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

Können Umweltchemikalien zur Verweiblichung führen?

Umweltchemikalien, die nach ökotoxikologischen Gesichtspunkten bisher als unbedenklich galten, können hormonähnliche Wirkungen entfalten. Diese sogenannten "endocrine disruptors" stören die normalen hormongesteuerten Abläufe im Körper und da ihre bisher beobachteten Effekte in den verschiedenen Vertebratenklassen hauptsächlich auf östrogenartigen Wirkungen beruhten, wurden all diese Phänomene unter dem Begriff der "Verweiblichung" zusammengefaßt. Ziel des hier vorgestellten Forschungsvorhabens ist es mit Amphibien als Studienmodell zu klären, ob in Baden-Württemberg die Gewässerbelastung mit Umweltchemikalien zum Phänomen der Verweiblichung führen kann, was auch ein Indikator für eine potentielle Gefährdung des Menschen wäre. Die Untersuchungen hierzu umfassen bei Xenopus laevis verschiedene Nachweisebenen zur östrogenen Potenz von Umweltchemikalien: (1) Nachweis der Bindung an den Östrogenrezeptor in der Leber, was die Voraussetzung für eine hormonähnliche Wirkung darstellt, (2) biologische Wirkung auf zellulärer Ebene durch Bestimmung der Vitellogeninsynthese in Hepatocyten-Primärzellkulturen, die durch Östrogene spezifisch induziert wird, und (3) Wirkung in vivo auf das Gesamttier mit Experimenten zum Nachweis der nduktion der Vitellogeninsynthese bei adulten Tieren und der Beeinflussung der Geschlechtsdifferenzierung bei der Kaulquappenentwicklung. Die Ergebnisse belegen mit Amphibien als Studienmodell die verschieden gute Eignung der 3 Nachweisebenen zur Bestimmung östrogenartiger Wirkungen. Recently several studies showed that environmental chemicals without toxical risks exhibit humoral effects. These so called "endocrine disruptors" disturb normal endocrine feed back mechanisms and mainly estrogenic effects were observed in all classes of vertebrates causing "feminization" phenomena. The aim of our present research using amphibians as a model is to study if environmental water pollution in Baden-Württemberg is able to cause feminization phenomena, which would also indicate a potential risk for humans. The assessment of estrogenic potencies of environmental chemicals includes several levels of investigation using the amphib Xenopus laevis: (1) binding of environmental chemicals to liver estrogen receptor, (2) biological significance at cellular level by assaying vitellogenin synthesis in primary cultured hepatocytes, and (3) in vivo effects on intact animals by experiments determining induction of vitellogenin synthesis in adult animals and influences of sexual determination during larval development. Our results indicate several suitabilities of all 3 levels of investigation for assessment of estrogenic effects using amphibs as model

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

Does Atrazine Influence Larval Development and Sexual Differentiation in Xenopus laevis?

Debate and controversy exists concerning the potential for the herbicide atrazine to cause gonadal malformations in developing Xenopus laevis. Following review of the existing literature the U.S. Environmental Protection Agency required a rigorous investigation conducted under standardized procedures. X. laevis tadpoles were exposed to atrazine at concentrations of 0.01, 0.1, 1, 25, or 100 μg/l from day 8 postfertilization (dpf) until completion of metamorphosis or dpf 83, whichever came first. Nearly identical experiments were performed in two independent laboratories: experiment 1 at Wildlife International, Ltd. and experiment 2 at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB). Both experiments employed optimized animal husbandry procedures and environmental conditions in validated flow-through exposure systems. The two experiments demonstrated consistent survival, growth, and development of X. laevis tadpoles, and all measured parameters were within the expected ranges and were comparable in negative control and atrazine-treated groups. Atrazine, at concentrations up to 100 μg/l, had no effect in either experiment on the percentage of males or the incidence of mixed sex as determined by histological evaluation. In contrast, exposure of larval X. laevis to 0.2 μg 17β-estradiol/l as the positive control resulted in gonadal feminization. Instead of an even distribution of male and female phenotypes, percentages of males:females:mixed sex were 19:75:6 and 22:60:18 in experiments 1 and 2, respectively. These studies demonstrate that long-term exposure of larval X. laevis to atrazine at concentrations ranging from 0.01 to 100 μg/l does not affect growth, larval development, or sexual differentiation

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

COMPRENDO: Focus and approach

Tens of thousands of man-made chemicals are in regular use and discharged into the environment. Many of them are known to interfere with the hormonal systems in humans and wildlife. Given the complexity of endocrine systems, there are many ways in which endocrine-disrupting chemicals (EDCs) can affect the body’s signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. A major concern is that some of these EDCs appear to be biologically active at extremely low concentrations. There is growing evidence to indicate that the guiding principle of traditional toxicology that “the dose makes the poison” may not always be the case because some EDCs do not induce the classical dose–response relationships. The European Union project COMPRENDO (Comparative Research on Endocrine Disrupters—Phylogenetic Approach and Common Principles focussing on Androgenic/Antiandrogenic Compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (AACs) to wildlife and humans by focusing on the commonalities and differences in responses to AACs across the animal kingdom (from invertebrates to vertebrates)

Dagstuhl-Manifest zur Strategischen Bedeutung des Software Engineering in Deutschland

Im Rahmen des Dagstuhl Perspektiven Workshop 05402 "Challenges for Software Engineering Research" haben führende Software Engineering Professoren den derzeitigen Stand der Softwaretechnik in Deutschland charakterisiert und Handlungsempfehlungen für Wirtschaft, Forschung und Politik abgeleitet. Das Manifest fasst die diese Empfehlungen und die Bedeutung und Entwicklung des Fachgebiets prägnant zusammen

Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of Xenopus laevis

Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17β-estradiol (E2), at concentrations from 0.2 to 6.0 μg E2 L−1 in Experiment 1A, and 0.015 to 2.0 μg E2 L−1 in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC50 of 0.12 μg E2 L−1. The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis