The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

Analytical and Environmental Sciences Division King's College London UK

Department of Environmental and Life Sciences Università del Piemonte Orientale Vercelli Novara Alessandria Alessandria Italy

Department of Environmental Engineering Technical University of Denmark Kgs Lyngby Denmark

Diabetes and Nutritional Sciences Division King's College London UK

Eawag Swiss Federal Institute of Aquatic Science and Technology Dübendorf Switzerland ETH Zürich Department of Environmental Systems Science Swiss Federal Institute of Technology Zürich Switzerland EPF Lausanne School of Architecture Civil and Environmental Engineering Lausanne Switzerland

European Commission DG Joint Research Centre Institute for Environment and Sustainability Via E Fermi 2749 21027 Ispra Italy

Faculty of Science Masaryk University RECETOX Brno Czech Republic

Institute of Life Sciences The Hebrew University of Jerusalem Israel

Istituto Superiore per la Protezione e la Ricerca Ambientale Rome Italy

Life Science Center Örebro University Sweden

Marine Biology Station Piran National Institute of Biology Slovenia

National Institute for Industrial Environment and Risks Verneuil en Halatte France

National Institute for Industrial Environment and Risks Verneuil en Halatte France Reims University France

National Institute of Nutrition and Seafood Research Bergen Norway

Norwegian University of Science and Technology Trondheim Norway

Swiss Centre for Applied Ecotoxicology Eawag EPFL Dübendorf Switzerland

University of Natural Resources and Life Sciences Vienna Austria

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