Jörg 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.

TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level

Tributyltin (TBT) exposure at different concentrations (5, 60, and 100 ng TBT as Sn/l) induces a concentration- and time-dependent imposex (=pseudohermaphroditism) development in femaleNucella lapillus andHinia reticulata. In both species the average imposex stage, termed as vas deferens sequence (VDS) index, and the average female penis length increases with increasing TBT concentration and duration of TBT exposure. Testosterone added at a concentration of 500 ng/l induces a faster and more intensive imposex development compared to that induced by the TBT concentrations used in the present experiments. Radioimmunological determination of endogenous steroid content reveals increasing testosterone titres in female gastropods exposed to TBT which correlate with the TBT concentration used and the duration of the experiment. The most marked and highest increase of the endogenous testosterone level is exhibited by females, of both species exposed to testosterone. Simulataneous exposure to TBT and to the antiandrogen cyproterone acetate which suppresses imposex development completely inN. lapillus and reduces imposex development strongly inH. reticulata proves that the imposex-inducing effects of TBT are mediated by an increasing androgen level and are not caused directly by the organotin compound itself. Further-more, TBT-induced imposex development can be suppressed in both snails by adding estrogens to the aqueous medium. These observations suggest that TBT causes an inhibition of the cytochrome P-450 dependent aromatase system which catalyses the aromatization of androgens to estrogens. The increase of the androgen content or the shift of the androgen-estrogen balance in favour of androgens induces the development of pseudohermaphroditism in marine prosobranchs. Artificial inhibition of the cytochrome P-450 dependent aromatase system using SH 489 (1-methyl-1,4-androstadiene-3,17-dione) as a steroidal aromatase inhibitor and flavone as a nonsteroidal aromatase inhibitor induces imposex development inN. lapillus as well as inH. reticulata.

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.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

The morphological expression of imposex in Hinia reticulata (Gastropoda: Buccinidae): a potential indicator of tributultin pollution

Specimens from the prosobranch Hinia reticulata collected along the coast of Brittany and Normandy from 1988 to 1991 exhibited imposex (occurrence of male parts in addition to the female genital duct) in response to tributyltin (TBT) pollution. Four stages of imposex development (1 to 4) with two different types in the Stages 1, 3 and 4 could be distinguished and have been documented with scanning electron micrographs for the first time. Furthermore, three additional alterations of the genital tract are shown. Neither TBT-induced sterilization nor sex change occurred. TBT accumulation in whole body and selected tissues is described and sex-related differences are shown. The VDS (vas deferens sequence) index, cubed and uncubed RPS (relative penis size) index and average female penis length of a population were analysed with regard to their quality as indices for TBT biomonitoring. Based on the way imposex develops in this species the VDS is recommended as the most valid index. Only in highly polluted areas should the uncubed RPS be used as a second index.

Tributyltin (TBT) effects on Ocinebrina aciculata (Gastropoda: Muricidae): imposex development, sterilization, sex change and population decline

Abstract The anatomy and histology of the male and female genital systems of the prosobranch Ocinebrina aciculata are described and compared with Nucella lapillus and Ocenebra erinacea. O. aciculata , collected at Roscoff (France) from 1989 to 1993, exhibited imposex (pseudohermaphroditism; occurrence of male parts in addition to the female genital duct) in response to tributyltin (TBT) pollution. Two stages of imposex development (4 and 5) with three different types (a, b, c) in stage 5 can be distinguished and are documented for the first time with scanning electron micrographs. Furthermore, three additional alterations of the genital tract are shown. Evidence for TBT-induced sterilization and protogyne sex change is given. This species exhibits a higher TBT-sensitivity than Nucella lapillus , the most sensitive TBT indicator species known previously. Due to TBT pollution, populations of Ocinebrina aciculata are declining in France and corresponding symptoms for this extremely endangered species are described: high numbers of sterilized females, male-biased sex ratios and poor reproductive performance and recruitment. The question of whether chemicals in the environment can influence the hormonal systems of humans and animals has been discussed for many years. At the centre of interest from early on were compounds with a known or supposed estrogenic activity and their possible impact on male reproductive health. However, another class of endocrine disruptors, environmental androgens such as TBT with their detrimental effects on females, may also cause serious problems for wildlife and for mankind.

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.