 000 ml in a v o l u m e t r i c flask. 1 ml contains 0 . 0 2 5 h m o l e POLI-P. T h i s s o l u t i o n should not be s t o r e d for f u t u r e use.
4.4.
4 . 4 . 1 . S p e c t r o p h o t o m e t e r o r f i l t e r photometer w i t h filter at o r close t o 8 8 2 nm. C e l l s o f 5 cm a n d / o r 1 0 cm l e n g t h . 4 . 4 . 2 . G l a s s s t o p p e r e d t e s t t u b e s , 3 0 - 5 0 ml v o l u m e , o r o t h e r suitable containers. 4 . 4 . 3 . Kipp pipette ( F i g u r e 2 ) o f 35 ml o r 2 5 m l volume for d i s p e n s i n g of s a m p l e s . T h i s h a n d y pipette i s marketed under a v a r i e t y of n a m e s e . g . p o u r - o u t d i s p e n s e r , TIPET, a u t o m a t i c transfer p i p e t t e , r e p e a t e r pipette.
Automatic s y r i n g e p i p e t t e s o f 1 ml or 2 ml v o l u m e for reagent additions.
T h e s e last t w o t y p e s o f pipettes are not n e c e s s a r y , but they m a k e the r o u t i n e h a n d l i n g o f a great number of s a m p l e s m u c h easier.
4.5.
SAMPLING AND PRETREATMENT
The a n a l y s i s should be c o m m e n c e d as soon a s possible after s a m p l i n g , p r e f e r a b l y within h a l f an h o u r , and definitely before two hours. S a m p l e s s h o u l d be k e p t i n a cool dark place a n d not w a r m e d to room t e m p e r a t u r e until the t i m e of analysis. I f the analysis has t o be delayed for m o r e t h a n a b o u t one hour, s t o r e the samples i n the r e f r i g e r a t o r . O n s h o r t t r i p s it m a y be possible t o a d d the r e a g e n t s t o the s a m p l e s a n d perform the measurements a s h o r e . There i s c o n f l i c t i n g e v i d e n c e c o n c e r n i n g the effect of different p r e s e r v a t i o n m e t h o d s and the f a t e o f phosphate m o l e c u l e s in glass and p l a s t i c b o t t l e s . No e n t i r e l y s a t i s f a c t o r y preservation method is k n o w n (for s a m p l e s c o l l e c t e d for nutrient analysis). H o w e v e r , quick f r e e z i n g i n a dry i c e / a c e t o n e bath has o f t e n been used when there is no alternative to storing samples.
19
Figure 2
Kipp Pipette
I ! .
20
6.
ANALYTICAL PROCEDURES Calibration --_--------
4.6.1.
A s e r i e s o f w o r k i n g s t a n d a r d s a r e prepared from t h e phosphate w o r k i n g s o l u t i o n by d i l u t i o n w i t h d i s t i l l e d water. If 50 m l volumetric f l a s k s are u s e d , T a b l e IV c a n be u s e d .
I I II
0.40
11
I1
It
0.80
It
II
It
It
II
II
2.00
II
I1
11
4.00
II
As described i n ( 4 . 6 . 2 ) b e l o w , 3 5 ml ( o r 25 m l ) portions o f t h e s e s o l u t i o n s a r e t r a n s f e r r e d t o t e s t tubes and reagents are added. F o r t h e c a l i b r a t i o n no t u r b i d i t y b l a n k s (samples w i t h only the acid m o l y b d a t e r e a g e n t ) a r e n e e d e d . A r e a g e n t blank i s prepared from the s a m e v o l u m e s o f distilled water a n d reagents.
After m e a s u r e m e n t t h e a b s o r b a n c e s - corrected for the reagent a r e plotted v e r s u s c o n c e n t r a t i o n on millimetre paper. This blank c a l i b r a t i o n s h o u l d give a s t r a i g h t l i n e up to at least 10 ,umoles/litre. o f *P04-P.
4.6.2.
Analysis
All g l a s s w a r e t o be used must be absolutely clean and s h o u l d n o t be used for o t h e r purposes. P o w d e r e d synthetic detergents contain p h o s p h a t e (large a m o u n t s ) i n m o s t c a s e s , and s u c h products should n o t be used. A s o l u t i o n of distilled water containing the detergent u s e d for c l e a n i n g mu.st be tested f o r phosphate c o n t e n t . T h i s is the o n l y s a f e way t o avoid u n n e c e s s a r y complications.
S e a water for t h e d i f f e r e n t a n a l y s e s is preferably poured d i r e c t l y from t h e water s a m p l e r s i n t o stoppered g l a s s bottles of s u i t a b l e v o l u m e . F o r r o u t i n e a n a l y s i s o f a great number o f samples i t i s v e r y convenient t o measure t h e different s u b s a m p l e s with a n a u t o m a t i c d i s p e n s i n g p i p e t t e ( K i p p p i p e t t e ) with a ground glass joint f i t t i n g t o t h e g l a s s s a m p l e bottle.
21
From each s a m p l e t w o 35-ml p o r t i o n s a r e t r a n s f e r r e d t o test tubes. One o f the portions is r e g a r d e d a s the s a m p l e a n d t h e o t h e r is the turbidity blank. To each o f the p o r t i o n s 1 m l o f t h e a c i d molybdate solution (4.3.4) i s a d d e d a n d t o t h e s a m p l e a l s o 1 m l o f the ascorbic acid s o l u t i o n (4.3.5). Mix w e l l b e t w e e n the a d d i t i o n s . In case 25 m l o f sample is a n a l y z e d use 0.7ml portions of the reagents.
After five m i n u t e s each s a m p l e i s measured a g a i n s t i t s turbidity blank in the photometer at 882 nm. I f t h i s w a v e - l e n g t h i s not p r o v i d e d , it i s possible t o m e a s u r e a t a shorteu. w a v e - l e n g t h but this r e s u l t s in decreased s e n s i t i v i t y . T h e colour o f t h e s a m p l e s i s s t a b l e for a t least 24 h o u r s . H o w e v e r , t h e reduction of t h e a r s e n a t e m o l y b d a t e c o m p l e x i s c o m p l e t e a f t e r one h o u r (see a l s o p a r a g r a p h 4.6.3.2.).
--_NOTE
: The u s e of a t u r b i d i t y blank may be o m i t t e d i n case o f h i g h c o n c e n t r a t i o n s of phosphate and - a t t h e s a m e t i m e insignificant turbidity. H o w e v e r , t o test the s i g n i f i c a n c e o f t h e t u r b i d i t y , i t i s recommended to a n a l y s e at l e a s t s o m e s a m p l e s w i t h and w i t h o u t a turbidity blank.
I f 5-cm cells a r e used i n s t e a d o f 10 c m , the s a m p l e v o l u m e s may be r e d u c e d to 25 ml a n d the r e a g e n t v o l u m e s t o 0.7 ml.
4.6.3.
Disturbances _____-_-_--Disturbance from s i l i c a t e
4.6.3.1.
It i s a well k n o w n fact t h a t t h e blue p h o s p h o m o l y b d e n u m c o m p l e x formec! with a s c o r b i c acid is s t a b l e f o r h o u r s . I f g r e a t amounts o f silicate are present, especially in some types o f river and l a k e w a t e r s and s t a g n a n t s e a w a t e r t h a t c o n t a i n s hydrogen s u l p h i d e a blue silicomolybdate c o m p l e x is g r a d u a l l y f o r m e d . With the s t a t e d c o n c e n t r a t i o n s of s u l p h u r i c acid a n d m o l y b d a t e , a n d by m e a s u r i n g t h e a b s o r p t i o n after f i v e m i n u t e s , L ~ Jt o 10 m g o f S i 0 4 - S i p e r l i t r e can be present without d i s t u r b a n c e .
4.6.3.2. Disturbance f r o m a r s e n a t e
T h e arsenate i o n gives w i t h s u l p h u r i c a c i d , m o l y b d a t e a n d ascorbic a c i d , a blue c o m p l e x . H o w e v e r , t h e r e a c t i o n i s r a t h e r s l o w , being complete a f t e r one h o u r . By m e a s u r i n g t h e a b s o r p t i o n a f t e r five m i n u t e s the d i s t u r b a n c e from a r s e n a t e i s a v o i d e d . I t m a y be mentioned that with s t a n n o u s c h l o r i d e a s t h e r e d u c t a n t t h e blue a r s e n a t e c o m p l e x is formed i m m e d i a t e l y .
22
4.6.3.3.
I n f l u e n c e from t h e c o n c e n t r a t i o n of s u l p h u r i c a c i d
T o a c h i e v e a rapid c o l o u r development a n d to depress the i n t e r f e r e n c e from s i l i c a t e , i t i s o f fundamental i m p o r t a n c e that the f i n a l r e a c t i o n t a k e s place a t a pH l o w e r than 1 a n d that t h e ratio b e t w e e n s u l p h u r i c a c i d (in m o l e s / l i t e r ) and molybate (in per c e n t ) i s kept between 2.0 2 . 5 . W i t h the r e a g e n t s described the concent r a t i o n o f s u l p h u r i c a c i d i n t h e s a m p l e i s about 0 . 1 mole/liter. I t h a s been s h o w n e x p e r i m e n t a l l y that w i t h the c o n c e n t r a t i o n o f a n t i m o n y i o n s used h e r e ( 0 . 0 0 0 6 8 per c e n t ) the a c i d concentration m a y v a r y between 0.06 and 0 . 1 5 moles/liter whereas a t 0.18 moles/liter t h e a b s o r b a n c e d e c r e a s e s by about 30 per cent.
4.7.
T h e c o r r e c t e d a b s o r b a n c e i s used for evaluation of t h e r e s u l t , u s i n g the c a l i b r a t i o n g r a p h described i n paragraph 4.6.1).

