Marine Pollution Bulletin 51 (2005) 811816

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

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 eects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days
resulted in a statistically signicant decrease in cell viability at concentrations of 0.5 lg/l and above. TBT had no eect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically signicant 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 (Oberdorster 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: jhagger@plymouth.ac.uk (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

812

J.A. Hagger et al. / Marine Pollution Bulletin 51 (2005) 811816

of the endocrine disrupting chemical, tributyltin, by

investigating cytotoxic, immunological and genotoxic
eects 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-specic 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 ora 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, ltered, 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
eects 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 o 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 10,000g. Fifty
microlitres of supernatant in duplicate was pipetted into
microtitre plate wells. Aqueous solutions of know FeII
concentrations in the range of 100500lmol/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 200g at 4 C for 23 min and the
supernatant was removed. Eighty ve 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 Na2EDTA, 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 lled with fresh electrophoresis buer
(300 mM NaOH, 1 mM Na2EDTA) 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 buer
(0.4 M Tris, pH 7.5). The slides were stained with 20 ll
of 5lg/ml ethidium bromide solution, viewed under
ultraviolet uorescence 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) 811816

813

4.5

4.0

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 signicant dierence 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 signicantly dierent from 1 and 5,
and 0.5 was signicantly dierent from 5.
Fig. 2 indicates that there was no signicant 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 dierent concentrations of TBTO for 7 days (+ = mean) (* = signicant dierence from
the control).

60
50
40
30
20
10
0
Control Solvent

Tributyltin was determined using a HPLC method

described by Kleibohmer and Cammann (1989). In brief
samples were passed through a C18 Sep-Pak cartridge
and the organotin compounds were eluted o 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 uorescence detection with an excitation
wavelength of 392 nm and an emission wavelength of
555 nm.

0.1

Concentration TBTO g/L

Zymosan particles per mg protein

Approximately 300500 ll of haemolymph was

placed on slides that had been pre-coated in 10% polyL-lysine solution. Slides were placed into a humidier
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 xed in methanol for
15 min, followed by staining in 5% giemsa/buer 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 identied 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

10

Concentration of TBTO g/L

Fig. 2. Phagocytosis of zymosan by haemocytes of mussels exposed to
dierent 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 signicant dierence 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 signicant dierence 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 signicant dierence
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

814

J.A. Hagger et al. / Marine Pollution Bulletin 51 (2005) 811816

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

ND = not detectable. Limits of detection 0.01 lg/l.

Concentration TBTO g/L

Fig. 3. Percentage of DNA damage in haemocytes of mussels exposed
to dierent concentrations of TBTO for 7 days (+ = mean) (* =
signicant dierence from the control).

12

MN per1000 cells

10

8
6

4
2
0
0.1

Control Solvent

0.5

Concentration of TBTO g/L

Percentage of DNA in comet tail

Fig. 4. Induction of micronuclei (MN) in haemocytes of mussels

exposed to dierent concentrations of TBTO for 7 days (+ = mean)
(* = signicant dierence from the control).

30
25

y = 4.03 + 1.58x
R2 = 0.6148

20
15
10
5
0
0

10

12

Micronuclei
Fig. 5. Linear regression analysis illustrating the correlation between
strand breaks and micronuclei in haemocytes of individual mussels
exposed to dierent 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 signicant 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 eect lysosomal integrity as measured using the
neutral red assay in the 6-armed seastar Leptasterias
polaris (Bekri and Pelletier, 2004). In addition Matozzo
et al. (2002) showed that lysosomal activity was signicant 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 (Bekri 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 signicant inhibition
of haemocyte eector functions including spontaneous
cytotoxicity of allogeneic cells and the production of free
radicals including nitric oxide. These dierences in
response may reect variability in the pre-existing
immune status of individual organisms.

J.A. Hagger et al. / Marine Pollution Bulletin 51 (2005) 811816

Both the comet assay and the micronucleus test

showed statistically signicant increases in DNA damage in haemocytes from mussels exposed to TBTO.
Ferraro et al. (2004) demonstrated that TBT produced
mutagenic eects with an increase in DNA damage in
the sh 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 nd 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 dierent organotin compounds. They
reported that tributyltin chloride (TBTC) and dibutyltin
chloride (DBTC) both had pronounced eects on the
tail length, tail intensity and tail moment but that
monobutyltin chloride (MBTC) was more ecient in
producing DNA damage. MBTC produced the fastest
genotoxic eect 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 signicant genotoxic eect
with the comet assay in erythocytes of the rainbow trout
(Salmo irideus). The genotoxic eect 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 sh H. malabaricus when exposed
to TBT (Ferraro et al., 2004). They demonstrated TBT
induced mutagenic eects both with an increase in
DNA damage as detected with the micronucleus assay
and with the incidence of chromosomal aberrations in
the sh.
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

815

inhibit sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA), which triggers release of endoplasmic
reticulum Ca2+ and activation of the store-dependent
Ca2+ inux. As a result of the prolonged inhibition of
SERCA, the activation of the inux 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 eects 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
aect marine organisms at many stages of the lifecycle
and through multiple mechanisms, conrming its deleterious eects to the marine environment.
Acknowledgments
This research was supported by a Leverhulme Trust
grant (F/00 568/D).
References
Bekri, K., Pelletier, E., 2004. Trophic transfer and in vivo immunotoxicological eects of tributyltin (TBT) in polar seastar Leptasterias polaris. Aquatic Toxicology 66, 3953.
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, 7076.
Bettin, C., Oehlmann, J., Stroben, E., 1996. TBT-induced imposex in
marine neogastropods is mediated by an increasing androgen level.
Helogander Meeresunters 50, 299317.
Boelsterli, U.A., 2003. Mechanistic Toxicology. Taylor and Francis,
London, pp. 148155.
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,
248254.
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 eect of tributyltin from antifouling
paints. Journal of the Marine Biological Association UK 66,
611640.
Cima, F., Ballarin, L., 2000. Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via
interaction with calmodulin. Aquatic Toxicology 48, 419429.
Cima, F., Dominici, D., Mammi, S., Ballarin, L., 2002. Butlytins and
calmodulin: Which interaction? Applied Organometallic Chemistry
164, 182186.

816

J.A. Hagger et al. / Marine Pollution Bulletin 51 (2005) 811816

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, 5965.
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, 23932399.
Countryman, P.I., Heddle, J.A., 1976. The production of micronuclei
from chromosomal aberrations in irradiated cultures of human
lymphocytes. Mutation Research 41, 321331.
Davis, A., Barale, R., Brun, G., Forster, R., Gunther, 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 eects of bis(tri-n-butyltin) oxide (TBTO), a broad
spectrum pesticide, in multiple in vivo and in vitro short term tests.
Mutation Research 188, 6595.
Fent, K., 1996. Ecotoxicology of organotin compounds. Critical
Reviews in Toxicology 26, 1117.
Ferraro, M.V.M., Fenocchio, A.S., Mantovani, M.S., Ribeiro, C.D.,
Cestari, M.M., 2004. Mutagenic eects of tributyltin and inorganic
lead (Pb II) on the sh H. malabaricus as evaluated using the comet
assay and the piscine micronucleus and chromosomal aberration
tests. Genetics and Molecular Biology 27, 103107.
Gabbianelli, R., Villarini, M., Falcioni, G., Lupidi, G., 2002. Eect of
dierent organotin compounds on DNA of gilthead sea bream
(Sparus aurata) erythrocytes assessed by the comet assay. Applied
Organometallic Chemistry 16, 163168.
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, 767777.
Gibbs, P.E., Pascoe, P.L., Bryan, G.W., 1991. Tributyltin-induced
imposex in stenoglossan gastropods: pathological eects on the
female reproductive system. Comparative Biochemistry and Physiology 1, 231235.
Goldberg, E.D., 1986. TBT: an environmental dilemma. Environment
28, 1744.
Hagger, J.A., Fisher, A.S., Hill, S.J., Depledge, M.H., Jha, A.N., 2002.
Genotoxic, cytotoxic and ontogenetic eects of tri-n-butyltin on the
marine worm, Platynereis dumerilii (Polychaeta: Nereidae). Aquatic Toxicology 57, 243255.
Heard, C.S., Walker, W.W., Hawkins, W.E., 1986. Aquatic toxicological eects of organotins: an overview. In: Proceedings of the
Organotin Symposium. Oceans 2, 554563.
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, 343350.

Kass, G.E., Orrenius, S., 1999. Calcium signalling and cytotoxicity.

Environmental Health Perspectives 107, 2535.
Kleibohmer, W., Cammann, K., 1989. Separation and determination
of organotin compounds by HPLC and uorimetric detection in
micellar systems. Fresenius Zeitschrift Fur Analytical Chemistry
335, 780784.
Lignot, J.H., Pannier, F., Trilles, J.P., Charmantier, G., 1998. Eects
of tributyltin oxide on survival and osmoregulation of the shrimp
Penaeus japonicus. Aquatic Toxicology 41, 277299.
Lowe, D.M., Pipe, R.K., 1994. Contamination induced lysosomal
membrane damage in marine mussel digestive cells: an in vitro
study. Aquatic Toxicology 30, 357365.
Matozzo, V., Ballarin, L., Del Favero, M., Cima, F., 2002. Eects 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, 243262.
Oberdorster, E., Rittschof, D., McClellan-Green, P., 1998. Testosterone metabolism in imposex and normal Ilyanassa obsoleta:
comparsion of eld and TBTA Cl-induced imposex. Marine
Pollution Bulletin 36, 144151.
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, S33S42.
Pipe, R.K., Farley, S.R., Coles, J.A., 1999. Copper induced immunomodulation in the marine mussel, Mytilus edulis. Aquatic Toxicology 46, 4354.
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, 184191.
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, 185190.
St Jean, S.D., Pelletier, E., Courtenay, S.C., 2002. Hemocyte functions
and bacterial clearance aected in vivo by TBT and DBT in the
blue mussel Mytilus edulis. Marine Environmental Research 236,
163178.
Thain, J.E., Waldock, M.J., 1986. The impact of tributyltin (TBT)
antifouling paints on molluscan sheries. Water Science and
Technology 18, 193202.
Tiano, L., Fedeli, D., Moretti, M., Falcioni, G., 2001. DNA damage
induced by organotins on trout-nucleated erythrocytes. Applied
Organometallic Chemistry 15, 575580.