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Accumulation of tin and tributyltin from anti-fouling paint by cultivated scallops (Pecten maximus) and Pacific oysters (Crassostrea gigas)
Aquaculture, 56 (1986) 103-114 Efsevier Science Publishers B.V., Amsterdam
103 - Printed in The Netherlands
ACCUMULATION OF TIN AND TRIBUTYLTIN FROM ANTI-FOULING PAINT BY CULTIVATED SCALLOPS (PlXX’EN MAX~MUS) AND PACIFIC OYSTERS (~~ASSOST~~A GIGAS)
I.M. DAVIES, J.C. McKIE and J.D. PAUL*
MarineLaboratory
Victoria Road, Aberdeen ABS SDB (Great ~~t~n~ *Sea Fish Industries Authority, Marine Farming Unit, Ardtoe, Acharacie, Argyll PH36 4LD (Great Britain)
(Accepted 8 April 1986)
ABSTRACT Davies, I.M., McKie, J.C. and Paul, J.D., 1986. Accumulation of tin and tributyltin from anti-fouling paint by cultivated scallops (Pecfen maximus) and Pacific oysters ~cr~sost~a gigas). Aquacu~ture, 65: 103--114. The accumulation of total tin and tributyltin by oysters ( Crassostrea gigas) and scdfop (Pecten maximus) from anti-fouling paint, and the subsequent depuration of these substances, have been observed over a period of 41 weeks. Oysters accumulated up to 1.41 mg/kg tin (0.87 mgjkg TBT-tin), and subsequently lost 90% of this during depum tion. Juvenile scahops accumulated 2.5 mgjkg total tin (1.86 mgjkg TBT), but lost only 20-40% of this. In adult scallops, indi~dual organs were analysed, and a progressive transfer of TBT to the adductor muscle was found. At the end of the experiment the adductor musclecontained0.53 mgjkg. There is evidence of a storagejdetoxification mechanism for TBT operating in both scallops and oysters, but which is more effective in scallops, and involves the adductor muscle.
INTRODLWI’ION
In an attempt to develop a successful system for the control of the growth of fouling organisms on she~fi~~o~g equipment, the properties of antifouling systems based on organo-tin and copper compounds have been investiiated. The results of growth trials using scallops [&Men mrrrcimus (L.)] and Pacific oysters [Crassmtwa gigas (Thunberg)] are being reported (Paul and Davies, 1986), as is the accumulation of copper and nickel from the anti-foulants (Davies and Paul, 1986). In general, anti-foulants are not used widely in shellfish cultivation, but the development of a suitable system would bring considerable commercial benefit, In a commercial system it is necessary to avoid any accumulation of toxic substances in the cultivated animals, to prevent their subsequent transfer to the consumer. The ac~um~ation of tin (measured as total tin) and tributyltin (TBT) by scallops and oysters from nets treated with a 0044~486/86j$O3.50
o 1986 Elsevier Science Publishers B.V.
104
‘free-association’ type organo-tin anti-fouling and evaluated. In this type of paint the
paint
has been
measured
MATERIALS AND METHODS
Standard commercial 6-mm pearl nets and Zl-mm lantern nets were coated with Flexgard (Flexabar Corp.) anti-fouling paint in accordance with the manufacturer’s instructions. This paint contains about 3.5% bis-(trin-butyltin) oxide (TBTO) as its active ingredient (Waldock and Thain, 1985). After drying, the nets were soaked in sea water for 3 weeks, in simulation of commercial practice, before being stocked with shellfish. Ten replicate pearl nets were each stocked with 50 one-year old scallop spat, and 10 lantern nets with 3-year-old scallops or 5-10 g oysters, at 25-30 per layer. In May 1984 the stocked nets were suspended from a raft in Loch Moidart, W. Scotland (Fig. l), and control untreated nets suspended from a second raft 200 m away.
7”
.su
5”
4w
3!4
ZY
Fig. 1. Location of bch Moidart on the west coast of Scotland.
The shellfish were kept in the nets for 31 weeks. Samples of 10 animals were periodically removed for assessment of growth (Paul and Davies, 1986), and for chemical analysis. Adult scallops were dissected into adductor muscle, gonad, digestive gland and gill/mantle, which were stored at -20°C until analysis. Organs were analysed as a combined homogenate from five individuals, two per sampling occasion. Scallop spat and oysters were analysed as combined homogenates of the whole soft parts from five individuals, two per sampling occasion.
105
After 31 weeks exposure, the remaining stock was transferred to untreated nets, and sampling continued for a further 10 weeks. Total tin in tissue was determined by graphite furnace atomic absorption spectrophotometry after digestion in concentrated nitric acid, employing ammonium dihydrogen orthophosphate as a matrix modifier. Tributyltin (TBT) compounds were separated from tissue dispersed in cold hydrochloric acid by extraction with hexane, which was subs~uently washed with sodium hydroxide. Dedication was as for total tin. These methods have detection limits of 30-50 ng/sample, and are to be described in detail by McKie (1986). TBT concentrations are expressed as tin throughout, and all concentrations are presented on a wet weight basis. All statements of significance have been tested (Student’s t) and all differences are significant at the 5% level or better. RESULTS
{a) ~oncen~~tions of tin and 7232’ in o~sie~ Total tin concentrations (Fig. 2) in the oysters kept in lantern nets treated with TBT paint increased rapidly during the early part of the experiment. Initial samples of five individuals contained 0.11, 0.11 mg/kg, which increased to 1.26, 1.30 mg/kg after 6 weeks and to 1.36, 1.45 mg/kg after 16 weeks. Concentrations then fell steadily over the next 15 weeks, and more rapidly following transfer to untreated nets at week 31. The final mean concentration (0.25 mg/kg) was approximately twice that in the control oysters.
D) f
(.2
;;
if
z
;;-’ 5
0.8
-
E
E 2
o.4
----*___
__--
-9
_*____~__~_~~~~~~~~*___~~~~~~~~~--~--~-
0.0
0
10
/
“g:___L:: ---
30
20
40
SO
WEEKS
Fig. 2. Total tin and TBT concentrations in control oysters and oysters kept in TBTtreated nets. x, TBT-treated nets, total tin concentrations; +, TBT-treated nets, TBT concentrations; 0, controfs, total tin concentration; 0, controls, TBT concentrations.
