Toxicity of copper, mercury and lead to a marine nematode

Volume17/Number 10/October1986 Marine Pollution Bulletin, Vol. 17. No. 10, pp. 453-457. 1986 Printed in Great Britain

11025-326X/86 $3.OO+O.111~ O 1986 Pergamon Journals Ltd.

Toxicity of Copper, Mercury and Lead to a Marine Nematode G. VRANKEN and C. HEIP

Marine Biology Section, Zoology Institute, State University of Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Toxicity of copper, lead and mercury to the nematode Diplolaimella spec 1 is studied. Mortality responses obtained demonstrate high resistance to heavy metals. Population growth parameters as the intrinsic rate of natural increase and net-reproductivity are significantly depressed at copper-concentrations which cause no juvenile mortality. The lowest concentrations tested caused significant inhibition of development rate in both sexes. For this particular nematode species suppression of fecundity and developmental inhibition are more reliable criteria, determining non-exceedable limits with regard to environmental safety. Our tests show that nematode productivity may be significantly depressed at copper levels found in some areas of the North Sea.

These tests are presented as a supplement to the few data available and are intended to check whether the resistance of nematodes to toxicants is indeed true. Second, we will also report on sublethal effects of copper on fecundity, because mortality is not a very sensitive index of toxicity. Fecundity, the number of eggs produced, has been used because it is known to be a sensitive criterion in toxicity testing (Bryan, 1980; Reish & Carr, 1978). As a combination of fecundity and development rate (generation time) the intrinsic rate of natural increase rm of the population can be calculated. This has been done by Tietjen & Lee (1984) to measure the impact of PCBs, PAHs and heavy metals on the nematodes Chromadorina germanica and Diplolaimella punicea. Because r m values relate also to the productivity of the population, ECs0 values, defined as the concentration at Marine nematodes are the most abundant animals in which there is a 50% inhibitory effect on population marine sediments and have several features that are growth, are excellent tools to predict the environmental advantageous in ecotoxicological research in the labora- impact of toxicants (Hummon & Hummon, 1975; Sabatory: they have a short life-span (Vranken & Heip, in tini & Marcotte, 1983; Vernberg & Coull, 1981). For press), a high fecundity (Vranken & Heip, 1983), repre- several nematodes, a good correlation between rm sent several trophic levels (herbivores, bacterial feeders values and the rate of increase in the field has been and carnivores) and at least some species are easily cul- found (Heip et aL, 1978; Smol et al., 1980; Romeyn et tured (Heip et aL, 1985). However, the effect of only a al., 1983). Since nematodes are part of the diet of very limited set of chemical agents to only a few species macrofauna, some fish and commercial crustaceans has so far been tested (Bogaert et al., 1984; Howell, such as Crangon crangon, and, since a significant part of 1984; Vranken et al., 1984b). From these few experi- the energy flow through benthic systems passes through ments it appears that nematodes are relatively resistant the nematodes (Platt & Warwick, 1980), any factor to pollutants. Thi~ is corroborated by evidence from influencing nematode productivity may be of more field surveys which have shown that nematode density is global importance. not affected substantially by raw domestic sewage (Vidakovic, 1983), heavy metals (Tietjen, 1977, 1980) M a t e r i a l s a n d M e t h o d s and acid iron waste (Lorenzen, 1974). The species Diplolaimella spec 1, previously misIn this paper we present results from tests on the species Diplolaimella spec 1, a new species described by identified as Monhystera microphthalma, is a new speJacobs & Vranken (in prep.) and previously misidenti- cies not as yet described. It was sampled in the Dievenfled as Monhystera microphthalma. First, a series of gat, a polyhaline water pond, situated near the Nature acute (static) and sublethal (chronic) tests on the toxicity Reserve 'het Zwin' in north-western Belgium. Adults of copper, mercury and lead is described, in which both are about 1 mm long and circa 0.5 lxg wet weight. Stockmortality and development rate are studied simultan- cultures were maintained agnotobiotically on 0.5% eously. Nematodes moult four times before becoming bacto-agar (DIFCO) enriched with 1% Vlasblomadult and the success in reaching a particular stage has medium (Vranken et al., 1984a) and 1% silicate been proposed as a sensitive indicator of toxicity (Na2SiO3.9H20; 0.053 M stock-solution). Stock-cul(Samoiloff et al., 1980). We used a slightly different tures were kept in vented petri-dishes of 5 cm diameter. Copper (CuSO4.5H20), lead (Pb(C2H302)2.3H20) approach and have determined generation time and preadult mortality as a function of metal concentration. and mercury (Hg2CI2) toxicity was studied using egg-, 453

