An integrated assessment of estrogenicity of complex environmental samples using in vivo and in vitro effect-based tools

Assessing the risk posed by complex environmental samples is a challenging task that requires reliable tools for detection and evaluation of effects. Among hazardous compounds, endocrine disrupting chemicals (EDC) in mixtures need to be identified given their potential to interfere with wild life reproduction. In this context, the aim of our work is to apply a set of effect-based tools, both in vivo and in vitro, to environmental samples in order to gain a wide overview of adverse outcomes related to estrogenic pathways in fish. As part of the FP7-SOLUTIONS project, we screened surface water extracts of Danube River (22 sites) and extracts of Rhine tributaries (water extracts of 3 STP effluents, upstream, and downstream sites in Switzerland) using an in vitro human ER reporter system (MELN cell line). Additionally, Rhine tributaries extracts were assessed in vivo for induction of CYP19a1b gene, which is strictly ER-regulated, in transgenic zebrafish embryos (EASZY test). Results obtained so far with the MELN assay show very low estradiol-equivalents (E2-EQ) levels at Danube sites (only 3 sites displaying E2-EQ above 0.1ng/L), while slightly higher estrogenic activity was measured at Swiss sites (E2-EQ ranged from 2.9-5.5 ng/L in effluents, and 0.1-0.3 and 0.5-1.5 at upstream and downstream sites, respectively). The estrogenic activity detected in vitro in effluents was correlated to estrogenicity in vivo for one site, based on CYP19a1b induction; the other effluents displayed developmental toxicity which impeded reporter gene activation detection. To gather further information on estrogenicity of these samples, work is under progress to assess their effects on zebrafish (zf) cell lines stably transfected with zebrafish estrogen receptors (i.e. zfERalpha, zfERbeta1 and zfERbeta2). Results will be interpreted in relation to targeted chemical analyses done at these sites. Eventually, the study should provide a comprehensive assessment of estrogenicity of given environmental samples using a combination of mammalian and zebrafish cell lines, as well as an in vivo fish embryo bioassay. This study is supported by INERIS and by SOLUTIONS Project from the European Union Seventh Framework Programme (FP7-ENV-2013-two-stage Collaborative project) under grant agreement 603437