Effects of BPA in snails : Oehlmann et al. respond

We welcome critical appraisals that help to provide balance; however, Dietrich et al. gave an unjustified reproach. We feel that Dietrich’s position is severely compromised because he serves as an expert for the bisphenol A (BPA) Industry Group (Brussels, Belgium). We would like to respond to the issues raised by Dietrich et al., as well as to their oversights and inappropriate interpretations of our findings..

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

Bisphenol A Induces Superfeminization in the Ramshorn Snail (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

Biological impact of organotin compounds on mollusks in marine and freshwater ecosystems

Organotin compounds, like tributyltin (TBT) and triphenyltin (TPT), degrade only slowly in the environment. Today, TBT is known to evoke a variety of effects in animals, even at very low environmental concentrations. In terms of sensitivity, none rivals that of two virilisation phenomena-intersex and imposex-in prosobranch gastropods. The effects of TBT and TPT on various marine and freshwater snails were investigated in newly developed sediment biotests in the laboratory. Most tested species showed very low effect concentrations for both organotin compounds (e. g. the freshwater mudsnail Potamopyrgus antipodarum with an EC10 of 0.03 μg TPT as Sn/kg and 0.98μg TBT as Sn/kg for the reduction of fertility). In order to assess the actual TBT contamination of coastal sediments, extensive surveys were carried out in France and Ireland, indicating that there is still a continuing threat for sensitive marine organisms. To obtain information on the contamination of fluvial and estuarine sediments in Germany, an effect monitoring was conducted using the biotests with the netted whelk Nassarius reticulatus and P. antipodarum. From our investigations we may conclude that a variety of benthic mollusks is strongly affected by low concentrations of organotin compounds in aquatic ecosystems. Due to their persistence in the environment, a continuing impact on aquatic wildlife has to be expected

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

No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals

This article is made available through the Brunel Open Access Publishing Fund. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.Estrogen receptor orthologues in molluscs may be targets for endocrine disruptors, although mechanistic evidence is lacking. Molluscs are reported to be highly susceptible to effects caused by very low concentrations of environmental estrogens which, if substantiated, would have a major impact on the risk assessment of many chemicals. The present paper describes the most thorough evaluation to-date of the susceptibility of Marisa cornuarietis ER and ERR gene transcription to modulation by vertebrate estrogens in vivo and in vitro. We investigated the effects of estradiol-17β and 4-tert-Octylphenol exposure on in vivo estrogen receptor (ER) and estrogen-related receptor (ERR) gene transcription in the reproductive and neural tissues of the gastropod snail M. cornuarietis over a 12-week period. There was no significant effect (p > 0.05) of treatment on gene transcription levels between exposed and non-exposed snails. Absence of a direct interaction of estradiol-17β and 4-tert-Octylphenol with mollusc ER and ERR protein was also supported by in vitro studies in transfected HEK-293 cells. Additional in vitro studies with a selection of other potential ligands (including methyl-testosterone, 17α-ethinylestradiol, 4-hydroxytamoxifen, diethylstilbestrol, cyproterone acetate and ICI182780) showed no interaction when tested using this assay. In repeated in vitro tests, however, genistein (with mcER-like) and bisphenol-A (with mcERR) increased reporter gene expression at high concentrations only (>10−6 M for Gen and >10−5 M for BPA, respectively). Like vertebrate estrogen receptors, the mollusc ER protein bound to the consensus vertebrate estrogen-response element (ERE). Together, these data provide no substantial evidence that mcER-like and mcERR activation and transcript levels in tissues are modulated by the vertebrate estrogen estradiol-17β or 4-tert-Octylphenol in vivo, or that other ligands of vertebrate ERs and ERRs (with the possible exception of genistein and bisphenol A, respectively) would do otherwise.BBSR

Superfeminization as an effect of bisphenol A in \u3ci\u3eMarisa cornuarietis\u3c/i\u3e & Response from Forbes et al. to Oehlmann et al.

