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Seasonal fluctuations of tributyltin (TBT) and dibutyltin (DBT) in the dogwhelk, Nucella lapillus (L.), and the blue mussel, Mytilus edulis L., in Icelandic waters
Pergamon
0025-326X(95)00204-9
Marine Pollution Bulletin, Vol. 32, No. 4, pp. 358-361, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0025-326X/96 $15.00 + 0.00
Seasonal Fluctuations of Tributyltin (TBT) and Dibutyltin (DBT) in the Dogwhelk, Nucella lapillus (L.), and the Blue Mussel, Mytilus edulis L., in Icelandic Waters H A L L D O R A SKARPHI~DINSDrTTIR*, KRISTiN 6 L A F S D r T T I R t , J(SRUNDUR SVAVARSSON* and TORKELL JOHANNESSON*
*Institute of Biology, University of Iceland, Grensdtsvegur 12, 108 ReykjavlTc, Iceland "fDepartment of Pharmacology, University of Iceland, Arm~li 30, 108 Reykjavik, Iceland
Seasonal levels of tributyltin (TBT) and dibutyltin (DBT) were analysed in the dogwhelk (NuceUa lapUlus) and the blue mussel (Mytilus edulis) from south-western Iceland. Seasonal fluctuations of TBT were observed in both dogwhelk and blue mussel, with summer levels being approximately five (dogwhelk) to ten (blue mussel) times the winter levels. These changes were related to seasonal activity in feeding and resting of the dogwhelk and feeding of the blue mussel, while seasonal changes in shipping activity were insignificant. This indicates that maximum levels of TBT pollution should mainly be monitored in these animals at high latitudes during late summer or early autumn. Copyright © 1996 Elsevier Science Ltd
Tributyltin (TBT) is a toxic compound that has been used as an antifouling agent in marine paints. A number of studies have demonstrated the deleterious effects of TBT on non-target marine organisms, even at very low concentrations. These include effects on oysters, in which the shell-forming mechanism is disturbed and reproduction is reduced (Alzieu, 1991). TBT also induces male characteristics, i.e. penis (pseudopenis) and vas deferens development on female gonochoristic gastropod molluscs, a phenomenon called imposex (Smith, 1971; Gibbs et al., 1987). Imposex can result in total failure of reproduction and is believed to account for the extinction of dogwhelk (Nucella lapillus) in severely contaminated areas (Bryan et al., 1986). Despite indications that TBT pollution is global (Ellis & Pattisina, 1990), very little has been reported on TBT levels and their effects at higher latitudes. Brick & Bolte (1994) recently found imposex in Buccinum undatum (L.) in Svalbard, but without detecting any TBT. The finding of imposex in dogwhelk in Icelandic waters (Svavarsson & Skarph66insd6ttir, 1995) indicates that TBT pollution may be widespread even in fairly remote, northern waters. Here we present an analysis of the 358
seasonal fluctuations in body burden of TBT in dogwhelk (N. lapillus) and blue mussel (M. edulis) from Icelandic sites.
Materials and Methods Dogwhelks and blue mussels were coUected several times during a year (July 1993-June 1994) at a single intertidal locality, Laugarnes, near Reykjavik harbour, Iceland (64°09'32"N, 21°52'90"W), where an analysis of imposex in the dogwhelk had previously indicated severe pollution (locality 16 in Svavarsson & Skarphr8insd6ttir, 1995). Dogwhelks were collected 11 times, and TBT and DBT measured in dogwhelks from eight sampling occasions. The frequency and the degree of imposex (development of vas deferens and penis on the female, VDS index, see Gibbs et al., 1987) was determined in dogwhelks from five sampling occasions. This index is 0 for normal individuals, at stage 1 a proximal section of vas deferens is formed on the females, stage 2 involves initiation of penis development and further development of vas deferens, stage 3 is characterized by formation of a small penis and development of the distal section of the vas deferens, stage 4 involves mainly fusion of the vas deferens, stage 5 involves overgrowth of vas deferens on the genital papilla and hence female sterility, and in stage 6 aborted capsules can be seen in the capsule gland (Gibbs et al., 1987). The length of the penis was measured on the females. Blue mussels were collected eight times during one year, and TBT and DBT analysed from all sampling occasions. Prior to analysis the length of the molluscs was measured and the molluscs stored frozen until tissue samples were analysed. Dogwhelks were also collected intertidally from September to November 1992 and from June to August 1993 at 35 sites in south-western, western and north-western Iceland (for locations, see Svavarsson & Skarph68insd6ttir, 1995). Blue mussels were collected at
Volume 32/Number4/April 1996 17 of those sites. Their length was measured and these were then stored frozen until tissue samples were analysed. The method of Hannah et al. (1989) was used for determining TBT and its biodegradation product DBT, with some modification. The following chemicals were used: tributyltinchlor±de (96%), dibutyltinchloride (96%), tetrabutyltin (TeBT, 93%), and tributyltinhydride. These were obtained from Aldrich Chemical Co., WI, USA, and 1,2,3,4-tetrachloronaphthalene (TCN) was obtained from Promochem, Wesel, Germany. Organotin solutions were prepared at concentrations of approximately 200 mg 1- ~ in hexane. TCN was prepared at 1 mg 1- i in hexane. Silica Sep-pak cartridges (Waters, Mill±pore Co., MA, USA) were prepared by eluting 1 ml of HC1 (conc.) followed by 20 ml of hexane through each cartridge just prior to use. Fresh solutions of 4% (w/v) sodium tetraborohydride (Merck) were prepared just prior to use in deionized water. All solvents were pro analysi from Merck, Germany. Frozen molluscs were thawed, deshelled, cut to pieces, mixed and weighted (5--10 g) into duplicate glass centrifuge tubes. The samples were homogenized in 7.5 ml of saturated NaCI (aq.) with a stainless steel Ultra Turrax homogenizer (Janke and Kunkel, IKA, Germany), and diluted with 7.5 ml HCI (1:1, aq.). Then 200 ng of TeBT, the recovery standard, was added, and the samples extracted by thorough mixing with 14 ml of hexane-diethylether (9:1) every 10 min for 1 h. After centrifugation the organic phases were transferred to separate glass centrifugation tubes and the homogenates washed once with 5 ml hexane-diethylether (9:1). The combined organic layers were concentrated under Nzgas at room temperature to approximately 3 ml. This extract was eluted through the acid-prepared silica Seppak cartridges followed by 8 ml of hexane-diethylether (9:1) into 2 ml of saturated NaC1 (aq.). The organic part of the eluate was concentrated under Nz to 1 ml, and then organotinchlorides were reduced to respective hydrides with 1 ml 4% NaBH4 (aq.) and the solution vortex-mixed every 10 min for 30 min. At this point a mixed standard of TBT, DBT and TeBT was prepared by combining 10 ~tg of each in 1 ml of hexane and reducing the respective chlorides to hydrides with NaBH4 as with the samples. After the addition of 4 ml of saturated NaC1 (aq.) and 1 ml of hexane, the samples and standard were centrifuged and the hexane layers transferred to separate tubes. Na2SO4 (1 g) was added, the samples mixed and centrifuged and the liquid transferred and combined with 100 ng of TCN, the instrument standard. Finally, the samples were concentrated under N2 to approximately 200 ~tl, being very careful not to dry them any'.further as that would cause losses of hydrides. Appropriate dilutions of the standard in TCN for a standard curve were made and run with the samples. The samples and standards (I l~l) were analysed by GC-MSD. An HP5890 GC with an Ultra-2 (25 m, 0.20 mm i.d., 0.33 Ixm film thickness) capillary column and an HP5970 MSD were used. Carrier gas was He (25 cm s-1), splitless injection of 1 min, injector temp. 270°C,
MSD interphase 280°C. Temperature programme: 40°C for 2 min, 25°C min -1 to ll0°C, 10°C min -1 to 260°C, 20°C min-i to 280°C and hold for 2 min. The limit of quantification was at 0.1 ng g-~. The chemicals were detected by single ion monitoring (121, 123, 177, 179, 235 and 237 amu for the tin compounds and 263.9, 265.9 and 267.9 amu for TCN) and quantified from ion chromatograms of the single most abundant ion for each chemical. Standard curves were prepared by first preparing a standard curve for TeBT and using that to estimate the content of TeBT in the mixed standard, giving the dilution error in the mixed standard to correct the concentrations of TBT and DBT. The recovery of TeBT, TBT and DBT was determined by fortifying 10 g of blue mussel from an uncontaminated site (Hvalfj6rrur) with 200 ng of TeBT (8 samples), TBT (5 samples) and DBT (3 samples). The recovery was 66+__4%, 27___2% and 53__6%, respectively (mean_S.D.). The recovery of TeBT, which was added to all samples in the extraction process, was used to correct the recovery of DBT and TBT, assuming the latter to be half the recovery of TeBT and the recovery of DBT the same as TeBT. Duplicate samples were analysed for each datapoint and their average is presented in the figures.