F O routine ~ analyses a calibration factor is calculated. S e l e c t from the g r a p h a n a b s o r b a n c e v a l u e (e.g. 0.170) and find i t s c o r r e s p o n d i n g c o n c e n t r a t i o n figure ( e . g . 1.65 umoles/litre 1 and calculate.
F5cm
= ----1.65 0.170
which g i v e s
F5cm
9.71
Then t h e c o n c e n t r a t i o n o f a s a m p l e , i n moles/litre, i s obtained by m u l t i p l y i n g i t s a b s o r b a n c e with t h e factor F.
4.8:
From a n u t r i e n t i n t e r c a l i b r a t i o n reported by Koroleff and P a l m o r k (see G r a s s h o f f 1976) a c c u r a c y was given a s + 15 per cent a t low l e v e l ( 0 . 2 umole/litre ), +5 per cent at a n i n t e r m e d i a t e - l e v e l (0.9 umo1e:Liter) a n d +2 per cent' at a h i g h level (2.8 umoles/litre,).
-23-
5.
DETERMINATION OF TOTAL PHOSPHORUS
A great number of sensitive analytical procedures for determination of total phosphorus have been published during recent years (see e.g. Koroleff 1976). All of these methods involve the use of peroxodisulfate as an oxidant to transform the various phosphorus fractions to dissolved orthophosphate.
However, in most marine chemistry programmes not only total phosphorus but also total nitrogen are determined. In 1977 Koroleff presented a practical method for the simultaneous determination of these parameters from the same sample and in 1981 Valderrama published a slightly modified variant of the same method. Therefore, nr, separate procedure for the determination of total phosphorus is presented here, but the analyst is guided to the technique described in section 10 "Simultaneous persulfate oxidation of phosphorus and nitrogen compounds in seawater".
6.
DETERMINATION OF
REACTIVE SILICATE
6.1 SCOPE A N D FIELD OF APPLICATION Depending on pH the yellow silicomolybdate complex that is formed in this analysis exists in two isomeric forms, which differ only in their hydration. The -silicomolybdic acid which is formed below a pH value of 2.5 is rather unstable, but has a higher molar absorbance than the -acid. In early work silicate was determined by visual or photometric estimation of the -complex. The literature has been reviewed by Mullin and Riley (1955), and in 1968 a careful study of the optimum conditions for this technique was performed by a research group at the Sagami Chemical Center in Japan. In their modified method the colour of the complex is stable from 5 to 20 minutes at room temperature, in both distilled water and sea water; the salt effect is diminished to 3 percent. Grasshoff (1984) has developed a method based on the formation of the -isomer at a pH of 3.7 to 4.0, and states that the colour in sea water is stable (after two hours) for days.
6.2 PRINCIPLE
The determination of dissolved silicon compounds in natural waters is based on the formation of a yellow silicomolybdic acid, when a more or less acidic sample is treated with a molybdate reagent. Only silicic acid and its dimer react with molybdate at any appreciable speed and the methods therefore give only the amount of "reactive" silicate, which is probably a reasonable measure of the silicate available to growing phytoplankton.
-24-
Since both of the yellow silicomolybdic acid isomers are rather weak in colour, several methods have been developed in which they are reduced to intensely coloured, blue complexes. A wide range of inorganic and organic reducing agents have been used. A mixture of metol and sulphite is commonly used. In the method described here ascorbic acid acts as the reductant,
The molar absorption of the coloured complex This implies that if the concentrations are given 1.000 is equal to 45 pmoles/litre Si with a 1 cm With this cell length up to about 80 poles/litre (absorbance about 1.7). With a 10 cm cell, 0.045 determined. As this corresponds to an absorbance as the limit for direct determination. 6.3
REAGENTS
is about 22 200 litres/mole/cm. as poles/litre, the absorbance cell used for the measurement. of Si can be determined pmoles/litre of Si can ke of 0.010, it can be regarded
All solutions should be prepared from reagent grade (pro analysi) chemicals, using good quality distilled water. The solutions should be stored in plastic bottles rather than glass bottles.
6.3.1 Ammonium heptamolybdate solution 0.16 mole/litre
l . . Disssolve 49.5 g (NH4)6M07024.4H20 and dilute to 250 m salt dissolves slowly, moderate heating may be applied.
As the
6.3.2
Sulphuric acid solution about 3,7 moles/litre
198 ml H2204 (concentrated sp.gr. 1.82) is carefully poured, under constant cooling and mixing, into distilled water and finally diluted to 1000 ml.
6.3.3 Molybdate reagent Add a measured volume of the molybdate solution (6.3.1) to an equal volume of the sulphuric acid solution (6.3.2) and mix. 6.3.4 Oxalic acid solution 0.7 mole/litre Dissolve 63 g C204H2 and dilute to 1000 ml. 6.3.5 Ascorbic acid solution 0.1 mole/litre Dissolve 4.4 g C6H806 and dilute to 250 ml. bottle, the reagent is stable for weeks. 6.3.6 Silicate stock solution Sodium silicofluoride, Na SiF6 p.a. is dried over concentrated sulphuric acid to constant weigzt. Weigh in 0.4701 g of the salt and dissolve in distilled water in aplastic beaker. Transfer to a 500-ml volumetric flask and dilute to the specified volume with d-istilledwater. Stored cold in a brown
-25-
Finally, transfer to a plastic bottle. 1 ml contains 5.0 umoles Si. The solution is stable for several months. 6.3.7 Silicate working solutions
50 ml of the stock solution (6.3.6) is diluted with distilled water or synthetic sea water of appropriate salinity (see below) to a final volume of 500 ml in a volumetric flask made of plastic materials. 1 ml contains 0.50 pole/litre Si. This solution should be used at once.
6.3.8
Synthetic sea water
25 g sodium chloride, NaCl p.a., and 8 g magnesium sulphate heptahydrate, MgS04.7H20 p.a. are dissolved in distilled water for every litre of solution. This water must be stored in a plastic bottle. For analytical purposes it is equivalent to a salinity of 28.10-3. For calibration work it may be diluted to the desired salinity. the silicate concentration should be below 1-2 ,moles Si/litre.
6.4
6.4.1 Test tubes. Stoppered tubes of glass may be used but plastic ones are preferred (inexpensive tubes made from polystyrene are commercially available). Small plastic bottles (25-50 ml) may also be used.. If glass tubes are used they must initially be carried through the analytical procedure at least five times, otherwise they will yield too low results.
6.4.2
Automatic syringe pipettes (3 pcs.), capacity 2 ml.
6.4.3 Spectrophotometer, or filter photometer with the filter at or close to 810 nm. If this is not provided, use a filter having a maximum transmission above 700 nm; in this case the sensitivity is reduced and a slight deviation from the Lambert-Beer's law may occur. Cells of 1,5 and 10 cm length as required.
6.5
Samples of sea water for silicate determination should not be stored in glass bottles or more than a short while prior to analysis and it is best, therefore, to transfer samples directly into containers of polyethylene or waxed glass. To minimize the effects of diatom multiplication, store samples in the dark and for nc longer than a day prior to analysis. Conflicting opinions and evidence exist as to whether samples can be stored or long periods or not, and if stored, whether they should be deep frozen or not. If deep frozen, the silicate concentration can be expected to be acceptable only for samples containing less than 50 poles/litre. When stored frozen, silicate tends to polymerize. Therefore, samples must be allowed to stand for at least three hours at room temperature after thawing. Thus it is recommended to perform the
-26-
analysis as soon as possible after sampling. If the sampler have to he stored, the tolerable storage time ought to be investigated and the possible size of the error estimated.
6 . 6
ANALYTICAL PROCEDURES
6 . 6 . 1
Calibration
From the working solution (6.3.7) a series of working standards are prepared by diluting with distilled water or synthetic sea water (6.3.8) in volumetric flasks. Table V below may be used. For routine analysis in sea water it is normally sufficient to prepare the calibration solutions in synthetic sea water of a salinity representing a mean value of the salinities in the working area. However, for the greatest accuracy, it should be more convenient to calibrate in distilled water and then correct for the salt error. This is of particular importance in areas where the silicate concentrations in surface water may become very low as a result of phytoplankton primary production.
TABLE
Preparation of working standards for silicate
ml of working solution/100 ml (dilute with synthetic sea water or distilled water)

