Background and objectives There is an urgent need to detect, assess, and reduce effects of hormonally active compounds and endocrine disrupters in aquatic systems, as reflected in national research programs like the Swiss NRP 50 "Endocrine Disruptors" and its consensus platforms. As a medium-term measure, the EU strategy on endocrine disruptors (SEC(2007)1635) uses the Endocrine Disruptor Testing and Assessment (EDTA) Task Force of the Organisation for Economic Co-operation and Development (OECD) along with other research activities. In particular the test methods of the OECD that are currently in validation or already validated may contribute to a better understanding of the extent of endocrine disruption, in particular if they are applied on environmental samples and in the context of risk-assessment strategies, for instance in waste water treatment. This article aims to give an overview and an evaluation on available and validated biological test systems for the detection of endocrine disruptive and reproductive effects in aquatic systems. Based on this a recommendation for a modular ecotoxicological test platform is given. The study focuses on test methods for sex hormone active substances.
Material and methods On the basis of an extensive literature search and ongoing international validation efforts by the OECD for methods to detect endocrine disruptive effects, 15 biological test methods (5 in vivo and 10 in vitro) were selected. Comprising, for example, of eight OECD methods and three out of five in-vitro methods mentioned in the Global Water Research Coalition (GWRC) report "Tools to detect estrogenic activity in environmental waters" (Leusch 2008). Experienced users and developers were then asked to rate the test according to given relevant criteria. The resulting criteria profiles were compiled, compared, and evaluated.
Results The methods were selected on the basis of validation status, distribution, their suitability for standardisation, and their proven sensitivity for environmental samples. We assumed that specific YES/YAS-procedures and the ER/AR Calux systems achieve the mentioned criteria. In the case that strong cytotoxicity of environmental samples affects the applicability of cellular reporter gene assays, alternatively a molecular receptor binding assay (e. g. ELRA) could be used. Additional molecular receptor binding assays are currently in validation by the OECD. Modulating effects on steroidgenesis and probably even on aromatase activity can be detected by the OECD validated H295R Steroidgenesis Assay. Its applicability for environmental samples is currently tested. As in-vivo methods different fish assays (e. g. Fish Screening Assay and the Zebrafish Embryo Test) were evaluated. For the detection of effects on the thyroid-associated hormone system the Amphibian Metamorphosis Assay (XEMA, for Xenopus Metamorphosis Assay) will soon be available as an OECD-Guideline. Because of trends in the international community to avoid tests with vertebrates in the future, only an optional recommendation was given for such tests. Reproduction toxicity, on the other hand, can be sensitively tested by the tests using the gastropod Potamopyrgus antipodarum, which, as invertebrate tests, do not require a permit to perform animal testing procedures . This test is also sensitive for many substances which are able to induce an endocrine disruption in vertebrates. Its validation is currently performed by the OECD. The applicability of the herein proposed test methods of the modular test platform for environmental-sample assessments were previously confirmed by several published scientific studies.
Discussion We propose several test methods to be included in an application-oriented modular test platform. These tests should be suitable to efficiently indicate different mechanisms of endocrine disruption and reproduction toxicity, and may be employed in different situations according to their respective advantages. The modular test platform is able to detect impacts on the reproduction relevant effects in invertebrates, amphibians, and fishes. Furthermore the different mode of actions of estrogenic and androgenic receptor binding, steroid genesis and perhaps even the modulation of aromatase are detectable. A research report on the relevance of endocrine substances and pharmaceuticals in aquatic environments (Moltmann et al. 2007) showed that endocrine end-points of in-vivo tests (31 of 71 tested substances) tend to be more sensitive than general ecotoxicological end-points, such as mortality and growth. Consequently, endocrine and general-toxic effects should be detected in an integrative manner by ecotoxicological test platforms.
Conclusions By combining literature research with a targeted query for information about the chosen test procedures it was possible to obtain a detailed overview about the current state-of-the-art of science and technology in the detection of hormone-active effects and reproduction toxicity. Ecotoxicological and regulative aspects were considered equally, and the applicability of the test procedures was evaluated. Because of the diversity of endocrine disrupting mechanisms, a modular combination of in-vivo and in-vitro methods in a joint test platform is needed to recognise and differentiate the transitions from hormone-active effects to endocrine disruption.
Recommendations and perspectives Our study leads to a proposal of a modular ecotoxicological test platform, which offers an integrative detection of hormone-active and reproduction-relevant effects in the aquatic environment. The modular system presented here allows the switching between test modules according to the continuously developing state-of-the-art of science and technology as well as the incorporation of novel developments. Further standardisation of such methods for regulative applications is recommended.