Results Considerable seasonal fluctuations in TBT levels were observed in both dogwhelk (N. lapillus) and blue mussel (M. edulis) at Laugarnes. The dogwhelk had a maximum in TBT levels in late summer and in autumn, but declining amounts were found toward a minimum in February-April (Fig. l(a)). Low DBT concentrations were found at all times, but no seasonal pattern was observed (Fig. l(b)). Imposex frequency was 100% during the year. The mean penis length of the female dogwhelks did not vary significantly (p>0.05; student t-test) during the year and was always around 5 mm (4.6-5.5) (Fig. l(c)). The mean VDS index was always close to 5 (4.4-4.9). In the blue mussels at Laugarnes TBT levels were high in July-January, but low in February-April (Fig. 2(a)). The DBT levels, which were always low, did not show any clear seasonal pattern (Fig. 2(b)), but the lowest values were seen in March and April. In the survey of TBT contamination in south-western, western and north-western Iceland body burdens were usually fairly low in both dogwhelk (N. lapillus: mean=8.5 ng TBT g - i wet wt ±8.3 S.D.; n=31, samples from June to November) and blue mussel (M. edulis: mean=14.7 ng TBT g-~ wet wt ±13.2 S.D.; n = 14; samples from June and August) in areas distant from Reykjavik harbour. At localities quite near Reykjavik harbour (excluding seasonal study site), concentrations were considerably higher (N. lapillus: mean=61.9 ng TBT g - i wet wt +33.8 S.D.; n=4, samples from September and November; M. edulis: mean=122.6 ng TBT g - i wet wt +92.5 S.D.; n=3, samples from November). These differences were significant for the dogwhelk (p<0.05; students t-test), but not for the blue mussel (p > 0.05; students t-test). 359
Marine Pollution Bulletin
60
Discussion
A
50
Seasonal changes have frequently been observed in TBT levels of the water column (Hall et al., 1987; Evans & Huggett, 1991; Oehlmann et al., 1993) and have been "= 30 , + t related to seasonal changes in boating activity in the ÷ respective areas. Page & Widdows (1991) found , 10 temporal variations of TBT levels in M . edulis in the I I I I I I I I I ' ' Lynher River, UK, and related this to seasonal input from recreational boat launching in spring and early summer, and a reduced flushing of the estuary in the 7 B spring and summer. 6 In this study, pronounced seasonal changes were 5 observed in TBT concentrations in both dogwhelk and ~4 blue mussel tissues near to Reykjavik harbour. There is, however, negligible seasonal variation in ship traffic in ~" 2 Reykjavik harbour, as pleasure crafts are relatively rare in comparison with other places. We therefore relate I I I I I I I I I I I these fluctuations to seasonal feeding and resting activity of the dogwhelk and feeding activity of the 7 C blue mussel. "-'6 Maximum TBT concentrations in dogwhelk tissue E E5 were seen during the late summer, while minimum concentrations were observed in late winter/early ~3 spring. This coincides fairly well with the water "~. 2 temperature, which is generally highest in August 1 ( ~ 1I°C), but lowest in February ( ~ I°C) (Gunnarsson I I I I I I I I I I & Ing61fsson, 1995). The dogwhelks aggregate in clefts Jan Feb Mar Apr May June July Aug Sep Oct Nov or under rocks during the coldest period in winter (Feare, 1971). Near Reykjavik they migrate into Month Fig. 1 Seasonal fluctuations in body burden of TBT (A) and DBT (B) aggregation sites in late October or early November, when the temperature is falling, and do not emerge until (rig g - i tissue wet wt +S.E.) in Nucella lapillus at Laugarnes, near Reykjavik harbour. Each datapoint is based on early April (Ing61fsson, A., pers. comm.). During that measurement of two samples, except for the April sample. period the individuals remain more or less immobile and (C) Female penis length (ram, +S.D.; n= 14-18) for Nucella do not feed for about 5 months. lapillus during 1 year. Despite seasonal changes in the body burdens, the mean penis length of the females and the VDS index of the dogwhelk did not change over the year. This is to be expected as the imposex of the gastropods is probably 80 A irreversible (Gibbs et al., 1987). It is thus not possible to 70 relate the degree of imposex to the levels of TBT found 60 in the gastropods. ~ 5o t Low levels of TBT were observed in blue mussel "~ 40 tissue in February-April, but high levels from July~ 3o January. Phytoplankton levels in the water in nearby 20 Hvalfj6rrur were at a minimum in November-March, 10 I I I I I I 1 I i 0 ' ' started to increase in early April and were high in late April and early May (Th6rarinsd6ttir & Antonsson, 1993). Consequently the blue mussels grow only slowly 7 B from October to March. Hence low levels of TBT are 6 ÷ found in the mussels during the coldest time of the year, ,~ 5 and some time after phytoplankton has reached low ~4 levels in the water column, The results indicate that both the blue mussel and " 2 0 the dogwhelk depurate themselves annually. The large l drop in body burden of TBT in the blue mussel I I I I I I I I I I I 0 occurred from January to February. Experimental Jan Feb Mar Apr May June July Aug Sept Oct Nov studies have shown that blue mussel can depurate Month quickly. Zuolian & Jensen (1989) observed that 50% Fig. 2 Seasonal fluctuations in body burden of TBT (a) and DBT Co) of organic tin was depurated after 40 days in clean (ng g - i tissue wet wt :t:S.E.) in Mytilus edulis at Laugarnes, seawater, while Laughlin et al. (1986) found depuranear Reykjavik harbour. Each datapoint is based on tion of 50% of tissue burdens after 14 days. There is, measurement of two samples, except for TBT in July. 40
+I
t
tt
+
t
'+t
360
Volume 32/Number 4/April 1996 h o w e v e r , n o d a t a o n the rate o f d e p u r a t i o n at different seasons. The seasonal fluctuation in body burden of TBT implies that the timing of monitoring TBT may be critical. O u r r e s u l t s i n d i c a t e t h a t the a p p r o p r i a t e t i m e for s a m p l i n g m o l l u s c s for t h e e s t i m a t i o n o f m a x i m u m
burdens is late in the summer or early in autumn at higher latitudes.
