- Email: [email protected]
Comparative toxicological pathology in mammals and fish: some examples with endocrine disrupters
Toxicology 205 (2004) 27–32
Comparative toxicological pathology in mammals and fish: some examples with endocrine disrupters Piet W. Wester∗ , Leo T.M. van der Ven, Joseph G. Vos Laboratory for Toxicology, Pathology and Genetics, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, Bilthoven 3720, The Netherlands
Abstract Toxicologic pathology is a classical discipline in the toxicology arena, and despite various emerging techniques, still is a major cornerstone in the process of hazard identification and risk characterization. Most knowledge derives from laboratory animal studies and, to a lesser extent, human data. Currently interest is growing in applying toxicological pathology for lower animals, in particular fish as being the most developed aquatic genus. This is triggered by the interest in so-called endocrine disrupting chemicals (endocrine disrupters, EDCs), xenobiotics that interfere with the endocrine system and thus may affect reproduction and/or development, and for which pathology is an essential technique in general in vivo studies. As the aquatic ecosystem is a major recipient of pollutants, fish constitute an important potential target and can be used as a research and bio-monitoring tool. For this goal knowledge of the pathological responses of fish to EDCs is essential and therefore we have studied the responses of laboratory fish to a set of reference endocrine modulating chemicals. In this paper, such effects are compared with known response patterns in mammals, thereby accounting for the specific aspects of anatomy and physiology in fish. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Mammals; Fish; Endocrine disrupters
1. Comparison of endocrine effects in rats and fish A major group of endocrine disrupters has estrogenic activity and includes natural estrogens, synthetic hormones, phyto-estrogens, various industrial chemicals, pesticides, etc. Also many effects observed in wildlife point towards estrogenic activity; therefore ap∗
Corresponding author. E-mail address: [email protected] (P.W. Wester).
parently estrogens have received much attention. On the other hand, as there are also many compounds, which have an anti-estrogen, androgen, anti-androgen or anti-thyroid activity, we also included reference compounds of these categories in our study. 2. Estrogens Estrogens are known to exert a vast number of endocrine and metabolic effects in mammals that even may vary between species. At least some
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.06.063
28
P.W. Wester et al. / Toxicology 205 (2004) 27–32
Fig. 2. Induction of intersex (testis–ova, feminisation) in male medaka (Oryzias latipes) following exposure to betahexachlorocyclohexane.
3. Other (anti)sex hormones Fig. 1. Ovaries from control, 1 and 100 nM ethynylestradiol treated zebrafish. There is increasing degeneration (atresia) of maturing eggs, resulting in complete absence of mature eggs in the 100 nM animals. (From: van den Belt et al., 2002).
of these effects are mimicked by pseudoestrogens, of which  hexachlorocyclohexane (–HCH) has been studied by our group in the eighties (Wester et al., 1985; Wester and Canton, 1986; van Velsen et al., 1986). For the present purpose we have focused on some major effects on the reproductive system. In mammals (rodents) estrogens are principally involved in the development and the (cyclic) driving force in the female reproductive tract activity. In males the typical sexual development and activity tends to be inhibited (e.g. feminisation). In our fish studies (guppies, medaka and zebrafish) with estrogens we observed analogous effects such as inhibited maturation of oocytes, intersexuality, pituitary alterations and induction of vitellogenin synthesis in both males and females. Vitellogenin is the precursor of yolk in egglaying animals that is produced by the liver and causes characteristic morphologic hepatic and blood plasma changes. Being a yolk precursor, VTG evidently has no mammalian counterpart, and conversely, endometrium changes characteristic for estrogen treatment in mammals was not present in fish. Other effects were inhibition of spermatogenesis and change in sex ratio (feminisation) in offspring (Wester et al., 2003) (Figs. 1–4).
A summary of the effects of estrogen, androgen, anti-estrogen and anti-androgen is presented in Table 1. It is clear that these compounds that interfere with receptor binding and/or hypothalamus/pituitary/gonadal axis, in one way or another effect sexual development and functioning. Normally these effects include (the results of) inhibition (e.g. atrophy, degeneration) or stimulation (e.g. hyperplasia, hypertrophy) of certain sensitive cell types. As the reproductive endocrine system is, to a large extent similar in mammals and fish (Kime, 1998), effects can be expected to be comparable, although significant differences are recognised, such as the above mentioned estrogen driven vitellogenesis and hormone dependent gender determination (in this respect it is worth mentioning that even within the class of mammals significant differences do exist (Alison et al., 1994)).