106
TBT did not accumulate (Fig. 2) as rapidly as total tin, but the maximum concentration (0.87 mg/kg) was again observed at week 16. Concentrations decreased only gradually until the oysters were transfered to untreated nets, when a more rapid reduction occurred. The final concentration was 0.19 mg/kg TBT. Control oysters (Fig. 2) did not show any well-defined pattern of tin concentration. TBT was always undetectable (<0.03 mg/kg), and total tin, initially 0.11 mg/kg, usually lay between <0.05 and 0.17 mg/kg (mean 0.12 mg/kg). No firm interpretation is made of the suggestion of higher concentrations early and late in the experiment, which may represent natural variability, or seasonality in tin levels. (b) Tin and TBT accumulation by juvenile scallops Juvenile scallops rapidly accumulated both TBT and total tin (Fig. 3) when grown in pearl nets treated with TBT anti-fouling paint. Initial concentrations of total tin and TBT of 0.16 and <0.05 mg/kg, respectively, increased rapidly to 2.50 and 1.86 mg/kg, respectively, after 16 weeks Some gradual reduction in concentrations then occurred. After exposure. transfer to untreated nets at 31 weeks, the total tin concentration fell rapidly from 1.73 to 1.14 mg/kg in 2 weeks. Concentrations were then maintained for the subsequent 8 weeks until the end of the experiment, when the scallop spat still contained approximately 20 times as much tin as the controls. The final TBT concentration (0.85 mg/kg) was not significantly different from that immediately prior to transfer to untreated nets.
WEEKS
Fig. 3. Total tin and TBT concentrations in scallop spat from control nets and nets treated with TBT anti-foulant. x, TBT-treated nets, total tin concentration; +, TBTtreated nets, TBT concentration; 0, controls, total tin concentrations; 0, controls, TBT concentrations.
107
The total tin concentration in control juvenile scallops (Fig. 3) varied irregularly during the experiment between <0.03 and 0.21 mg/kg (mean 0.12 mg/kg), whilst TBT was always undetectable (<0.03 mg/kg). fc) Tin and TBT concentrations in adult scallops
Control adult scallops contained low concentrations of tin (normally <0.15 mg/kg) in ah organs, except during the summer months (JuneAugust, weeks G-16). During this period some accumulation occurred, with the highest concentrations being found in the digestive gland (0.44 mgfkg) and the gills/mantle (0.41 mg/kg). Concentrations in all organs had fallen by week 16, and were very low at week 21. In the summer months TBT was undetectable in adductor muscle, digestive gland and gills/mantle, but was present at concentrations of up to 0.10 mg/kg in gonad tissue. During the rest of the year TBT con~ent~tions were low (<0.03-~.08 mg/kg, undetectable in 85% of the samples). The low proportion of TBT in the control scallops in the summer (particularly in the digestive gland and gills/mantle) suggests that the observed seasonal variation is natural, and not a result of cross~on~mination from treated nets. In the adult scallops kept in TBT-treated nets, accumulation of total tin and TBT occurred in all orgam (Table 1). When the concentrations of total tin in control scallops are subtracted from those in scallops in TBTtreated nets it can be seen (Fig. 4) that rapid accumulation of tin occurred in the digestive gland, gills/mantle and gonad during the first 2 weeks of the experiment. The highest accumulations (up to 0.86 mg/kg) were found in the digestive gland, and concentrations in this organ varied irregularly TABLE
7
Adult scallops: total tin and TBT concentrations TBT-treated nets
Sample No.
1 2 3 4 5 6 7 8 9 10
Weeks
0 2 6 10 16 21 31 33 35 41
Add&or
muscle
Gonad
(mg/kg wet weight) in scallops from
Digestive gland
Gills/mantle
Tin
TBT
Tin
TBT
Tin
TBT
Tin
TBT
0.07 0.27 0.27 0.42 0.33 0.28 0.33 0.44 0.53 0.56
<0.05 0.14 0.28 0.43 0.38 0.27 0.42 0.36 0.43 0.53
0.10 0.47 0.41 0.56 0.48 0.24 0.35 0.39 0.25 0.23
<0.05 0.32 0.33 0.44 0.40 0.28 0.29 0.19 0.16 0.17
0.14 0.86 0.84 0.99 1.03 0.60 0.78 0.94 0.56 0.43
<0.05 0.45 0.43 0.61 0.31 0.37 0.27 0.16 0.09 0.16
0.09 0.58 0.44 0.46 0.48 0.24 0.27 0.28 0.14 0.11
<0.05 0.42 0.43 0.56 0.27 0.17 0.17 0.07 0.09 0.04
108
WEEKS
Fig. 4. Differences between total tin concentrations in organs of adult scallops from TBT-treated nets and control nets. x, Adductor muscle; 0, gonad; 0, digestive gland; +, gills/mantle.
is a suggestion of a decrease 5--10 weeks after transfer to untreated nets (weeks 36-41). In the gills/mantle a maximum accumulation of 0.51 mg/kg total tin was attained at week 2. Tin was then gradually lost from this organ for the rest of the experiment, and the final concentration was less than 0.05 mg/kg. The maximum concentration in the gonad occurred at week 2 (0.42 mg/kg). Subsequently a slow gradual reduction occurred, with a final concentration of 0.20 mg/kg. Transfer to untreated nets at week 31 had no effect on the rate of loss of total tin from the gonad or gills/mantle. . The adductor muscle displayed a different pattern of accumulation. Following initial accumulation of 0.21 mg/kg total tin after 2 weeks in the TBT-treated nets, further gradual accumulation to 0.28 mg/kg after 31 weeks occurred. After transfer to untreated nets, the concentration increased more rapidly and the final concentration was 0.49 mg/kg. Maximum TBT concentrations (Fig. 5) in the gills/mantle (0.56 mg/kg), digestive gland (0.51 mg/kg), and gonad (0.44 mg/kg) occurred after 10 weeks exposure. In all three organs the concentration then fell gradually until the transfer to untreated nets at week 31. Some increase in the rate of loss of TBT then occurred, and final concentrations were in gills/mantle 0.04 mg/kg, digestive gland 0.16 mg/kg, and gonad 0.17 mg/kg. In contrast, the adductor muscle accumulated TBT relatively slowly in the initial weeks of the experiment, and the concentration passed through a maximum of 0.43 mg/kg at week 10. Concentrations then fell to 0.27 mg/kg at week 21, and subsequently increased during the remainder of the exposure period, and after transfer to untreated nets. The final concentration
109
0.004 0
10
20
30
40
50
WEEKS
Fig. 5. TBT concentrations in organs of adult scallops from TBT-treated ductor muscle; 0, gonad; 0, digestive gland; +* gills/mantle.