We wish to thank M. Amacker and B. P/dsson for their help with collecting dogwhelks, and A. Ing61fsson for his valuable comments. This study was supported by the Ministry for the Environment, Iceland. HaUd6ra Skarph66insd6ttir was guest scientist at the Department of Pharmacology during the progress of this work.
Alzieu, C. (1991). Environmental problems caused by TBT in France: assessment, regulations, prospects. Mar. Environ. Res. 32, 7-17. Brick, M. & BoRe, M. (1994). Cytology of the outer penis epithelium of Buccinum undatum (L.) from the arctic region--an observation of the imposex phenomenon. Helgol. Meeresunters. 411, 123-131. Bryan, G. W., Gibbs, P. E , Hummerstone, L. G. & Burt, G. R. (1986). The decline of the gastropod Nucella lapillus around southwest England: evidence for the effect of tributyltin from antifouling paints. J. Mar. BioL Ass. UK 66, 611-640. Ellis, D. V. & Pattisina, L. A. (1990). Widespread neogastropod imposex: a biological indicator of global TBT contamination? Mar. Pollut. Bull. 21, 248-253. Evans, D. A. & Huggett, R. J. (1991). Statistical modeling of intensive TBT monitoring data in two tidal creeks of the Chesapeake Bay. Mar. Environ. Res. 32, 169-186.
Feare, C. J. (1971). The adaptive significance of aggregation behaviour in the dogwhelk Nucella lapillus (L.). Oecologia 7, 117-126. Gibbs, P. E., Bryan, G. W., Pascoe, P. L. & Burt, G. R. (1987). The use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin (TBT) contamination. J. Mar. Biol. Ass. UK 67, 507-523. Gunuarsson, K. & Ingrlfsson, A. (1995). Seasonal changes in the abundance of intertidal algae in southwestern Iceland. Bot. Mar. 38, 69-77. Hall, L. W. Jr., Lenkevich, M. J., Hall, W. S., Pinkney, A. E. & Bushong, S. J. (1987). Evaluation of butyltin compounds in Maryland waters of Chesapeake Bay. Mar. Pollut. Bull. 18, 7883. Hannah, D. J., Page, T. L., Pickston, L. & Taucher, J. A. (1989). Analysis of tributylin compounds in shellfish by using gas chromatography-mass spectrometry. Bull. Environ. Contam. Toxicol. 43, 22-27. Laughlin, R. B., French, W. & Guard, H. E. (1986). Accumulation of bis(tributyltin)oxide by the marine mussel Mytilus edulis. Environ. Sci. Technol. 20, 884-890. Oehlmann, J., Stroben, E. & Fioroni, P. (1993). Frrquence et degr6 d'expression du pseudohermaphrodisme chez quelques Prosobranches Strnoglosses des c6tes fran~aises (surtout de la baie de Morlaix et de la Manche). 2. Situation jusqu'au printemps de 1992. Cah. Biol. Mar. 34, 343-362. Page, D. S. & Widdows, J. (1991). Temporal and spatial variation in levels of alkyltins in mussel tissues: a toxicological interpretation of field data. Mar. Environ. Res. 32, 113-129. Smith, B. S. (1971). Sexuality in the American mud snail, Nassarius obsoletus Say. Proc. Malac. Soc. Lond. 39, 377-378. Svavarsson, J. & Skarph66insd6ttir, H. (1995). Imposex in the dogwhelk Nucella lapillus (L.) in Icelandic waters. Sarsia 80, 35-40. Th6rarinsd6ttir, G. G. & Antonsson, U. (1993). Experimental culturing of mussel in Hval0/Srdur. Ntitt~rufrcefJingurinn 63, 243251. (In Icelandic). Zuolian, C. & Jensen, A. (1989). Accumulation of organic and inorganic tin in blue mussel, Mytilus edulis, under natural conditions. Mar. Pollut Bull. 20, 281-286.
