Lead in Port Phillip Bay mussels

Marine Pollution Bulletin

ated with carbon particlesmcarcinogens bound in this manner may not be biologically available. BRUCE P. DUNN*

Cancer Research Centre, § Department o f Zoology, 2075 Wesbrook Place, University o f British Columbia, Vancouver, B. C., Canada V6T 1 W5 DAVID R. YOUNG

Southern California Coastal Water Research Project, 1500 East Imperial Highway, EI Segundo, CA 90245, U.S.A. *Research Fellow of the National Cancer Institute of Canada. §To which correspondence should be addressed.

Blumer, M. & Youngblood, W. W. (1975). Polycyclic aromatic hydrocarbonsin soilsand recent sediments. Science, 188, 53-55. Dunn, B. P. (1976). Techniques for determination of benzo(a)pyrene in marine organisms and sediments. Env. Sci. Technol., (in press). Dunn, B. P. & Stich, H. F. (1975a). The use of mussels in estimating benzo(a)pyrene contamination of the marine environment. Proc. Soc. Exptl. Biol. Med., 150, 49-51. Dunn, B. P. & Stich, H. F. (1975b). The use of accumulator organisms

in a monitoring system for chemical carcinogens in coastal waters. Proc. Thirteenth Pacific ScienceCongress, I, 20. Dunn, B. P. & Stich, H. F, (1976a). Release of the carcinogen benzo(a)pyrene from environmentally contaminated mussels. Bull. Env. Contain. Toxicol., 15,398--401. Dunn, B. P. & Stich, H. F. (1976b). Monitoring procedures for chemical carcinogens in coastal waters. J. Fish. Res. Bd. Can., (in press). Grimmer, G. & Duvel, D. 0970). Untersuchungen zur endogenen Bildung yon Polycyclischen Kohlenwasserstoffen in hoheren Pflanzen 8. Mitt.; Cancergogene Kohlenwasserstoffe in der Umgebung des Menschen. Z. Naturforsch., 25b, 1171-1175. Goldberg, E. D. (1975). The Mussel Watch. A first step in global marine monitoring. Mar. Pollut. Bull., 6, l 11. Morton, B. (1975). Pollution of Hong Kong's commercial oyster beds. Mar. Poll. Bull., 6, 117-122. Pancirov, R. J. & Brown, R. A. (1975). Analytical methods for polynuclear aromatic hydrocarbons in crude oils, heating oils, and marine tissues, pp. 103-113, Proceedings 1975 Conference on Prevention and Control o f Oil Pollution. American Petroleum Institute, Washington, D.C. SCCWRP (1973). The ecology of the Southern California Bight: implications for water quality management. TR 104, p. 68, Southern California Coastal Water Research Project, El Segundo, CA 90245. Stich, H. F., Acton, A. B. & Dunn, B. P. (1975). Carcinogens in estuaries, their monitoring and possible hazard to Man. Symposium on Environmental Pollution and Carcinogenic Risks. The International Agency for Research on Cancer, Lyon, France, (in press). ZoBell, C. E. (1971). Sources and biodegradation of carcinogenic hydrocarbons, pp. 441-451, Proceedings Joint Conference on Prevention and Control o f Oil Spills, American Petroleum Institute, Washington, D.C.

Lead in Port Phillip Bay Mussels Lead levels in the indicator mussel Mytilus edulis, exceeds the WHO food standards in 19 of the 22 locations sampled in Port Phimp Bay, Melbourne. While this seriously reflects contamination of the food chain, LDs0 experiments on Mytilus indicate that the lethal effect of lead on this organism is much less severe than excessive doses of mercury, cadmium, copper or zinc. Little is known about the long term effects of lead on Mytilus. Lead is the largest contaminant of the environment. About 1 kg/day is discharged from cars per capita in the United States (Schroeder, 1974). The effect of lead poisoning on human beings vary from the inhibition of the haem synthesis (Gibson & Goldberg, 1970) to hindering the metabolism of some neurotransmitters hence reducing conduction velocity in the peripheral nerves (Catton et al., 1970; Galzigna et al., 1964). Most of the major problems of lead poisoning are not immediately recognisable, hence any build up of tffis element in the food chain is extremely undesirable.

and 2 ml HCIO4 and evaporated to dryness. The final residue was again dissolved in 10 ml of 1 M HNO3 or an appropriate volume depending on concentration of metal. The resulting solution was determined by a Varian (AAS) atomic absorption spectrometer. Blanks were run to determine background correction for reagents, glassware and filter paper while molecular absorption was R. Mori byrnong ~ ~Melbourne "~,l ~#'~-.~ .~3Yorro River