[Oehlmann et al.]: Recently, we had the opportunity to read two articles in volume 66 of Ecotoxicology and Environmental Safety. Forbes et al. (2007a, , 2007b) declare “to explore the reproducibility of prior work” showing that bisphenol A (BPA) induces superfeminization in the freshwater snail Marisa cornuarietis (Oehlmann et al., 2000, 2006; Schulte- Oehlmann et al., 2001). Based on the outcome of a toxicity test with the same species, the authors conclude that their results “do not support previous claims of enhanced reproduction in M. cornuarietis in response to exposure to BPA.” We take issue with the declaration of exploring the reproducibility of our challenged work and the validity of the conclusions made by Forbes et al. (2007a, 2007b). Furthermore, we feel the toxicity test is flawed because its experimental design and the selected exposure conditions result in an irresponsiveness of test animals to the tested compound. ... [Forbes et al.]:We thank the editor for giving us the opportunity to respond to Oehlmann et al.’s comments (Oehlmann et al., 2008) on our papers (Forbes et al., 2007a,b). In response to the criticisms of our studies we would first like to emphasize that it was not our aim to exactly repeat the experiments of Oehlmann and colleagues, but rather to produce robust and reproducible results that were statistically valid and that therefore could be used in the risk assessment of bisphenol A (BPA). We are disturbed by the highly speculative suggestion by Oehlmann et al. that we may have studied a different ‘cryptic’ species of Marisa cornuarietis, as there is no evidence to support such suggestion. Furthermore, the snails used in our studies were collected from a documented pristine field site and identified to species by Dr. Sharon File-Emperador from University of Puerto Rico, a recognized snail expert. They were reared for a known number of generations under tightly controlled and documented conditions. In contrast, the snails used in Oehlmann et al.’s studies (e.g., Oehlmann et al., 2006) came from the Dusseldorf Zoo (original site of field collection not indicated) and were periodically supplemented with snails from Florida (location not indicated). There is likewise very little information in the published studies about the culture and husbandry conditions of Oehlmann et al.’s snails, whereas a major portion of our research aimed to identify appropriate husbandry and culture conditions for M. cornuarietis. It is on the basis of such detailed study that our toxicity tests with BPA were conducted

Hydrobia ulvae imposex levels at Ria de Aveiro (NW Portugal) between 1998 and 2007: a counter-current bioindicator?

Imposex expression in prosobranch gastropods has been widely used as a biomarker of tributyltin (TBT) pollution. Estuaries have been described as the most affected areas by this problem since they usually enclose the main TBT sources—ports, dockyards and marinas—resulting from the compound’s application as a biocide in antifouling paints on ships. Using Hydrobia ulvae as a bioindicator, the current work addresses the most reliable methods to reduce the influence of critical variables, such as the animals’ size, on imposex levels assessment for TBT pollution monitoring and presents its temporal trends from 1998 to 2007 in Ria de Aveiro (NW Portugal) to evaluate the effectiveness of recent legislation applied to reduce TBT environmental levels. H. ulvae imposex levels did not decrease in this estuarine system during the last decade despite the implementation of the EU Regulation No. 782/2003. Instead, there was a global significant increase in the percentage of females affected by imposex and a slight increase of the vas deferens sequence index (VDSI), contrasting with what has been described for other bioindicators in the same study area. These results show that different biology/ecology traits determine distinct routes of TBT uptake and/or bioaccumulation, pointing the importance of choosing the bioindicator depending on the compartment that is being monitored (sediment vs. water). Sediment ingestion as feeding habit is discussed and pointed as a reason to choose H. ulvae as a bioindicator of TBT pollution persistence in sediment. It is therefore predicted that the response of different prosobranch species around the world may diverge according to the compartment that is being monitored and that female masculinisation may not be completely eradicated in the near future due to TBT persistence in sediments