nets. x, Ad-
DISCUSSION
The maximum concentrations of tin and TBT found in oysters in this experiment are in general higher than those reported by Waldock and Thain (1983) from English estuaries subject to high pleasure craft activity. Comparable concentrations were occasionally reported from areas with high numbers of boats in summer (e.g., Brighton Marina in August, 11.3 mg/kg Sn, and 4.5 mg/kg TBT dry weight). Laboratory experimental studies of the uptake of TBT from sea water by Cmssostrea gigus (Waldock et al., 1983) have suggested that equilibrium concentrations of TBT in tissue were attained after 2-3 weeks exposure at 14°C. In contrast, oysters in the current experiment required 16 weeks exposure to reach their maximum TBT concentration, during which time water temperatures increased gradually from 10°C to 14.5%. Applying the bio-concentration factor of 6000 obtained by Waldock et al,, (1983) to this maximum value indicates a mean TBT concentration in the sea water inside the treated nets of 0.35 E.cg/l TBTO (0.14 fig/l Sn). If the same bio-concentration factor is applied to the concentration observed after 2 weeks exposure, 0.18 pg/l TBTO (0.07 @g/l Sn) in the sea water is suggested. Waldock et al. (1983) also observed the depuration of TBT from oysters, and estimated 50% loss in 23 days. In the depuration period of the current experiment, following transfer to untreated nets at week 31, the TBT concentration fell by a factor of 3 (0.57 to 0.19 mg/kg) in 70 days, which indicates a less rapid release in the field at 6-9”C than observed by Waldock et al. (1983) at 14°C under laboratory conditions. Other differences arising from different locality, oyster stocks, and type of food, etc., may also have been significant.
110
The proportion of tin present as TBT in the oysters during the main period of accumulation (O-21 weeks) was 52.2% (SD 12.5%) and is rather higher than the 2-25s reported by Waldock and Miller (1983) from cultivated oysters in English estuaries. This proportion increased significantly to 72.3% (SD 4.6%) in weeks 31-41. This preferential loss of non-TBT-tin after removal of the source of TBT suggests that depuration is influenced by the availability of the TBT in the oyster for elimination either as TBT or as its decomposition products. In the aquatic environment, it has been shown that TBT decomposes by progressive loss of butyl groups leaving the tin finally present as inorganic compounds (Blunden et al., 1984). Maguire (1984) has demonstrated that some methylation can occur before all the butyl groups have been lost, resulting in tin species containing both butyl and methyl groups. In oysters, if the main mechanism of TBT elimination involved a similar slow initial decomposition of the TBT to less heavily alkylated species, and subsequent excretion, an apparent preferential loss of non-TBT-tin could result. However, the high concentrations of non-TBT-tin earlier in the experiment suggest a greater availability of TBT for decomposition/excretion at that time than in the depuration phase. Therefore, whilst much of the absorbed tin is readily eliminated, a proportion may be stabilized by some mechanism and more firmly retained by the oyster. Such processes are recognised and involved in the detoxification of other contaminants, and include the incorporation of metals in membrane-lined vesicles or granules (Thomson et al., 1985), and the association of metals with specific proteins (e.g., metallothioneins) (Roesijadi, 1981). The maximum concentrations of both tin and TBT in oysters were observed at week 16. Body burdens of these contaminants (Table 2) can be TABLE 2 Pacific oysters: whole body contents of total tin and tributyltin from nets treated with TBT paint
Sample no.
1 2 3 4 5 6 7 8 9 10
Weeks
0
2 6 10 16 21 31 33 35 41
Total tin MI
TBT (rg)
0.11 1.95 2.60 3.65 4.08 4.48 1.72 1.94 0.85 0.33
<0.05 1.24 1.11 1.61 1.87 1.71 1.26 1.48 0.72 0.22
compounds of oysters
As % of maximum burden Tin %
TBT %
2.5 43.5 58.0 81.5 91.1 100 38.4 43.3 19.0 7.4
< 2.7 66.3 59.4 86.1 100 91.4 67.4 79.1 38.5 11.8
111
growth data from Paul and (1986), and show that maximum during the weeks 16-21 of the accumulated tin by the end of the experiment. The proportion of tin present as TBT in juvenile
(0.19 mgfkg, 11.8%). Paul and Davies (1986) reported severe inhibition of growth of the oysters in this experiment, and a lesser effect on juvenile scallop growth. The juvenile scallop may therefore utilise a more effective detox~ication process than the oyster, and be able to isolate a larger proportion such dramatic biological effects as the oysters. Combination of the analytical data for whole animals (Tables 4 and 5). ~ax~urn body burdens of TBT and total tin were attained after 10 and 16 weeks, respectively. Subsequent declines were not rapid, and at the end of the experiment 70% of the total tin and 79% of the TBT remained in the TABLE 3 Juvenile scallops: whole body contents of total tin and tributyltin kept in nets treated with TBT paint
Sample no.
1 2 3 4 5 6 7 8 9 10
Weeks
0
2 6 10 16 21 31 33 35 41
Total tin (pa) 0,045 1.32 2.19 3.60 6.70 9.11 9.31 6.31 4.97 5.84
compounds of scalbps
As % of maximum burden
<0.014 0.62 1.51 2.59 4.96 4.27 5.25 3.45 3.19 4.13
Tin%
TBT%
0.48 14.2 23.5 38.7 72.0 97.9 100 57.0 53.4 62.7
< 0.27 11.8 28.8 49.3 94.5 81.3 100 65.7 60.8 78.7
112 TABLE 4 Adult scallops: total tin contents (rg) of individual organs and whole nets treated with TBT paint
scallops kept in
Sample Weeks no.