%

~Sondringhorn

Port Phillip BOy

Methods and Materials Samples were collected in June 1975 by diving for continuously submerged mussels 5.5 cm in length (Fig. 1). For comparison, continuously submerged oysters from the seabed and partially submerged mussels from the inter-tidal zones of piers were also collected. For analysis, all apparatus was acid washed. Each tissue was digested by adding 0.1 g of dried homogenate to 10ml of A.R. grade 7 N HNO~ and reducing the volume at 80°C to a residue. It was redissolved in 8 ml of 3.5 HNO~ 234

~lwuromona Rosebud I0

20

I

i

Kilometres

Fig. 1 Sampling locations for mussels and oysters in Port Phillip and Corio Bays.

Volume 7/Number 12/December 1976 determined using all2 lamp. Results were calculated using both calibration and standard addition curves for comparison (Elwell & Gidley, 1966). An experiment to determine the LDs0 values for Pb in Mytilus was carried out in acid washed glass tanks. The volume of seawater used being 5 l./tank. Each tank contained 10 mussels and the lead was added in the chloride form. Death was defined by the failure of the animal to close its valves on interference. Water analysis was carried out to monitor any loss to the container, or precipitated solids using a standard method of A P D C MIBK (Kinrade & Van Loon, 1974). Mussels were analysed in a similar manner.

Results and Discussion This element exceeds the W H O standards for food in 19 of the 22 locations sampled. It appears more widespread but less intense than the cadmium pollution recently reported (Talbot et al., 1976). Table 1 gives the mean value for five observations from both single tissues and total soft portion of the mussels. Oyster results are

3020 to

E o. o.

5 4 :3-

G

2

restricted to the total soft portion of the animal (Table 3). The oyster sampled was the indigenous species Ostrae angasi. Table 3 indicates that oysters do not concentrate this metal to the same extent as Mytilus. Table 2 shows that mussels collected from the intertidal zones of piers have a lower lead content. This can be explained by the fact that they are only partially submerged and that most trace metals in seawater bind to solid matter and is eventually sedimented (Bloom, 1975). The distribution of lead in the bay can be explained by the fact that stormwater runoff accounts for a large proportion of lead present in the vicinity of built up areas and that the tidal movements in the bay tend to sweep out Port Melbourne in a south east direction as indicated by the arrows in Fig. 1. These arrows are a simplified representation of total water movement calculated from a mathematical model simulation (M.M.B.W., 1973). Trace metals often accumulate in the gills and digestive system of mussels (Broo~s & Rumsby, 1965). So it is with lead. However, higher levels appear in the muscle tissue and valves than would be expected from the distribution of other trace metals in this animal. The presence of noticable amounts of lead in the valves as PbCO3 is understandable. Calcium and lead both form divalent ions. A pH model for speciation of Pb in seawater at a pH of 8.1 constructed from available and estimated thermodynamic stability constants and activity coefficients indicate that 87°70 of Pb is present as the PbCO~ form (Zirino & Yamamoto, 1972). The same workers predicted that Cd, Zn and Cu, are in the CdCl~, Zn (OH)~ and CU(OH)~ forms respectively. TABLE 1 Levels of Pb in Port Phillip and Corio Bay mussels. Location

Foot

Gills

Stomach

Mantle

Muscle

Shell

Bulk

6.4 1.3 8.3 2.8 8.2 3.2 7.3 2.2 4.3 2.4 3.7 2,8 4.2 2.8 8.3 4.6 5.1 2.1 8.3 4,9 7.4 2.1 3,7 2.1 4.7 2.3 5.7 3.2 7.3 4.1 7.8 3.4

32.6 13.8 16.3 4.8 14.6 4.8 24.6 10.8 15.9 4.1 19.4 6.4 18.7 6.8 15.3 3.8 12.4 3.6 I 1,2 2,1 23,8 6.7 18,8 3.6 18.7 2.6 26.8 8.4 12.8 5,5 17,8 4.7

18.9 9.7 8.9 4.2 9.6 3.1 22.9 8.1 9.3 4.4 8.9 2.3 9.4 2.1 12.6 4.1 11.6 2.9 9.6 3.2 14.3 5.9 16.9 4,7 13.9 4.8 29,4 9,2 13,4 5.3 20.6 4.3

