Ecotoxicology, 9, 383᎐397, 2000 ᮊ 2000 Kluwer Academic Publishers. Manufactured in The Netherlands. Effects of Endocrine Disruptors on Prosobranch Snails ()Mollusca: Gastropoda in the Laboratory. Part I: Bisphenol A and Octylphenol as Xeno-Estrogens JORGÈ OEHLMANN,* ULRIKE SCHULTE-OEHLMANN, MICHAELA TILLMANN AND BERND MARKERT Internationales Hochschulinstitut Zittau, Lehrstuhl Umwelt¨erfahrenstechnik, Fachgruppe Human- und Okotoxikologie,È Markt 23, D-02763 Zittau, Germany Abstract. The effects of suspected endocrine disrupting chemicals on freshwater and marine proso- branch 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-estro- genic model compounds bisphenol AŽ. BPA and octylphenol Ž. OP are presented at nominal concentra- tion ranges between 1 and 100 ␮grL. 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 concentrationsŽ. 1 ␮g BPA or OPrL , 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. Keywords: endocrine disruptors; xeno-estrogens; bisphenol A; octylphenol; snails *To whom correspondence should be addressed: IHI Zittau, Markt 23, D-02763 Zittau, Germany. E-mail: oehlmann@ihi- zittau.de. 384 J. Oehlmann et al. Introduction as endocrine modulators on freshwater and ma- rine prosobranch species in the laboratory. Most Recent reports have shown that a number of of the results were obtained during a research xenobiotics in the environment are capable of project for the German Federal Environmental interfering with the normal endocrine function in AgencyŽ. project code 216 02 001r04 between a variety of animals and also in humans. Some of September 1997 and May 2000. The objective was the reported effects of suspected endocrine dis- to develop an organismic invertebrate test system ruptors in humans include decreased sperm for the simultaneous identification of either an- counts, increased cases of breast, testicular and drogen- or estrogen-mimicking chemicals. Re- other forms of reproductive cancers, genital ab- cently, gonochoristic prosobranchs were rated as normalitiesŽ. e.g. hypospadia, cryptorchidism , pre- the most promising candidates for this purpose mature puberty in females, and increased cases of next to insects and crustaceansŽ deFur et al., endometriosisŽ. Gist, 1998 . The overwhelming 1999. This first publication is focussed on the majority of the studies on the effects of hormone- effects of two suspected xeno-estrogens, bisphe- mimetic industrial chemicals were focussed on nol AŽ. BPA and octylphenol Ž. OP . The next findings in vertebrates. More detailed informa- papers will be dedicated to the xeno-androgen tion about the effects on and mechanisms of triphenyltin and the xeno-antiandrogen vinclo- action in invertebrates has only been obtained zolin. from a few cases although invertebrates represent The first evidence that bisphenol A and more than 95% of the known species in the alkylphenols could be estrogenic was published in animal kingdomŽ. deFur et al., 1999 . The limited the 1930s on the basis of feeding experiments number of examples for endocrine disruption in with BPA and 4-propylphenol to ovariectomised invertebrates is partially due to the fact that their ratsŽ. Dodds and Lawson, 1936, 1938 . More re- hormonal systems are rather poorly understood in cent research has highlighted the implications of comparison with vertebrates. Deleterious en- these effects. The growth of human breast cancer docrine changes following an exposure to certain cellŽ. MCF-7 cultures is affected by octylphenol compounds may therefore easily be missed or at concentrations as low as 0.1 ␮M20Ž.␮grL simply be unmeasurable at present, even though a Ž. number of field investigations and laboratory e.g. Soto et al., 1991 . Estrogenic effects have studies show that endocrine disruption has proba- also been shown in tissue and cell culture experi- bly occurredŽ. for review: deFur et al., 1999 . The ments with rainbow trout hepatocytes, chicken example of tributyltinŽ. TBT compounds and their embryo fibroblasts and in mammalian estrogen masculinising effects in about 150 species of receptor assaysŽ Jobling and Sumpter, 1993; White prosobranch molluscs shows that apparently triv- et al., 1994; Yamakoshi et al., 2000. ial biochemical changesŽ inhibition of aromatase Bisphenol A is manufactured for the plastics activity according to Bettin et al.