Marine Pollution Bulletin

juvenile- and pooled pre-adult mortality as criteria. Experiments were performed in the dark in vented petri-dishes (0:3.5 cm) at 20°C, 20%o salinity and an initial pH--7.5. Salinity was measured with a refractometer. The medium used for the experiments consisted of 0.5% bacto-agar (DIFCO) made in Dietrich & Kalle (1957) seawater, enriched with 1% Walne-Provasoli medium, plus 1% amino-acids solution with glucose enrichment and 0.5% silicate (see Vranken et al., 1984a for details). At each concentration 5 replicate experiments were run in which 125 gravid females were allowed to deposit eggs for 1 day. An unidentified bacterial mixture grown separately on bottoms free from metals was given as food. The number of eggs studied varied between 268 and 348 for copper, 248 and 427 for mercury, and 248 and 407 for lead. Fecundity was studied under similar conditions as the lethal tests. At copper concentrations of 100 Ixg 1-1 and 1 mg 1-1, 60 females and males, which matured in the previous acute tests, were divided at random over 5 replicates. Every 6 days eggs were counted and adults transferred to fresh cultures. At 500 Ixg 1-1 copper no mortality-assay was performed. The adults (60 males and females) for the fecundity-test at this concentration were taken from the 100 lxg 1-1 experiment. Population growth rm was calculated with Lotka's equation:

Mercury (Table 1) For both egg- and pre-adult mortality, MEC = 100 ~tg 1-1. At 1 mg 1-1 0.3% of the juveniles did not reach adulthood. For the juveniles, MEC -- 5 mg 1-1. At 10 mg 1-1 pre-adult mortality is 42%: 31% of the eggs did not hatch and 16% of the juveniles died before reaching maturity. Lead(Table 1) The MEC for non-hatching and pre-adult mortality is 5 mg 1-1. For juvenile mortality MEC is 10 mg 1-1. Developmental assay Development time prolongs significantly (P< 0.001; Nested Anova) with increasing metal concentration (Table 2). The smallest levels tested caused a significant lengthening for the three metals. Developmental retardation of both sexes, with an exception at 20 ~tg 1-1 Cu (0.001 < P < 0.01 ; Anova) is similar. Development time at the highest Cu level is significantly longer when compared with the other two metals. Fecundity and demographic analysis Egg-production is significantly

influenced

TABLE 1

Diplolaimella spec 1. Egg mortality (e), juvenile mortality (/) and preadult mortality in percentages at different metal concentrations

¢o

Z e"~.U~=l

(1)

X=0

Metal

Significance (G/q test) * •

Life stage e j preadult

0 3 1 3

Concentration (mg 1- i) 0.1 1 5 10 50 12t 17 25 100 0 0 10"t 12-~ 17 33 100

mercury

* • •

e j preadult

3 0 3

10t 0 10t

11 0 11

15 5+ 19

31 16 42

lead

* • •

e ] preadult

3 0 3

5 0 5

8 0 8

10~ 0 10~

17 9f 25

copper

U~ is the number of female-newborns produced per female of the preceding generation, when that generation is in the age-interval (x, x + 1). Ux is calculated as U~= Nex.p. Ix, where Nex is fecundity of a female aged x, p the proportion females in the population (-- 0.5) and lx the survival probability form the egg-stage onwards until age x. Net-reproductivity Ro, the multiplication rate per generation is calculated as: go = ~. U~

(2)

•: P < 0.001

f: MEC-value (P ~<0.05) -: not tested TABLE 2

,trio

Diplolaimella spec 1. Development time (Train) ill days of females and males (with 95% confidence interval) at different copper, mercury and lead levels.

