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Toxicity of tributyltin in the marine mollusc Mytilus edulis
Marine Pollution Bulletin 51 (2005) 811–816 www.elsevier.com/locate/marpolbul
Toxicity of tributyltin in the marine mollusc Mytilus edulis Josephine A. Hagger a
a,*
, Michael H. Depledge b, Tamara S. Galloway
a
Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth, Devon PL4 8AA, UK b Environment Agency, Rio House, Waterside Drive, Aztec West, Almondsbury, Bristol BS12 4UD, UK
Abstract Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 lg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 lg/l of TBTO (P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei (P = 0.0005; R2 = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis. 2005 Elsevier Ltd. All rights reserved. Keywords: Tributyltin; Genotoxicity; Immunotoxicity; Cytotoxicity; Mytilus edulis; Endocrine disruption; Biomarker
1. Introduction Tributyltin (TBT) is a biocide and catalyst used worldwide (Oberdo¨rster et al., 1998). TBT compounds have particularly been used as biocides in antifouling paints and wood preservatives (Bettin et al., 1996). Leachate of these compounds has contaminated both marine and freshwater habitats and it has been considered to be one of the most toxic agents entering the environment (Goldberg, 1986). TBT has been demonstrated to cause impairments in growth, development, reproduction and survival of many marine species (Heard et al., 1986). Of particular concern has been the induction of reproductive abnormalities and sterilisation of female marine prosobranch snails caused by tributyltin based compounds. This phenomenon known as imposex
*
Corresponding author. Tel.: +44 1752 232797; fax: +44 1752 232970. E-mail address: [email protected] (J.A. Hagger). 0025-326X/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.06.044
is characterised by the development of male sex organs, notably a penis and sperm duct (vas deferen), by the female gastropods (Gibbs et al., 1991). An increase in the severity of imposex in gastropods ultimately leads to reproductive failure as the pallial oviduct becomes blocked leading to sterilisation and premature death of the female snails (Gibbs and Bryan, 1986). Population levels of these gastropod molluscs have shown a marked decline in areas of high TBT contamination and a high degree of imposex (Bryan et al., 1986). As well as being an endocrine disrupting agent TBT has proven to be extremely toxic to a number of aquatic organisms, in particular to sensitive early life stages (Thain and Waldock, 1986; Fent, 1996; Lignot et al., 1998). Our recent studies suggest that TBT can induce cytogenetic damage in embryo-larvae of the marine mollusc Mytilus edulis and the polychaete worm Platynereis dumerilii (Jha et al., 2000; Hagger et al., 2002). However relatively little work has been carried out on the genotoxic potential of tributyltin to adult marine organisms. Thus the purpose of this study was to investigate the toxicological potential
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of the endocrine disrupting chemical, tributyltin, by investigating cytotoxic, immunological and genotoxic effects in the adult life stages of the marine mollusc M. edulis. Cytotoxicity was measured using the neutral red assay that measures lysosomal stability in viable cells. Phagocytosis was measured as an indication of immunotoxicity as it is a non-specific defence mechanism. In addition, the ferric reducing ability of plasma (FRAP) assay, a measure of the antioxidant capacity, was deduced. Genotoxicity was determined using micronucleus formation and the comet assay to detect single strand DNA breaks.
2. Methods and materials Mussels were collected from Whitsand Bay, Cornwall, UK. All fauna and flora were removed from the mussels, which were then left for 2 weeks to depurate. Eight mussels per concentration (4 per replica) were placed into 1 l of clean, filtered, aerated seawater (36&, 15 ± 1 C). Mussels were exposed to 0.1, 0.5, 1 and 5 lg/l tributyltin oxide (TBTO) obtained from Lancaster Synthesis UK (1 lg/l TBTO 0.973 lg/l of TBT), in addition to a solvent and seawater control. Water was renewed and re-dosed every day for 7 days and organotin concentrations were measured at the beginning and end of the exposure period. Following the 1-week exposure to TBTO, haemolymph was extracted from the posterior adductor muscle and gill tissue was removed to determine the toxicological effects using the following biomarkers. 2.1. Neutral red assay The neutral red assay was performed as described by Coles et al. (1995) and Pipe et al. (1999). In brief 50 ll of haemolymph was pipetted into duplicate wells of a microtitre plate. The plate was left for 45 min after which the excess solution was thrown away. Two hundred microlitres of 0.33% neutral red was pipetted into each well and left for 3 h, after which time the neutral red solution was removed. The cells were carefully washed and 200 ll of 1% acetic acid in 50% ethanol were added to all the wells and the plate was read at 550 nm. The results were presented as the optical density/mg protein. Protein concentration was determined following the method of Bradford (1976). 2.2. Phagocytosis The phagocytosis assay was performed as described by Coles et al. (1995) and Pipe et al. (1999). In brief 50 ll of haemolymph was pipetted in duplicate wells of a microtitre plate. The plate was left for 60 min until the cells had adhered. Ten minutes before the end of
the designated time 100 ll of bakers formol calcium was added to the negative control wells. After the 60 min, excess solution was tipped away and 50 ll of dyed zymosan suspension (50 · 107) was added to every well including two blanks containing 50 ll of physiological saline. The plate was incubated for 30 min at 10 C after which time the reaction was stopped, by adding 100 ll of bakers formol calcium to all the wells for 10 min. The plate was spun at 60 g (700 rpm) for 5 min, the supernatant pipetted off and the cells washed several times. One hundred microlitre of standards containing 1.56, 3.125, 6.25, 12.5, 25 · 107 zymosan particles were added in duplicate to the plate. Two hundred microlitre of 1% acetic acid in 50% ethanol were added to all the wells and then read at 550 nm. Results were presented as the zymosan particles/mg protein (Coles et al., 1995; Pipe et al., 1999). Protein concentration was determined following the method of Bradford (1976). 