4. Other endocrine disrupters In the past we have conducted some comparative toxicity studies with topical toxicants that happened to manifest endocrine activity (Table 2). These included -HCH, (sodium) bromide, and tributyltinoxide (TBTO). -HCH is the persistent -isomer in the technical mixture of the insecticide lindane and was shown to exert estrogenic activity in both fish and rats (see above).
P.W. Wester et al. / Toxicology 205 (2004) 27–32
29
Fig. 3. Testis of a control (left) and ethynylestradiol treated rat (right, higher magnification) (top) and zebrafish (bottom) Note inhibition of spermatogenesis, absence of mature sperm and atrophy of interstitial cells in the treated rat. In the zebrafish clusters of spermatogenic cells (“cysts”) are predominant earlier stages (spermatogonia) compared to the control, indicating inhibition of spermatogenesis.
Fig. 4. Pituitary of control (upper panels) guppy (left, PAS stained) and rat (right, anti-prolactin stained). Lower panels are -HCH treated animals. Note increased density of prolactin cells in the rat and the swollen gonadotrophs compartment (bottom, center) in the guppy.
30
P.W. Wester et al. / Toxicology 205 (2004) 27–32
Table 1 Comparison fish–rat for some endocrine compounds Zebrafish
Rat
Estrogen (17estradiol, ethynylestradiol)
VTG ⇑; ovarian regression; inhibited spermatogenesis; (possible∗ ) Leydig cell atrophy; skewed sex ratio (F)
␣-Estrogen (tamoxifen)
Oocyte degeneration; disturbed spermatogenesis; Leydig cell stimulation; skewed sex ratio (M) Inhibited ovulation; enhanced spermatogenesis; Sertoli cell stimulation; skewed sex ratio (M) Inhibited sperm maturation; intersex (testis–ova); Leydig cell stimulation; Sertoli cell stimulation
Ovarian regression; endometrial transformation; inhibited spermatogenesis; Leydig cell atrophy Atrophy of gonads
Androgen (17␣methyldihydrotestosterone) ␣-Androgen (flutamide, vinclozolin, phtalate) ∗
Trophic effects for male characteristics Atrophy of gonads; feminisation in males; Leydig cell stimulation
:difficult to assess.
Table 2 Comparison fish–rat for some endocrine disrupters Guppy
Medaka
Rat
-HCH
VTG ⇑; gonad changes; pit; gonadotrophs ⇑
VTG ⇑; testis–ova; thyroid ⇑
TBT/DBT
Thymus atrophy; glycogen storage; local irritant; retina changes Goiter; locomotor disturb
Glycogen storage; local irritant; retina changes; thyroid stimulation
Endometrium transform; gonad changes; pituitary; prolactin cells ⇑ Thymus atrophy; local irritant; thyroid inhibition
Goiter
Goiter; locomotor disturb
Bromide
Fig. 5. Thyroid stimulation after exposure to the goitrogens bromide (rat, upper panels) and propylthiouracyl (zebrafish, lower panels). Thyroid stimulation in the right panels is evident by increased follicle cell height and decrease in follicle.
P.W. Wester et al. / Toxicology 205 (2004) 27–32
31
Fig. 6. TBTO induced thymus atrophy in guppy (upper right) and rat (lower right). Left panels are controls.
Bromide was tested as the use of methylbromide as applied in glasshouse fumigation could result in residues in the aquatic environment or in food commodities. Studies revealed that bromide was a powerful thyroid inhibitor in both rats (Loeber et al., 1983) and fish (Wester et al., 1988). This was not surprising as the thyroid axis is also well preserved following the same principles in either class. A practical difficulty, however, is the fact that the thyroid is not an encapsulated organ in fish and therefore is not available for organ weight determination. Thus the only approach for detecting thyroid active compounds is analysis of histopathology or circulating hormone levels. In a recent study with the thyroid inhibitor propylthiouracil (Fig. 5) we demonstrated that the sensitivity of both methods was in the same order of magnitude (Wester et al., 2003). TBTO was tested in the eighties for its potential risk as residue in the aquatic food chain. TBTO is nowadays classified as endocrine disrupter mainly because of its masculinisation potential in prosobranch
snails due to inhibition of aromatisation (Oehlmann and Schulte-Oehlmann, 2002). Despite various endocrine effects at higher concentrations, the significant atrophy of the thymus at lower doses received more attention (Fig. 6) both in rats (Krajnc et al., 1984) and fish (guppies) (Wester and Canton, 1987). As is the case with the thyroid, also the thymus in fish is not accessible for organ weight determination and thus this diagnosis also depends on careful histopathology.