Mean generation time Tis determined as T= In gffr m

(3)

Minimum generation time, Train is estimated as the period between identical stages of successive generations, equalling approximately the sum of embryonic and postembryonic time.

Train

metal (mgl -I)

females

N

males

N

copper

0 0.02 0.04 0.06 0.1 I 10

12.9 (5: 0.23) 14.0 (5: 0.27) 15.3 ( ± 0.28) 14.8 ( 5: 0.23) 14.8 ( 5: 0.24) 17.0 (5: 0.42) 17.6 ( ± 0.51)

156 114 121 142 120 -107 102

12.8 (5: 0.20) 14.6 (5: 0.22) 15.4 (± 0.29) 15.0 ( ± 0.22) 14.8 (5: 0.21) 17.0 (5: 0.46) 17.9 (± 0.44)

180 131 134 149 121 116 114

mercury

0 0.1 1 5 10 0 0.1 1 5 10

12.7 (+ 0.19) 14.3 ( 5: 0.29) 15.2 (+_ 0.28) 15.0 (+ 0.35) 16.1 (5: 0.61)

213 126 186 109 69

13.0 (-+ 0.22) 14.2 (5: 0.24) 15.1 (5: 0.25) 15.2 (5: 0.37) 16.3 (± 0.60)

181 140 194 119 75

213 183 123 115 90

13.0(+ 0.22) 14.0 ( 5: 0.23) 14.6 (5: 0.29) 15.3 (5: 0.33) 16.2 (+ 0.47)

181 195 129 146 96

Results Acute toxicity tests

Copper(Table 1) The lowest concentration inducing a significantly different hatchability (P= 0.05) when compared with 12.7 (+ 0.19) the blank ( = MEC: minimum effective concentration) is lead 14.3 ( 5: 0.25) 100 Ixg 1-1. For the juveniles MEC is 10 mg 1-1 and for 14.5 (5: 0.32) the pooled pre-adult mortality MEC is 100 ~tg 1-1. At 15.0 (+_ 0.39) 50 mg 1-1 Cu, 5 eggs were deposited; none of these 16.3 (5: 0.44) hatched. N = number of individuals studied. 454