2.3. Ferric reducing ability of plasma (FRAP) assay The FRAP assay was carried out as described by Benzie and Strain (1996). In brief 200 ll of gill suspension was centrifuged for 10 min at 10,000·g. Fifty microlitres of supernatant in duplicate was pipetted into microtitre plate wells. Aqueous solutions of know FeII concentrations in the range of 100–500lmol/l, were used for calibration. 200 ll of reagent (300 mM acetate buffer, TBTZ (2,4,6-tripyridyl-s-triazine) and 20 mM iron chloride in a ratio on 10:1:1) was placed into each well. The plate was incubated at 25 C for 10 min and read at 593 nm. The change in absorbency between time 0 and 10 min was used as an indicator of the amount of antioxidant activity. 2.4. Comet assay The comet assay was adapted from a protocol described by Singh et al. (1988). Slides were pre-coated in high melting point (HMP) agarose. One hundred microlitres of haemolymph was placed into 200 ll of 4 C physiological saline (pH 7.36). The cell suspension was centrifuged at 200·g at 4 C for 2–3 min and the supernatant was removed. Eighty five microlitre of low melting point (LMP) agarose was pipetted on to the cell pellet and then placed onto the HMP agarose covered slides. After the gel had set the slides were placed into a freshly prepared lysing solution at 4 C for 1 h (2.5 M NaCl, 100 mM Na2–EDTA, 10 mM Tris, 1% Na sarconisate, pH 10.0, 1% Triton X-100, 10% DMSO). The slides were then removed from the lysis solution and placed in a horizontal gel electrophoresis tank. The tank was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM Na2–EDTA) and the DNA was allowed to unwind for 40 min before electrophoresis,
which was carried out at 25 V for 30 min. After electrophoresis the slides were washed in neutralising buffer (0.4 M Tris, pH 7.5). The slides were stained with 20 ll of 5lg/ml ethidium bromide solution, viewed under ultraviolet fluorescence light and scored using the KometTM software, Kinetic Imaging Ltd. A total of 100 randomly chosen cells were scored per each individual, 50 per replicate. 2.5. Micronucleus assay (MN)
OD of neutral red per mg protein
J.A. Hagger et al. / Marine Pollution Bulletin 51 (2005) 811–816
813
4.5
*
4.0
*
*
1
5
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
2.6. TBT analysis
Control Solvent
3. Results The data for the neutral red retention was not normally distributed and therefore the non-parameter Kruskal Wallis statistical test was applied. There was a statistically significant difference between the viability of the cells (P = 0.0003) as shown in Fig. 1. There was a statistical increase in cytotoxicity at the 0.5, 1 and 5 lg/l of TBTO in comparison with the controls. In addition 0.1 was significantly different from 1 and 5, and 0.5 was significantly different from 5. Fig. 2 indicates that there was no significant change in phagocytotic activity of haemocytes from mussels
0.5
Fig. 1. Cell viability as measure by the uptake of neutral red (OD mg of protein) by haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean) (* = significant difference from the control).
60 50 40 30 20 10 0 Control Solvent
Tributyltin was determined using a HPLC method described by Kleibo¨hmer and Cammann (1989). In brief samples were passed through a C18 Sep-Pak cartridge and the organotin compounds were eluted off with methanol. HPLC conditions were 1.0 ml/min of 75:25 methanol:water, passed through a 250 mm · 4, 5SA Nucleosil column. A 100 ll aliquot of sample was injected into the column and the organotin compounds detected by fluorescence detection with an excitation wavelength of 392 nm and an emission wavelength of 555 nm.
0.1
Concentration TBTO µg/L
Zymosan particles per mg protein
Approximately 300–500 ll of haemolymph was placed on slides that had been pre-coated in 10% polyL-lysine solution. Slides were placed into a humidifier for 30 min to allow the cells to adhere to the slide, after this time the excess solution was removed. The slides were left to air dry and then fixed in methanol for 15 min, followed by staining in 5% giemsa/buffer solution for 20 min. Slides were scored blind under ·40 and MN validated under oil immersion. A total of 1000 cells were analysed per mussel. The MN were identified according to the following criteria: (1) diameter smaller than one-third of the main nucleus but greater than one-tenth, (2) no contact with nucleus (absence of chromatid bridge), (3) colour and texture resembling the nucleus, (4) spherical cytoplasmic inclusions with sharp contour (Countryman and Heddle, 1976).
0.5
1
5
10
Concentration of TBTO µg/L Fig. 2. Phagocytosis of zymosan by haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean).
exposure to TBTO (P = 0.667). Although the variation between individual mussels did seem to increase with increasing TBTO concentrations, particularly at 5 lg/l. There was also no statistically significant difference in antioxidant status between any of the mussels exposed to TBTO and those of the controls (P = 0.926). Fig. 3 shows the percentage of DNA damage that occurred in haemocytes of M. edulis exposed to TBTO. There was a statistically significant difference in the percentage of DNA damage present in the comet tail between the controls and 0.5, 1 and 5 lg/l of TBTO (P = 0.0005). There was no significant difference between 0.1 lg/l and the control. Fig. 4 indicates an increase in micronuclei with increasing concentrations of TBTO (P = 0.00005). All the concentrations of TBTO produced an increase in the induction of MN when compared to the controls and there was a dose dependent increase in the levels of MN with increasing concentration of TBTO. In addition there was a strong correlation between the percentage of strand break DNA damage detected using the
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Percentage of DNA in tail
30
*
*
Table 1 Mean and standard deviation concentrations of tributyltin of replicate water samples before and after the exposure period following renewal and re-dosing every 24 h
*
25 20
Concentration (lg/l)
Before exposure
After exposure
15
Seawater control Solvent control 0.1 0.5 1 5
ND ND 0.09 ± 0.05 0.41 ± 0.13 1.09 ± 0.09 4.65 ± 0.63
ND ND 0.04 ± 0.02 0.30 ± 0.06 0.54 ± 0.27 2.87 ± 0.99
10 5 0 0.1
Control Solvent
0.5
1
ND = not detectable. Limits of detection 0.01 lg/l.
5
Concentration TBTO µg/L Fig. 3. Percentage of DNA damage in haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean) (* = significant difference from the control).