Adductor muscle
Gonad
Digestive gland
Gills/ Mantle
Total
Total as % of maximum
1 2 3 4 5 6 7 8 9 10
0.45 3.18 2.13 4.17 4.63 4.26 4.95 6.41 6.43 6.64
0.31 0.96 1.35 1.47 1.84 0.48 1.09 1.17 0.77 0.96
0.24 1.75 1.95 2.85 3.78 3.21 3.49 2.92 1.81 1.43
0.82 3.09 1.82 2.59 4.53 2.10 3.04 3.76 1.60 1.28
1.72 8.98 7.25 11.08 14.78 10.05 12.57 14.26 10.61 10.31
11.6 60.8 49.1 75.0 100 68.0 85.0 96.5 71.8 69.8
0 2 6 10 16 21 31 33 35 41
TABLF, 5 Adult scallops: tributyltin contents (fig) of individual organs and whole scallops kept in nets treated with TBT paint
Sample no. 1 2
Weeks
0 2
Adductor muscle
1.65
Gonad
0.66
Digestive gland
-
Gills/ Mantle
-
Total
0.97
Total as % of maximum
<
0.92
scallops. These percentages are similar to those found for juvenile scallops (63%, 79%) and much greater than for Pacific oysters (7%, 12%). Early in the experiment, all four organs analysed increased in total tin content and, in all except the adductor muscle, maxima were attained at week 10. The gills/mantle, digestive gland, and gonad lost 72%, 62% and 48%, respectively, of this tin by the end of the experiment. In contrast, the tin content of the adductor muscle continued to rise throughout the experiment, even after transfer to untreated nets. The data for TBT content of individual organs show a similar pattern, with increasing TBT burden in the adductor muscle,
113
The proportion of TBT in the total tin burden of individual organs varied between organs. In the exposure period (31 weeks) on average 83.5%, 84.6% and 47.5% of the total tin was TBT in gonad, gills/mantle and digestive gland, respectively. These proportions decline significantly in the depuration period to 52.3%, 45.7% and 23.3%, respectively. In adductor muscle, the percentages were higher (93%, 85.3%) and did not fall significantly. These data indicate an internal transfer of TBT to the adductor muscle from the other organs, and suggest that storage/detoxification of TBT may take place in the adductor muscle. TBT as measured will be present as a non-polar compound. It may be speculated that the mechanism operating in the adductor muscle may be different from granule formation or metallothionein induction, which are commonly the result of exposure to inorganic metallic species (e.g., Roesijadi, 1981; Thomson et al., 1985). A closer analogy may possibly be drawn with the storage of methyl-mercury compounds which also tend to accumulate in muscle, and which bind to sulphydryl groups (cysteine residues) in proteins. In trout (Olsen et al., 1978), methyl-mercury did not induce the formation of significant amounts of metallothionein. The methyl-mercury did become associated with metallothionein and other proteins, but the action of the metallothionein was as a binding scavenger, rather than a response to methyl-mercury exposure. The underlying cause of the broad spectrum of acute toxicity of triorganotin compounds is thought to be through interference with mitochondrial functions (Blunden et al., 1984), and there is indication that interaction with cysteine and histidine residues occurs (e.g., in cats, reviewed by Blunden et al., 1984). The commercial application of TBT-based anti-foulants of the type tested to oyster cultivation is unadvisable, bearing in mind the field (Alzieu et al., 1982), and experimental (Waldock and Thain, 1983; Paul and Davies, 1986) evidence of adverse effects of TBT on oyster growth. TBT reduced the growth of juvenile scallops, but did not affect adult scallop growth. The evidence presented here of progressive accumulation of TBT in the adductor muscle (the organ of prime importance for human consumption) must put in some doubt the application of TBT-based paints in scallop cultivation. CONCLUSIONS
(1) Juvenile and adult scallops, and Pacific oysters accumulated and other tin compounds from nets treated with anti-fouling paint taining TBT. (2) Oysters were able to depurate 90% of the accumulated tin and on transfer to untreated nets. (3) In contrast, scallops under the same exposure times retained 80% of the accumulated tin and TBT. (4) In both species a mechanism for the storage/detoxification of
TBT conTBT 60part
114
of the absorbed TBT may exist, and this operates more effectively in the scallop. (5) In adult scallops, TBT was progressively transferred during the experiment to adductor muscle the other analysed. (6) caused moderate and reduced growth juvenile scallops, enhanced survival adult scallops. oysters showed little
Crassostrea gigas. Rev. Trav. Inst. Peches Marit., 45: 100-116. Blunden, S.J., Hobbs, L.A. and Smith, P.J., 1984. The environmental chemistry of organotin compounds. In: H.J.M. Brown (Editor), Environmental Chemistry, Vol. 51-77. Davies, I.M. and Paul, J.D., 1986. Accumulation of copper and nickel from anti-fouling compounds during cultivation of scallops (Pecten maximus L.) and Pacific oysters (Cr~os~~a gigas Thun.). Aquaculture, 55: 93-102. Maguire, R.J., 1984. Butyltin compounds and inorganic tin in sediments in Ontario. Environ. Sci. Technol., 18: 291-294. tin and tributyltin in fish and shellfish tissue by graphite furnace atomic absorption spectrophotometry. ICES CM 1986. Olsen, K.R., Squibb, K.S. and Cousins, R.J., 1978. Tissue uptake, sub-cellular distribution, and metabolism of 14CH,HgCl and CH, zOHgCl by rainbow trout, Salmo gairdneri. J. Fish. Res. Board Can., 35(4): 381-390. Paul, and and oysters. Aquaculture, 54: of low molecular weight metallothionein-like G., 1981. The significance invertebrates: current status. Mar. Environ. Res., 4: 167-179. Thomson, J.D., Pirie, B.J.S. and George, S.G., 1985. Cellular metal dist~bution in the Pacific oyster, (Thun.) determined by quantitative X-ray mieroprobe analysis. J. Exp. Mar. Biol. Ecol., 85: 37-45. Waldock, M.J. and Miller, D., 1983. The determination of total and tributyl-tin in seawater and oysters in areas of high pleasure craft activity. ICES CM 1983/E:12, 17 pp. Waldock, M.J. and Thain, J.E., 1983. Shell thickening in Crassostrea gigas: organotin antifouling or sediment induced? Mar. Pollut. Bull., 14: 411-415. Waldock, M.J. and Thain, J.E., 1985. The comparative leach rates and toxicity of two fish net antifouling preparations. ICES CM 1985/E: 29, 6 pp. Waldock, M.J., Thain, J.E. and Miller, D., 1983. The accumulation and depuration of bi~tributyltin~ oxide in oysters: a comparison between the Pacific oyster (C~Ssostrea g&as) and the European flat oyster (Ostrea edulis). ICES, CM 1983/E:52, 9 PP.
103 - Printed in The Netherlands
ACCUMULATION OF TIN AND TRIBUTYLTIN FROM ANTI-FOULING PAINT BY CULTIVATED SCALLOPS (PlXX’EN MAX~MUS) AND PACIFIC OYSTERS (~~ASSOST~~A GIGAS)
I.M. DAVIES, J.C. McKIE and J.D. PAUL*
MarineLaboratory
Victoria Road, Aberdeen ABS SDB (Great ~~t~n~ *Sea Fish Industries Authority, Marine Farming Unit, Ardtoe, Acharacie, Argyll PH36 4LD (Great Britain)
(Accepted 8 April 1986)
ABSTRACT Davies, I.M., McKie, J.C. and Paul, J.D., 1986. Accumulation of tin and tributyltin from anti-fouling paint by cultivated scallops (Pecfen maximus) and Pacific oysters ~cr~sost~a gigas). Aquacu~ture, 65: 103--114. The accumulation of total tin and tributyltin by oysters ( Crassostrea gigas) and scdfop (Pecten maximus) from anti-fouling paint, and the subsequent depuration of these substances, have been observed over a period of 41 weeks. Oysters accumulated up to 1.41 mg/kg tin (0.87 mgjkg TBT-tin), and subsequently lost 90% of this during depum tion. Juvenile scahops accumulated 2.5 mgjkg total tin (1.86 mgjkg TBT), but lost only 20-40% of this. In adult scallops, indi~dual organs were analysed, and a progressive transfer of TBT to the adductor muscle was found. At the end of the experiment the adductor musclecontained0.53 mgjkg. There is evidence of a storagejdetoxification mechanism for TBT operating in both scallops and oysters, but which is more effective in scallops, and involves the adductor muscle.
INTRODLWI’ION
In an attempt to develop a successful system for the control of the growth of fouling organisms on she~fi~~o~g equipment, the properties of antifouling systems based on organo-tin and copper compounds have been investiiated. The results of growth trials using scallops [&Men mrrrcimus (L.)] and Pacific oysters [Crassmtwa gigas (Thunberg)] are being reported (Paul and Davies, 1986), as is the accumulation of copper and nickel from the anti-foulants (Davies and Paul, 1986). In general, anti-foulants are not used widely in shellfish cultivation, but the development of a suitable system would bring considerable commercial benefit, In a commercial system it is necessary to avoid any accumulation of toxic substances in the cultivated animals, to prevent their subsequent transfer to the consumer. The ac~um~ation of tin (measured as total tin) and tributyltin (TBT) by scallops and oysters from nets treated with a 0044~486/86j$O3.50
o 1986 Elsevier Science Publishers B.V.
104
‘free-association’ type organo-tin anti-fouling and evaluated. In this type of paint the
paint
has been
measured
MATERIALS AND METHODS
Standard commercial 6-mm pearl nets and Zl-mm lantern nets were coated with Flexgard (Flexabar Corp.) anti-fouling paint in accordance with the manufacturer’s instructions. This paint contains about 3.5% bis-(trin-butyltin) oxide (TBTO) as its active ingredient (Waldock and Thain, 1985). After drying, the nets were soaked in sea water for 3 weeks, in simulation of commercial practice, before being stocked with shellfish. Ten replicate pearl nets were each stocked with 50 one-year old scallop spat, and 10 lantern nets with 3-year-old scallops or 5-10 g oysters, at 25-30 per layer. In May 1984 the stocked nets were suspended from a raft in Loch Moidart, W. Scotland (Fig. l), and control untreated nets suspended from a second raft 200 m away.
7”
.su
5”
4w
3!4
ZY
Fig. 1. Location of bch Moidart on the west coast of Scotland.
The shellfish were kept in the nets for 31 weeks. Samples of 10 animals were periodically removed for assessment of growth (Paul and Davies, 1986), and for chemical analysis. Adult scallops were dissected into adductor muscle, gonad, digestive gland and gill/mantle, which were stored at -20°C until analysis. Organs were analysed as a combined homogenate from five individuals, two per sampling occasion. Scallop spat and oysters were analysed as combined homogenates of the whole soft parts from five individuals, two per sampling occasion.
105
After 31 weeks exposure, the remaining stock was transferred to untreated nets, and sampling continued for a further 10 weeks. Total tin in tissue was determined by graphite furnace atomic absorption spectrophotometry after digestion in concentrated nitric acid, employing ammonium dihydrogen orthophosphate as a matrix modifier. Tributyltin (TBT) compounds were separated from tissue dispersed in cold hydrochloric acid by extraction with hexane, which was subs~uently washed with sodium hydroxide. Dedication was as for total tin. These methods have detection limits of 30-50 ng/sample, and are to be described in detail by McKie (1986). TBT concentrations are expressed as tin throughout, and all concentrations are presented on a wet weight basis. All statements of significance have been tested (Student’s t) and all differences are significant at the 5% level or better. RESULTS
{a) ~oncen~~tions of tin and 7232’ in o~sie~ Total tin concentrations (Fig. 2) in the oysters kept in lantern nets treated with TBT paint increased rapidly during the early part of the experiment. Initial samples of five individuals contained 0.11, 0.11 mg/kg, which increased to 1.26, 1.30 mg/kg after 6 weeks and to 1.36, 1.45 mg/kg after 16 weeks. Concentrations then fell steadily over the next 15 weeks, and more rapidly following transfer to untreated nets at week 31. The final mean concentration (0.25 mg/kg) was approximately twice that in the control oysters.
D) f
(.2
;;
if
z
;;-’ 5
0.8
-
E
E 2
o.4
----*___
__--
-9
_*____~__~_~~~~~~~~*___~~~~~~~~~--~--~-
0.0
0
10
/
“g:___L:: ---
30
20
40
SO
WEEKS
Fig. 2. Total tin and TBT concentrations in control oysters and oysters kept in TBTtreated nets. x, TBT-treated nets, total tin concentrations; +, TBT-treated nets, TBT concentrations; 0, controfs, total tin concentration; 0, controls, TBT concentrations.