Volume 17/Number 10/October1986 metals. This may reduce toxicity as it is known that some bacteria accumulate metals (Patrick & Loutit, 1976; Doyle et al., 1975; Berk & Colwell, 1981). The uptake of other bacteria, on the contrary, like Escherichia coli (Doyle et al., 1975) and gutbacteria of the nematode Mesorhabditis monhystera (Doelman et al., 1984) is very limited. In additional experiments with the closely related nematode species Monhystera disjuncta we could enhance mercury (II) toxicity when juveniles were fed E. coli cells previously exposed to 2.5 mg 1-1 Hg(lI). Mortality in this experiment was 56%. When mercury was only added to the substratum mortality was 6%. Hence it is clear that microbial activity influences toxicity in the Diplolaimella assays and contriDiscussion butes almost certainly to the discrepancies found with Three different responses: mortality during the pre- the Enoplus assay. Several studies indicate that the free metal ion is the adult stages, development retardation, and suppression of fecundity, were studied. For the first two responses a toxic species (see Borgmann, 1983; Moore & Ramamophysiological standard, development time, was chosen orthy, 1984 for review). Canterford & Canterford as the duration of the experiment. As generation time (1980) have demonstrated this with the marine diatom increases with metal concentration (Table 2), exposure Ditylum brightwellii: Cu, Zn, Cd and Pb toxicity detime in our experiments increases simultaneously. Using creased as the concentration of EDTA, a strong chelating agent, increased. Mercury-toxicity, on the other hand mortality as a toxicity ranking criterion, Diplolaimella spec 1, appears rather insensitive, even when compared was independent of EDTA, which is expected as in with larger marine invertebrates such as polychaetes seawater Hg(II) forms strong covalent compounds with (Reish, 1984), crustaceans (Ahsanullah et al., 1981) and chloride ions (Moore & Ramamoorthy, 1984). In freshbivalve molluscs (Bryan, 1984). For copper an LCs0 of water mercury complexation by E D T A lowers its toxi12 mg l- t (95% CI: 10.4-13.7), exceeding considerably city (Whitton, 1967). The medium used to culture Diplolaimella spec 1 is rich in substances, such as phosits seawater solubility, was calculated from the pooled phates, nitrates and organics like amino-acids, vitamins pre-adult mortality with the R e e d - M u e n c h method and sodium E D T A (Vranken et al., 1984a) which bind a (Woolf, 1968). For mercury (I) the LCs0 is close to 10 reduced toxicity of heavy metals (Ramamoorthy & mg 1-1 and for lead it is higher than 10 mg l -t, a value Kushner, 1975; Knezovich et al., 1981). With the musalso more than 1 order higher than its maximal solubisel Mytilus edulis, on the contrary, prior complexation lity. These figures are in contrast with those presented of cadmium enhances its uptake (George & Coombs, by Howell (1984) for two large free-living marine 1977) and therefore probably its toxicity. Concerning omnivorous/predacious nematodes, Enoplus brevis and the uptake of dissolved organics by nematodes, conflictE. communis. For a similar exposure duration as in our ing results exist in the literature. Two predacious/ experiments (312-430 h) the LC50s of Cu, Pb and Hg(II) of Enoplus brevis, the most resistant of the two omnivorous nematodes Pontonema vulgaris (Chia & enoplids, lies somewhere between 10 and 100 lxg 1-1. Warwick, 1969) and Adoncholaimus thalassophygas Howell (1984), however did not mention whether he (Lopez et al., .1979) are able to assimilate labelled fed his animals during exposure. For other organisms glucose, whereas the bactivorous PeUioditis marina is such as Daphnia magna metal-toxicity drops by adding not capable of doing it (Tietjen & Lee, 1975). Howell food (Biesinger & Christenen, 1972). We fed our ani- (1983) suggested that, besides food intake, a significant mals on bacteria previously grown on bottoms without route of metal uptake is via the cuticle. Without having quantitative knowledge on the influence of complexaTABLE 3 Diplolaimella spec 1. Total and daily egg-production per female at tion on metal-toxicity to nematodes and without knowdifferentcopper concentrations. ing the exact route of metal-uptake we cannot assess the importance of these factors in relation to the low toxiCu Total Daily R2 n (rag 1-1) egg-production egg-production city levels observed. Nevertheless we may conclude that 0 147 4.9 ( + 0.7) 0.99 6 Diplolaimella spec 1 is a rather insensitive species. 0.1 132 4.2(5: 0.9) 0.98 6 Experimental evidence exists that this also holds for 0.5 112 3.8 (+ 0.9) 0.97 6 other nematode species (Pitcher & McNamara, 1972; 1 97 3.5 (5: 1.0) 0.96 6 Samoiloff et al., 1980; Haight et al., 1982). Field R2~coefficientof determination;in parentheses: + 95% confidence limit, n anumber of observations. observations again corroborate this statement as in

(0.025 < P < 0.05; Anova) in the range tested. Maximum production was reached after 31 days. Total fecundity drops from 147 eggs per female in the blank to 97 at 1 mg 1-1 Cu (Table 3). Daily egg-production at 0.5 mg 1-1 and 1 mg 1-1 is significantly less (P--0.05) when compared with the blank (Table 3). Both netreproductivity (0.005 < P < 0.01 ; Anova) and rm (0.025 < P < 0.05; Anova) decreased significantly with increasing concentrations, r,, decreased 9.7% at 100 ixg 1-~ and 29% at 1 m g l -~ Cu. At 1 m g l - t this corresponds with a 43% decrease in net-reproductivity (Table 4).