12
*
MN per1000 cells
10
*
*
8 6
*
4 2 0 0.1
Control Solvent
0.5
1
5
Concentration of TBTO µg/L
Percentage of DNA in comet tail
Fig. 4. Induction of micronuclei (MN) in haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean) (* = significant difference from the control).
30 25
y = 4.03 + 1.58x R2 = 0.6148
20 15 10 5 0 0
2
4
6
8
10
12
Micronuclei Fig. 5. Linear regression analysis illustrating the correlation between strand breaks and micronuclei in haemocytes of individual mussels exposed to different concentrations of TBTO for 7 days.
comet assay and formation of micronuclei as indicated in Fig. 5 (P = 0.0005; R2 = 61.5%). Table 1 indicates the concentrations of tributyltin in water samples before and after the exposure period.
4. Discussion This study, designed to provide an integrated assessment of the toxicological impact of TBTO to adult bivalves has revealed a significant level of genotoxic damage to adult marine molluscs at environmentally realistic concentrations. This is surprising, as despite its documented toxicity to reproductive processes and immune and endocrine systems, TBTO has not been widely documented as a genotoxin in mammalian test systems (Davis et al., 1987). During the present study there was a decrease in the viability of haemocytes in mussels exposed to TBTO. Within the haemocytes, lysosomes are involved in sequestering and metabolising natural toxins and it has been shown that organic xenobiotics and metals can cause destabilisation of their membrane (Lowe and Pipe, 1994). Accumulated butyltins have been shown to adversely effect lysosomal integrity as measured using the neutral red assay in the 6-armed seastar Leptasterias polaris (Be´kri and Pelletier, 2004). In addition Matozzo et al. (2002) showed that lysosomal activity was significant reduced at 0.05 lM TBT in the marine worm Sipunclus nudus and that the percentage of coelomocytes with ingested neutral red dye was also inhibited at 1 lM. The endpoint used to assess immunotoxicity in this study, phagocytosis, showed no alteration in response to TBTO. It has previously been reported that sublethal doses of TBT can cause phagocytes to lose their ability to move towards and ingest foreign particles (Cima et al., 2002). Phagocytosis was also shown to decrease in amoebocytes of the 6-armed seastar L. polaris after exposure to TBT (Be´kri and Pelletier, 2004). St Jean et al. (2002) found that phagocytic activity was reduced in haemocytes of M. edulis by TBT at concentrations >10 ng/l Sn. Whilst Smith et al. (2000) found that similar concentrations of TBT caused significant inhibition of haemocyte effector functions including spontaneous cytotoxicity of allogeneic cells and the production of free radicals including nitric oxide. These differences in response may reflect variability in the pre-existing immune status of individual organisms.
J.A. Hagger et al. / Marine Pollution Bulletin 51 (2005) 811–816
Both the comet assay and the micronucleus test showed statistically significant increases in DNA damage in haemocytes from mussels exposed to TBTO. Ferraro et al. (2004) demonstrated that TBT produced mutagenic effects with an increase in DNA damage in the fish Hoplias malabaricus using the comet assay. Micic et al. (2002) found that TBT induced DNA fragmentation in human cells and gill cells of the mussel Mytilus galloprovincialis (Micic et al., 2002). However they did not find any correction between DNA strand breaks in natural populations of the mussels and DNA fragmentation leading to apoptosis. Gabbianelli et al. (2002) found that the comet assay was able to show differential sensitivity in the degree of DNA damage it detected between different organotin compounds. They reported that tributyltin chloride (TBTC) and dibutyltin chloride (DBTC) both had pronounced effects on the tail length, tail intensity and tail moment but that monobutyltin chloride (MBTC) was more efficient in producing DNA damage. MBTC produced the fastest genotoxic effect in cells from the gilthead sea bream (Sparus aurata) and in adition the degree of damage did not change with incubation time (Gabbianelli et al., 2002). In contrast Tiano et al. (2001) suggested that only TBTC produced a significant genotoxic effect with the comet assay in erythocytes of the rainbow trout (Salmo irideus). The genotoxic effect was less pronounced for DBTC and completely absent for MBTC. In the present study there was a good correlation between DNA damage detected by the comet assay and that resulting in micronucleus formation. No work has been carried out linking these two genotoxic biomarkers but both micronucleus formation and the comet assay produced results in the fish H. malabaricus when exposed to TBT (Ferraro et al., 2004). They demonstrated TBT induced mutagenic effects both with an increase in DNA damage as detected with the micronucleus assay and with the incidence of chromosomal aberrations in the fish. One of the mechanisms by which TBT may be genotoxic is through induction of endonucleases via disruption of intracellular calcium homeostasis (Orrenius et al., 1992). An increase in calcium can cause activation of various Ca2+-dependent degradative enzymes such as phospholipases, proteases and endonucleases which have been known to contribute to cell death (Orrenius et al., 1992). Furthermore it has been demonstrated that the production of calcium induced endonucleases can lead to DNA damage as a result of DNA fragmentation (Collins et al., 1996; Mattioli et al., 2003). A variety of mechanisms have been proposed to determine how TBT causes disruption to calcium homeostasis. It is hypothesised that TBT alters intracellular calcium homeostasis by directly interacting with endogenous calmodulin, a calcium modulator protein (Cima and Ballarin, 2000; Cima et al., 2002). TBT is also reported to
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inhibit sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA), which triggers release of endoplasmic reticulum Ca2+ and activation of the store-dependent Ca2+ influx. As a result of the prolonged inhibition of SERCA, the activation of the influx pathway leads to a massive accumulation of Ca2+ (Kass and Orrenius, 1999). Boelsterli (2003) suggests that organotin compounds can directly interact with thiol groups of the calcium pump that results in disruption to calcium homeostasis. TBT was shown to cause a rapid depletion of thiols and several TBT induced effects were prevented by various thiol reducing agents. Damage to the calcium pump by TBT was shown to cause a rapid increase in the levels of intracellular calcium to approximately 500 or 600 nM depending on the dose of TBT (Boelsterli, 2003). In conclusion, this study has shown TBTO to be cytotoxic and genotoxic to adult mussels. In addition there was a correlation between the incidence of strand breaks and that of micronuclei formation. When taken together with our previous observations of genotoxic activity towards early life stages, it is clear that TBT may adversely affect marine organisms at many stages of the lifecycle and through multiple mechanisms, confirming its deleterious effects to the marine environment. Acknowledgments This research was supported by a Leverhulme Trust grant (F/00 568/D). References Be´kri, K., Pelletier, E., 2004. Trophic transfer and in vivo immunotoxicological effects of tributyltin (TBT) in polar seastar Leptasterias polaris. Aquatic Toxicology 66, 39–53. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Analytical Biochemistry 239, 70–76. Bettin, C., Oehlmann, J., Stroben, E., 1996. TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helogander Meeresunters 50, 299–317. Boelsterli, U.A., 2003. Mechanistic Toxicology. Taylor and Francis, London, pp. 148–155. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Bryan, G.W., Gibbs, P.E., Hummerstone, L.G., Burt, G.R., 1986. The decline of the gastropod Nucella lapillus around the south-west England: evidence for the effect of tributyltin from antifouling paints. Journal of the Marine Biological Association UK 66, 611–640. Cima, F., Ballarin, L., 2000. Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin. Aquatic Toxicology 48, 419–429. Cima, F., Dominici, D., Mammi, S., Ballarin, L., 2002. Butlytins and calmodulin: Which interaction? Applied Organometallic Chemistry 164, 182–186.