0 . 0 0.5 1 . 0 2 . 0 5.0 1 0 . 0 20.0 25.0 30.0
poles/litre si
0.0 2.5 5.0 LO. 0 25.0 50.0 100.0 125.0 150.0
I
Some of the standards are chosen depending on the expected concentration range in the samples. Samples are pipetted from the working standards to the test tubes. The distilled water or synthetic sea water of the chosen salinity respectively, should be used as the blank. The reagent blank must be prepared together with every calibration.
-27-
The blank sample compensates for the silicate content in the reagents, as well as in the distilled water (or synthetic sea water) used for the dilution of the standards. However, the same blank value cannot be used for the analysis of the natural water samples. In this latter case a reagent blank has to be used. Preparation of reagent blank and analysis of samples is described in the section below. The absorbance is plotted versus concentration on millimetre paper. The result should be a straight line over the entire range of concentration.
6.6.2
Analysis
To 35 ml of the sample in a plastic test tube 1 . 0 ml of the molybdate reagent (6.3.3) is added. After 10 to 20 minutes (for distilled water) or 5 to 10 minutes (for sea water), 1.0 ml of the oxalic acid solution (6.3.4) is added followed immediately by 1.0 ml of the reductant ascorbic Swirl gently during the reagent additions. After at least acid (6.3.5). 30 minutes the absorbance of the sample is measured against distilled water in a cell of suitable length at a wave-length of 810 nm. Correct the measured absorbancy of the sea water samples by substracting that of the reagent blank. The reference sample is used for correcting the absorbances of the calibration samples. The reagent blank is prepared by carrying out the above procedure using 35 ml of distilled water. The absorbance is denoted Al. Repeat the determination, but with only 0.5 mL of the molybdate reagent. In this case measure the absorbance after one hour (A2). Absorbance of reagent blank =
2(A1
- A2)
The absorbance caused by the silicate content of the reagents only is thereby obtained. This reagent blank should be performed every time any of the reagent solutions are prepared. In addition, it is advisable to check the reagent blank from time to time. For the most precise estimate of low amounts of silicate using a 10 cm cell, a reference absorbance should be measured for every sample to compensate for its natural turbidity. The reference solution is prepared by adding 3 ml of 0.25 mole/litre sulphuric acid solution (e.g., prepared by diluting 7 ml of the 3.7 moles/litre solution to 100 ml with distilled water) to 35 ml of sample. Thus the absorbance of the sample is:
A
sample
= A
measured
- Areference
- Areagent blank
6.7
CALCULATIONS AND REPORTING OF RESULTS
For the routine analyses a calibration factor is calculated. Select from the graph (described in section 6.6.1) an absorption value, corrected as described in section 6.6.2 (e.g., 0.730) and find its corresponding concentration figure (e.g., 37,8 umoles/litre) and calculate: F1 cm
37,8 0,730
which gives F
1 cm
58,8
-28-
Then the concentration of a sample, in poles/litre, is obtained However, for the most by multiplying its absorbance with the factor F. accurate work in marine waters a correction must be applied for the salt error. It is known that the dissolved salts of sea water reduce the final colour intensity to some extent. It has been found that the variation of the salt error can be expressed as a linear function of the sea water salinity: Atrue , distilled water
= A
observed
. (1 +
0.0045
. sample salinity)
Thus, if the calibration factor F determined for distilled water is F dist:

F sample
Fdist.
(1 f 0.0045
. sample
salinity)
Note that these figures cannot be guaranteed for general use. Because of the varying quality of reagents, etc., every laboratory should construct a correction formula of its own, by calibrating at several salinities and then calculating from the observed variations of the absorbances.
6.8
ESTIMATION OF PWCISION AND ACCURACY
From a nutrient intercalibration reported by Koroleff and Palmork (see Grasshoff 1976) accuracy was given at &4 per cent at a low level (4.5 poles/litre), 2 2 , 5 per cent at an intermediate level (45 pmoles/litre) and 26 per cent at a high level (150 poles/litre).
6.9
6.9.1
REMARKS
Effect of the oxalic acid. Oxalic acid is added for two reasons:
(a) To avoid the reduction of any excess molybdate reagent. The stability of the silicomolybdic acid in the presence of oxalic acid is limited, and the reductant is therefore added immediately after the acid; (b) to eliminate the influence of any phosphate present. Trials have been made with up to 10 umoles/litre of P and no increase in colour intensity was observed.
6.9.2
Time for colour development and colour stability
For up to about 30 poles/litre of Si, the colour reaches its maximum after about 30 minutes and is then - provided any evaporation stable for several days. However, for practical purposes is prevented it may be advisable to regard thecoloured complex as being stable for several hours. For higher concentrations, up to an absorbance of 1.7 (1 cm cell) , the intensity increases extremely slowly after 30 minutes, only about 0.1 percent per hour.
-29-
7.
7.1
DIRECT DETERMINATION OF AMMONIA WITH THE INDOPHENOL BLUE METHOD

SCOPE AND FIELD OF APPLICATION
This method, which is specific for ammonia, is applicable to all kinds of natural waters and is the recommended method for sea water. It is four times as fast as the Nessler reaction. "Ammonia" here refers to the sum of ammonia and ammonium iens, because the original proporitons of them in a water sample are pH dependent. 7.2 PRINCIPLE
In a weakly alkaline solution ammonia reacts with hypochlorite to form monochloramine, which in the presence of phenol, catalytic amounts of nitroprusside ions and an excess of hypochlorite yields indophenol blue. By increasing the concentrations of the reagents used in this method, labile organic nitrogen compounds may also react, while the reaction time is decreased. The reaction mechanism leading to the coloured complex is complicated and not yet fully understood. Probably, a quinone chlorimide is formed in an intermediate step. The intensity of the coloured complex is measured in a photometer. 7.3 7.3.1 REAGENTS
"moria-free" water
There is not a standard procedure for the preparation of water with a very low ammonia content, as the results obtained depend on the equipment and working facilities as well as the quality of the water used. Each laboratory has therefore to find the method that gives the most satisfactory result. Deionized water may sometimes be used, with or without subsequent distillation, but it must be remembered that some ion exchange resins may leach out ammonia containing organic substances. Ordinary distilled water may be used after a second distillation. In this second step, 2 ml concentrated sulphuric acid and 1 q potassium peroxodisulphate (K2S298) are added per litre. Freshly distilled, such water should contain less than 0.3 p o l e of nitrogen per litre. Another alternative is to use surface water samples in an open sea area, preferably shortly after a plankton bloom. This water usually contains very little ammonia and it should be stored in tightly closed plastic containers with thick walls. 7.3.2 Reagent A, phenol
- nitroprusside solution
Dissolve 35 grams of analytical grade phenol, C6H50H, (a slightly pink quality may be accepted) and 400 mg of sodium nitsoprusside dihydrate (Na2Fe(CN)5N0.2H20) in ammonia free distilled water and dilute to 1 litre. If the solution is stored in a dark bottle in arefriqerator it is
-30-
stable for several months. When the reagent becomes greenish it should be discarded. (It is usually a good idea to store also the pure phenol in the refrigerator). 7.3.3 Reagent 3, alkaline solution with free chlorine, variant I
Prepare a solution of hypochlorite containing 0.14 per cent of free chlorine in 0.5 pole/litre sodium hydroxide in the following way. Commercial bleaching substance or concentrated sodium hypochlorite, NaC10, can be used as stock solution. the hypochlorite content is tested as follows. Dissolve 0.5 g potassium iodide (KI) in 50 ml distilled water and acidify with 5 ml sulphuric acid, 9 poles/litre (as used in the determination of oxygen) and titrate the liberated iodine with thiosulphate . 1 pole/litre, using starch as indicator. 1 ml of the thiosulphate solution, 0 solution is equivalent to 3.54 mg active chlorine. Prepare the reagent B by dilutirg the required volume with sodium hydroxide, 0.5 )unoLe/litre (20 g NaOH dissolved in 50 ml ammonia-free distilled water and then made up to l litre with the same water to yield a solution with 140 mg chlorine per litre. Store the reagent in the refrigerator in a dark bottle. It is stable for weeks. 7.3.4 Reagent B, variant I1
The reagent can be conveniently prepared from a solid substance instead of the hypochlorite solution. Trion is the commercial name for dichloroisocyanuric acid (dichloro-s-triazine-2,4,6 (lH, 3H, SH)-trione). Trion is available in at least technical grade. The product has to be tested for its actual content of available chlorine using the procedure described above. A typical figure may be that 1 g dissolved in the . 6 per cent sodium hydroxide, l)xnole/litre, produces a soluton containing 0 chlorine. Thus, roughly 2 g Trion should be dissolved per litre of the sodium hydroxide solution (prepared as in variant I) to yield a solution with 140 mg chlorine per litre. The reagent should be stored in a dark bottle in the refrigerator and it is stable for weeks. The advantage with variant is that the solution is easy to prepare without having to analyze the Trion at every occasion. However, the reactions in the analysis will proceed somewhat slower with this reagent. 7.3.5 Buffer solution
Dissolve 66.7 g trisodium citrate dihydrate, Na3C6H507.2H20, 34 g boric acid, H3BO3, and 19.4 g citric acid dihydrate, C6H807.H20 in m o n i a free distillea water and dilute to 1 litre. 7.3.6 Ammonia stock solution
As standard substance, either ammonium sulphate or ammonium chloride of p.a. quality can be used. Before weighing, the salts must be dried to constant weight at 100C and then preferably stored in a dessicator.
-31-
Dissolve 0.1320 g of (NH4)2S04 or 0.0535 g NH 4c1 in ammonia-free water and dilute to 100 ml. Add a drop of chloroform as preservant. The solutions should be stored in glass bottles. They are stable if protected from direct sunlight and evaporation. Each solution contains 10.0 p o l e s of NH3-N per ml. 7.4
7.4.1
APPARATUS AND EQUIPMENT Test tubes with ground glass (or plastic) stoppers and a volume of 30 ml or more Automatic syringe pipettes of 2 m l capacity (3 pcs.) Spectrophotometer or filter photometer with a filter having maximum transmission at (or close to) 630 nm. Photometric cells of 1, 5 or 10 cm length as required. SAMPLING AND PRETREATMENT
7.4.2 7.4.3
7.5
The samples may not be preserved with sublimate (HgC1 ) . Acid preservation is possible, but in that case every sample must 2 e carefully neutralized with sodium hydroxide before the analysis is started, and corrections made or the ammonia content in acid and hydroxide. This makes the analysis uncertain and the acid preservation should be avoided if at all possible. Preferably, the samples should be analyzed within a short time after the sampling, and any necessary storage should be in a dark, cold place. Polluted waters are often turbid and may be filtered through washed glass wool filters. However, such waters frequently contain high concentrations of ammonia and may therefore be diluted before the analysis. Any residual turbidity can be compensated with a similarly diluted sample in the reference cell. Thus, filtration should be avoided, if possible.
7.6
a
7 . 6 . 1
Calibration
All work with ammonium analysis must be carried out where smoking is not permitted,in order to avoid disturbances from ammonium. Before the test tubes can be used for calibration or analysis, they must be carefully cleaned; it is not sufficient just to wash them, but the following subsequent cleaning procedure must be carried out. To every tube about 25 ml distilled water (not necessarily ammonia-free water) and reagents are added as described in paragraph 7.6.2. Let the reaction proceed for at least two hours and shake the tubes from time to time during the reaction period. A11 ammonia contained in the tubes (dissolved in the water or adhered to the glass wall) will react. then the tubes are rinsed with ammonia-free water and kept stoppered when not in use. The tubes should not be washed between the different sets of calibrations or analyses, but just rinsed with ammonia-free water.
-32-
The analysis of ammonia is under the influence of disturbances from variations in pH and salinity of the samples. To account for this, the calibration can be carried out in either of two ways. For work in true oceanic areas, where the salinity variations are small, the ammonia working solution and the working standards should be diluted with ammonia-free sea surface water instead of distilled water For work in e.q., estuaries where the brackish water displays large salinity variations, a calibration in distilled water, followed by corrections for the salinity of each sample, is preferred.
With the guidance of Table VI, the given volumes of ammonia working solution are pipetted into 100 ml volumetric flasks and diluted to the mark with distilled water, or sea surface water, that is free from ammonia.
TABLE
VI
Preparation of working standards for ammonium
ml of working solution to be diluted to 1OC ml