5. Conclusion Comparative pathology of small fish and mammals using endocrine active chemicals has learned us that effects are largely similar; obviously, class and species specific anatomical, physiological and pathological pathways need to be considered. This allows us to use fish as an alternative test animal, for instance when aquatic studies are required, and as a suitable second laboratory animal species in hazard identification.
32
P.W. Wester et al. / Toxicology 205 (2004) 27–32
Introduction of histopathology is essential in this respect (van der Ven et al., 2003) and consequently, in order to foster training and education we have created a digital histopathology atlas of small fish that is available through the Internet. This atlas contains most of the fish studies described here (van der Ven and Wester, 2002).
References Alison, R.H., Capen, C.C., Prentice, D.E., 1994. Neoplastic lesions of questionable significance to humans. Toxicol. Pathol. 22, 179–186. Kime, D.E., 1998. Endocrine Disruption in Fish. Kluwer Academic Publishers, London. Krajnc, E.I., Wester, P.W., Loeber, J.G., van Leeuwen, F.X.R., Vos, J.G., Vaessen, H.A.M.G., van der Heijden, C.A., 1984. Toxicity of Bis(tri-n-butyltin)oxide in the rat. I. Short-term effects on general parameters and on the endocrine and lymphoid system. Toxicol. Appl. Pharmacol. 75, 363–386. Loeber, J.G., Franken, M.A.M., van Leeuwen, F.X.R., 1983. Effect of sodium bromide on endocrine parameters in the rat as studied by immunocytochemistry and radioimmunoassay. Fd Chem. Toxic. 21 (4), 391–404. Oehlmann, J., Schulte-Oehlmann, U., 2002. Endocrine disruptors: Invertebrates. In: Keller, R., Dircksen, H., Sedlmeier, D., Vaudry, H. (Eds.), Proceedings of the 21st Conference of European Comparative Endocrinologists. Bonn, Germany, 26–30 August 2002. Monduzzi Editore, Bologna, pp. 63–69.
van den Belt, K., Wester, P., van der Ven, L.T.M., Verheyen, R., Witter, H., 2002. Time-dependent effects of ethynylestradiol on the reproductive physiology in zebrafish (Danio rerio). Environ. Toxicol. Chem. 21, 767–775. van Velsen, F.L., Danse, L.H., van Leeuwen, F.X., Dormans, J.A.M.A., van Logten, M.J., 1986. The subchronic oral toxicity of the beta-isomer of hexachlorocyclohexane in rats. Fundam. Appl. Toxicol. 6, 697–712. van der Ven, L.T.M., Wester, P.W., 2002. Toxicological pathology atlas of small laboratory fish (http://www.rivm.nl/fishtoxpat/). van der Ven, L.T.M., Wester, P.W., Vos, J.G., 2003. Histopathology as a tool for the evaluation of endocrine disruption in zebrafish. Environ. Toxicol. Chem. 22, 908–913. Wester, P.W., Canton, J.H., Bisschop, A., 1985. Histopathological study of Poecilia reticulata (guppy) after long-term -hexachlorocyclohexane exposure. Aquat. Toxicol. 6, 271– 296. Wester, P.W., Canton, J.H., 1986. Histopathological study of Oryzias latipes (medaka) after long-term -hexachlorocyclohexane exposure. Aquat. Toxicol. 9, 21–45. Wester, P.W., Canton, J.H., 1987. Histopathological study of Poecilia reticulata (guppy) after long-term exposure to bis(trin-butyltin)oxide (TBTO) and di-n-butyltinchloride (DBTC). Aquat. Toxicol. 10, 143–165. Wester, P.W., Canton, J.H., Dormans, J.A.M.A., 1988. Pathological effects in freshwater fish Poecilia reticulata (guppy) and Oryzias latipes (medaka) following methyl bromide and sodium bromide exposure. Aquat. Toxicol. 12, 323–344. Wester, P.W., van der Ven, L.T.M., van den Brandhof, E.J., Vos, J.H., 2003. Identification of Endocrine Disruptive Effects in te Aquatic Environment: a Partial Life Cycle Study in Zebrafish. Report 640920. RIVM, the Netherlands, p. 112.
Comparative toxicological pathology in mammals and fish: some examples with endocrine disrupters Piet W. Wester∗ , Leo T.M. van der Ven, Joseph G. Vos Laboratory for Toxicology, Pathology and Genetics, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, Bilthoven 3720, The Netherlands
Abstract Toxicologic pathology is a classical discipline in the toxicology arena, and despite various emerging techniques, still is a major cornerstone in the process of hazard identification and risk characterization. Most knowledge derives from laboratory animal studies and, to a lesser extent, human data. Currently interest is growing in applying toxicological pathology for lower animals, in particular fish as being the most developed aquatic genus. This is triggered by the interest in so-called endocrine disrupting chemicals (endocrine disrupters, EDCs), xenobiotics that interfere with the endocrine system and thus may affect reproduction and/or development, and for which pathology is an essential technique in general in vivo studies. As the aquatic ecosystem is a major recipient of pollutants, fish constitute an important potential target and can be used as a research and bio-monitoring tool. For this goal knowledge of the pathological responses of fish to EDCs is essential and therefore we have studied the responses of laboratory fish to a set of reference endocrine modulating chemicals. In this paper, such effects are compared with known response patterns in mammals, thereby accounting for the specific aspects of anatomy and physiology in fish. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Mammals; Fish; Endocrine disrupters
1. Comparison of endocrine effects in rats and fish A major group of endocrine disrupters has estrogenic activity and includes natural estrogens, synthetic hormones, phyto-estrogens, various industrial chemicals, pesticides, etc. Also many effects observed in wildlife point towards estrogenic activity; therefore ap∗
Corresponding author. E-mail address: [email protected] (P.W. Wester).
parently estrogens have received much attention. On the other hand, as there are also many compounds, which have an anti-estrogen, androgen, anti-androgen or anti-thyroid activity, we also included reference compounds of these categories in our study. 2. Estrogens Estrogens are known to exert a vast number of endocrine and metabolic effects in mammals that even may vary between species. At least some
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.06.063
28
P.W. Wester et al. / Toxicology 205 (2004) 27–32
Fig. 2. Induction of intersex (testis–ova, feminisation) in male medaka (Oryzias latipes) following exposure to betahexachlorocyclohexane.
3. Other (anti)sex hormones Fig. 1. Ovaries from control, 1 and 100 nM ethynylestradiol treated zebrafish. There is increasing degeneration (atresia) of maturing eggs, resulting in complete absence of mature eggs in the 100 nM animals. (From: van den Belt et al., 2002).
of these effects are mimicked by pseudoestrogens, of which  hexachlorocyclohexane (–HCH) has been studied by our group in the eighties (Wester et al., 1985; Wester and Canton, 1986; van Velsen et al., 1986). For the present purpose we have focused on some major effects on the reproductive system. In mammals (rodents) estrogens are principally involved in the development and the (cyclic) driving force in the female reproductive tract activity. In males the typical sexual development and activity tends to be inhibited (e.g. feminisation). In our fish studies (guppies, medaka and zebrafish) with estrogens we observed analogous effects such as inhibited maturation of oocytes, intersexuality, pituitary alterations and induction of vitellogenin synthesis in both males and females. Vitellogenin is the precursor of yolk in egglaying animals that is produced by the liver and causes characteristic morphologic hepatic and blood plasma changes. Being a yolk precursor, VTG evidently has no mammalian counterpart, and conversely, endometrium changes characteristic for estrogen treatment in mammals was not present in fish. Other effects were inhibition of spermatogenesis and change in sex ratio (feminisation) in offspring (Wester et al., 2003) (Figs. 1–4).
A summary of the effects of estrogen, androgen, anti-estrogen and anti-androgen is presented in Table 1. It is clear that these compounds that interfere with receptor binding and/or hypothalamus/pituitary/gonadal axis, in one way or another effect sexual development and functioning. Normally these effects include (the results of) inhibition (e.g. atrophy, degeneration) or stimulation (e.g. hyperplasia, hypertrophy) of certain sensitive cell types. As the reproductive endocrine system is, to a large extent similar in mammals and fish (Kime, 1998), effects can be expected to be comparable, although significant differences are recognised, such as the above mentioned estrogen driven vitellogenesis and hormone dependent gender determination (in this respect it is worth mentioning that even within the class of mammals significant differences do exist (Alison et al., 1994)).