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Coles, J.A., Farley, S.R., Pipe, R.K., 1995. Alterations of the immune response of the common marine mussel Mytilus edulis resulting from exposure to cadmium. Diseases of Aquatic Organisms 22, 59–65. Collins, M.K.L., Furlong, I.J., Malde, P., Ascaso, R., Oliver, J., Rivas, A.L., 1996. An apoptotic endonuclease activated either by decreasing pH or by increasing calcium. Journal of Cell Science 109, 2393–2399. Countryman, P.I., Heddle, J.A., 1976. The production of micronuclei from chromosomal aberrations in irradiated cultures of human lymphocytes. Mutation Research 41, 321–331. Davis, A., Barale, R., Brun, G., Forster, R., Gu¨nther, T., Hautefeuill, H., van der Heijden, C.A., Knapp, A.G.A.C., Krowke, R., Kuroki, T., Loprieno, N., Malaveille, C., Merker, H.J., Monaco, M., Mosesso, P., Neubert, D., Norppa, H., Sorsa, M., Vogel, E., Umeda, M., Bartsch, H., 1987. Evaluation of the genetic and embryonic effects of bis(tri-n-butyltin) oxide (TBTO), a broad spectrum pesticide, in multiple in vivo and in vitro short term tests. Mutation Research 188, 65–95. Fent, K., 1996. Ecotoxicology of organotin compounds. Critical Reviews in Toxicology 26, 1–117. Ferraro, M.V.M., Fenocchio, A.S., Mantovani, M.S., Ribeiro, C.D., Cestari, M.M., 2004. Mutagenic effects of tributyltin and inorganic lead (Pb II) on the fish H. malabaricus as evaluated using the comet assay and the piscine micronucleus and chromosomal aberration tests. Genetics and Molecular Biology 27, 103–107. Gabbianelli, R., Villarini, M., Falcioni, G., Lupidi, G., 2002. Effect of different organotin compounds on DNA of gilthead sea bream (Sparus aurata) erythrocytes assessed by the comet assay. Applied Organometallic Chemistry 16, 163–168. Gibbs, P.E., Bryan, G.W., 1986. Reproductive failure in populations of the dogwhelk, Nucella lapillus, caused by imposex induced by tributyltin from antifouling paints. Journal of Marine Biological Association UK 66, 767–777. Gibbs, P.E., Pascoe, P.L., Bryan, G.W., 1991. Tributyltin-induced imposex in stenoglossan gastropods: pathological effects on the female reproductive system. Comparative Biochemistry and Physiology 1, 231–235. Goldberg, E.D., 1986. TBT: an environmental dilemma. Environment 28, 17–44. Hagger, J.A., Fisher, A.S., Hill, S.J., Depledge, M.H., Jha, A.N., 2002. Genotoxic, cytotoxic and ontogenetic effects of tri-n-butyltin on the marine worm, Platynereis dumerilii (Polychaeta: Nereidae). Aquatic Toxicology 57, 243–255. Heard, C.S., Walker, W.W., Hawkins, W.E., 1986. Aquatic toxicological effects of organotins: an overview. In: Proceedings of the Organotin Symposium. Oceans 2, 554–563. Jha, A.N., Hagger, J.A., Hill, S.J., 2000. Tributyltin induces cytogenetic damage in the early life stages of the marine mussel, Mytilus edulis. Environmental and Molecular Mutagenesis 35, 343–350.
