Michaela Tillmann

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 μg/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 μg 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 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 μg 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 ≥250 ng TPT-Sn/L (mean measured ±SD: ≥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.

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 µg 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.

Entwicklung eines Sedimentbiotests mit der ZwergdeckelschneckePotamopyrgus antipodarum (Gastropoda: Prosobranchia)

ZusammenfassungDerzeit bestehen nur wenige etablierte organismische Testverfahren zur Erfassung von reproduktionstoxischen Effekten, die eine Exposition gegenüber Gesamtsedimenten berücksichtigen. Ein hierfür gut geeigneter Testorganismus ist die parthenogenetische ZwergdeckeischneckePotamopyrgus antipodarum. Sie gehört zu den ovoviviparen (lebendgebärenden) Prosobranchiern und lebt in den obersten Schichten von Süß- und Brackwassersedimenten. Als empfindlicher Parameter zur Indikation reproduktionstoxisch wirkender Sedimentkomponenten erwies sich die Anzahl der (neugebildeten) Embryonen im Brutraum — dieser Endpunkt vermag zudem auf eventuell vorhandene östrogene Wirkstoffe im Sediment hinzuweisen. Mit Hilfe dieses 28 Tage umfassenden Biotests wurden in Laboruntersuchungen die Wirkungen verschiedener Umweltchemikalien, wie z.B. Triphenylzinn und Bisphenol A, aufP. antipodarum in umweltrelevanten Konzentrationsreihen getestet. Darüber hinaus wurde die Eignung des Biotests für die Ermittlung und Untersuchung der Wirkung komplexer Kontaminanten anhand von verschiedenen realen Flussedimenten unterschiedlichen Belastungsgrades — aus Neiße und Oder — erprobt.AbstractCurrently, only few organismic biotest systems are available for the assessment of effects on reproduction, which consider an exposure toward whole sediments. A well-suited test organism is the parthenogenetic mudsnailPotamopyrgus antipodarum. This ovoviviparous prosobranch snail lives in the upper layers of freshwater and brackish sediments. The number of (unshelled) embryos turned out to be a very sensitive parameter for the indication of sediment components affecting reproduction. Additionally, this endpoint allows to signal possible estrogenic sediment compounds.With this 28-day bioassay, laboratory tests on the effects of various chemicals onP. antipodarum were performed, e.g. triphenyltin and bisphenol A, in relevant environmental concentration ranges. Furthermore, the suitability of our test system for the investigation of complex and multiple level ranged contaminations was examined by means of various natural sediments from the Neiße and Odra rivers.