4.6 3.2 4.6 3,8 8.2 2.9 5.3 2.4 4".I 2.7 8.6 2.4 6.8 2.4 9.1 2.8 8.6 2.7 4.8 2,9 7.4 4.3 7.5 2.7 6.5 3.1 14.5 4.9 6.9 2.4 7.8 3.1

12.3 3.9 10.1 3.4 9.8 2.1 15.3 4.2 4.2 3.1 10.1 2,9 6.1 2.3 10.3 3.4 8.7 2.9 5.4 3.3 12.1 6.4 8.5 2.9 10.2 3.8 17,8 6.7 7.7 3.2 13.5 4.6

27.4 3.6 24.3 4.7 27.3 4.3 36.3 5.1 24.8 15.4 81 12.6 35.8 5.8 18.9 6.9 37.1 9,6 16,3 5.3 8.7 4.1 33.4 I 1.8 36,4 8.6 17.4 5.4 36 7.8 61.7 18.9

15,2" 4.3 10.7+ 2,9 13,3 + 33 19,8" 3,9 II,I + 4,2 18,9" 3,8 17.9" 4.1 11.3+ 3.7 10.3+ 4.2 9.7-2.8 16.4" 4.7 15.2" 4.9 16.8" 5,1 22.4* 2.8 8.6-3,4 14.1 + 4.8

17

83 4.6

20,6 4.3

18

6.4

18.7

3.3 7.3 2.9 4.9 2. I 8.4 3,6 6.2 2.1

4.5 23.8 7.6 13.5 5.4 19,1 6.7 14.3 4.7

19.3 3.9 17.3 6.8 21.8 5.8 12.6 4,9 23.6 5.8 12.6 3.4

8.7 2,8 8.9 4.6 13,7 4.3 7.2 3.8 7.3 2.3 5.4 2,1

15.1 4.6 9.4 4,4 14.6 4.8 10.4 5.1 14.8 3.8 7.2 3.8

53.4 16.7 46.0 13.5 29.4 9.9 36.1 12.9 52.0 17.4 26.1 9.7

17.9" 5.2 16.8" 6,2 18.9" 5,2 13.6+ 4.6 18.9" 7.2 8 .3 - 2.9

05--

1

Metal Pb

ppm s.d.

2 3 O~

I

2

i II

3 4 5

I

I

I II

10

205040.50

Time,

days

I

100

I

4

200

5

curves for C d ( O ) and P b ( X ) for the mussel, Mytilus edulis at a p H o f 8 . 1 a n d a temperature range o f 1 4 - 1 7 ° C .

Fig. 2a

LD50

6 7

8 9 3C

10 x~ x

2C

%x

II 12

E o.

13

5-

14 G c ou o

15 16

I 05-

i

19 20

I~ 0 ,~ 2

Conc of

I i0 3 metal m

L 104

Myt//u5

a f t e r half

I 2 x I0 ~

I0 5

the population had dted

Fig. 2b Cd(O) and Pb(X) levels detected in mussels on completion of the LDso experiment

at various concentrations.