106
TBT did not accumulate (Fig. 2) as rapidly as total tin, but the maximum concentration (0.87 mg/kg) was again observed at week 16. Concentrations decreased only gradually until the oysters were transfered to untreated nets, when a more rapid reduction occurred. The final concentration was 0.19 mg/kg TBT. Control oysters (Fig. 2) did not show any well-defined pattern of tin concentration. TBT was always undetectable (<0.03 mg/kg), and total tin, initially 0.11 mg/kg, usually lay between <0.05 and 0.17 mg/kg (mean 0.12 mg/kg). No firm interpretation is made of the suggestion of higher concentrations early and late in the experiment, which may represent natural variability, or seasonality in tin levels. (b) Tin and TBT accumulation by juvenile scallops Juvenile scallops rapidly accumulated both TBT and total tin (Fig. 3) when grown in pearl nets treated with TBT anti-fouling paint. Initial concentrations of total tin and TBT of 0.16 and <0.05 mg/kg, respectively, increased rapidly to 2.50 and 1.86 mg/kg, respectively, after 16 weeks Some gradual reduction in concentrations then occurred. After exposure. transfer to untreated nets at 31 weeks, the total tin concentration fell rapidly from 1.73 to 1.14 mg/kg in 2 weeks. Concentrations were then maintained for the subsequent 8 weeks until the end of the experiment, when the scallop spat still contained approximately 20 times as much tin as the controls. The final TBT concentration (0.85 mg/kg) was not significantly different from that immediately prior to transfer to untreated nets.
WEEKS
Fig. 3. Total tin and TBT concentrations in scallop spat from control nets and nets treated with TBT anti-foulant. x, TBT-treated nets, total tin concentration; +, TBTtreated nets, TBT concentration; 0, controls, total tin concentrations; 0, controls, TBT concentrations.
107
The total tin concentration in control juvenile scallops (Fig. 3) varied irregularly during the experiment between <0.03 and 0.21 mg/kg (mean 0.12 mg/kg), whilst TBT was always undetectable (<0.03 mg/kg). fc) Tin and TBT concentrations in adult scallops
Control adult scallops contained low concentrations of tin (normally <0.15 mg/kg) in ah organs, except during the summer months (JuneAugust, weeks G-16). During this period some accumulation occurred, with the highest concentrations being found in the digestive gland (0.44 mgfkg) and the gills/mantle (0.41 mg/kg). Concentrations in all organs had fallen by week 16, and were very low at week 21. In the summer months TBT was undetectable in adductor muscle, digestive gland and gills/mantle, but was present at concentrations of up to 0.10 mg/kg in gonad tissue. During the rest of the year TBT con~ent~tions were low (<0.03-~.08 mg/kg, undetectable in 85% of the samples). The low proportion of TBT in the control scallops in the summer (particularly in the digestive gland and gills/mantle) suggests that the observed seasonal variation is natural, and not a result of cross~on~mination from treated nets. In the adult scallops kept in TBT-treated nets, accumulation of total tin and TBT occurred in all orgam (Table 1). When the concentrations of total tin in control scallops are subtracted from those in scallops in TBTtreated nets it can be seen (Fig. 4) that rapid accumulation of tin occurred in the digestive gland, gills/mantle and gonad during the first 2 weeks of the experiment. The highest accumulations (up to 0.86 mg/kg) were found in the digestive gland, and concentrations in this organ varied irregularly TABLE
7
Adult scallops: total tin and TBT concentrations TBT-treated nets
Sample No.
1 2 3 4 5 6 7 8 9 10
Weeks
0 2 6 10 16 21 31 33 35 41
Add&or
muscle
Gonad
(mg/kg wet weight) in scallops from
Digestive gland
Gills/mantle
Tin
TBT
Tin
TBT
Tin
TBT
Tin
TBT
0.07 0.27 0.27 0.42 0.33 0.28 0.33 0.44 0.53 0.56
<0.05 0.14 0.28 0.43 0.38 0.27 0.42 0.36 0.43 0.53
0.10 0.47 0.41 0.56 0.48 0.24 0.35 0.39 0.25 0.23
<0.05 0.32 0.33 0.44 0.40 0.28 0.29 0.19 0.16 0.17
0.14 0.86 0.84 0.99 1.03 0.60 0.78 0.94 0.56 0.43
<0.05 0.45 0.43 0.61 0.31 0.37 0.27 0.16 0.09 0.16
0.09 0.58 0.44 0.46 0.48 0.24 0.27 0.28 0.14 0.11
<0.05 0.42 0.43 0.56 0.27 0.17 0.17 0.07 0.09 0.04
108
WEEKS
Fig. 4. Differences between total tin concentrations in organs of adult scallops from TBT-treated nets and control nets. x, Adductor muscle; 0, gonad; 0, digestive gland; +, gills/mantle.
is a suggestion of a decrease 5--10 weeks after transfer to untreated nets (weeks 36-41). In the gills/mantle a maximum accumulation of 0.51 mg/kg total tin was attained at week 2. Tin was then gradually lost from this organ for the rest of the experiment, and the final concentration was less than 0.05 mg/kg. The maximum concentration in the gonad occurred at week 2 (0.42 mg/kg). Subsequently a slow gradual reduction occurred, with a final concentration of 0.20 mg/kg. Transfer to untreated nets at week 31 had no effect on the rate of loss of total tin from the gonad or gills/mantle. . The adductor muscle displayed a different pattern of accumulation. Following initial accumulation of 0.21 mg/kg total tin after 2 weeks in the TBT-treated nets, further gradual accumulation to 0.28 mg/kg after 31 weeks occurred. After transfer to untreated nets, the concentration increased more rapidly and the final concentration was 0.49 mg/kg. Maximum TBT concentrations (Fig. 5) in the gills/mantle (0.56 mg/kg), digestive gland (0.51 mg/kg), and gonad (0.44 mg/kg) occurred after 10 weeks exposure. In all three organs the concentration then fell gradually until the transfer to untreated nets at week 31. Some increase in the rate of loss of TBT then occurred, and final concentrations were in gills/mantle 0.04 mg/kg, digestive gland 0.16 mg/kg, and gonad 0.17 mg/kg. In contrast, the adductor muscle accumulated TBT relatively slowly in the initial weeks of the experiment, and the concentration passed through a maximum of 0.43 mg/kg at week 10. Concentrations then fell to 0.27 mg/kg at week 21, and subsequently increased during the remainder of the exposure period, and after transfer to untreated nets. The final concentration
109
0.004 0
10
20
30
40
50
WEEKS
Fig. 5. TBT concentrations in organs of adult scallops from TBT-treated ductor muscle; 0, gonad; 0, digestive gland; +* gills/mantle.