0.0 0.5 1.0 2.0 6.0
resulting concentration moles/litre

0.00 0,25 0.5
1.0
3.0 5.0 10.0
10.0 20.0
1
Note that the concentration pole/litre refers to NH3-N, which means nitrogen coming from ammonia (and not the concentration of ammonia itself). From each of the standard concentration solutions above, 26 ml (preferably three portions of 25 ml) transferred to the test tubes. These samples are then analyzed as described in paragraph 7.6.2.
The following is an example oE a calibration series (measured in a 1 cm cell) where the absorbances have not been corrected for the blank value:
-33-
poles/li tre
A
0
0.022
7.14
0.171
17.85 0.397
35.7 0.772
53.6
1.105
The blank sample here corrects for the absorbance caused by the ammonia in the reagents and also the residual ammonia in the ammonia-free water. After subtraction of the blank absorbance, a calibration graph is constructed, showing corrected absorbance versus concentration. The calibration graph is reproducible within the entire range for direct determination and it is linear up to a concentration of about 36 poles/litre. For concentrations up to 14 pmoles/litre, a 5 cm cell can be used, and with this cell, concentrations down to 0.04 pole/litre can be determined.
A new calibration is required every time any of the reagents is exchanged for a new solution.
The reagents , especially the buff er solution (7.3.5) , contain ammonium ions and hence the abosrbance caused by the ammonium in the reagents - the reagent blank - must be determined. To 22 ml of ammoniafree water (preferably three portions of 22 ml), the normal reagent volumes are added 1 ml of each. To a second set of 22 ml portions, double the reagent volumes - 2 ml of each are added. After two hours, 3 m l of ammonia-free water is added to each sample in the first set, and the absorbances of all samples are measured immediately. The difference between the mean values of the two sets is the reagent blank. This difference is caused only by the ammonia content of single volumes of the reagents.
For example: the mean of the set with 2 ml of A = 0.040 and the mean of the second set is A = 0.022. the reagent blank is then 0.018 (for 1 cm cell).
each reagent is The difference -
If this is compared with the blank value given in the calibration series it can be noticed that the nitrogen content in the water used for dilution of the calibration samples gives an absorbance of 0,022-0.018 ED 0.004, which corresponds to a concentration of about 0.2 pnole/litre. 7.6.2 Analysis
Before the analysis and between analysis of different sets of samples the test tubes must be cleaned as described in the beginning of section 7.6.1. For each sample a volume of 25 ml is measured and transferred to a test tube. Add 1 ml of the buffer solution (7.3.5) Mix and 1 ml each of the reagents A (7.3.2) and B (7.3.3 or 7.3.4). properly, e.g., by swirling the test tube, after each addition. Stopper the tubes and keep them in a dark place duxing the reaction period (2 to 6 hours; see also below in paragraph 7.9.2).
-34Measure the abosorbances of the samples in the photometer using cells of suitable length depending on the colour intensity of the samples. Use distilled water as reference and take the measurements at the wavelength 630 nm. Depending on how the calibration was done, the result of the analysis has to he calculated according to one of the procedures in the following section (7.7).
7.7
CALCULATION AND REPORTING For routine
OF RESULTS
analysis a calibration factor is calculated.
Select
from the graph an absorption value (e.g., 0.240) and find its corresponding concentration figure (e.g., 2.85 pmoles/litre and calculate:
5cm
2.85 0.240
which gives
5cm
11.88
How this calibration factor should be used in the calculations is shown below. The absorbance should be corrected with the absorbance of the reagent blank that is described in paragraph 7.6.1. If the calibsation has been made, using sea surface water of about the same salinity as the sample has, the calculation is made using the calibration factor F, thus:
NH4-N
poles/litre
F .
Areagent blank 1
If, however, the calibration was made with solutions of distilled water it is necessary to correct for the actual salinity of each sample. Choose for each sample the salinity correction factor, Fs, from the table in paragraph 7.9.1. Thus we have: NH -N 4 poles/litre
(Asamp1e
Areagent blank)
FS
7.8
High quality analysis of ammonia with good precision and accuracy is highly dependent on how successful one can avoid contamination of samples by tobacco smoke and other airborne ammonia as well as contamination of reagents and glassware. An intercalibration exercise described by Dah1 (see Grasshoff 1976) ' 4.8 per cent and an accuracy of 25.5 per cent. yielded a precision of f
-35-
7.9 7.9.1
INFLUENCING FACTORS AND DISTURBING SUBSTANCES Salinity and pH -
It can be noted from the calibration factors found for distilled water and sea water solutions that in sea water the blue colour produced will be less intense for any given concentration of ammonia. This depends on the fact that in the final sample solution the pH will be a function of the sample salinity. The reaction is complete and quantitative ,in the pH-range between 10.4 and 11.3. Thus, for each Sample a correction has to be made with respect to its salinity and the resulting pH. The correction factor is selected from Table VII.
TABLE VI1
Salinity correction factors for ammonia and analysis
Salinity
( .10 1
3
0-8
11
10.6
14
10.5
1.02
17
10.4
1.03
20 10.3
1.04
23
27
30
9.95
1.07
33
9.90
36
9.80
PH
11.4-10.8
1.00
10.2
10.0
1.06
1.01
1.05
1 . 0 8
1.09
Fs
7.9.2
Reaction time
At room temperature the reaction is complete after two hours in distilled water and sea water with salinity below 15 if the reagent B used contains hypochlorite instead of Trion. In ocean water the reaction requires four hours. If the reagent
B with Trion is used the reaction will proceed slower and another two hours reaction time should be added. However, as the blue colour produced is
stable for many hours it is convenient in routine analysis to let the samples react over night. The reaction time may be shortened by using a higher concentration of hypochlorite. This cannot be recommended as some organic substances containing nitrogen may decompose and form monochloramine, thus giving too high results.
-367.9.3
Disturbing substances
With the recommended concentration of hypochlorite the method is specific for ammonia and other nitrogen compounds do not react. Among inorganic substances, Hg-ions are capable of impeding the reaction. Consequently, samples should never be preserved with sublimate (HqCl2). Likewise it has been observed that iron, in concentrations higher than 2 mg/l causes disturbance. Stagnant water ofen contains hydrogen sulphide and if its concentration exceeds 2 mg/l a disturbing effect is obtained. In such waters the concentration of armnonia is normally rather high, and thus the disturbance may be eliminated by diluting the samples.
8.
DETERMINATION OF NITRITE SCOPE AND FIELD OF APPLICATION
8 . 1
This method is applicable for the determination of nitrite nitrogen (N02-N) in most types of waters (including sea water and waste water). It constitutes a modification of the well known Bendschneider and Robinson method (Koroleff, 1973)
The colorimetric reaction is almost specific for nitrite ions. Interferences may be caused by aromatic amines, copper (more than 0.5 mg/l), . 1 mg/l). Suspended matter and strong colour iodide ion (more than 0 in the sample also interfere. This method is not appreciably affected by salinity, small changes in reagent concentration 01: volume, or by temperature. Of the interfering compounds, none are normally present in significant amounts in the ocean, inshore or estuarine waters unless excessive pollution from land drainage is encountered.
8.2
PRINCIPLE
The determination of nitrite is based on the classical Griess' reaction, in which the nitrite ion at pH 1.5-2.0 is diazotized with sulphanilamide, resulting in a diazo compound, which in turn is coupled with N-(1-naphthyl)-ethylenediamine to form a highly coloured azo dye with an absorption maxima at 545 nm whichis measured colorimetrically.
8.3
8.3.1
REAGENTS Masnesium sulphate solution, about 2 moles/litre Dissolve 50 g MgS04
. 7H20
in distilled water and dilute
to 100 ml.
8.3.2
Sodium hydroxide solution 4 moles/litre Dissolve 16 g NaOH in distilled water and dilute to 100 ml.
-37-
8.3.3
Sulphanilamide reagent
Dissolve 8 g sulphanilamide, NH2C6H4S02NH2, in a mixture of 80 ml concentrated hydrochloric acid (HC1, 37 percent sp. qr. 1.19) and 400 ml water. Dilute to 500 ml. The reagent is stable for several months. 8.3.4 Diamine solution
N- (1-naphthy 1)-et& lenediamine dihydrochloride. Dissolve 0.8 q of C10H7NHCH2CH2~H .2HC1 in distilled water and dilute to 500 ml. Store the solution in a iark bottle. Renew the solution about once a month, or as soon as it develops a strong brown colour.
8.3.5
Nitrite stock solution