4. Other endocrine disrupters In the past we have conducted some comparative toxicity studies with topical toxicants that happened to manifest endocrine activity (Table 2). These included -HCH, (sodium) bromide, and tributyltinoxide (TBTO). -HCH is the persistent -isomer in the technical mixture of the insecticide lindane and was shown to exert estrogenic activity in both fish and rats (see above).
P.W. Wester et al. / Toxicology 205 (2004) 27–32
29
Fig. 3. Testis of a control (left) and ethynylestradiol treated rat (right, higher magnification) (top) and zebrafish (bottom) Note inhibition of spermatogenesis, absence of mature sperm and atrophy of interstitial cells in the treated rat. In the zebrafish clusters of spermatogenic cells (“cysts”) are predominant earlier stages (spermatogonia) compared to the control, indicating inhibition of spermatogenesis.
Fig. 4. Pituitary of control (upper panels) guppy (left, PAS stained) and rat (right, anti-prolactin stained). Lower panels are -HCH treated animals. Note increased density of prolactin cells in the rat and the swollen gonadotrophs compartment (bottom, center) in the guppy.
30
P.W. Wester et al. / Toxicology 205 (2004) 27–32
Table 1 Comparison fish–rat for some endocrine compounds Zebrafish
Rat
Estrogen (17estradiol, ethynylestradiol)
VTG ⇑; ovarian regression; inhibited spermatogenesis; (possible∗ ) Leydig cell atrophy; skewed sex ratio (F)
␣-Estrogen (tamoxifen)
Oocyte degeneration; disturbed spermatogenesis; Leydig cell stimulation; skewed sex ratio (M) Inhibited ovulation; enhanced spermatogenesis; Sertoli cell stimulation; skewed sex ratio (M) Inhibited sperm maturation; intersex (testis–ova); Leydig cell stimulation; Sertoli cell stimulation
Ovarian regression; endometrial transformation; inhibited spermatogenesis; Leydig cell atrophy Atrophy of gonads
Androgen (17␣methyldihydrotestosterone) ␣-Androgen (flutamide, vinclozolin, phtalate) ∗
Trophic effects for male characteristics Atrophy of gonads; feminisation in males; Leydig cell stimulation
:difficult to assess.
Table 2 Comparison fish–rat for some endocrine disrupters Guppy
Medaka
Rat
-HCH
VTG ⇑; gonad changes; pit; gonadotrophs ⇑
VTG ⇑; testis–ova; thyroid ⇑
TBT/DBT
Thymus atrophy; glycogen storage; local irritant; retina changes Goiter; locomotor disturb
Glycogen storage; local irritant; retina changes; thyroid stimulation
Endometrium transform; gonad changes; pituitary; prolactin cells ⇑ Thymus atrophy; local irritant; thyroid inhibition
Goiter
Goiter; locomotor disturb
Bromide
Fig. 5. Thyroid stimulation after exposure to the goitrogens bromide (rat, upper panels) and propylthiouracyl (zebrafish, lower panels). Thyroid stimulation in the right panels is evident by increased follicle cell height and decrease in follicle.
P.W. Wester et al. / Toxicology 205 (2004) 27–32
31
Fig. 6. TBTO induced thymus atrophy in guppy (upper right) and rat (lower right). Left panels are controls.
Bromide was tested as the use of methylbromide as applied in glasshouse fumigation could result in residues in the aquatic environment or in food commodities. Studies revealed that bromide was a powerful thyroid inhibitor in both rats (Loeber et al., 1983) and fish (Wester et al., 1988). This was not surprising as the thyroid axis is also well preserved following the same principles in either class. A practical difficulty, however, is the fact that the thyroid is not an encapsulated organ in fish and therefore is not available for organ weight determination. Thus the only approach for detecting thyroid active compounds is analysis of histopathology or circulating hormone levels. In a recent study with the thyroid inhibitor propylthiouracil (Fig. 5) we demonstrated that the sensitivity of both methods was in the same order of magnitude (Wester et al., 2003). TBTO was tested in the eighties for its potential risk as residue in the aquatic food chain. TBTO is nowadays classified as endocrine disrupter mainly because of its masculinisation potential in prosobranch
snails due to inhibition of aromatisation (Oehlmann and Schulte-Oehlmann, 2002). Despite various endocrine effects at higher concentrations, the significant atrophy of the thymus at lower doses received more attention (Fig. 6) both in rats (Krajnc et al., 1984) and fish (guppies) (Wester and Canton, 1987). As is the case with the thyroid, also the thymus in fish is not accessible for organ weight determination and thus this diagnosis also depends on careful histopathology.