21 22

* = Exceeds W. H.O Standards. + = Borderline.-- = Under. 235

Marine Pollution Bulletin TABLE 2

Lead analysis for musselsfrom the inter-tidezone of piers. Location 4

Met~ Pb

8 10 11

F~t 3.4 1.1 3.1 1.4 2.1 1.5 2.4 0.9

Gills

Stomach

Muscle

Mantle

Shell

Bulk

4.9 1.4 4.7 2.1 4.8 2.7 5.1 2.1

4.8 1.7 5.1 2.2 4.6 2.2 4.8 1.6

2.9 1.3 3.3 1.8 3.9 2.6 3.8 1.6

2.2 1.2 2.6 1.4 2.7 1.4 3.1 1.3

13.4 2.8 12.6 3.1 11.9 2.8 11.4 3.1

4.7 1.4 4.6 2.4 3.7 1.1 3.4 1.4

The relative affinity o f lead to accumulate to a minor extent in muscle tissue may be explained by the ability by Pb to form mercaptides with the - S H group of compounds like cysteine and amino acid side chains (Vallee & Ulmer, 1972). Proteins with large numbers o f free - S H groups, e.g. thionein, bind Pb firmly (Ulmer & Vallee, 1969). However, differential selection o f selected metals with sites has been noted (Gurd & Murray, 1954). They showed that lead binds carboxylate groups of human serumalbumin while Zn and Cd competed with each other for the imidazole nitrogens. Many workers have implied that metallothionein shows far greater affinities for Cd, Zn, and Cu, in that order, than it does for lead (Olafson & Thompson, 1974; Bouquegneau et aL, 1975). The LDs0 experiment indicated that cadmium is far more lethal than lead (Fig. 2a). Thus 1.5 ppm Cd had the same lethal affect as 30 ppm Pb. The levels o f lead accumulated in the population when half the population had died at any particular concentration was far higher than that for Cd (Fig. 2b). The experimental levels of lead in seawater are 300 times that of the bay so it may be concluded that lead does not pose an immediate lethal effect. It has been pointed out that very often sublethal effects pose more o f a threat to a population (Bengtsson et al., 1975). The authors have isolated metallothionein from mussels collected in the cadmium polluted areas o f Corio Bay (Fig. 1) and showed its association with cadmium and zinc. However, lead was virtually undetected. Circumstantial evidence suggests that this protein has a specific function to absorb toxic trace metals and render them inert (Rugstad &Norseth, 1975; Pulido et aL, 1966; Suada et al., 1974). It may be more than a coincidence that it absorbs Hg, Cd, Zn and Cu in preference to Pb while the latter has been shown to have by far the least lethal effect on Mytilus o f the five metals mentioned (Unpublished work). Figure 2b shows that the level o f lead accumulated in the animals after the first LDs0 observation had a mean value o f 7600 ppm. This is about 300 times the highest value encountered in the bay. This observation occurred after 37 days in the 30 ppm Pb seawater solution. The first observation for Cd in Fig. 2a (30 ppm) contrasts with the corresponding figure for lead. Other work by the authors has shown that the lethal effect o f Cd is more similar to excessive doses o f Hg, Zn and Cu. After 2.25

days in 30 ppm Cd seawater solution, half the population had died and yielded a mean Cd level of 410 ppm with a S.D. o f 86. This figure is only twice the highest recorded mean value for Cd pollution in Corio Bay mussels in 1974 (Vie. Parl. Hansards, 1975). However, the mussels in Corio Bay appeared to be healthy. The geometry o f the curve in Fig. 2a suggests that the level o f excess cadmium in the seawater is extremely lethal above 2 ppm. This, coupled with the fact that mussels in Corio Bay had 200 ppm Cd, suggests that the kinetics o f the uptake is the factor governing whether the animals will survive. It appears that the rate with which metallothionein is synthesized to combat uptake o f toxic quantities o f heavy metals is the key factor. Hence, any concentration of Cd in seawater above which metallothionein cannot cope, will yield the same lethal effect. Recently, the existence o f metaUothionein was reported in mussels fed with unnatural levels o f cadmium and it was concluded that this was a low molecular weight protein synthesized solely to prevent trace metal intoxication (NOel-Lambot, 1976). In conclusion, the lead levels in mussels in Port Phillip Bay indicate that that part of the food chain that inhabits the sea floor in this area should be closely watched. The existence o f metallothionein, which detoxifies trace metal uptake in Mytilus could be the very agent which allows a build up o f trace metals in this part o f the food chain. V. T A L B O T R. J. M A G E E Department o f Inorganic and Analytical Chemistry, L a Trobe University, Bundoora, Victoria, Australia, 3083 M. HUSSAIN Environment Protection Authority, 240 Victoria Parade, East Melbourne, Victoria, Australia, 3002

Bengtsson, B. E., Carlin, C. H., Larsson, A. & Svanberg, O. (1975). Vertebral damage in minnows, Phoxinus phoxinus L., exposed to cadmium.Ambio, 4, 166-168. Bloom, H. (1975). Heavy Metals in the Derwent Estuary, Report by Chem. Dept., Univ. of Tasman. Bouquegneau, J. M., Gerday, C. & Disteche, A. (1975). Fish mercurybinding thionein related to adaption mechanisms. FEBS Lett., 55,173.

