Histological alterations in ovaries of pubertal female rats induced by triphenyltin.

Triphenyltin is an organotin compound that has been used extensively as an antifouling biocide and as an agricultural pesticide. Certain organotin compounds act as endocrine-active agents and have been reported to affect reproduction in mollusks and mammals. Here we studied the histopathological effects of 2 or 6mg triphenyltin chloride (TPTCl)/kg b.w. on the reproductive tissue and the thymus of female pubertal rats as part of a comprehensive pubertal assay. Beginning at postnatal day (PND) 23 female Wistar rats were treated daily per gavage until their first estrus after PND 53. Reproductive organs were removed and histologically evaluated. While no histological changes were observed in oviduct, uterus, vagina and mamma, an increase in the number of all follicle stages occurred at both dose levels. Furthermore, exposure to 2mg TPTCl/kg b.w. led to a significant reduction in the diameter of tertiary follicles. A significant increase in the number of atretic follicles was observed in tertiary and preovulatory follicles after exposure to 6mg TPTCl. The thymus displayed a decreased number of apoptotic cells in both dose groups. We conclude that peripubertal administration of 2 and 6mg TPTCl/kg b.w. caused effects on ovarian follicles of female rats.

The effect of organotin compounds on gender specific androstenedione metabolism in the freshwater ramshorn snail Marisa cornuarietis.

In a recent study, we demonstrated that androstenedione was mainly converted to testosterone (T) and 5alpha-dihydrotestosterone (DHT) by digestive gland/gonad complex microsomal fractions isolated from male Marisa cornuarietis, whereas it was primarily metabolized to 5alpha-dihydroandrostenedione (DHA) by females. In the present work, the sexual dimorphic metabolism of androstenedione was further investigated, and attributed to a higher 17beta-hydroxysteroid dehydrogenase activity in males than in females. Thereafter, the hypothesis was tested that the metabolism of androstenedione might be affected by exposure to tributyltin (TBT) and triphenyltin (TPT), which are known to induce the development of imposex in several gastropod species. The in vitro metabolism of androstenedione, particularly the formation of DHA and DHT, was inhibited by both compounds. However, in vivo experiments showed no significant alteration in the metabolism of androstenedione in males, but a marginal (TBT) and a significant (TPT) inhibition of the formation of DHA in females exposed for 150 days to concentrations that had significantly induced the development of imposex. The ratio DHT+T/DHA, a possible indicator of metabolic androgenization, tended to increase (0.43 versus 0.35, p=0.06) in TPT exposed females. However, this ratio never reached values comparable to those found in males (11+/-1).

Tributyltin biomonitoring using prosobranchs as sentinel organisms.

Tributyltin (TBT) compounds, some of the most toxic xenobiotics, produce a variety of pathological reactions in animals. A reliable biomonitoring method to assess the degree of environmental TBT pollution has been described based on investigations of virilization phenomena in prosobranch snails (Mollusca: Gastropoda). Examples are the imposex phenomenon in marine and freshwater species, the intersex reaction in littorinids and the reduction of female sexual glands and offspring numbers in further species resulting mainly in a sterilization of females. The degree of imposex or intersex in populations is determined by different biomonitoring indices which allow to assess the TBT pollution of the environment at low costs with high precision. The effectiveness of TBT legislations is analysed by extensive surveys in France and Ireland indicating that there is still a continuing threat to sensitive marine organisms. TBT disturbs the biosynthesis of steroid hormones on the level of estrogen biosynthesis. The observed virilization phenomena seem due to an inhibition of the cytochrome P-450 dependent aromatase by this organotin compound.

Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent.

It is now well established that there is a diverse array of chemical discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wild life groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological chemicals (17alpha-ethinylestradiol; EE2, bisphenol-A, and 4-tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum, were studied and compared with the effects of EE2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas, rainbow trout (Oncorhynchus mykiss); Cyprinus carpio, carp; Cyprinus carpio). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum. Furthermore, exposure to EE2 induced similar reproductive responses in the snails as in the fathead minnow (Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 week exposure period in treated snails. Plasma vitellogenin concentrations in two species of male fish (the rainbow trout and the carp) also increased over the same time period. These data indicate that both the nature of the response and the relative sensitivities to environmental estrogens in P. antipodarum and three different fish species fish are comparable. P. andtipodarum is thus, potentially a sensitive test organism for assessing estrogenicity of chemicals with a relevance to their activity in vertebrates.

Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent.

It is now well established that there is a diverse array of chemicals discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wildlife groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological importance of endocrine disruption in wildlife. In this study, the effects of exposure to individual estrogenic chemicals (17alpha-ethinylestradiol; EE2, bisphenol-A, and 4-tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum, were studied and compared with the effects of EE2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas, rainbow trout (Oncorhynchus mykiss); Cyprinus carpio, carp; Cyprinus carpio). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum. Furthermore, exposure to EE2 induced similar reproductive responses in the snails as in the fathead minnow (Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 weeks exposure period in treated snails. Plasma vitellogenin concentrations in two species of male fish (the rainbow trout and the carp) also increased over the same time period. These data indicate that both the nature of the response and the relative sensitivities to environmental estrogens in P. antipodarum and three different fish species fish are comparable. P. antipodarum is thus, potentially a sensitive test organism for assessing estrogenicity of chemicals with a relevance to their activity in vertebrates.

The use of Littorina littorea for tributyltin (TBT) effect monitoring - Results from the German TBT survey 1994/1995 and laboratory experiments.

Field investigations and laboratory experiments were performed to test the applicability of the intersex phenomenon in Littorina littorea for biological TBT-effect monitoring and to give further insights into toxicological aspects of intersex development. The intensity of the phenomenon is correlated with the degree of TBT contamination in the environment. Statistically highly significant correlations of biological indices and TBT concentrations in tissue and sediment exist. Intersex can be induced only in juvenile and sexually immature females and its intensity depends on the ontogenetic stage of development during TBT exposure. For the first time, malformations in male periwinkles closely related to the TBT contamination can be described; the reduction of male mamilliform penial glands shows highly significant correlations to TBT concentrations in tissues. The intersex phenomenon in Littorina littorea is proposed for a biological TBT-effect monitoring in those areas where the dogwhelk Nucella lapillus, as the more sensitive species in European surveys, is absent. The intersex index (ISI) as the mean value of the intersex stages in a sample should be used as the most sensitive biological parameter for the assessment of the TBT contamination in Littorina littorea.

A multispecies approach for monitoring persistent toxic substances in the Gulf of Gdansk (Baltic sea).

Bivalve mussels are usually used for biomonitoring persistent toxic substances (PTS) in coastal ecosystems. Nevertheless, these organisms, which live attached on hard substrates, can be found along the sandy coasts only on human manufactured products. In this work different species collected in the Gulf of Gdansk were compared to evaluate their suitability for monitoring PTS pollution at a local scale. The clam Mya arenaria seems to represent an excellent indicator of sediment pollution, mainly for organotin compounds which are selectively bioaccumulated. Organochlorine compounds are bioaccumulated in the different species mainly in function of their lipid body burden. Habitat conditions (salinity, substrate, pollution), however, strongly limited the occurrence of different species in the sampling sites; the most ubiquitous species, the common shrimp Crangon crangon, resulted therefore the most suitable to be used for the comparison of PTS pollution in this aquatic environment. Although the blue mussel (Mytilus trossulus) was confirmed to be a very useful sentinel species to compare pollution level inside and outside the Gulf of Gdansk, we recommend the use of other species to give a more detailed picture of the pollution situation in coastal areas.

Some chemical contaminant of surface sediments at the Baltic Sea coastal region with special emphasis on androgenic and anti-androgenic compounds.