TABLE 4

Copper (mgl-I) Control 0.1 0.5 1

Ro 71 58 50 41

Diplolaimellaspec 1: demographiccharacteristicsat differentcopper concentrations Estimated r., T(days) Estimated Ro r,, (day-I) + 95% CI 70 + 7.0 60 + 4.7 49 5:4.8 41 + 6.9

0.186 0.168 0.159 0.132

+ 95% CI 0.181 (0.158--0.207) 0.176 (0.156--0.198) 0.155 (0.141--0.171) 0.133 (0.112--0.158)

23 24 25 28

Estimated T -+-95% CI 23.15:2.6 23.6 5:2.3 25.45:1.9 27.75:3.3 455

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Recent trends in research on heavy metal contamithe blank, whereas the others died very quickly. So far it nation in the sea. Helgoliinder wiss. Meeresunters. 33, 6-25. seems advisable to supplement d e v e l o p m e n t a l tests b y Bryan, G. W. (1984). In Marine Ecology, Vol. V, Ocean Management, Part 3 (O. Kinne, Ed.), pp. 1289-1431. Wiley,Chichester, New York. other s h o r t - t e r m assays. A possible alternative is to Canterford, G. S. & Canterford, D. R. (1980). Toxicity of heavy metals count the n u m b e r s reaching a d u l t h o o d in a fixed p e r i o d to the marine diatom Ditylum brightwellii (West) Grunow: correlation of time, e.g. when at least 5 0 % o f the individuals between toxicity and metal speeiation. J. mar. biol. Ass. U.K. 60, 227-242. matured in the blank. T h e s e figures are typical for two Chia Fu-Shiang & Warwick, R. M. (1969). Assimilation of labelled gludifferent responses: mortality and d e v e l o p m e n t a l inhicose from seawater by marine nematodes. Nature 224, 720-721. bition. W h e n using this as m e a s u r e we obtain ECs0s Dictrich, G. &. Kallc, K. (1957). AIIgemeine Meereskumle. Eine Eip!l~ihrung in die Ozeanographie. Gcbrfidcr Borntraegcr, Bcrlin-Niko(concentrations which induce a 5 0 % m a t u r a t i o n inhibilassec. tion when c o m p a r e d with the blank) o f 28 lxg 1-~ Doelman, R, Nieboer, G., Schrooten, J. & Visser, M. (1984). Antagoniscopper, 93 ~tg 1-~ m e r c u r y (I) and 60 Ixg 1- t lead. tic and synergistic toxic effects on Pb and Cd in simple food chain: For D i p l o l a i m e l l a spec 1 significant r e d u c t i o n in Nematodes feeding on bacteria or fungi. Bull. environ, contain. ToMcol. 32,717-723. fecundity o c c u r s at levels m o r e than o n e o r d e r o f m a g Doyle, J. J., Marshall, R. T., Pfander, W. H. (1975). Effects of cadmium nitude less than the LCs0, e.g. for the n e t - r e p r o d u c t i v i t y on the growth and uptake of cadmium by microorganisms. Appl. the ECs0 is 1.4 mg 1- t C u 2+ ( 9 5 % C I : 0 . 7 - 3 . 0 m g 1-~ Microbiol. 29, 562-564. George, G. S. & Coombs, T. L (1977). The effects of chelating agents CuZ+). Reish & Carr (1978) f o u n d for two p o l y c h a e t e s on the uptake and accumulation of cadmium by Mytilus edulis. Mar. differences of the same m a g n i t u d e b e t w e e n these two Biol. 39, 261-268. types of tests, except for c o p p e r a n d m e r c u r y w h e r e the Haight, M., Mudry, T. & Pasternak, J. (1982). Toxicity of seven heavy metals on Panagrellus silusiae: the efficacy of the free-living nemadifferences were rather small. A n a l o g o u s small differtode as an in vivo toxigologicalbioassay. Nematologica 28, 1-11. ences were obtained by Biesinger & Christensen (1972) Heip, C., Herman, R. & Vincx, M. (1984). Variability and productivity of meiobenthos in the southern bight of the North Sea. Rapp. P.-v. and W i n n e r et al. (1977) for D a p h n i a m a g n a . F o r this REun. Cons. int. Explor. Mer. 183, 51-56. species the 72 h LCs0 value o f c o p p e r was not m u c h difHeip, C., Smol, N. & Absillis, V. (1978). Influence of temperature on the ferent f r o m the c o n c e n t r a t i o n which inhibited the intrinreproductive potential of Oncholaimus oxyuris (Nematoda, Oncholsic rate of natural increase. This is highly in contrast aimidae). Mar. Biol. 45,255-260. Heip, C., Vincx, M. & Vranken, G. (1985). The ecology of marine nemwith o u r findings. Others (Blaylock et al., 1985) w o r k i n g atodes. Oceanogr. Mar. Biol. Ann. Rev. 23,399-489. with D a p h n i a magna, also u n d e r c o p p e r stress, Howell, R. (1983). Heavy metals in marine nematodes: uptake, tissue observed large differences b e t w e e n mortality and r e p r o distribution and loss of copper and zinc. Mar. Poll Bull 14, 263268. duction, with the latter the m o s t sensitive biological Howell, R. (1984). Acute toxicity of heavy metals to two species of endpoint. Similar results were o b t a i n e d b y M o r a i t o u marine nematodes. Mar. Environ. Res. 11,153-161. A p o s t o l o p o u l o u & Verriopoulos ( 1 9 7 9 ) for the m a r i n e Hummon, W. D. & Hummon, M. R. (1975). Use of life-table data in tolerance experiments. Cab. Biol. mar. 16,743-749. c o p e p o d A c a r t i a clausi again u n d e r c o p p e r stress. Jacobs, L. J. & Vrankcn, G. (in prcss). Dcscription of Dil~lolaimella dieW h e n pre-adult mortality is not too high, rm approxivengatensis n.sp. (Ncmatoda, Monhystcridac), an aquatic nematode mates daily weight-specific productivity, n a m e l y the proof the Bclgian coast. Nematologica. Knezovich, J. R, Harrison, E L. & Tucker, J. S. (1981). The influence of d u c t i o n - b i o m a s s ratio (P/B) (Vranken & Heip, in press). organic chelators on the toxicity of copper to embryos of the Pacific M e a n annual temperature during the yearly growth oyster, Crassostrea gigas. Arch. Environm. Contain. ToxicoL 10, 241period o f D i p l o l a i m e l l a spec 1 is a b o u t 16°C. A t this 249. t e m p e r a t u r e daily P/B equals 0.112 J o u l e . J o u l e - L d a y -~, Lopez, G., Riemann, E & Schrage, M. (1979). Feeding biology of the brackish water oncholaimid nematode Adoncholaimus thalassophyresulting in a yearly P/B of a p p r o x i m a t e l y 22. A t a C u gas. Mar. Biol. 54,311-318, c o n c e n t r a t i o n o f 1 mg 1-a, a level f o u n d in s o m e sedi- Lorenzen, S. (1974). Die Nematodenfauna der sublittoralen Region der Deutschen Bucht, insbesondere im Titan-Abwassergebiet bei Helgoments of the Belgian coastal area o f the N o r t h Sea (Heip land. Vertff. Inst. Meeresforsch. Bremerh. 14,305-327. et al., 1984), yearly P/B r e d u c e s to 15.5. C o n s e q u e n t l y it Moore, J. W. & Ramamoorthy, S. (1984). Heavy metals in Natural is clear that heavy-metal load m a y lower the p r o d u c Waters. Springer-Verlag, New York, Berlin. Moraitou-Apostolopoulou, M. & Verriopoulous, G. (1979). Some tivity o f b o t t o m - d w e l l i n g organisms such as nematodes, effects of sub-lethal concentrations of copper on a marine copepod. Mar Poll. Bull. 10, 88-92. Patrick, E M. & Loutit, M. (1976). Passage of metals in effluents, through bacteria to higher organisms. Water Res. 10,333-335. This research is conducted under contract no. ENV-767-B of the Pitcher, R. S. & McNamara, D. G. (1972). The toxicity of low concentrations of silver and cupric ions to three species of plant-parasitic environmental programme of the CEC. C. Heip acknowledges a grant nematodes. Nematologica 18,385-390, of the Belgian National Science Foundation (NFWO). We thank Mr. W. Platt, M. 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MarinePollutionBulletin.Vol.17.No. I(1.pp.457-46I. 1986 PrintedinGreatBritain