0
Anhydrous sodium nitrite, NaN02 p.a. is dried at 110 C for several hours. Dissolve 0.3449 g of the dry salt in distilled water and dilute to 1,000 ml. Store the solution in a dark bottle with 1 ml chloroform as preservant. The solution is stable for at least 1-2 months. 1 ml contains 5 m o l e s as N02-N. 8.3.6 Nitrite working solution
10 ml of the stock solution is transferred to a volumetric flask and diluted to 1,000 ml with distilled H20. The solution must be used the same day. 1 ml contains 0.05 umole NO -N. 2
8.4.
8.4.1
Test tubes with glass or plastic stoppers and with a capacity of 25 ml or more. Automatic syringe pipettes or piston pipettes for dispensing of reagents (not necessary, but very handy equipment). Colorimetric equipment, (i) one of the following is required: cm cells and
8.4.2
8.4.3
Spectrophotometer for use at 545 nm, with 1 longer as required;
(ii) Filter photometer, equipped with a yellow-green filter having maximum transmittance near 545 nm. 1 cm cells or longer as required.
8.5
Nitrite is an intermediate compound in the simplified redox chain ammonia - nitrite - nitrate. Samples for analysis of nitrite cannot be properly preserved. Storage in a dark and cold place can be tolerated for up to about five hours. If possible, samples should be analyzed without delay.
-38-
Avoid filtering the turbid samples, and use coprecipitation as pretreatment in extreme cases: Add 0.2 ml magnesium sulphate solution and 0.2 ml 4 moles/litre sodium hydroxide solution per 100 ml sample; agitate, and after 30 minutes, take the decanted part of the sample for analysis. Samples that are slightly turbid and contain no other disturbing substances, as sea water from near-shore areas, may be analyzed together with turbidity blanks instead of being pretreated.
8 . 6
8.6.1
Calibration
As there is no salinity effect in the formation of the azo dye, the calibration can be done in solutions made with distilled water.
Using the nitrite working solution (1 ml = 0.05 pole) a series of standard solutions is prepared by diluting with distilled water. If 100-ml volumetric flasks are used Table VI11 below can be followed:
TABLE VI11
r0.20
I1
Preparation of working standards for nitrite

0.05 pmole/litre N02-N
0.10 0.25
I '
I1 I1
0.10 ml working solution/100 ml

II
0.50
1.00 2.00
I1
11
11
11
0.50
I1
I1
I1
a. 00
I1
From the flasks, 25 ml samples are transferred to stoppered test tubes. Distilled water is used as the blank. The reagents are added and the analysis is performed as described below. The absorbances (corrected for the blank) are plotted versus concentration on a millimetre paper. In a 10 cm cell, the concentration 1 pmole/litre NO2-N gives an absorbance of about 0.500.
-39-
8.6.2
Analysis
For precise analysis of low concentrations of nitrite in sea water, any turbidity in the sample must either be removed (as described in section 8.5) or compensated for by a turbidity blank.
25 ml of the possibly pretreated sample is transferred to a stoppered test tube. If a turbidity blank is to be analyzed, another 25 ml is transferred to a second tube. Add 0.5 ml of the sulphanilamide solution (8.3.3) to the sample and turbidity blank, then mix well. After not less than three minutes, but not longer than eight minutes, 0.5 ml of the diamine solution (8.3.4) is added to the sample but not to the turbidity blank. Mix the sample once again.
After ten minutes, the absorbances of the sample and the turbidity blank are measured in a photometer at 545 in a cell length as required. The colour intensity is constant for two hours. The concentration of the sample is evaluated from the calibration graph, when the abosrbance of the turbidity blank has been subtracted from that of the sample. As the addition of acid to a water sample usually changes its turbidity, it is important that the acidic sulphanilmi.de solution is added not only to the sample, but to the turbidity blank as well.
a. 7
For routine analysis a calibration factor is calculated. Select from the graph an absorption value (e.g., 0.230) and find its corresponding concentration figure (.e.g., 0.904 pole/litre) and calculate:
which gives F
5 m
. 9
Then the concentration of a sample, in pnoles/litre, is found by multiplying its absorbance, corrected for absorbance caused by turbidity and the reagent blank, by the factor F.
8.8
Turbidity of the sample may introduce a considerable systematic error, particularly at low concentration levels. For highest accuracy and turbidity should, therefore, be removed as-described in section 8.5 or compensated for as described in section 8.6.2.
about
2 0.02
In routine analytical work the precision can be expected to be pole/litre.
-40-
9 . 9.1
DETERMINATION OF NITRATE SCOPE AND FIELD OF APPLICATION
This method based on a previous publication (Koroleff, 1973) is applicable for the determination of the sum of nitrate and nitrite, in natural waters. If the sample contains considerable amounts of nitrite, a separate determination of nitrite must be conducted and the result subtracted from that obtained with this method. Nitrate concentrations up to about 40 poles/litre of nitrogen (NO -N) can be determined without prior dilution of the samples. 3 The colorimetric reaction is almost specific for nitrite ions. Interferences may occur, caused by aromatic amines, copper (more than 0.5 mg/l), and iodide ion (more than 0.1 mg/litre). Suspended matter and a strong colour in the sample also interfere. Instead of filtering the sample, use the precipitation technique. However, moderate amounts of suspended matter can be tolerated as it will be trapped in the reduction column.
At concentrations higher than about 15 )unoles/litre of NO3-N in sea water, the dissolved salts cause a deviation from the linear relationship between concentration and the intensity of the coloured nitrite complex.
9.2
P RINC IPLE
Nitrate is reduced to nitrite almost quantitatively (about 90-95 percent) by amalgamated cadmium. The nitrite is then determined according to the classical Griess' reaction as described in the method for nitrite analysis (section 8 ) . The reduction is carried out at a pH of about 8.5. An ammonium chloride buffer is added to the sample to control the pH and to complex the liberated cadmium ions.
9.3
REAGENTS
Generally, the ordinary distilled or de-ionized water of the laboratory should be suitable for the analysis of nitrite as well as nitrate. However, if problems are encountered with the quality of the water, "nitrogen-free" water has to be prepared. Distilled (or de-ionized) water is distilled in all-glass equipment. For this distillation 2 ml concentrated sulphuric acid and 1 g potassium peroxodisulphate (K s 0 ) are added per litre. 2 2 8 All chemicals should be of reagent grade (E analysi).
-41-
9.3.1
Hydrochloric acid, about 0.5 moles/litre Dilute

10 ml concentrated HC1 (density l.l9/ml) to 250 ml with water.
9.3.2
Mercuric chloride solution, 1 percent. distilled water.
Dissolve 1 g HgC12 in LOO ml
9.3.3
Hydrochloric acid, 2 moles/litre. Dilute 166 ml concentrated HC1 (density 1.19 g/ml) to 1 litre with distilled water. Concentrated buffer solution
9.3.4
Dissolve 250 g ammonium chloride, NH C1, in distilled water, add 25 ml concentrated ammonium hydroxide, NH40H (%ensity 0.91 g/ml) and dilute to 1 litre. 9.3.5 Dilute buffer solution Dilute 20 ml of the concentrated buffer to 1 litre with distilled Renew daily. Acid washing solution
water. 9.3.6
Dissolve 4 g NH CJ. in distilled water, add 15 ml hydrochloric 4 acid, 2 moles/litre, and dilute to 1 litre with water. 9.3.7 Sulphanilamide reagent (same reagent as for nitrite determinations)
Dissolve 8 g sulphanilamide, NH2C6H4S02NH2, in a mixture of 80 ml concentrated hydrochloric acid (sp. gr. 1.19) and 400 ml water. Dilute to 500 ml with water. The reagent is stable for several months. 9.3.8 1-naphthyl)-ethylenediamine dihydrochloride solution N- ( (same reagent as for nitrite determinations)
Dissolve 0.8 g C10H7NHCH2CH2NH2.2HC1 in water and dilute to 500 ml. Store the solution in a dark bottle. Renew the solution about once a month, or immediately if it develops a strong brown colour. 9.3.9 Magnesium sulphate solution, about 2 moles/litre Dissolve 50 g M SO4.7 H20 in water and dilute 9 9.3.10 Sodium hydroxide solution 4 moles/litre Dissolve 160 g NaOH in water 9.3.11 Nitrate stock solution and dilute to 1 litre. to 100 ml.
Dissolve 1.0111 g potassium nitrate, KN03, in distilled water and dilute to 1,000 ml. Add 1 m l chloroform as preservant. The solution is stable for several months if evaporation is prevented. 1 ml = 10.0 ugat (140 ug) NO3-N.
-42-
9.3.12
Diluted nitrate solution