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0025-326X(95)00204-9
Marine Pollution Bulletin, Vol. 32, No. 4, pp. 358-361, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0025-326X/96 $15.00 + 0.00
Seasonal Fluctuations of Tributyltin (TBT) and Dibutyltin (DBT) in the Dogwhelk, Nucella lapillus (L.), and the Blue Mussel, Mytilus edulis L., in Icelandic Waters H A L L D O R A SKARPHI~DINSDrTTIR*, KRISTiN 6 L A F S D r T T I R t , J(SRUNDUR SVAVARSSON* and TORKELL JOHANNESSON*
*Institute of Biology, University of Iceland, Grensdtsvegur 12, 108 ReykjavlTc, Iceland "fDepartment of Pharmacology, University of Iceland, Arm~li 30, 108 Reykjavik, Iceland
Seasonal levels of tributyltin (TBT) and dibutyltin (DBT) were analysed in the dogwhelk (NuceUa lapUlus) and the blue mussel (Mytilus edulis) from south-western Iceland. Seasonal fluctuations of TBT were observed in both dogwhelk and blue mussel, with summer levels being approximately five (dogwhelk) to ten (blue mussel) times the winter levels. These changes were related to seasonal activity in feeding and resting of the dogwhelk and feeding of the blue mussel, while seasonal changes in shipping activity were insignificant. This indicates that maximum levels of TBT pollution should mainly be monitored in these animals at high latitudes during late summer or early autumn. Copyright © 1996 Elsevier Science Ltd
Tributyltin (TBT) is a toxic compound that has been used as an antifouling agent in marine paints. A number of studies have demonstrated the deleterious effects of TBT on non-target marine organisms, even at very low concentrations. These include effects on oysters, in which the shell-forming mechanism is disturbed and reproduction is reduced (Alzieu, 1991). TBT also induces male characteristics, i.e. penis (pseudopenis) and vas deferens development on female gonochoristic gastropod molluscs, a phenomenon called imposex (Smith, 1971; Gibbs et al., 1987). Imposex can result in total failure of reproduction and is believed to account for the extinction of dogwhelk (Nucella lapillus) in severely contaminated areas (Bryan et al., 1986). Despite indications that TBT pollution is global (Ellis & Pattisina, 1990), very little has been reported on TBT levels and their effects at higher latitudes. Brick & Bolte (1994) recently found imposex in Buccinum undatum (L.) in Svalbard, but without detecting any TBT. The finding of imposex in dogwhelk in Icelandic waters (Svavarsson & Skarph66insd6ttir, 1995) indicates that TBT pollution may be widespread even in fairly remote, northern waters. Here we present an analysis of the 358
seasonal fluctuations in body burden of TBT in dogwhelk (N. lapillus) and blue mussel (M. edulis) from Icelandic sites.
Materials and Methods Dogwhelks and blue mussels were coUected several times during a year (July 1993-June 1994) at a single intertidal locality, Laugarnes, near Reykjavik harbour, Iceland (64°09'32"N, 21°52'90"W), where an analysis of imposex in the dogwhelk had previously indicated severe pollution (locality 16 in Svavarsson & Skarphr8insd6ttir, 1995). Dogwhelks were collected 11 times, and TBT and DBT measured in dogwhelks from eight sampling occasions. The frequency and the degree of imposex (development of vas deferens and penis on the female, VDS index, see Gibbs et al., 1987) was determined in dogwhelks from five sampling occasions. This index is 0 for normal individuals, at stage 1 a proximal section of vas deferens is formed on the females, stage 2 involves initiation of penis development and further development of vas deferens, stage 3 is characterized by formation of a small penis and development of the distal section of the vas deferens, stage 4 involves mainly fusion of the vas deferens, stage 5 involves overgrowth of vas deferens on the genital papilla and hence female sterility, and in stage 6 aborted capsules can be seen in the capsule gland (Gibbs et al., 1987). The length of the penis was measured on the females. Blue mussels were collected eight times during one year, and TBT and DBT analysed from all sampling occasions. Prior to analysis the length of the molluscs was measured and the molluscs stored frozen until tissue samples were analysed. Dogwhelks were also collected intertidally from September to November 1992 and from June to August 1993 at 35 sites in south-western, western and north-western Iceland (for locations, see Svavarsson & Skarph68insd6ttir, 1995). Blue mussels were collected at