5. Conclusion Comparative pathology of small fish and mammals using endocrine active chemicals has learned us that effects are largely similar; obviously, class and species specific anatomical, physiological and pathological pathways need to be considered. This allows us to use fish as an alternative test animal, for instance when aquatic studies are required, and as a suitable second laboratory animal species in hazard identification.
32
P.W. Wester et al. / Toxicology 205 (2004) 27–32
Introduction of histopathology is essential in this respect (van der Ven et al., 2003) and consequently, in order to foster training and education we have created a digital histopathology atlas of small fish that is available through the Internet. This atlas contains most of the fish studies described here (van der Ven and Wester, 2002).
References Alison, R.H., Capen, C.C., Prentice, D.E., 1994. Neoplastic lesions of questionable significance to humans. Toxicol. Pathol. 22, 179–186. Kime, D.E., 1998. Endocrine Disruption in Fish. Kluwer Academic Publishers, London. Krajnc, E.I., Wester, P.W., Loeber, J.G., van Leeuwen, F.X.R., Vos, J.G., Vaessen, H.A.M.G., van der Heijden, C.A., 1984. Toxicity of Bis(tri-n-butyltin)oxide in the rat. I. Short-term effects on general parameters and on the endocrine and lymphoid system. Toxicol. Appl. Pharmacol. 75, 363–386. Loeber, J.G., Franken, M.A.M., van Leeuwen, F.X.R., 1983. Effect of sodium bromide on endocrine parameters in the rat as studied by immunocytochemistry and radioimmunoassay. Fd Chem. Toxic. 21 (4), 391–404. Oehlmann, J., Schulte-Oehlmann, U., 2002. Endocrine disruptors: Invertebrates. In: Keller, R., Dircksen, H., Sedlmeier, D., Vaudry, H. (Eds.), Proceedings of the 21st Conference of European Comparative Endocrinologists. Bonn, Germany, 26–30 August 2002. Monduzzi Editore, Bologna, pp. 63–69.
van den Belt, K., Wester, P., van der Ven, L.T.M., Verheyen, R., Witter, H., 2002. Time-dependent effects of ethynylestradiol on the reproductive physiology in zebrafish (Danio rerio). Environ. Toxicol. Chem. 21, 767–775. van Velsen, F.L., Danse, L.H., van Leeuwen, F.X., Dormans, J.A.M.A., van Logten, M.J., 1986. The subchronic oral toxicity of the beta-isomer of hexachlorocyclohexane in rats. Fundam. Appl. Toxicol. 6, 697–712. van der Ven, L.T.M., Wester, P.W., 2002. Toxicological pathology atlas of small laboratory fish (http://www.rivm.nl/fishtoxpat/). van der Ven, L.T.M., Wester, P.W., Vos, J.G., 2003. Histopathology as a tool for the evaluation of endocrine disruption in zebrafish. Environ. Toxicol. Chem. 22, 908–913. Wester, P.W., Canton, J.H., Bisschop, A., 1985. Histopathological study of Poecilia reticulata (guppy) after long-term -hexachlorocyclohexane exposure. Aquat. Toxicol. 6, 271– 296. Wester, P.W., Canton, J.H., 1986. Histopathological study of Oryzias latipes (medaka) after long-term -hexachlorocyclohexane exposure. Aquat. Toxicol. 9, 21–45. Wester, P.W., Canton, J.H., 1987. Histopathological study of Poecilia reticulata (guppy) after long-term exposure to bis(trin-butyltin)oxide (TBTO) and di-n-butyltinchloride (DBTC). Aquat. Toxicol. 10, 143–165. Wester, P.W., Canton, J.H., Dormans, J.A.M.A., 1988. Pathological effects in freshwater fish Poecilia reticulata (guppy) and Oryzias latipes (medaka) following methyl bromide and sodium bromide exposure. Aquat. Toxicol. 12, 323–344. Wester, P.W., van der Ven, L.T.M., van den Brandhof, E.J., Vos, J.H., 2003. Identification of Endocrine Disruptive Effects in te Aquatic Environment: a Partial Life Cycle Study in Zebrafish. Report 640920. RIVM, the Netherlands, p. 112.