10 ml of the stock solution is diluted with distilled water 0.1 Pgat (1.4~9) NO3-N. to 1,000 ml. 1 ml
9.3.13
Synthetic sea water
Dissolve 310 g sodium chloride, NaC1, IO0 g magnesium sulphate, MgSO4,7H 0 and 0.50 g sodium hydrogen carbonate, NaHC03.H20 in 10 litres of distifled water. The salinity of this water is about 33.7 percent.
9.3.14
Amalgamated cadmium
WARNING: Cadmium is a poisonous metal. For reasons of occupational health and safety it should be handled with care. Perform all operations on the dry metal, particularly the granules, in a well ventilated area e.g., a fume cupboard. Never inhale the dust. If granulated cadmium (for instance, Merck Cadmium grob gepulvert, zur Fiillunq von Reduktoren) is not available, one has to prepare it in the following way: File sticks of pure cadmium metal (reagent grade) with a coarse metal hand file (about second cut) and collect the fraction which passes a sieve with 1-mm openings and is retained on a sieve with 0.5-mm openingsl). Calculate the required amount of cadmium about 35 g per reductor column (see Figure 3). Rinse the filings quickly with 0.5 mole/litre hydrochloric acid (9.3.1) and then with distilled water until the rinse water does not give the chloride reaction with silver nitrate. Transfer about 35 g of the washed metal into a 100 ml glass bottle and fill the bottle with the mercuric chloride solution (9.3.2) so that all air is excluded from the bottle. Seal the bottle with a glass stopper. After this step avoid all contact between air and the metal. Rotate the bottle for 90 minutes in a horizontal position with suitable equipment. Open the bottle and rinse out the turbid sublimate solution with distilled water which is introduced through a glass tube a ' the bottom of the bottle. Collect the sublimate solution. If a rotary evaporator carried out. Transfer or 250 ml round bottom is allowed to turn for is available the treatment can be conveniently the cadmium and the sublimate solution to a 100 ml flask that is aligned on the evaporator where it the required time.
Do not pour the used mercuric chloride (sublimate) solution into the sewer! When a suitable vDlume is coliected, add 25 ml concentrated HC1 per litre and precipitate with hydrogen sulphide (H2S) or sodium sulphide (Na2S). Filter the liquid. Store the precipitate and discard the clear filtrate.
1) There are different sieves available commercially. the suitable sieves. TABLE
IX
Table IX gives
Comparison of sieve dimensions according to different systems Opening : upper sieve Lower sieve
mm 1 0.5
DIN 6 12
Mesh/cm 7 13
Mesh/ inch 17.2 32
Sieve No.
18
35
-43-
30
75
f l
a
$2
180
Figure 3.
Reduction column for analysis of nitrate and total nitrogen (all measures given in millimetres)
(a) glass wool (b) Cd-amalgam (c) copper wire
-44-
9.4
APPARATUS AND EOUIP?ENT Reduction columns (see Figure 3) Volumetric flasks or test tubes with a capacity of 25 ml. The tubes should either be graduated or marked at the 25 ml volume. following is required:
9.4.1
9.4.2
9.4.3
Colorimetric equipment; one of the (a)

(b)
u n and provided with spectrophotometer, for use at 545 r cells of 1 cm (and 5 cm) length, or
filter photometer, equippped with a filter having maximum transmittance at (or near) 545 nm, with cells of 1 cm (and 5 an) length.
9.5
SAMPLING AND PRETREATllENT
The analysis should preferably be undertaken immediately after the sampling. If this is not possible, the samples may be preserved by the addition of 1 ml sulphuric acid 4 moles/litre per litre. The sample has to be neutralized again before the analysis of this preservation is applied. Suspended matter and a strong colouration in the sample are removed by using a precipitation technique: Add 0.2 ml of magnesium sulphate solution and 0.2 ml of sodium hydroxide solution per 100 ml sample. Agitate, and after 30 minutes, use the decanted solution for analysis.
9.6 9.6.1
ANALYTICAL PROCEDURES Preparation of the reduction columns
Place a small ball of thin copper wire at the bottom of the reduction column and fill it entirely with water. Open the bottle containing the amalgamated cadmium fillings and put a finger (or a rubber plate) on the opening. turn the bottle obliquely downward and place the opening under the water surface in the reductor column. The metal should flow down into the reductor column without coming into contact with air. Make sure that no cavities are formed in the reductor. Fill the column with the metal to about 1 cm below the reservoir and plug the column with glass wool. This operation should be carried out so that all liquid that flows from the column etc. can be collected and precipitated as described above. Flush the column several times with the acid washing solution
(9.3.6) and with the dilute buffer solution (9.3.5) until the effluent shows
a weakly alkaline reaction (for instance, blue colouration with bromothymol blue). The mlumnshould never be left with the acid washing solution. Flush the column weekly when it is used regularly or more often if the flow decreases through the column. A newly prepared column reduces nitrate almost immediately with an efficiency of 95-100 percent (see section 9.7 for further details). If the reduction efficiency decreases below 85 percent, empty the column, wash the filings quickly with 2 moles/litre HC1 and rinse very thoroughly with distilled water. Dry the filings, sieve again, and reamalgamate as described above.
-45-
If a column does not give a proper reduction when it is newly prepared it may be "started" by running through a nitrate standard solution, e.g., 10 pmoles/litre, or common tap water. In most cases, less than one litre will suffice.
9.6.2
Calibration
A series of standard concentrations is prepared from the nitrate stock solution (9.3.11) and the diluted nitrate solution (9.3.12)' by diluting with distilled water or synthetic sea water. As there is no salt error at concentrations below about 15 poles/litre the synthetic sea water will probably seldom be required. As several columns are likely to be calibrated at the same time, the standard solutions are prepared in at least 1 litre quantities according to Table x.
TABLE .
Preparation of working standaxds or n i t r a s
m l diluted - nitrate solution/l,OOO
ml
pmoles/litre 0.10 0.50 1 . 0 0 5.00 pmoles/litre