Volume 32/Number4/April 1996 17 of those sites. Their length was measured and these were then stored frozen until tissue samples were analysed. The method of Hannah et al. (1989) was used for determining TBT and its biodegradation product DBT, with some modification. The following chemicals were used: tributyltinchlor±de (96%), dibutyltinchloride (96%), tetrabutyltin (TeBT, 93%), and tributyltinhydride. These were obtained from Aldrich Chemical Co., WI, USA, and 1,2,3,4-tetrachloronaphthalene (TCN) was obtained from Promochem, Wesel, Germany. Organotin solutions were prepared at concentrations of approximately 200 mg 1- ~ in hexane. TCN was prepared at 1 mg 1- i in hexane. Silica Sep-pak cartridges (Waters, Mill±pore Co., MA, USA) were prepared by eluting 1 ml of HC1 (conc.) followed by 20 ml of hexane through each cartridge just prior to use. Fresh solutions of 4% (w/v) sodium tetraborohydride (Merck) were prepared just prior to use in deionized water. All solvents were pro analysi from Merck, Germany. Frozen molluscs were thawed, deshelled, cut to pieces, mixed and weighted (5--10 g) into duplicate glass centrifuge tubes. The samples were homogenized in 7.5 ml of saturated NaCI (aq.) with a stainless steel Ultra Turrax homogenizer (Janke and Kunkel, IKA, Germany), and diluted with 7.5 ml HCI (1:1, aq.). Then 200 ng of TeBT, the recovery standard, was added, and the samples extracted by thorough mixing with 14 ml of hexane-diethylether (9:1) every 10 min for 1 h. After centrifugation the organic phases were transferred to separate glass centrifugation tubes and the homogenates washed once with 5 ml hexane-diethylether (9:1). The combined organic layers were concentrated under Nzgas at room temperature to approximately 3 ml. This extract was eluted through the acid-prepared silica Seppak cartridges followed by 8 ml of hexane-diethylether (9:1) into 2 ml of saturated NaC1 (aq.). The organic part of the eluate was concentrated under Nz to 1 ml, and then organotinchlorides were reduced to respective hydrides with 1 ml 4% NaBH4 (aq.) and the solution vortex-mixed every 10 min for 30 min. At this point a mixed standard of TBT, DBT and TeBT was prepared by combining 10 ~tg of each in 1 ml of hexane and reducing the respective chlorides to hydrides with NaBH4 as with the samples. After the addition of 4 ml of saturated NaC1 (aq.) and 1 ml of hexane, the samples and standard were centrifuged and the hexane layers transferred to separate tubes. Na2SO4 (1 g) was added, the samples mixed and centrifuged and the liquid transferred and combined with 100 ng of TCN, the instrument standard. Finally, the samples were concentrated under N2 to approximately 200 ~tl, being very careful not to dry them any'.further as that would cause losses of hydrides. Appropriate dilutions of the standard in TCN for a standard curve were made and run with the samples. The samples and standards (I l~l) were analysed by GC-MSD. An HP5890 GC with an Ultra-2 (25 m, 0.20 mm i.d., 0.33 Ixm film thickness) capillary column and an HP5970 MSD were used. Carrier gas was He (25 cm s-1), splitless injection of 1 min, injector temp. 270°C,
MSD interphase 280°C. Temperature programme: 40°C for 2 min, 25°C min -1 to ll0°C, 10°C min -1 to 260°C, 20°C min-i to 280°C and hold for 2 min. The limit of quantification was at 0.1 ng g-~. The chemicals were detected by single ion monitoring (121, 123, 177, 179, 235 and 237 amu for the tin compounds and 263.9, 265.9 and 267.9 amu for TCN) and quantified from ion chromatograms of the single most abundant ion for each chemical. Standard curves were prepared by first preparing a standard curve for TeBT and using that to estimate the content of TeBT in the mixed standard, giving the dilution error in the mixed standard to correct the concentrations of TBT and DBT. The recovery of TeBT, TBT and DBT was determined by fortifying 10 g of blue mussel from an uncontaminated site (Hvalfj6rrur) with 200 ng of TeBT (8 samples), TBT (5 samples) and DBT (3 samples). The recovery was 66+__4%, 27___2% and 53__6%, respectively (mean_S.D.). The recovery of TeBT, which was added to all samples in the extraction process, was used to correct the recovery of DBT and TBT, assuming the latter to be half the recovery of TeBT and the recovery of DBT the same as TeBT. Duplicate samples were analysed for each datapoint and their average is presented in the figures.