Androgenic and anti-androgenic compounds including p,p'-DDE, Diuron, Linuron, Fenarimol, Vinclozolin, 1-(3,4-dichlorophenyl) urea (DCPU), 1-(3,4-dichlorophenyl)-3-methylurea, (DCPMU), tributyltin (TBT) and triphenyltin (TPT) and their metabolites (DBT, MBT, DPT, MPT) as well as metallic elements (Ni, Cu, Zn, As, Cd, Pb, Co, Tl, Cr, Fe, Mn, Al, K, Mg, Na, Ca, Ba, Ti, Sn), PAHs (16 indicator compounds), DDTs and PCBs have been quantified in top layer (0-10 cm) of up to 37 surface sediment samples collected from several sites in costal zone of the Gulf of Gdansk, an inland freshwater area of Brdyujscie in Poland and the tidal flats of the Norderney Island, Wadden Sea in 2002-2003. These sites differed in the degree of anthropogenic activities, including chemical pollution and related impact on biota. Especially in sediments near shipyards, ship repair facilities, harbours, other industrial activities or close to municipal sewage treatment plant outlets butyltins, PAHs and some metallic elements were found at high concentrations. Diuron, Linuron and DCPMU were detected at a few sites, Fenarimol only once, while Vinclozolin and DCPU were not detected. DDT concentrations in the sediments from the Gdansk and Gdynia region of the Gulf show a stepwise decrease following the ban for production and use, while diffusion of PCBs at some industrial sites seems to continue. Elevated PAH concentrations in sediments seem to be mainly due to pyrogenic and less to mixed pyrogenic and petrogenic sources, while for a few sites rather petrogenic sources dominated. The reference sites in the Norderney Island, Wadden Sea showed similar or slightly higher loads of DDTs, BTs, PAHs, PCBs and metallic elements when compared to sediments from the least contaminated sites in the coastal Gulf of Gdansk area, while phenyltins were not detected at both spatially distant European areas.

Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part I: Bisphenol A and octylphenol as xeno-estrogens.

The effects of suspected endocrine disrupting chemicals on freshwater and marine prosobranch species were analysed in laboratory experiments. In this first publication, the responses of the freshwater snail Marisa cornuarietis and of the marine prosobranch Nucella lapillus to the xeno-estrogenic model compounds bisphenol A (BPA) and octylphenol (OP) are presented at nominal concentration ranges between 1 and 100 micrograms/L. Marisa was exposed during 5 months using adult specimens and in a complete life-cycle test for 12 months. In both experiments, the xeno-estrogens induced a complex syndrome of alterations in female Marisa referred to as "superfemales" at the lowest concentrations. Affected specimens were characterised by the formation of additional female organs, an enlargement of the accessory pallial sex glands, gross malformations of the pallial oviduct section resulting in an increased female mortality, and a massive stimulation of oocyte and spawning mass production. The effects of BPA and OP were comparable at the same nominal concentrations. An exposure to OP resulted in inverted U-type concentration response relationships for egg and spawning mass production. Adult Nucella from the field were tested for three months in the laboratory. As in Marisa, superfemales with enlarged accessory pallial sex glands and an enhancement of oocyte production were observed. No oviduct malformations were found probably due to species differences in the gross anatomical structure of the pallial oviduct. A lower percentage of exposed specimens had ripe sperm stored in their vesicula seminalis and additionally male Nucella exhibited a reduced length of penis and prostate gland when compared to the control. Because statistically significant effects were observed at the lowest nominal test concentrations (1 microgram BPA or OP/L), it can be assumed that even lower concentrations may have a negative impact on the snails. The results show that prosobranchs are sensitive to endocrine disruption at environmentally relevant concentrations and that especially M. cornuarietis is a promising candidate for a future organismic invertebrate model to identify endocrine-mimetic test compounds.

Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part III: Cyproterone acetate and vinclozolin as antiandrogens.