10.0 20.0 30.0
1.00 5.00 10.00 50.00 ml nitrate stock solution/1,000 ml
1.00 2.00 3.00 4 . 0 0
40.0
I
The standard solutions are analyzed as described below. of each solution must be analyzed.
Duplicates
The absorbances are plotted versus concentration for each of the reduction columns. If the results are very uniform from column to column, one calibration graph may be used or the subsequent evaluation of the samples. However, most probably one graph will be required for every column. If samples have been treated according to the precipitation procedure in paragraph 9.5, then the calibration solutions and blank samples should be treated in the same way.
-46-
9.6.3 9.6.3.1
Analysis Reduction of nitrate
Neutralize acid preserved samples with 0.1 or 0.5 rnole/litre NaOH after addition of two drops of bromothymol blue indicator. (Calculate prospective dilution) .
(1) Fill the resexvoir of the reduction column with the dilute buffer solution (9.3.5) and let it flow through. Discard the solution. (2)
Fill the reservoir again with 50 ml distilled water and 1 ml concentrated buffer solution (9.3.4). Discard the first 20 ml which flows through the column and collect 25 ml in a volumetric flask or test tube. Proceed in the same manner with all the columns in order to obtain a blank value. These blank samples are then analyzed for nitrite as described in (9) and onward. If samples have been treated according to the precipitation procedure in paragraph 9.5, then blank samples and the calibration solutions should be treated in the same way. The reduction efficiency of each column must be controlled for every analytical batch. The standard concentration used for this control depends on the types of concentration range of the samples. Analyze duplicates of this control standard as described in (4) to (10). Take a 50 ml sample or less containing up to 15 pmoles/litre NO -N) 3 and adjust the volume to 50 ml. Add 1 ml of concentrated buffer solution (9.3.4) and mix; use an automatic pipette if possible. Draw off the excess solution in the reductor by using a capillary-tipped glass tube connected to a suction pump and proceed immediately with (7). Transfer the entire sample (4) to the reservoir of the reductor. Discard the first 10 ml flowing through the column and use 10 mL to rinse a 25 m l volumetric flask (or test tube). Then collect 25 ml in the flask or tube. 50 ml should flow through the column in ten to twelve minutes. Proceed as described in (9). (The determination of nitrite should be carried out within one hour.) Draw off the remaining sample from the reductor and begin directly with the next sample.
(3)
The reductor column must be washed immediately after every analytical batch. F 11 the reservoirs twice with the dilute buffer solution (9.3.5). Cover the reductors and make sure that they do not dry.
9.6.3.2
Determination of nitrite
(3) To 25 ml of the reduced sample, coming
from the reductor, add to samples, standard solutions and blank samples 0.5 ml sulphanilamide solution (9.3.7) and mix. After not less than 3 minutes, and not longer than 8 miwtes, add 0.5 m l of N-(1-naphthyl)-ethylenediamine solution (9.3.8) and mix again.
-47-
(10)
The absorbance of the coloured sample i.- measured after l? minuter, but within two hours, against the reference sample at 545 nm. Use a cell of suitable length depending on the intensity of the colour. (The absorbance oE the blank sample against water should not exceed 0.010, measured in a 1 cm cell.)
9.7
Subtr.rlct the absc-rbznceof the blank sample from the absorbances of the sa11p1.esa2d evaluate their Concentrations from the czlihration gra7h. For routine analysis a calibration factor is calculated. Select from the graph an absorption value (e.g., 0.715) and find its corresponding concentration figure (e.g., 13.9 poles/litre) and calculate:
Ian
13.9 0,715
which gives F
lcm
19.44
Then the concentration of a sample, in poles/litre, is found by multiplying its absorbance, corrected for the absorbance caused by the blank, by the factor F. NOTE : If the nitrate determination is part of the determination of total nitrogen the calculation is carried out as described in section 10.7.
The result obtained is the sum of the concentrations of nitrite and nitrate nitrogen in each sample. If a sample contains a significant concentration of nitrite, this must be compensated for by subtracting the result found from a separate determination for nitrite according to the previous method (Section 8). (If the sample was dilutedprior to the reduction this must be taken into account in the calculation: poles/litre N03-N
A x B
V
where :
= v =
A
nitrate concentration, pnoles/litre, as obtained from the calibration graph. ml of original sample before dilution final volume, after dilution.
For example: A 25 ml sample was diluted to 100 ml and of this 50 ml was analyzed. The result ob ained from the calibration graph was 26 umoles. Then
26
x
25
100
104 poles/litre N 0 3 - ~
To check the reduction efficiency of the reductor (4) a nitrate standard solution of suitable concentration is analyzed. For instance, a solution of 1 pole/litre is analyzed and an absorbance of 0.241 is obtained when using a 5 cm cell. An analysis of the nitrite solution with the same concentration gives the absorbance 0.258 under the same conditions. The reduction efficiency is then: 0.241 x 100 93 percent 0.258
9.8
Deviation between duplicate samples analyzed with the aid of one and the same reductor is c O . 1 pole/litre in the range 0 - 5 poles/litre, c0.2 umole/litre in the range 5 - 10 pmoles/litre and 0.5 pmole/litre at higher concentrations. Deviation of results from different columns depend on the reduction efficiency (yield factors) of the columns. When systematic errors are avoided an accuracy o f i 3 per cent can be assumed in the range between 0 - 10 poles/litre.
10.
SIMULTANEOUS PERSULPHATE OXIDATION OF PHOSPHORUS AND NITROGEN CONTAINING COMPOUNDS IN WATER SCOPE AND FIELD OF APPLICATION
10.1
Commonly used names of the parameters determined with this method are total phosphorus and total nitrogen. Earlier methods published for their determination have been designed for either of these parameters. In 1977 Koroleff presented a method for a simultaneous determination of total phosphorus and total nitrogen in the same sample. The method was published in 1981 by Valderrama in a slightly modified version. The method exhibits improved reliability and precision and it allows for the storage of samples to be analyzed later. The yields obtained by oxidation of various nitrogen compounds depend on the form of nitrogen linkage. Thus, or example, nitrate, nitrite, ammonia, urea, some aliphatic amino acids and some proteins give yields over 92%. Pronouncedly poor yields have been obtained from compounds containing nitrogen-to-nitrogen bonds, while a double bond between nitrogen atoms seems to prevent their oxidation to nitrate entirely (Nydahl, 1978). The digestion time, a function of the autodecomposition of peroxodisulphate, needs to be no longer than 30 minutes, provided that the temperature attained in the pressure cooker is 110-115C.
10.2
PRINCIPLE
To allow nitrogen compounds to become oxidized, it is necessary to use an alkaline medium, otherwise. nitrate is not produced in quantifiable amounts. Conversely, the Oxidation of phosphorus compounds must be performed on an acidified sample. In the simultaneous oxidation the reaction starts at pH 9.7 and ends at pH 5-6. These conditions are obtained by a boric acid-sodium hydroxide system. In seawater samples no precipitate is formed when the oxidation reagent is added. At elevated temperature (100-120C) a precipitate is formed, which, however, almost dissolves as oxidation proceeds. After the oxidation the remaining small amount dissolves upon swirling. The free chlorine, which is formed in seawater samples, is reduced by adding ascorbic acid before the molybdate reagent (Koroleff, 1977).
-49-
After oxidation the nitrogen compounds are determined as nitrate according to the procedure in Section 9 and the phosphorus compounds are determined as inorganic phosphate according to the procedure in Section 4 .
10.3
REAGENTS
In addition to the reagents needed for determintion of dissolved inorganic phosphate (Section 4) and nitrate (Section 9) the following items are needed.
10.3.1
Dilute
hydrochloric acid, about 0.2 umoles/litre
Dilute 17 ml concentrated hydrochloric acid, HC1 (density 1.19 g/ml) with distilled water to 1 litre. 10.3.2 Sodium hydroxide, 1 umole/litre
Dissolve 40 g sodium hydroxide, NaOH, in about 50-60 ml distilled water and then dilute to 1 litre. 10.3.3 Oxidation reagent
Dissolve 50 g sodium peroxodisulphate, K2S208, and 30 g boric acid, H3B03, in 350 ml of sodium hydroxide (1 mole/litre) and make up to 1 litre with distilled water. The peroxodisulphate has to have a very low content of nitrogen compounds. The product with No. 5092 from Merck is usually of very satisfactory quality. If the oxidation reagent is stored in dark glass bottle and protected from direct light in room remperature it is stable for at least six to eight months. 10.3.4 Concentrated standard solution
Dissolve 0.4505 g dried glycine, C2H502N (reagent grade) and 0.1361 g of potassium dihydrogen phosphate , KH2P04 (reagent grade), previously dried at 1lOOC and kept in a desiccator, with distilled water and make up to a final volume of 100 ml. 1 ml contains 10.0 ,poles of P and 60.0 p o l e s of N. 10.3.5 Dilute standard solution
Pipette 1.00 m l of the concentrated standard solution (10.3.4) to a 100 ml volumetric flask and make up to the mark with distilled water. 1 ml contains 0.10 p o l e s of P and 0.60 p o l e s of N. 10.4 APPARATUS AND EQUIPMENT
In addition to the equipment needed or the determination of dissolved inorganic phosphate (Section 4) and nitrate (Section 9) the following items are needed.
-50-
Oxidation bottles of alkali resistent glass with screw caps of non-nitrogen containing material and about 50 ml volume. (Suitable bottles are Sovirel graduated bottles with screw caps. However, as these caps, contain nitrogen compounds they have to be replaced. The replacement must not be of polyethylene as they will become brittle and crack as a result of the free chlorine that is developed in the oxidation. Suitable replacements are polypropylene caps from Nalgene Labware, Nalge Corporation).
10.4.1
10.4.2
Stainless steel pressure cooker of any ordinary kitchen type that will maintain an internal temperature of 110-1L5c. SAMPLING AND PRETFLEATPG3NT
10.5
Soon after the sampling, e.g., in connection with the analysis of the other nutrient parameters, 30 ml portions of the samples are transferred to oxidation bottles. TO each portion 4 ml of the oxidation reagent (10.3.3) are added with an automatic syringe pipette and the bottles tightly stoppered. Boil the samples for 30 minutes at 110-115C in suitable batches (e.g., 30-40 bottles) to fill the pressure cooker. Together with each batch boil two oxidation reagent blank samples consisting of 4 ml reagent and no extra distilled water. After boiling, the samples should be gently swirled to promote dissolution of the precipitate thay may have formed. Then the bottles are allowed to cool down to room temperature. The bottles should not be opened until the analysis of phosphate and nitrate is started. It has been shown (Valderrama, 1981) that it is preferential to add the oxidation reagent and boil the samples soon after sampling and then store the samples in oxidized form. If samples are stored before oxidation instead the analyses will receive a higher coefficient of variation. The storage has been successfully tested or at least three months. 10.6
10.6.1
ANALYTICAL PROCEDURES Calibration
Using the dilute standard solution, prepare a series of standards with the concentrations given in Table XI, below:
TABU
XI
Preparation of working standards for total phosphorus and total nitrogen

ml dilute standard/200 ml
phosphorus poles/ Litre

7.0 5.0 3.0 1.0
nitrogen p o les/ 1itre 42.0 30.0 18.0 6.0
14.0 1 0 . 0 6.0 2.0
-51-
Analyze 30 ml portions in duplicate from each standard according to the description in section 10.5 (oxidation) and then section 10.6.2 (procedure) . Note that for the calibration the blank samples must consist of 30 ml of the distilled water used with addition of 4 ml of oxidation reagent (10.3.3) to each blank. This will compensate for any phosphorus and nitrogen present in the distilled water and the oxidation reagent. Correct the two absorbance figures obtained for each solution with the corresponding blank absorbance and construct calibration graphs as described previously in paragraph 13.6 for nitrate and paragraph 8.5 for phosphate. The calibration factors, F, can also be calculated as described. As the original sample taken for analysis (30 ml) is diluted to 35 ml in the procedure, the calibration factors will be about 1.167 times (35/30) higher than those or nitrate and phosphate. Thus a factor, FN, of about 24 for nitrogen and Fp, 11 for phosphorus can be considered as representative. Note that although the oxidized nitrogen sample is diluted from 10 ml to 50 ml in the analysis this is compensated for by using a 5 cm cell for the photometric reading as compared with the 1 cm cell that is (usually) used for nitrate. Thus the difference in the calibration factors becomes rather small.
10.6.2
Analysis
Open the bottles containing the oxidized, cool, samples. As each sample is to be used for two determinations it has to be divided into two separate parts. Transfer each sample to a measuring cylinder, preferably a stoppered one, and adjust the volume to 35 ml and mix well. Continue as described below for each analysis. After each batch of samples that have been oxidized it is convenient to fill the oxidation bottles with the diluted hydrochloric acid (10.3.1) and let them stand for a couple of hours before rinsing them with distilled water. 10.6.2.1 Procedure for nitrogen compounds (total nitrogen) Prepare the reduction columns for nitrate analysis as described in steps (1) and (2) of section 9.6.3 Nitrate analysis. Pipette 10 m l of the sample, adjusted to volume as described above in section 14.7, and continue the analysis as for a nitrate sample according to the steps (4) through (10) in section 9.6.3 Nitrate analysis. In most cases a 5 cm cell is suitable for the photometric determination as indicated above concerning calibration.
-5210.6.2.2 Procedure for phosphorus compounds (total phosphurus)
The remaining 25 ml from the sample in section 10.6.2 is used for the analysis of phosphorus compounds. Transfer the sample to a test tube and add 0.7 mL of the ascorbic acid solution (4.3.5). Mix well and let the sample rest 1-2 minutes so any remaining free chlorine, that was liberated during the oxidation, is completely destroyed. Finally, add 0 . 7 ml of the acid-molybdate reagent (4.3.4) and mix the sample well again. (The preparation of these reagents were described in Section 4.3). After five minutes read the absorbance of the sample in a
5 cm cell at 882 nm using distilled water as a reference. See also paragraphs 4.6.2 and 4.6.3 concerning the stability of the coloured
complex and possible disturbances. 1 0 . 7 CALCULATION AND REPORTING OF RESULTS
The absorbance measured for each sample, As has to be corrected for the absorbance of the blank that contains the oxidation reagent, Aox. The corrected absorbance is then used for evaluation of the result from the calibration graph. If a calibration factor, F ,is used for the calculation use the formulas: Total nitrogen, poles/litre Total phosphorus, poles/litre
FN
(As,N
Px
- Aox.N 1 (A 1 s,P Aox,P.
1 0 . 8
The actual capability of the method is summarized in Table XI1 below (from Valderrama 1981).
TABLE XI1
Accuracy and precision
__
Total nitrogen
.
.~
Total phosphorus
30.0
8
_~
1.00 8 1.02
3.0 8
True concentration values (pmolI-' )

