Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17β-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17β-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

ARPA Lazio Regional Agency for Environmental Protection Via G Saredo 52 00173 Rome Italy

ARPA Lombardia Regional Agency for Environmental Protection Via Rosellini 17 20124 Milan Italy

Center for Environmental and Human Toxicology Department of Physiological Sciences College of Veterinary Medicine University of Florida Gainesville FL USA

Department Cell Toxicology Helmholtz Centre for Environmental Research UFZ Permoserstraße 15 04318 Leipzig Germany

Department of Biology Norwegian University of Science and Technology NO 7491 Trondheim Norway

Department of Plant and Environmental Sciences The Alexander Silberman Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem 91904 Israel

European Commission Joint Research Centre Via E Fermi 2749 21027 Ispra VA Italy

Federal Institute of Hydrology Am Mainzer Tor 1 D 56068 Koblenz Germany

French National Institute for Industrial Environment and Risks UMR 1 02 SEBIO 60550 Verneuil en Halatte France

Institute for the Study of Anthropic Impacts and Sustainability in Marine Environment National Research Council Via de Marini 6 Genova 16149 Italy

ISPRA Environmental Metrology Unit Via di Castel Romano 100 00128 Rome Italy

ISS National Health Institute Viale Regina Elena 299 00161 Rome Italy

Laboratório de Análises Instituto Superior Tecnico Universidade de Lisboa Av Rovisco Pais 1049 001 Lisboa Portugal

Metal Metabolism Group Department of Nutritional Sciences Faculty of Life Sciences and Medicine King's College London 150 Stamford St London SE1 9NH UK

RECETOX Faculty of Science Masaryk University Kamenice 5 CZ62500 Brno Czech Republic

School of Population Health and Environmental Sciences Faculty of Life Sciences and Medicine King's College London 150 Stamford Street London UK

Swiss Centre for Applied Ecotoxicology Überlandstrasse 133 8600 Dübendorf Switzerland

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