Kass, G.E., Orrenius, S., 1999. Calcium signalling and cytotoxicity. Environmental Health Perspectives 107, 25–35. Kleibo¨hmer, W., Cammann, K., 1989. Separation and determination of organotin compounds by HPLC and fluorimetric detection in micellar systems. Fresenius Zeitschrift Fu¨r Analytical Chemistry 335, 780–784. Lignot, J.H., Pannier, F., Trilles, J.P., Charmantier, G., 1998. Effects of tributyltin oxide on survival and osmoregulation of the shrimp Penaeus japonicus. Aquatic Toxicology 41, 277–299. Lowe, D.M., Pipe, R.K., 1994. Contamination induced lysosomal membrane damage in marine mussel digestive cells: an in vitro study. Aquatic Toxicology 30, 357–365. Matozzo, V., Ballarin, L., Del Favero, M., Cima, F., 2002. Effects of TBT on functional responses of coelomocytes in the marine worm Sipunculus nudus. Fresenius Environmental Bulletin 11, 568– 572. Mattioli, M., Barboni, B., Luisa, G., Loi, P., 2003. Cold induced calcium elevation triggers DNA fragmentation in immature pig oocytes. Molecular Reproduction and Development 65, 289– 297. Micic, M., Bihari, N., Jaksic, Z., Muller, W.E.G., Batel, R., 2002. DNA damage and apoptosis in the mussel Mytilus galloprovincialis. Marine Environmental Research 53, 243–262. Oberdo¨rster, E., Rittschof, D., McClellan-Green, P., 1998. Testosterone metabolism in imposex and normal Ilyanassa obsoleta: comparsion of field and TBTA Cl-induced imposex. Marine Pollution Bulletin 36, 144–151. Orrenius, S., Burkitt, M.J., Kass, G.E.N., Dypbukt, J.M., Nicotera, P., 1992. Calcium-ions and oxidative cell injury. Annals of Neurology 32, S33–S42. Pipe, R.K., Farley, S.R., Coles, J.A., 1999. Copper induced immunomodulation in the marine mussel, Mytilus edulis. Aquatic Toxicology 46, 43–54. Singh, N.P., McCoy, M., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–191. Smith, K.L., Galloway, T.S., Depledge, M.H., 2000. Neuro-endocrine biomarkers of pollution-induced stress in marine invertebrates. The Science of the Total Environment 262, 185–190. St Jean, S.D., Pelletier, E., Courtenay, S.C., 2002. Hemocyte functions and bacterial clearance affected in vivo by TBT and DBT in the blue mussel Mytilus edulis. Marine Environmental Research 236, 163–178. Thain, J.E., Waldock, M.J., 1986. The impact of tributyltin (TBT) antifouling paints on molluscan fisheries. Water Science and Technology 18, 193–202. Tiano, L., Fedeli, D., Moretti, M., Falcioni, G., 2001. DNA damage induced by organotins on trout-nucleated erythrocytes. Applied Organometallic Chemistry 15, 575–580.
Toxicity of tributyltin in the marine mollusc Mytilus edulis Josephine A. Hagger a
a,*
, Michael H. Depledge b, Tamara S. Galloway
a
Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth, Devon PL4 8AA, UK b Environment Agency, Rio House, Waterside Drive, Aztec West, Almondsbury, Bristol BS12 4UD, UK
Abstract Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 lg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 lg/l of TBTO (P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei (P = 0.0005; R2 = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis. 2005 Elsevier Ltd. All rights reserved. Keywords: Tributyltin; Genotoxicity; Immunotoxicity; Cytotoxicity; Mytilus edulis; Endocrine disruption; Biomarker
1. Introduction Tributyltin (TBT) is a biocide and catalyst used worldwide (Oberdo¨rster et al., 1998). TBT compounds have particularly been used as biocides in antifouling paints and wood preservatives (Bettin et al., 1996). Leachate of these compounds has contaminated both marine and freshwater habitats and it has been considered to be one of the most toxic agents entering the environment (Goldberg, 1986). TBT has been demonstrated to cause impairments in growth, development, reproduction and survival of many marine species (Heard et al., 1986). Of particular concern has been the induction of reproductive abnormalities and sterilisation of female marine prosobranch snails caused by tributyltin based compounds. This phenomenon known as imposex
*
Corresponding author. Tel.: +44 1752 232797; fax: +44 1752 232970. E-mail address: [email protected] (J.A. Hagger). 0025-326X/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.06.044
is characterised by the development of male sex organs, notably a penis and sperm duct (vas deferen), by the female gastropods (Gibbs et al., 1991). An increase in the severity of imposex in gastropods ultimately leads to reproductive failure as the pallial oviduct becomes blocked leading to sterilisation and premature death of the female snails (Gibbs and Bryan, 1986). Population levels of these gastropod molluscs have shown a marked decline in areas of high TBT contamination and a high degree of imposex (Bryan et al., 1986). As well as being an endocrine disrupting agent TBT has proven to be extremely toxic to a number of aquatic organisms, in particular to sensitive early life stages (Thain and Waldock, 1986; Fent, 1996; Lignot et al., 1998). Our recent studies suggest that TBT can induce cytogenetic damage in embryo-larvae of the marine mollusc Mytilus edulis and the polychaete worm Platynereis dumerilii (Jha et al., 2000; Hagger et al., 2002). However relatively little work has been carried out on the genotoxic potential of tributyltin to adult marine organisms. Thus the purpose of this study was to investigate the toxicological potential
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of the endocrine disrupting chemical, tributyltin, by investigating cytotoxic, immunological and genotoxic effects in the adult life stages of the marine mollusc M. edulis. Cytotoxicity was measured using the neutral red assay that measures lysosomal stability in viable cells. Phagocytosis was measured as an indication of immunotoxicity as it is a non-specific defence mechanism. In addition, the ferric reducing ability of plasma (FRAP) assay, a measure of the antioxidant capacity, was deduced. Genotoxicity was determined using micronucleus formation and the comet assay to detect single strand DNA breaks.