Brooks, R. R. & Rumsby, M. G. (1965). The biogeochemistryof trace element uptake by some New Zealand bivalves. Limnol. Oceanogr., 10, 521-527. Catton, M. J., Harrison, M. J. G., Fuilerton, P. M. & Kazantis, G. (1970). Subclinicalneuropathy in lead workers. Brit. Med. J., 2, 80. Elwell, W. T. & Gidley, J. A. F. (1966). Atomic-Absorption Spectrophotometry. Oxford: Pergamon Press. Galzigna, L., Brugnone, E. & Corsi, G. C. (1964). Cited by Bryce-Smith,D. &Waldron, H. A. (1974). Lead, behaviourand TABLE3 criminality.Ecol., 4, 367-377. Gibson, S. L. M. & Goldberg, A. (1970). Defects in haem synthesis in Location Man S.D. Location Man S.D. Location ~.~n S.D. mammalian tissues in experimental lead poisoning and experi2 5.4 2.7 3 8.0 2.6 6 4.0 1.8 mental porphyria. Ciin. Sci., 38, 63-72. Lead analysis for mud oysters from continuouslysubmergedlocations. Gurd, F. R. N. & Murry, G. R. (1954). Preparation and properties of

236

Volume 7/Number 12/December 1976 serum and plasma proteins. The interaction of human serum albumin with plumous ions. J. Am. Chem. Soc., 76, 187. Kinrade, 3. D. & Van Loon, J. C. (1974). Solvent extraction for use with flame atomic absorption spectrometry. Anal Chem., 46, 1894. Melbourne Metropolitan Board Works (1973). Environmental study of Port Phillip Bay. Report on Phase I: 1968-1971. M.M.B.W. Melbourne. NOel-Lambot, F. (1976). Distribution of cadmium, zinc and copper in the mussel Mytilus edulis. Existence of cadmium-binding proteins similar to metallo thioneins. Experientia, 32, 324-326. Olafson, R. W. & Thompson, J. A. J. (1974). Isolation of heavy metal binding protein from marine vertebrates. Mar. Biol., 28, 83. Pulido, P., Kagi, J. H. R. & Vallee, B. L. (1966). Isolation and some properties of human metallothionein. Biochem., 5, 1768. Rugstad, H. E. & Norseth, T. (1975). Cadmium resistance and content of cadmium binding protein in cultured human ceils. Nature, Lond., 257, 136.

Schroeder, H. A. (1974). The Poisons Around Us. Indiana Univ. Press. Suda, T., Horiuchi, N., Ogata, E., Ezawa, I., Otaki, N. & Kimura, M. (1974). Prevention by metallothionein of cadmium-induced inhibition of vitamin D activation reaction in kidney FEBS Lett., 42, 23. Talbot, V. W., Magee, R. J. & Hussain, M. (1976). Cadmium in Port Phillip Bay mussels. Mar. Pollut. Bull., 7, 84-85. Ulmer, D. D. & Vallee, B. L. (1969). Trace substances in Environmental Health--II. Proc. Univ. Missouri Ann. Conf. Trace substances Environ. Health, 2nd ed. D. D. Hemphill, 7. Columbia: Univ. Missouri. Valley, L. B. & Ulmer, D. D. (1972). Biochemical effects of mercury, cadmium and lead. Ann Rev. Biochem., 41,91-128. Victorian Parliamentary Hansards (1975). Trace metals in sea food of Victorian waters. 46th Parl. 2nd session, 27,7881. Zirino, A. & Yamamoto, S. (!972). A pH dependent model for the chemical speciation of copper, zinc, cadmium and lead in seawater. LimnoL Oceanogr., 17,661-671.

Mercury Content of Mussels from the St. Lawrence Estuary and Northwestern Gulf of St. Lawrence, Canada A first survey o f the mercury content o f intertidal mussels in the St. Lawrence Estuary and part o f the Gulf of St. Lawrence shows means values ranging from 0.160 to 0.629 ppm (dry weight). Highest values (grand mean 0.430 ppm) were f o u n d in the upper part o f the lower estuary, lowest values (grand mean 0.172 ppm) on the north shore o f the estuary and Gulf, and intermediate values along the Gasp~ Peninsula. The results indicate a decreasing gradient in mercury levels from the m o u t h of the Saguenay Fjord seaward. T h e St. L a w r e n c e E s t u a r y receives f r e s h w a t e r d r a i n a g e f r o m t h e G r e a t L a k e s a n d the St. L a w r e n c e Basin t h r o u g h the St. L a w r e n c e River. T h e e s t u a r y ' s d i s c h a r g e is i n c r e a s e d b y the i n f l o w o f t h e S a g u e n a y F j o r d a n d a n u m b e r o f rivers, p a r t i c u l a r l y t h o s e f r o m the M a n i c o u a g a n - O u t a r d e s - B e t s i a m i t e s c o m p l e x (Fig. 1). A l l these w a t e r s r u n t h r o u g h t h e m o s t i n d u s t r i a l i z e d a r e a o f E a s t e r n C a n a d a a n d a r e r e c e i p i e n t o f m a n y effluents i n c l u d i n g c h l o r a l k a l i p l a n t s a n d p a p e r mills ( F i m r e i t e , 1970). R e c e n t studies in the S a g u e n a y F j o r d s h o w t h a t m e r c u r y levels a r e p a r t i c u l a r l y high in the w a t e r , suspended particles, sediments and animals. For instance m e r c u r y levels as high as 12 a n d 15 p p m were f o u n d in s e d i m e n t s ( L o r i n g , 1975) a n d s h r i m p s ( G u b e l i et al., 1976) respectively. In the St. L a w r e n c e E s t u a r y , m e r c u r y levels in the s e d i m e n t r e a c h 0.9 p p m ( L o r i n g , 1975) b u t no results a r e a v a i l a b l e for a n i m a l s . Studies c a r r i e d o u t so far s h o w t h a t an i m p o r t a n t s o u r c e o f m e r c u r y c o n t a m i n a t i o n o f the e s t u a r y is l o c a t e d n e a r the h e a d o f the S a g u e n a y River. T h e p r e s e n t survey was c a r r i e d o u t to e v a l u a t e the a c t u a l levels o f c o n t a m i n a t i o n o f the l o w e r e s t u a r y ( f r o m the m o u t h o f the S a g u e n a y to G r o s s e s - R o c h e s ) a n d p a r t

o f the G u l f o f St. L a w r e n c e (Fig. 1). T h e l o w e r e s t u a r y is p a r t i c u l a r l y i m p o r t a n t e c o l o g i c a l l y as a source o f n u t r i e n t (Steven, 1974; Sinclair et al., 1976) w h e r e a s the n o r t h - w e s t e r n p a r t o f the G u l f is r e c o g n i z e d t o be a highly p r o d u c t i v e a r e a ( P l a t t , 1972; Steven, 1974).

Materials and M e t h o d s D u e to its w i d e d i s t r i b u t i o n in the s t u d y a r e a a n d its w i d e s p r e a d a c c e p t a n c e as a n i n d i c a t o r o f p o l l u t i o n ( G o l d b e r g , 1975), the mussel M y t i l u s edulis L. was c h o s e n in this survey. In o r d e r t o r e d u c e p o s s i b l e p h y s i o l o g i c a l effects o n m e t a l levels (De W o l f , 1975), all s a m p l e s were collected w i t h i n a s h o r t p e r i o d o f 2 weeks at the e n d o f M a y 1976. A d u l t s p e c i m e n s r a n g i n g f r o m 2.5 to 4.0 c m were collected o n r o c k y s h o r e at m i d tide level at 30 s t a t i o n s d i s t r i b u t e d o v e r 1000 k m a l o n g t h e e s t u a r y a n d G u l f c o a s t s (Fig. 1). A t P o i n t e - M 6 t i s , h o w e v e r , s a m p l e s were also collected o n m u d d y a n d r o c k y s u b s t r a t a at e x t r e m e (lowest) h i g h - w a t e r o f n e a p tides a n d m e a n low w a t e r o f n e a p tides. In the l a b o r a t o r y , a n i m a l s were m e a s u r e d a n d freed f r o m their shells. T h e s o f t p a r t s were f r e e z e - d r i e d a n d weighed. E a c h s a m p l e a n a l y s e d (0.5 to 1.0 g, d r y wt) included 3-5 individuals. A n a l y s e s were p e r f o r m e d using flameless a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( C o l e m a n M A S 50) as initially d e s c r i b e d b y H a t c h & O t t (1968) a n d s u b s e q u e n t l y m o d i f i e d b y U t h e et al. (1970) a n d T h i b a u d (1975). T h e sensibility o f the m e t h o d (1 °7o a b s o r b a n c e ) is 0.020 p p m a n d d e t e c t i o n limit 0.004 p p m . T h e s t a n d a r d e r r o r for 105 d e t e r m i n a t i o n s was 0.008 p p m . F o r v e r i f i c a t i o n o f the a n a l y t i c a l m e t h o d t w o h o m o g e n i z e d s a m p l e s were f r a c t i o n e d a n d sent for analysis to t w o o t h e r l a b o r a t o r i e s . T h e results p r e s e n t e d in T a b l e 1 s h o w 237