nets. x, Ad-
DISCUSSION
The maximum concentrations of tin and TBT found in oysters in this experiment are in general higher than those reported by Waldock and Thain (1983) from English estuaries subject to high pleasure craft activity. Comparable concentrations were occasionally reported from areas with high numbers of boats in summer (e.g., Brighton Marina in August, 11.3 mg/kg Sn, and 4.5 mg/kg TBT dry weight). Laboratory experimental studies of the uptake of TBT from sea water by Cmssostrea gigus (Waldock et al., 1983) have suggested that equilibrium concentrations of TBT in tissue were attained after 2-3 weeks exposure at 14°C. In contrast, oysters in the current experiment required 16 weeks exposure to reach their maximum TBT concentration, during which time water temperatures increased gradually from 10°C to 14.5%. Applying the bio-concentration factor of 6000 obtained by Waldock et al,, (1983) to this maximum value indicates a mean TBT concentration in the sea water inside the treated nets of 0.35 E.cg/l TBTO (0.14 fig/l Sn). If the same bio-concentration factor is applied to the concentration observed after 2 weeks exposure, 0.18 pg/l TBTO (0.07 @g/l Sn) in the sea water is suggested. Waldock et al. (1983) also observed the depuration of TBT from oysters, and estimated 50% loss in 23 days. In the depuration period of the current experiment, following transfer to untreated nets at week 31, the TBT concentration fell by a factor of 3 (0.57 to 0.19 mg/kg) in 70 days, which indicates a less rapid release in the field at 6-9”C than observed by Waldock et al. (1983) at 14°C under laboratory conditions. Other differences arising from different locality, oyster stocks, and type of food, etc., may also have been significant.
110
The proportion of tin present as TBT in the oysters during the main period of accumulation (O-21 weeks) was 52.2% (SD 12.5%) and is rather higher than the 2-25s reported by Waldock and Miller (1983) from cultivated oysters in English estuaries. This proportion increased significantly to 72.3% (SD 4.6%) in weeks 31-41. This preferential loss of non-TBT-tin after removal of the source of TBT suggests that depuration is influenced by the availability of the TBT in the oyster for elimination either as TBT or as its decomposition products. In the aquatic environment, it has been shown that TBT decomposes by progressive loss of butyl groups leaving the tin finally present as inorganic compounds (Blunden et al., 1984). Maguire (1984) has demonstrated that some methylation can occur before all the butyl groups have been lost, resulting in tin species containing both butyl and methyl groups. In oysters, if the main mechanism of TBT elimination involved a similar slow initial decomposition of the TBT to less heavily alkylated species, and subsequent excretion, an apparent preferential loss of non-TBT-tin could result. However, the high concentrations of non-TBT-tin earlier in the experiment suggest a greater availability of TBT for decomposition/excretion at that time than in the depuration phase. Therefore, whilst much of the absorbed tin is readily eliminated, a proportion may be stabilized by some mechanism and more firmly retained by the oyster. Such processes are recognised and involved in the detoxification of other contaminants, and include the incorporation of metals in membrane-lined vesicles or granules (Thomson et al., 1985), and the association of metals with specific proteins (e.g., metallothioneins) (Roesijadi, 1981). The maximum concentrations of both tin and TBT in oysters were observed at week 16. Body burdens of these contaminants (Table 2) can be TABLE 2 Pacific oysters: whole body contents of total tin and tributyltin from nets treated with TBT paint
Sample no.
1 2 3 4 5 6 7 8 9 10
Weeks
0
2 6 10 16 21 31 33 35 41
Total tin MI
TBT (rg)
0.11 1.95 2.60 3.65 4.08 4.48 1.72 1.94 0.85 0.33
<0.05 1.24 1.11 1.61 1.87 1.71 1.26 1.48 0.72 0.22
compounds of oysters
As % of maximum burden Tin %
TBT %
2.5 43.5 58.0 81.5 91.1 100 38.4 43.3 19.0 7.4
< 2.7 66.3 59.4 86.1 100 91.4 67.4 79.1 38.5 11.8
111
growth data from Paul and (1986), and show that maximum during the weeks 16-21 of the accumulated tin by the end of the experiment. The proportion of tin present as TBT in juvenile
(0.19 mgfkg, 11.8%). Paul and Davies (1986) reported severe inhibition of growth of the oysters in this experiment, and a lesser effect on juvenile scallop growth. The juvenile scallop may therefore utilise a more effective detox~ication process than the oyster, and be able to isolate a larger proportion such dramatic biological effects as the oysters. Combination of the analytical data for whole animals (Tables 4 and 5). ~ax~urn body burdens of TBT and total tin were attained after 10 and 16 weeks, respectively. Subsequent declines were not rapid, and at the end of the experiment 70% of the total tin and 79% of the TBT remained in the TABLE 3 Juvenile scallops: whole body contents of total tin and tributyltin kept in nets treated with TBT paint
Sample no.
1 2 3 4 5 6 7 8 9 10
Weeks
0
2 6 10 16 21 31 33 35 41
Total tin (pa) 0,045 1.32 2.19 3.60 6.70 9.11 9.31 6.31 4.97 5.84
compounds of scalbps
As % of maximum burden
<0.014 0.62 1.51 2.59 4.96 4.27 5.25 3.45 3.19 4.13
Tin%
TBT%
0.48 14.2 23.5 38.7 72.0 97.9 100 57.0 53.4 62.7
< 0.27 11.8 28.8 49.3 94.5 81.3 100 65.7 60.8 78.7
112 TABLE 4 Adult scallops: total tin contents (rg) of individual organs and whole nets treated with TBT paint
scallops kept in
Sample Weeks no.