2. Methods and materials Mussels were collected from Whitsand Bay, Cornwall, UK. All fauna and flora were removed from the mussels, which were then left for 2 weeks to depurate. Eight mussels per concentration (4 per replica) were placed into 1 l of clean, filtered, aerated seawater (36&, 15 ± 1 C). Mussels were exposed to 0.1, 0.5, 1 and 5 lg/l tributyltin oxide (TBTO) obtained from Lancaster Synthesis UK (1 lg/l TBTO 0.973 lg/l of TBT), in addition to a solvent and seawater control. Water was renewed and re-dosed every day for 7 days and organotin concentrations were measured at the beginning and end of the exposure period. Following the 1-week exposure to TBTO, haemolymph was extracted from the posterior adductor muscle and gill tissue was removed to determine the toxicological effects using the following biomarkers. 2.1. Neutral red assay The neutral red assay was performed as described by Coles et al. (1995) and Pipe et al. (1999). In brief 50 ll of haemolymph was pipetted into duplicate wells of a microtitre plate. The plate was left for 45 min after which the excess solution was thrown away. Two hundred microlitres of 0.33% neutral red was pipetted into each well and left for 3 h, after which time the neutral red solution was removed. The cells were carefully washed and 200 ll of 1% acetic acid in 50% ethanol were added to all the wells and the plate was read at 550 nm. The results were presented as the optical density/mg protein. Protein concentration was determined following the method of Bradford (1976). 2.2. Phagocytosis The phagocytosis assay was performed as described by Coles et al. (1995) and Pipe et al. (1999). In brief 50 ll of haemolymph was pipetted in duplicate wells of a microtitre plate. The plate was left for 60 min until the cells had adhered. Ten minutes before the end of
the designated time 100 ll of bakers formol calcium was added to the negative control wells. After the 60 min, excess solution was tipped away and 50 ll of dyed zymosan suspension (50 · 107) was added to every well including two blanks containing 50 ll of physiological saline. The plate was incubated for 30 min at 10 C after which time the reaction was stopped, by adding 100 ll of bakers formol calcium to all the wells for 10 min. The plate was spun at 60 g (700 rpm) for 5 min, the supernatant pipetted off and the cells washed several times. One hundred microlitre of standards containing 1.56, 3.125, 6.25, 12.5, 25 · 107 zymosan particles were added in duplicate to the plate. Two hundred microlitre of 1% acetic acid in 50% ethanol were added to all the wells and then read at 550 nm. Results were presented as the zymosan particles/mg protein (Coles et al., 1995; Pipe et al., 1999). Protein concentration was determined following the method of Bradford (1976). 2.3. Ferric reducing ability of plasma (FRAP) assay The FRAP assay was carried out as described by Benzie and Strain (1996). In brief 200 ll of gill suspension was centrifuged for 10 min at 10,000·g. Fifty microlitres of supernatant in duplicate was pipetted into microtitre plate wells. Aqueous solutions of know FeII concentrations in the range of 100–500lmol/l, were used for calibration. 200 ll of reagent (300 mM acetate buffer, TBTZ (2,4,6-tripyridyl-s-triazine) and 20 mM iron chloride in a ratio on 10:1:1) was placed into each well. The plate was incubated at 25 C for 10 min and read at 593 nm. The change in absorbency between time 0 and 10 min was used as an indicator of the amount of antioxidant activity. 2.4. Comet assay The comet assay was adapted from a protocol described by Singh et al. (1988). Slides were pre-coated in high melting point (HMP) agarose. One hundred microlitres of haemolymph was placed into 200 ll of 4 C physiological saline (pH 7.36). The cell suspension was centrifuged at 200·g at 4 C for 2–3 min and the supernatant was removed. Eighty five microlitre of low melting point (LMP) agarose was pipetted on to the cell pellet and then placed onto the HMP agarose covered slides. After the gel had set the slides were placed into a freshly prepared lysing solution at 4 C for 1 h (2.5 M NaCl, 100 mM Na2–EDTA, 10 mM Tris, 1% Na sarconisate, pH 10.0, 1% Triton X-100, 10% DMSO). The slides were then removed from the lysis solution and placed in a horizontal gel electrophoresis tank. The tank was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM Na2–EDTA) and the DNA was allowed to unwind for 40 min before electrophoresis,
which was carried out at 25 V for 30 min. After electrophoresis the slides were washed in neutralising buffer (0.4 M Tris, pH 7.5). The slides were stained with 20 ll of 5lg/ml ethidium bromide solution, viewed under ultraviolet fluorescence light and scored using the KometTM software, Kinetic Imaging Ltd. A total of 100 randomly chosen cells were scored per each individual, 50 per replicate. 2.5. Micronucleus assay (MN)
OD of neutral red per mg protein
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813
4.5
*
4.0
*
*
1
5
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
2.6. TBT analysis
Control Solvent
3. Results The data for the neutral red retention was not normally distributed and therefore the non-parameter Kruskal Wallis statistical test was applied. There was a statistically significant difference between the viability of the cells (P = 0.0003) as shown in Fig. 1. There was a statistical increase in cytotoxicity at the 0.5, 1 and 5 lg/l of TBTO in comparison with the controls. In addition 0.1 was significantly different from 1 and 5, and 0.5 was significantly different from 5. Fig. 2 indicates that there was no significant change in phagocytotic activity of haemocytes from mussels
0.5
Fig. 1. Cell viability as measure by the uptake of neutral red (OD mg of protein) by haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean) (* = significant difference from the control).
60 50 40 30 20 10 0 Control Solvent
Tributyltin was determined using a HPLC method described by Kleibo¨hmer and Cammann (1989). In brief samples were passed through a C18 Sep-Pak cartridge and the organotin compounds were eluted off with methanol. HPLC conditions were 1.0 ml/min of 75:25 methanol:water, passed through a 250 mm · 4, 5SA Nucleosil column. A 100 ll aliquot of sample was injected into the column and the organotin compounds detected by fluorescence detection with an excitation wavelength of 392 nm and an emission wavelength of 555 nm.
0.1
Concentration TBTO µg/L
Zymosan particles per mg protein
Approximately 300–500 ll of haemolymph was placed on slides that had been pre-coated in 10% polyL-lysine solution. Slides were placed into a humidifier for 30 min to allow the cells to adhere to the slide, after this time the excess solution was removed. The slides were left to air dry and then fixed in methanol for 15 min, followed by staining in 5% giemsa/buffer solution for 20 min. Slides were scored blind under ·40 and MN validated under oil immersion. A total of 1000 cells were analysed per mussel. The MN were identified according to the following criteria: (1) diameter smaller than one-third of the main nucleus but greater than one-tenth, (2) no contact with nucleus (absence of chromatid bridge), (3) colour and texture resembling the nucleus, (4) spherical cytoplasmic inclusions with sharp contour (Countryman and Heddle, 1976).