01986

Low Levels of Tributyl Tin Reduce Growth of Marine Micro-Algae A , R. B E A U M O N T a n d P. B. N E W M A N D e p a r t m e n t o f M a r i n e Biology, University College o f North Wales, M a r i n e Science Laboratories, M e n a i Bridge, G w y n e d d L L 5 9 5 E H , U K

Three species of micro-algae, Pavlova lutheri, Dunaliella tertiolecta and Skeletonema costatum were cultured in a range of concentrations of tributyl tin (TBT). Death occurred within 2 days in 5 [~g I-I TBT and growth was much reduced by I pg !-t TBT in all three species. Virtually no growth occurred in S. costatum cultured in 0.1 pg 1-t TBT, whereas growth of the other two species was slightly, though significantly, reduced, compared to controls at this concentration. These results suggest that primary production in certain areas may be significantly affected by elevated levels of TBT.

T h e r e c e n t i n c r e a s e in t h e u s e o f t r i b u t y l tin ( T B T ) as the b i o c i d e in a n t i - f o u l i n g p a i n t s f o r p l e a s u r e craft h a s led to e l e v a t e d levels o f T B T , f r o m 0.1 0g 1-1 to 2.0 pg 1-1 , in c e r t a i n e s t u a r i e s a n d m a r i n a s , m a i n l y in t h e S o u t h E a s t o f U K 0 V a l d o c k & Miller, 1983). A c c o r d i n g to C l e a r y & S t e b b i n g ( 1 9 8 5 ) a r e a s o f high p l e a s u r e craft activity in t h e S o u t h W e s t s e e m to b e less affected a l t h o u g h E b d o n et al. ( 1 9 8 5 ) r e p o r t T B T c o n c e n t r a -

t i o n s in t h e r e g i o n o f 0.5 og 1-1 in P l y m o u t h h a r b o u r . N e v e r t h e l e s s , high levels o f T B T have b e e n i m p l i c a t e d in t h e s l o w g r o w t h a n d shell m a l f o r m a t i o n ('bailing') in t h e o y s t e r Crassostrea gigas ( A l z i e u et al., 1982; W a l d o c k & T h a i n , 1983) a n d B e a u m o n t & B u d d ( 1 9 8 4 ) h a v e s h o w n that veliger larvae o f t h e c o m m o n m u s s e l Mytilus edulis suffer high m o r t a l i t y after 15 d a y s at c o n c e n t r a t i o n s o f tributyl tin o x i d e ( T B T O ) as low as 0.1 0g 1-1. S i m i l a r m e d i u m t e r m studies b y L a u g h l i n et al. ( 1 9 8 4 ) h a v e s h o w n significant r e d u c t i o n s in survival o f G a m m a r u s oceanicus l a r v a e at 0.3 Og l-1 T B T O a n d U ' r e n ( 1 9 8 3 ) h a s s h o w n that 0.4 pg 1-1 T B T O has a significant effect o n the c o p e p o d Acartia tonsa after 6 d a y s . H a l l & P i n k n e y ( 1 9 8 4 ) a n d S t e b b i n g ( 1 9 8 5 ) have r e c e n t l y r e v i e w e d t h e l i t e r a t u r e c o n c e r n i n g t h e analysis, t o x i c i t y a n d a c c u m u l a t i o n o f TBT. A l t h o u g h o t h e r s o u r c e s o f f o o d a r e available, members of both the holoplankton and the meroplankt o n rely m a i n l y o n m i c r o a i g a e f o r their n u t r i t i o n . It is o f c o n s i d e r a b l e i m p o r t a n c e , therefore, to e s t a b l i s h n o t o n l y w h e t h e r a t o x i n affects t h e z o o p l a n k t o n d i r e c t l y b u t a l s o w h e t h e r it m a y significantly r e d u c e t h e a m o u n t 457