Ulrike Schulte-Oehlmann

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.

A critical analysis of the biological impacts of plasticizers on wildlife

This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol A (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng l−1 to µg l−1 range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.

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.

Bisphenol A induces superfeminization in the ramshorn snail Marisa cornuarietis(Gastropoda: Prosobranchia) at environmentally relevant concentrations.

Previous investigations have shown that bisphenol A (BPA) induces a superfeminization syndrome in the freshwater snail Marisa cornuarietis at concentrations as low as 1 μg/L. Superfemales are characterized by the formation of additional female organs, enlarged accessory sex glands, gross malformations of the pallial oviduct, and a stimulation of egg and clutch production, resulting in increased female mortality. However, these studies were challenged on the basis of incomplete experimentation. Therefore, the objective of the current approach was to bridge several gaps in knowledge by conducting additional experiments. In an initial series of experiments, study results from the reproductive phase of the snails were evaluated in the sub-micrograms per liter range. Before and after the spawning season, superfemale responses were observed [NOEC (no observed effect concentration) 7.9 ng/L, EC10 (effective concentration at 10%) 13.9 ng/L], which were absent during the spawning season. A further experiment investigated the temperature dependence of BPA responses by exposing snails at two temperatures in parallel. The adverse effect of BPA was at least partially masked at 27°C (EC10 998 ng/L) when compared with 20°C (EC10 14.8 ng/L). In M. cornuarietis, BPA acts as an estrogen receptor (ER) agonist, because effects were completely antagonized by a co-exposure to tamoxifen and Faslodex. Antiandrogenic effects of BPA, such as a significant decrease in penis length at 20°C, were also observed. Competitive receptor displacement experiments indicate the presence of androgen- and estrogen-specific binding sites. The affinity for BPA of the estrogen binding sites in M. cornuarietis is higher than that of the ER in aquatic vertebrates. The results emphasize that prosobranchs are affected by BPA at lower concentrations than are other wildlife groups, and the findings also highlight the importance of exposure conditions.

Endocrine disruption in invertebrates

Recent reports have shown that a number of xenobiotics in the environment are capable of interfering with the normal endocrine function in a variety of animals. The overwhelming majority of the studies on the effects of hormone-mimetic industrial chemicals were focused on findings in vertebrates. More detailed information about the effects on and mechanisms of action in invertebrates has only been obtained from a few cases, although invertebrates represent more than 95 % of the known species in the animal kingdom and are extremely important with regard to ecosystem structure and function. The limited number of examples for endocrine disruption (ED) in invertebrates is partially due to the fact that their hormonal systems are rather poorly understood in comparison with vertebrates. Deleterious endocrine changes following an exposure to certain compounds may easily be missed or simply be unmeasurable at present, even though a number of studies show that endocrine disruption has probably occurred. The well-documented case studies of tributyltin effects in mollusks and of insect growth regulators, the latter as purposely synthesized endocrine disruptors, are explained to support this view. According to our present knowledge, there is no reason to suppose that such far-reaching changes are in any sense unique. The additional existing evidence for ED in invertebrates from laboratory and field studies are summarized as an update and amendment of the EDIETA report from 1998. Finally, conclusions about the scale and implications of the observed effects are drawn and further research needs are defined.

Evidence for Endocrine Disruption in Invertebrates

The issue of endocrine disruption (ED) in invertebrates has generated remarkably little interest in the past compared to research with aquatic vertebrates in this area. However, with more than 95% of all known species in the animal kingdom, invertebrates constitute a very important part of the global biodiversity with key species for the structure and function of aquatic and terrestrial ecosystems. Despite the fact that ED in invertebrates has been investigated on a smaller scale than in vertebrates, invertebrates provide some of the best documented examples for deleterious effects in wildlife populations following an exposure to endocrine-active substances. The article provides an overview of the diversity in endocrine systems of invertebrates. The principal susceptibility of invertebrates to endocrine-active compounds is demonstrated with the case studies of tributyltin effects in mollusks and of insect growth regulators, the latter as purposely synthesized endocrine disrupters. The additional evidence for ED in invertebrates from laboratory and field studies is summarized as an update and amendment of the EDIETA report from 1998. Finally, conclusions about the scale and implications of the observed effects are drawn and research needs are defined.

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.

Spoilt for choice: A critical review on the chemical and biological assessment of current wastewater treatment technologies.

The knowledge we have gained in recent years on the presence and effects of compounds discharged by wastewater treatment plants (WWTPs) brings us to a point where we must question the appropriateness of current water quality evaluation methodologies. An increasing number of anthropogenic chemicals is detected in treated wastewater and there is increasing evidence of adverse environmental effects related to WWTP discharges. It has thus become clear that new strategies are needed to assess overall quality of conventional and advanced treated wastewaters. There is an urgent need for multidisciplinary approaches combining expertise from engineering, analytical and environmental chemistry, (eco)toxicology, and microbiology. This review summarizes the current approaches used to assess treated wastewater quality from the chemical and ecotoxicological perspective. Discussed chemical approaches include target, non-target and suspect analysis, sum parameters, identification and monitoring of transformation products, computational modeling as well as effect directed analysis and toxicity identification evaluation. The discussed ecotoxicological methodologies encompass in vitro testing (cytotoxicity, genotoxicity, mutagenicity, endocrine disruption, adaptive stress response activation, toxicogenomics) and in vivo tests (single and multi species, biomonitoring). We critically discuss the benefits and limitations of the different methodologies reviewed. Additionally, we provide an overview of the current state of research regarding the chemical and ecotoxicological evaluation of conventional as well as the most widely used advanced wastewater treatment technologies, i.e., ozonation, advanced oxidation processes, chlorination, activated carbon, and membrane filtration. In particular, possible directions for future research activities in this area are provided.

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.

Imposex in Nucella lapillus and intersex in Littorina littorea: interspecific comparison of two TBT-induced effects and their geographical uniformity

Two different tributyltin (TBT)-induced virilisation phenomena in prosobranch snails — intersex in Littorina littorea and imposex in Nucella lapillus — are compared in order to facilitate their simultaneous use in geographical large scale effect monitoring surveys. Imposex in dogwhelks is a more sensitive biomarker and should be used in areas that are only slightly or moderately contaminated with TBT (ambient TBT concentrations < 2.0 ng as Sn 1−1). The assessment of intersex intensities in periwinkle populations has considerable advantages in areas with higher TBT concentrations and should be used also wherever dogwhelks are absent irrespective of the TBT exposure level. The intersex index (ISI) and vas deferens sequence (VDS) index are proposed as the most suited parameters for effect monitoring purposes. The geographical uniformity of intersex and imposex is analysed and proven for the coasts of Ireland, France, and Germany. A relative loss of TBT sensitivity in females can be found, but to a varying extent in both species. The implications of this result for biological TBT effect monitoring programmes are discussed in light of the fact that intersex and imposex have both been found to be irreversible. Because it is the objective of these programmes to assess current TBT contaminations and resulting biological effects, only relatively young specimens should be considered in the sampling strategy.