0.5
1
5
10
Concentration of TBTO µg/L Fig. 2. Phagocytosis of zymosan by haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean).
exposure to TBTO (P = 0.667). Although the variation between individual mussels did seem to increase with increasing TBTO concentrations, particularly at 5 lg/l. There was also no statistically significant difference in antioxidant status between any of the mussels exposed to TBTO and those of the controls (P = 0.926). Fig. 3 shows the percentage of DNA damage that occurred in haemocytes of M. edulis exposed to TBTO. There was a statistically significant difference in the percentage of DNA damage present in the comet tail between the controls and 0.5, 1 and 5 lg/l of TBTO (P = 0.0005). There was no significant difference between 0.1 lg/l and the control. Fig. 4 indicates an increase in micronuclei with increasing concentrations of TBTO (P = 0.00005). All the concentrations of TBTO produced an increase in the induction of MN when compared to the controls and there was a dose dependent increase in the levels of MN with increasing concentration of TBTO. In addition there was a strong correlation between the percentage of strand break DNA damage detected using the
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Percentage of DNA in tail
30
*
*
Table 1 Mean and standard deviation concentrations of tributyltin of replicate water samples before and after the exposure period following renewal and re-dosing every 24 h
*
25 20
Concentration (lg/l)
Before exposure
After exposure
15
Seawater control Solvent control 0.1 0.5 1 5
ND ND 0.09 ± 0.05 0.41 ± 0.13 1.09 ± 0.09 4.65 ± 0.63
ND ND 0.04 ± 0.02 0.30 ± 0.06 0.54 ± 0.27 2.87 ± 0.99
10 5 0 0.1
Control Solvent
0.5
1
ND = not detectable. Limits of detection 0.01 lg/l.
5
Concentration TBTO µg/L Fig. 3. Percentage of DNA damage in haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean) (* = significant difference from the control).
12
*
MN per1000 cells
10
*
*
8 6
*
4 2 0 0.1
Control Solvent
0.5
1
5
Concentration of TBTO µg/L
Percentage of DNA in comet tail
Fig. 4. Induction of micronuclei (MN) in haemocytes of mussels exposed to different concentrations of TBTO for 7 days (+ = mean) (* = significant difference from the control).
30 25
y = 4.03 + 1.58x R2 = 0.6148
20 15 10 5 0 0
2
4
6
8
10
12
Micronuclei Fig. 5. Linear regression analysis illustrating the correlation between strand breaks and micronuclei in haemocytes of individual mussels exposed to different concentrations of TBTO for 7 days.
comet assay and formation of micronuclei as indicated in Fig. 5 (P = 0.0005; R2 = 61.5%). Table 1 indicates the concentrations of tributyltin in water samples before and after the exposure period.
4. Discussion This study, designed to provide an integrated assessment of the toxicological impact of TBTO to adult bivalves has revealed a significant level of genotoxic damage to adult marine molluscs at environmentally realistic concentrations. This is surprising, as despite its documented toxicity to reproductive processes and immune and endocrine systems, TBTO has not been widely documented as a genotoxin in mammalian test systems (Davis et al., 1987). During the present study there was a decrease in the viability of haemocytes in mussels exposed to TBTO. Within the haemocytes, lysosomes are involved in sequestering and metabolising natural toxins and it has been shown that organic xenobiotics and metals can cause destabilisation of their membrane (Lowe and Pipe, 1994). Accumulated butyltins have been shown to adversely effect lysosomal integrity as measured using the neutral red assay in the 6-armed seastar Leptasterias polaris (Be´kri and Pelletier, 2004). In addition Matozzo et al. (2002) showed that lysosomal activity was significant reduced at 0.05 lM TBT in the marine worm Sipunclus nudus and that the percentage of coelomocytes with ingested neutral red dye was also inhibited at 1 lM. The endpoint used to assess immunotoxicity in this study, phagocytosis, showed no alteration in response to TBTO. It has previously been reported that sublethal doses of TBT can cause phagocytes to lose their ability to move towards and ingest foreign particles (Cima et al., 2002). Phagocytosis was also shown to decrease in amoebocytes of the 6-armed seastar L. polaris after exposure to TBT (Be´kri and Pelletier, 2004). St Jean et al. (2002) found that phagocytic activity was reduced in haemocytes of M. edulis by TBT at concentrations >10 ng/l Sn. Whilst Smith et al. (2000) found that similar concentrations of TBT caused significant inhibition of haemocyte effector functions including spontaneous cytotoxicity of allogeneic cells and the production of free radicals including nitric oxide. These differences in response may reflect variability in the pre-existing immune status of individual organisms.