No. measurements
Measured concentrations M e a n values in (prnoiI-' Range Standard deviation Variation coefficient (%) Relative error (%)
1.5
8
1.8
1.7-1.9
0.063
6.0 8
0.25
8
6.7
6.4-6.9 0.217 3.2 11.7
31.2 29.9-3 2.7 0.988 3.2

4.0
0.21
0.2 54.29
.1.94 4.52-4.95
0.056 1.1
0.98-1.0 4
0.020 2.0 2.0
0.014 5.2 8.0
3.5
20.0
-1.2
-53-
REFERENCES
Bates, R.G., 1963: Determination of pH; Theory and Practice. John Wiley and Sons, Inc., New York, 435 pp. Grasshoff, K. (editor); 1976 : Methods of sea water analysis, Verlag Chemie, Weinheim, 317 pp. FAO, 1975: Manual of methods in aquatic environment research, Part 1. Methods for detection, measurement and monitoring of water pollution. FAO Fish.Tech.Pap., (137): 238 pp. Gieskes, J.M., 1969: Intercalibration of pH measurements. Unesco Techn.Paper.Mar.Sci. 9, 908. Koroleff, F., 1976a: Determination of phosphorus. In Grasshoff, K. (editor), Methods of sea water anazsis, Verlag Chemie, Weinheim. Koroleff, F., 1976b: Determination of ammonia. Ibidem.
Koroleff, F., 1977: Simultaneous persulphate oxidation of phosphorus and nitrogen compounds in water. In: Grasshoff, K., Report of the Baltic Intercalibration Workshop, Annex. Interim Commission for the Protection of the Environment of the Baltic Sea.
Nydahl, F., 1978: On the peroxodisulphate oxidation of total nitrogen in waters to nitrate. Water Research 12; 1123-30.
Valderrama, J.C., 1981: The simultaneous analysis of total nitrogen and total phosphorus in natural waters. Marine Chemistry, 1 0 p p 109-122.

Chemical Methods USE in Marine Environmental Monitoring: Commission Manuals

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Chemical Methods USE in Marine Environmental Monitoring: Commission Manuals

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Intergovernmental Oceanographic Commission

Manuals and guides

CHEMICAL METHODS FOR USE I N MARINE ENVIRONMENTAL MONITORING

....................... Determination of Dissolved Oxygen and Oxygen Saturation ....

Determination of dissolved inorganiz phosphate

............ Determination of total phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . Determination of reactive silicate ........................

Figure Figurz Figure

SCOPE AND FIELD OF APPLICATION

Table I ------Standard p H v a l u e s o f t h e buffer solutions

- 3 1.4. APPARATUS AND EQUIPMENT

1.4.1. Water b a t h , t h e r m o s t a t e d , w i t h pump for e x t e r n a l c i r c u l a t i o n o f t h e thermosted water. S h o u l d be c a p a b l e o f h o l d i n g a s e l e c t e d temperature t o within + 0.5'.

For t h e exact h a n d l i n g o f the pH m e t e r , t h e i n s t r u c t i o n manual supplied with t h e i n s t r u m e n t s h o u d be c o n s u l t e d .

CALCULATION AND EXPRESSION OF RESULTS value) be sample for

P H------in situ where : t l t2

t e m p e r a t u r e __----in Situ measurement t e m p e r a t u r e

1 . Thennostated pH-cell and Lrat-c>xcharLgPr

6 ) s n m p l e overflow 7) t:irrnmostaterl hater inlet 8 ) ) t h e n o c t a t e d water outlet (9) to waste

ESTIMATION OF PRECISION AND ACCURACY

2.1. SC-OPE AND

are d e t e r m i n e d by t i t r a t i o n with thiosulphate i o n s :

solution about 9 moles/liter.

2 . 3 . 8 . ' S t a r c h solution. 1 g s o l u b l e s t a r c h i s dissolved in 100 m l

2 . 4 . 1 . Calibrated b u r e t t e , f o r i n s t a n c e a 12 m l Derona burette w i t h a n a u t o m a t i c z e r o a d j u s t e r , o r a motor-driven piston burette.

SAMPLING AND PRE-TREATMENT

Standardization o f the t h i o s u l p _____________ hate solution. ___-______________________

The exact titre o f t h i s s o l u t i o n c a n be d e t e r m i n e d by t i t r a t i o n a g a i n s iodate i o n s in a c i d s o l u t i o n :

the c o n c e n t r a t i o n o f the t h i o s u l p h a t e s o l u t i o n , moles/litre

the c o n c e n t r a t i o n o f the KIO

T o c a l c u l a t e t h e c o n c e n t r a t i o n o f dissolved oxygen , use t h e f o r m u l a below. a v o l u m e o f t h e t h i o s u l p h a t e consumed i n m l c o n c e n t r a t i o n o f t h e t h i o s u l p h a t e s o l u t i o n , moles/litre

the c a l c u l a t i o n s i t m a y be p r a c t i c a l t o calculate the term a c o n s t a n t f a c t o r ( w h i c h i s close to 112 for a s o l u t i o n 4 t h a t c o n t a i n s 0,02 moles/litre

If t h e s m a l l s a m p l e b o t t l e (50-6Onl)is used the v o l u m e o f e a c h a d d e d r e a g e n t is 0 . 5 m l . I n t h i s c a s e the ( V - 2 ) in t h e formula i s r e p l a c e d by ( V - 1 ) .

DETERMINATION OF HYDROGEPJ SUL,PHIDE SCOPE AND FIELD QE APPI.ICATION

___________________ N , N - d i m e t h y l p-phenylene _____ di _a _m _ i_ n_ e _d _ i_ h_ yd _r _ o _ c_ h_ lo _r ide _ _ _ _ _ vL _ ~ ~ ~ .

__ O x y g_______________________ e n - free di st i 1 led water

--_ S u l p ___-______h i d e w o r k i n g______-__ s o l u t i o n (about 0,156 !Jmoles/ml S 7

1 g per 100ml distilled water or Thyodene indicator as for the dissolved o x y g e n d e t e r m i n a t i o n .

Potassium iodide K I , p.a. c r y s t a l s .

APPARATUS AND EQUIPMENT

ANALYTICAL PROCEDURE Calibration ----------_ S t a n d a r d i z a t i o n o f the thiosul-phate _solution. P r o c e e d a s described i n s e c t i o n 2.6.2. a b o v e .

mean of the t i t r a t i o n s o f t h r e e s o l u t i o n s with n o added s u l p h i d e , i n m l . m e a n of the t i t r a t i o n s o f the t h r e e s o l u t i o n s c o n t a i n i n g sulphide, in ml.

concentration o f the t h i o s u l p h a t e s o l u t i o n , moles/litre

(the individual t i t r a t i o n s c o n s t i t u t i n g a t r i p l i c a t e s h o u l d agree t o within 0.05 ml).

From t h e w o r k i n g s o l u t i o n (3.3.5) the following standard series is prepared.

CALCULATION AND REPORTING OF RESULTS

ESTIMATION OF PRECISION AND ACCURACY

A t a c o n c e n t r a t i o n level o f 25 p m o l e s / l i t e r , the precision ~ d be n e x p r e s s e d a s +0.3%lnu;;;F;er o f r e p l i c a t e s a m p l e s .

DETERMINATION OF DISSOLVED INORGANIC PHOSPHATE

4.1. SCOPE AND FIELD OF APPLICATION

__-___________L_-------L_______________ Sulphuric acid about moles/litre

_________________ Potassium a n t i m o n x________________ l t a r t r a t e s o l u t i o-L_---------_----_ n 0.1 moles/litre

- molybdate reagent __________ ___-_------

Phosphate stock solution

APPARATUS AND EQUIPMENT

Automatic s y r i n g e p i p e t t e s o f 1 ml or 2 ml v o l u m e for reagent additions.

SAMPLING AND PRETREATMENT

ANALYTICAL PROCEDURES Calibration --_--------

A s e r i e s o f w o r k i n g s t a n d a r d s a r e prepared from t h e phosphate w o r k i n g s o l u t i o n by d i l u t i o n w i t h d i s t i l l e d water. If 50 m l volumetric f l a s k s are u s e d , T a b l e IV c a n be u s e d .

I f 5-cm cells a r e used i n s t e a d o f 10 c m , the s a m p l e v o l u m e s may be r e d u c e d to 25 ml a n d the r e a g e n t v o l u m e s t o 0.7 ml.

Disturbances _____-_-_--Disturbance from s i l i c a t e

CALCULATION AND REPORTING OF RESULTS

T h e c o r r e c t e d a b s o r b a n c e i s used for evaluation of t h e r e s u l t , u s i n g the c a l i b r a t i o n g r a p h described i n paragraph 4.6.1).

Then t h e c o n c e n t r a t i o n o f a s a m p l e , i n moles/litre, i s obtained by m u l t i p l y i n g i t s a b s o r b a n c e with t h e factor F.

Standardization o f the t h i o s u l p ___________ hate solution. _-______________________

_______________ N , N - d i m e t h y l p-phenylene _ di _a _m _ i_ n_ e _d _ i_ h_ yd _r _ o _ c_ h_ lo _r ide _ _ _ _ _ vL _ ~ ~ ~ .

O x y g_____________________ e n - free di st i 1 led water

--_ S u l p _-h i d e w o r k i n g- s o l u t i o n (about 0,156 !Jmoles/ml S 7

-_________L_-------L_______________ Sulphuric acid about moles/litre

_ Potassium a n t i m o n x l t a r t r a t e s o l u t i o-L_---------_----_ n 0.1 moles/litre

- molybdate reagent ________ _-_------