Adductor muscle
Gonad
Digestive gland
Gills/ Mantle
Total
Total as % of maximum
1 2 3 4 5 6 7 8 9 10
0.45 3.18 2.13 4.17 4.63 4.26 4.95 6.41 6.43 6.64
0.31 0.96 1.35 1.47 1.84 0.48 1.09 1.17 0.77 0.96
0.24 1.75 1.95 2.85 3.78 3.21 3.49 2.92 1.81 1.43
0.82 3.09 1.82 2.59 4.53 2.10 3.04 3.76 1.60 1.28
1.72 8.98 7.25 11.08 14.78 10.05 12.57 14.26 10.61 10.31
11.6 60.8 49.1 75.0 100 68.0 85.0 96.5 71.8 69.8
0 2 6 10 16 21 31 33 35 41
TABLF, 5 Adult scallops: tributyltin contents (fig) of individual organs and whole scallops kept in nets treated with TBT paint
Sample no. 1 2
Weeks
0 2
Adductor muscle
1.65
Gonad
0.66
Digestive gland
-
Gills/ Mantle
-
Total
0.97
Total as % of maximum
<
0.92
scallops. These percentages are similar to those found for juvenile scallops (63%, 79%) and much greater than for Pacific oysters (7%, 12%). Early in the experiment, all four organs analysed increased in total tin content and, in all except the adductor muscle, maxima were attained at week 10. The gills/mantle, digestive gland, and gonad lost 72%, 62% and 48%, respectively, of this tin by the end of the experiment. In contrast, the tin content of the adductor muscle continued to rise throughout the experiment, even after transfer to untreated nets. The data for TBT content of individual organs show a similar pattern, with increasing TBT burden in the adductor muscle,
113
The proportion of TBT in the total tin burden of individual organs varied between organs. In the exposure period (31 weeks) on average 83.5%, 84.6% and 47.5% of the total tin was TBT in gonad, gills/mantle and digestive gland, respectively. These proportions decline significantly in the depuration period to 52.3%, 45.7% and 23.3%, respectively. In adductor muscle, the percentages were higher (93%, 85.3%) and did not fall significantly. These data indicate an internal transfer of TBT to the adductor muscle from the other organs, and suggest that storage/detoxification of TBT may take place in the adductor muscle. TBT as measured will be present as a non-polar compound. It may be speculated that the mechanism operating in the adductor muscle may be different from granule formation or metallothionein induction, which are commonly the result of exposure to inorganic metallic species (e.g., Roesijadi, 1981; Thomson et al., 1985). A closer analogy may possibly be drawn with the storage of methyl-mercury compounds which also tend to accumulate in muscle, and which bind to sulphydryl groups (cysteine residues) in proteins. In trout (Olsen et al., 1978), methyl-mercury did not induce the formation of significant amounts of metallothionein. The methyl-mercury did become associated with metallothionein and other proteins, but the action of the metallothionein was as a binding scavenger, rather than a response to methyl-mercury exposure. The underlying cause of the broad spectrum of acute toxicity of triorganotin compounds is thought to be through interference with mitochondrial functions (Blunden et al., 1984), and there is indication that interaction with cysteine and histidine residues occurs (e.g., in cats, reviewed by Blunden et al., 1984). The commercial application of TBT-based anti-foulants of the type tested to oyster cultivation is unadvisable, bearing in mind the field (Alzieu et al., 1982), and experimental (Waldock and Thain, 1983; Paul and Davies, 1986) evidence of adverse effects of TBT on oyster growth. TBT reduced the growth of juvenile scallops, but did not affect adult scallop growth. The evidence presented here of progressive accumulation of TBT in the adductor muscle (the organ of prime importance for human consumption) must put in some doubt the application of TBT-based paints in scallop cultivation. CONCLUSIONS
(1) Juvenile and adult scallops, and Pacific oysters accumulated and other tin compounds from nets treated with anti-fouling paint taining TBT. (2) Oysters were able to depurate 90% of the accumulated tin and on transfer to untreated nets. (3) In contrast, scallops under the same exposure times retained 80% of the accumulated tin and TBT. (4) In both species a mechanism for the storage/detoxification of
TBT conTBT 60part
114
of the absorbed TBT may exist, and this operates more effectively in the scallop. (5) In adult scallops, TBT was progressively transferred during the experiment to adductor muscle the other analysed. (6) caused moderate and reduced growth juvenile scallops, enhanced survival adult scallops. oysters showed little
Crassostrea gigas. Rev. Trav. Inst. Peches Marit., 45: 100-116. Blunden, S.J., Hobbs, L.A. and Smith, P.J., 1984. The environmental chemistry of organotin compounds. In: H.J.M. Brown (Editor), Environmental Chemistry, Vol. 51-77. Davies, I.M. and Paul, J.D., 1986. Accumulation of copper and nickel from anti-fouling compounds during cultivation of scallops (Pecten maximus L.) and Pacific oysters (Cr~os~~a gigas Thun.). Aquaculture, 55: 93-102. Maguire, R.J., 1984. Butyltin compounds and inorganic tin in sediments in Ontario. Environ. Sci. Technol., 18: 291-294. tin and tributyltin in fish and shellfish tissue by graphite furnace atomic absorption spectrophotometry. ICES CM 1986. Olsen, K.R., Squibb, K.S. and Cousins, R.J., 1978. Tissue uptake, sub-cellular distribution, and metabolism of 14CH,HgCl and CH, zOHgCl by rainbow trout, Salmo gairdneri. J. Fish. Res. Board Can., 35(4): 381-390. Paul, and and oysters. Aquaculture, 54: of low molecular weight metallothionein-like G., 1981. The significance invertebrates: current status. Mar. Environ. Res., 4: 167-179. Thomson, J.D., Pirie, B.J.S. and George, S.G., 1985. Cellular metal dist~bution in the Pacific oyster, (Thun.) determined by quantitative X-ray mieroprobe analysis. J. Exp. Mar. Biol. Ecol., 85: 37-45. Waldock, M.J. and Miller, D., 1983. The determination of total and tributyl-tin in seawater and oysters in areas of high pleasure craft activity. ICES CM 1983/E:12, 17 pp. Waldock, M.J. and Thain, J.E., 1983. Shell thickening in Crassostrea gigas: organotin antifouling or sediment induced? Mar. Pollut. Bull., 14: 411-415. Waldock, M.J. and Thain, J.E., 1985. The comparative leach rates and toxicity of two fish net antifouling preparations. ICES CM 1985/E: 29, 6 pp. Waldock, M.J., Thain, J.E. and Miller, D., 1983. The accumulation and depuration of bi~tributyltin~ oxide in oysters: a comparison between the Pacific oyster (C~Ssostrea g&as) and the European flat oyster (Ostrea edulis). ICES, CM 1983/E:52, 9 PP.