J.A. Hagger et al. / Marine Pollution Bulletin 51 (2005) 811–816
Both the comet assay and the micronucleus test showed statistically significant increases in DNA damage in haemocytes from mussels exposed to TBTO. Ferraro et al. (2004) demonstrated that TBT produced mutagenic effects with an increase in DNA damage in the fish Hoplias malabaricus using the comet assay. Micic et al. (2002) found that TBT induced DNA fragmentation in human cells and gill cells of the mussel Mytilus galloprovincialis (Micic et al., 2002). However they did not find any correction between DNA strand breaks in natural populations of the mussels and DNA fragmentation leading to apoptosis. Gabbianelli et al. (2002) found that the comet assay was able to show differential sensitivity in the degree of DNA damage it detected between different organotin compounds. They reported that tributyltin chloride (TBTC) and dibutyltin chloride (DBTC) both had pronounced effects on the tail length, tail intensity and tail moment but that monobutyltin chloride (MBTC) was more efficient in producing DNA damage. MBTC produced the fastest genotoxic effect in cells from the gilthead sea bream (Sparus aurata) and in adition the degree of damage did not change with incubation time (Gabbianelli et al., 2002). In contrast Tiano et al. (2001) suggested that only TBTC produced a significant genotoxic effect with the comet assay in erythocytes of the rainbow trout (Salmo irideus). The genotoxic effect was less pronounced for DBTC and completely absent for MBTC. In the present study there was a good correlation between DNA damage detected by the comet assay and that resulting in micronucleus formation. No work has been carried out linking these two genotoxic biomarkers but both micronucleus formation and the comet assay produced results in the fish H. malabaricus when exposed to TBT (Ferraro et al., 2004). They demonstrated TBT induced mutagenic effects both with an increase in DNA damage as detected with the micronucleus assay and with the incidence of chromosomal aberrations in the fish. One of the mechanisms by which TBT may be genotoxic is through induction of endonucleases via disruption of intracellular calcium homeostasis (Orrenius et al., 1992). An increase in calcium can cause activation of various Ca2+-dependent degradative enzymes such as phospholipases, proteases and endonucleases which have been known to contribute to cell death (Orrenius et al., 1992). Furthermore it has been demonstrated that the production of calcium induced endonucleases can lead to DNA damage as a result of DNA fragmentation (Collins et al., 1996; Mattioli et al., 2003). A variety of mechanisms have been proposed to determine how TBT causes disruption to calcium homeostasis. It is hypothesised that TBT alters intracellular calcium homeostasis by directly interacting with endogenous calmodulin, a calcium modulator protein (Cima and Ballarin, 2000; Cima et al., 2002). TBT is also reported to
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inhibit sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA), which triggers release of endoplasmic reticulum Ca2+ and activation of the store-dependent Ca2+ influx. As a result of the prolonged inhibition of SERCA, the activation of the influx pathway leads to a massive accumulation of Ca2+ (Kass and Orrenius, 1999). Boelsterli (2003) suggests that organotin compounds can directly interact with thiol groups of the calcium pump that results in disruption to calcium homeostasis. TBT was shown to cause a rapid depletion of thiols and several TBT induced effects were prevented by various thiol reducing agents. Damage to the calcium pump by TBT was shown to cause a rapid increase in the levels of intracellular calcium to approximately 500 or 600 nM depending on the dose of TBT (Boelsterli, 2003). In conclusion, this study has shown TBTO to be cytotoxic and genotoxic to adult mussels. In addition there was a correlation between the incidence of strand breaks and that of micronuclei formation. When taken together with our previous observations of genotoxic activity towards early life stages, it is clear that TBT may adversely affect marine organisms at many stages of the lifecycle and through multiple mechanisms, confirming its deleterious effects to the marine environment. Acknowledgments This research was supported by a Leverhulme Trust grant (F/00 568/D). References Be´kri, K., Pelletier, E., 2004. Trophic transfer and in vivo immunotoxicological effects of tributyltin (TBT) in polar seastar Leptasterias polaris. Aquatic Toxicology 66, 39–53. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Analytical Biochemistry 239, 70–76. Bettin, C., Oehlmann, J., Stroben, E., 1996. TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helogander Meeresunters 50, 299–317. Boelsterli, U.A., 2003. Mechanistic Toxicology. Taylor and Francis, London, pp. 148–155. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Bryan, G.W., Gibbs, P.E., Hummerstone, L.G., Burt, G.R., 1986. The decline of the gastropod Nucella lapillus around the south-west England: evidence for the effect of tributyltin from antifouling paints. Journal of the Marine Biological Association UK 66, 611–640. Cima, F., Ballarin, L., 2000. Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin. Aquatic Toxicology 48, 419–429. Cima, F., Dominici, D., Mammi, S., Ballarin, L., 2002. Butlytins and calmodulin: Which interaction? Applied Organometallic Chemistry 164, 182–186.
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Kass, G.E., Orrenius, S., 1999. Calcium signalling and cytotoxicity. Environmental Health Perspectives 107, 25–35. Kleibo¨hmer, W., Cammann, K., 1989. Separation and determination of organotin compounds by HPLC and fluorimetric detection in micellar systems. Fresenius Zeitschrift Fu¨r Analytical Chemistry 335, 780–784. Lignot, J.H., Pannier, F., Trilles, J.P., Charmantier, G., 1998. Effects of tributyltin oxide on survival and osmoregulation of the shrimp Penaeus japonicus. Aquatic Toxicology 41, 277–299. Lowe, D.M., Pipe, R.K., 1994. Contamination induced lysosomal membrane damage in marine mussel digestive cells: an in vitro study. Aquatic Toxicology 30, 357–365. Matozzo, V., Ballarin, L., Del Favero, M., Cima, F., 2002. Effects of TBT on functional responses of coelomocytes in the marine worm Sipunculus nudus. Fresenius Environmental Bulletin 11, 568– 572. Mattioli, M., Barboni, B., Luisa, G., Loi, P., 2003. Cold induced calcium elevation triggers DNA fragmentation in immature pig oocytes. Molecular Reproduction and Development 65, 289– 297. Micic, M., Bihari, N., Jaksic, Z., Muller, W.E.G., Batel, R., 2002. DNA damage and apoptosis in the mussel Mytilus galloprovincialis. Marine Environmental Research 53, 243–262. Oberdo¨rster, E., Rittschof, D., McClellan-Green, P., 1998. Testosterone metabolism in imposex and normal Ilyanassa obsoleta: comparsion of field and TBTA Cl-induced imposex. Marine Pollution Bulletin 36, 144–151. Orrenius, S., Burkitt, M.J., Kass, G.E.N., Dypbukt, J.M., Nicotera, P., 1992. Calcium-ions and oxidative cell injury. Annals of Neurology 32, S33–S42. Pipe, R.K., Farley, S.R., Coles, J.A., 1999. Copper induced immunomodulation in the marine mussel, Mytilus edulis. Aquatic Toxicology 46, 43–54. Singh, N.P., McCoy, M., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–191. Smith, K.L., Galloway, T.S., Depledge, M.H., 2000. Neuro-endocrine biomarkers of pollution-induced stress in marine invertebrates. The Science of the Total Environment 262, 185–190. St Jean, S.D., Pelletier, E., Courtenay, S.C., 2002. Hemocyte functions and bacterial clearance affected in vivo by TBT and DBT in the blue mussel Mytilus edulis. Marine Environmental Research 236, 163–178. Thain, J.E., Waldock, M.J., 1986. The impact of tributyltin (TBT) antifouling paints on molluscan fisheries. Water Science and Technology 18, 193–202. Tiano, L., Fedeli, D., Moretti, M., Falcioni, G., 2001. DNA damage induced by organotins on trout-nucleated erythrocytes. Applied Organometallic Chemistry 15, 575–580.