Results Considerable seasonal fluctuations in TBT levels were observed in both dogwhelk (N. lapillus) and blue mussel (M. edulis) at Laugarnes. The dogwhelk had a maximum in TBT levels in late summer and in autumn, but declining amounts were found toward a minimum in February-April (Fig. l(a)). Low DBT concentrations were found at all times, but no seasonal pattern was observed (Fig. l(b)). Imposex frequency was 100% during the year. The mean penis length of the female dogwhelks did not vary significantly (p>0.05; student t-test) during the year and was always around 5 mm (4.6-5.5) (Fig. l(c)). The mean VDS index was always close to 5 (4.4-4.9). In the blue mussels at Laugarnes TBT levels were high in July-January, but low in February-April (Fig. 2(a)). The DBT levels, which were always low, did not show any clear seasonal pattern (Fig. 2(b)), but the lowest values were seen in March and April. In the survey of TBT contamination in south-western, western and north-western Iceland body burdens were usually fairly low in both dogwhelk (N. lapillus: mean=8.5 ng TBT g - i wet wt ±8.3 S.D.; n=31, samples from June to November) and blue mussel (M. edulis: mean=14.7 ng TBT g-~ wet wt ±13.2 S.D.; n = 14; samples from June and August) in areas distant from Reykjavik harbour. At localities quite near Reykjavik harbour (excluding seasonal study site), concentrations were considerably higher (N. lapillus: mean=61.9 ng TBT g - i wet wt +33.8 S.D.; n=4, samples from September and November; M. edulis: mean=122.6 ng TBT g - i wet wt +92.5 S.D.; n=3, samples from November). These differences were significant for the dogwhelk (p<0.05; students t-test), but not for the blue mussel (p > 0.05; students t-test). 359
Marine Pollution Bulletin
60
Discussion
A
50
Seasonal changes have frequently been observed in TBT levels of the water column (Hall et al., 1987; Evans & Huggett, 1991; Oehlmann et al., 1993) and have been "= 30 , + t related to seasonal changes in boating activity in the ÷ respective areas. Page & Widdows (1991) found , 10 temporal variations of TBT levels in M . edulis in the I I I I I I I I I ' ' Lynher River, UK, and related this to seasonal input from recreational boat launching in spring and early summer, and a reduced flushing of the estuary in the 7 B spring and summer. 6 In this study, pronounced seasonal changes were 5 observed in TBT concentrations in both dogwhelk and ~4 blue mussel tissues near to Reykjavik harbour. There is, however, negligible seasonal variation in ship traffic in ~" 2 Reykjavik harbour, as pleasure crafts are relatively rare in comparison with other places. We therefore relate I I I I I I I I I I I these fluctuations to seasonal feeding and resting activity of the dogwhelk and feeding activity of the 7 C blue mussel. "-'6 Maximum TBT concentrations in dogwhelk tissue E E5 were seen during the late summer, while minimum concentrations were observed in late winter/early ~3 spring. This coincides fairly well with the water "~. 2 temperature, which is generally highest in August 1 ( ~ 1I°C), but lowest in February ( ~ I°C) (Gunnarsson I I I I I I I I I I & Ing61fsson, 1995). The dogwhelks aggregate in clefts Jan Feb Mar Apr May June July Aug Sep Oct Nov or under rocks during the coldest period in winter (Feare, 1971). Near Reykjavik they migrate into Month Fig. 1 Seasonal fluctuations in body burden of TBT (A) and DBT (B) aggregation sites in late October or early November, when the temperature is falling, and do not emerge until (rig g - i tissue wet wt +S.E.) in Nucella lapillus at Laugarnes, near Reykjavik harbour. Each datapoint is based on early April (Ing61fsson, A., pers. comm.). During that measurement of two samples, except for the April sample. period the individuals remain more or less immobile and (C) Female penis length (ram, +S.D.; n= 14-18) for Nucella do not feed for about 5 months. lapillus during 1 year. Despite seasonal changes in the body burdens, the mean penis length of the females and the VDS index of the dogwhelk did not change over the year. This is to be expected as the imposex of the gastropods is probably 80 A irreversible (Gibbs et al., 1987). It is thus not possible to 70 relate the degree of imposex to the levels of TBT found 60 in the gastropods. ~ 5o t Low levels of TBT were observed in blue mussel "~ 40 tissue in February-April, but high levels from July~ 3o January. Phytoplankton levels in the water in nearby 20 Hvalfj6rrur were at a minimum in November-March, 10 I I I I I I 1 I i 0 ' ' started to increase in early April and were high in late April and early May (Th6rarinsd6ttir & Antonsson, 1993). Consequently the blue mussels grow only slowly 7 B from October to March. Hence low levels of TBT are 6 ÷ found in the mussels during the coldest time of the year, ,~ 5 and some time after phytoplankton has reached low ~4 levels in the water column, The results indicate that both the blue mussel and " 2 0 the dogwhelk depurate themselves annually. The large l drop in body burden of TBT in the blue mussel I I I I I I I I I I I 0 occurred from January to February. Experimental Jan Feb Mar Apr May June July Aug Sept Oct Nov studies have shown that blue mussel can depurate Month quickly. Zuolian & Jensen (1989) observed that 50% Fig. 2 Seasonal fluctuations in body burden of TBT (a) and DBT Co) of organic tin was depurated after 40 days in clean (ng g - i tissue wet wt :t:S.E.) in Mytilus edulis at Laugarnes, seawater, while Laughlin et al. (1986) found depuranear Reykjavik harbour. Each datapoint is based on tion of 50% of tissue burdens after 14 days. There is, measurement of two samples, except for TBT in July. 40
+I
t
tt
+
t
'+t
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Volume 32/Number 4/April 1996 h o w e v e r , n o d a t a o n the rate o f d e p u r a t i o n at different seasons. The seasonal fluctuation in body burden of TBT implies that the timing of monitoring TBT may be critical. O u r r e s u l t s i n d i c a t e t h a t the a p p r o p r i a t e t i m e for s a m p l i n g m o l l u s c s for t h e e s t i m a t i o n o f m a x i m u m
burdens is late in the summer or early in autumn at higher latitudes.
We wish to thank M. Amacker and B. P/dsson for their help with collecting dogwhelks, and A. Ing61fsson for his valuable comments. This study was supported by the Ministry for the Environment, Iceland. HaUd6ra Skarph66insd6ttir was guest scientist at the Department of Pharmacology during the progress of this work.
Alzieu, C. (1991). Environmental problems caused by TBT in France: assessment, regulations, prospects. Mar. Environ. Res. 32, 7-17. Brick, M. & BoRe, M. (1994). Cytology of the outer penis epithelium of Buccinum undatum (L.) from the arctic region--an observation of the imposex phenomenon. Helgol. Meeresunters. 411, 123-131. Bryan, G. W., Gibbs, P. E , Hummerstone, L. G. & Burt, G. R. (1986). The decline of the gastropod Nucella lapillus around southwest England: evidence for the effect of tributyltin from antifouling paints. J. Mar. BioL Ass. UK 66, 611-640. Ellis, D. V. & Pattisina, L. A. (1990). Widespread neogastropod imposex: a biological indicator of global TBT contamination? Mar. Pollut. Bull. 21, 248-253. Evans, D. A. & Huggett, R. J. (1991). Statistical modeling of intensive TBT monitoring data in two tidal creeks of the Chesapeake Bay. Mar. Environ. Res. 32, 169-186.
Feare, C. J. (1971). The adaptive significance of aggregation behaviour in the dogwhelk Nucella lapillus (L.). Oecologia 7, 117-126. Gibbs, P. E., Bryan, G. W., Pascoe, P. L. & Burt, G. R. (1987). The use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin (TBT) contamination. J. Mar. Biol. Ass. UK 67, 507-523. Gunuarsson, K. & Ingrlfsson, A. (1995). Seasonal changes in the abundance of intertidal algae in southwestern Iceland. Bot. Mar. 38, 69-77. Hall, L. W. Jr., Lenkevich, M. J., Hall, W. S., Pinkney, A. E. & Bushong, S. J. (1987). Evaluation of butyltin compounds in Maryland waters of Chesapeake Bay. Mar. Pollut. Bull. 18, 7883. Hannah, D. J., Page, T. L., Pickston, L. & Taucher, J. A. (1989). Analysis of tributylin compounds in shellfish by using gas chromatography-mass spectrometry. Bull. Environ. Contam. Toxicol. 43, 22-27. Laughlin, R. B., French, W. & Guard, H. E. (1986). Accumulation of bis(tributyltin)oxide by the marine mussel Mytilus edulis. Environ. Sci. Technol. 20, 884-890. Oehlmann, J., Stroben, E. & Fioroni, P. (1993). Frrquence et degr6 d'expression du pseudohermaphrodisme chez quelques Prosobranches Strnoglosses des c6tes fran~aises (surtout de la baie de Morlaix et de la Manche). 2. Situation jusqu'au printemps de 1992. Cah. Biol. Mar. 34, 343-362. Page, D. S. & Widdows, J. (1991). Temporal and spatial variation in levels of alkyltins in mussel tissues: a toxicological interpretation of field data. Mar. Environ. Res. 32, 113-129. Smith, B. S. (1971). Sexuality in the American mud snail, Nassarius obsoletus Say. Proc. Malac. Soc. Lond. 39, 377-378. Svavarsson, J. & Skarph66insd6ttir, H. (1995). Imposex in the dogwhelk Nucella lapillus (L.) in Icelandic waters. Sarsia 80, 35-40. Th6rarinsd6ttir, G. G. & Antonsson, U. (1993). Experimental culturing of mussel in Hval0/Srdur. Ntitt~rufrcefJingurinn 63, 243251. (In Icelandic). Zuolian, C. & Jensen, A. (1989). Accumulation of organic and inorganic tin in blue mussel, Mytilus edulis, under natural conditions. Mar. Pollut Bull. 20, 281-286.
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