Ž. 1996 , can have industry as an intermediate in the production of drastic effects up to the population and commu- polycarbonate and epoxy resins. A smaller amount nity levels by a final sterilisation of affected fe- is used in the manufacture of thermopaper, tyres, males. According to Matthiessen and GibbsŽ. 1998 dental composites and sealantsŽ. BUA, 1997 . The there is no reason to suppose that such far-re- total BPA consumption in Germany was approxi- aching changes are in any sense unique. The main mately 163,000 t in 1994. In 1993, the correspond- endocrine effects of TBT in these molluscs are ing amounts for Western Europe were 347,000 t, the induction of imposex, an additional formation for the USA 552,000 t and Japan 214,000 tŽ BUA, of male sex characters like penis andror vas 1997. deferens on femalesŽ Gibbs et al., 1987; BPA is not readily biodegradableŽ less than Oehlmann, 1994. and of intersex which is charac- 1% transformation in 28 days in sewage treat- terised by a modification or supplanting of female ment plants according to Howard, 1989. although by male sex organsŽ. Bauer et al., 1995, 1997 . with sufficiently adapted microorganisms, it can This publication is the first in a series which be eliminated to more than 90% in the laboratory investigates effects of compounds suspected to act and in industrial sewage treatment plants. Under Endocrine Effects on Prosobranchs. I: Xeno-Estrogens 385 environmental conditions neither hydrolysis nor of tens of ␮grL of a wide range of alkylphenolic photolysis is likelyŽ. BUA, 1997 . compoundsŽ. Ahel et al., 1994b . Low levels were There is evidence from estrogen receptor reported for drinking water in the USA, with a Ž.Greim, 1998 and MCF-7 assays Ž Krishnan et al., total concentration of alkylphenols of almost 1993. that BPA exhibits an estrogen-mimetic ac- 1 ␮grL and a concentration of OP2EO of 32 tion at concentrations as low as 2᎐5 ␮grL. The ngrLŽ. Clark et al., 1992 . results for in vivo studies with mammals are con- flictingŽ. see Discussion . Materials and methods The occurrence of BPA in aquatic ecosystems is summarised by RippenŽ. 1999 . In effluents from The experiments were performed with two dif- sewage treatment plants in BerlinŽ. Germany ferent gonochoristic prosobranch species, the concentrations of up to 160 ngrL were detected freshwater ramshorn snail, Marisa cornuarietis in 1997Ž mean: 80 ngrL; median: 60 ngrL; ns Ž.Mesogastropoda: Ampullariidae , and the ma- 12. The highest concentrations in rivers were rine dogwhelk Nucella lapillus ŽNeogastropoda: reported from JapanŽ. Tokyo region with values Muricidae. Marisa specimens came from our own between 10 and 1,900 ngrL. In smaller rivers laboratory breeding stock which was built up with near Berlin concentrations of up to 410 ngrL specimens obtained from the breeding stock of were reported for 1997Ž mean: 23 ngrL; median: Aquazoo DusseldorfŽ. Germany in 1991. Dog- 6ngrL; ns41. In the same river system sedi- È whelks came directly from the field and were ments were contaminated with - 5᎐150 ␮g collected at Mean Melen, Brittany, in March 1999. BPArkgŽ.Ž dry wt. mean and median: 42 ␮grkg; ÂÂ For all laboratory experiments, a 24 h ns19.Ž. In the Rhine estuary The Netherlands Ž.weekends: 48 h semi-static renewal system in 60 concentrations were between -10 and 119 ngrL litre glass aquaria filled with tap waterŽ. for Marisa in 1989. The measured bioconcentration factors or artificial seawaterŽ. for Nucella; salinity 35½ for BPA in carp Ž.-100 , calculated values Ž BCF and provided with an Eheim power filter was up to 366. and the log P value of 3.32᎐3.4 ow used. The tests were performed under constant suggest a low bioaccumulation potential in aquatic conditions with a temperature of 22"1ЊC for organismsŽ. BUA, 1997 . freshwater and 14"1ЊC for marine snails; the NonylphenolŽ. NP and octylphenol are the most light dark cycle was adjusted to 12:12 h. important high production volume alkylphenols Three different series of exposure experiments with nonylphenol ethoxylatesŽ. NPnEO taking ap- were conducted with the test compounds bisphe- proximately 80% of the world market, and nol AŽ.