The effects of suspected endocrine disrupting chemicals on freshwater and marine prosobranch species were analysed in laboratory experiments. In this last of three publications, the responses of the fresh water snail Marisa cornuarietis and of two marine prosobranchs (Nucella lapillus, Nassarius (Hinia) reticulatus) to the antiandrogenic model compounds cyproterone acetate (CPA) and vinclozolin (VZ) are presented. The snails were exposed to nominal CPA concentrations of 1.25 mg/L alone and simultaneously to a potent synthetic estrogen (ethinylestradiol), androgen (methyltestosterone) or an indirectly acting xeno-androgen (tributyltin) in experiments with adult specimens and in a life cycle test for 12 months. Marisa and Nucella were furthermore exposed to nominal concentrations of 0.03-1.0 microgram VZ/L for up to 5 months. The antiandrogens induced a number of biological responses in all three species. The length of the penis and of accessory male sex organs (e.g., penis sheath, prostate) were significantly reduced. For Marisa, this effect occurred only in sexually immature specimens and was reversible as the males attained puberty. Typical androgen-mediated responses (imposex development, delayed spermatogenesis, tubulus necrosis of the testis with orchitis and Leydig cell hyperplasia) were partially or totally suppressed by a simultaneous administration of CPA. In the two marine species even adult, sexually mature males responded to antiandrogens with a reduction of the male sex organs and an advancement of the sexual repose phase. The results for CPA and VZ are compared with the effects of an exposure to xeno-estrogens (bisphenol A, octylphenol) and xeno-androgens (triphenyltin, tributyltin) in the same species. Each group of endocrine disruptors induces a characteristic set of toxicological effects in prosobranch snails which can be used as endpoints in an organismic invertebrate test for the identification of endocrine mimetic test compounds. Estrogens cause primarily an induction of superfemales resulting in an increased female mortality by the enhancement of spawning mass and egg production. The main effects of androgens are a virilization of females by imposex development and a marked decrease of the fecundity. Compared with estrogens and androgens, the antiandrogen responses seem to be less drastic and might have--in contrast to the two other disruptor classes--no biologically significant effects at the population level.

Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part II: Triphenyltin as a xeno-androgen.

In laboratory experiments the effects of suspected endocrine disrupting chemicals on freshwater and marine prosobranch species were analysed. In this second of three publications the responses of the freshwater ramshorn snail Marisa cornuarietis and of two marine prosobranchs (the dogwhelk Nucella lapillus and the netted whelk Hinia reticulata) to the xeno-androgenic model compound triphenyltin (TPT) are presented. Marisa and Nucella were exposed via water (nominal concentrations 5-500 ng TPT-Sn/L) and Hinia via sediments (nominal concentrations 50-500 micrograms TPT-Sn/kg dry wt.) for up to 4 months. Female ramshorn snails but not the two marine species developed imposex in a time and concentration dependent manner (EC10 4 months: 12.3 ng TPT-Sn/L) with a comparable intensity as described for tributyltin. TPT reduced furthermore the fecundity of Marisa at lower concentrations (EC10 4 months: 5.59 ng TPT-Sn/L) with a complete inhibition of spawning at nominal concentrations > or = 250 ng TPT-Sn/L (mean measured +/- SD: > or = 163 +/- 97.0 ng TPT-Sn/L). The extension of the pallial sex organs (penis with accessory structures and prostate gland) of male ramshorn snails and dogwhelks were reduced by up to 25% compared to the control but not in netted whelks. Histopathological analyses for M. cornuarietis and H. reticulata provide evidence for a marked impairment of spermatogenesis (both species) and oogenesis (only netted whelks). The test compound induced a highly significant and concentration independent increase in the incidence of hyperplasia on gills, osphradia and other organs in the mantle cavity of N. lapillus indicating a carcinogenic potential of TPT. The results show that prosobranchs are sensitive to endocrine disruption at environmentally relevant concentrations of TPT. Also, M. cornuarietis is a promising candidate for a future organismic invertebrate system to identify endocrine-mimetic test compounds.

Marisa cornuarietis (Gastropoda, prosobranchia): a potential TBT bioindicator for freshwater environments.

: The ramshorn snail Marisa cornuarietis (L., 1767) exhibits imposex (occurrence of male parts in addition to the female genital duct) under the influence of TBT (tributyltin) in laboratory experiments and accumulates this biocide in a time-and concentration-dependent manner. A comparison of BCF (bioconcentration factors) demonstrates that this limnic species accumulates more TBT than marine species. Evidence is given that TBT causes hormonal disorders which are responsible for imposex development. The testosterone/oestradiol quotient reflects the imposex development which can be described by a classification scheme differentiating four stages in Marisa cornuarietis. In the background of the constantly increasing organotin concentration in the limnic environment Marisa cornuarietis could be a well-suited bioindicator for TBT pollution. No other limnic species with a lower threshold concentration for measurable TBT effects is reported within the literature.