A multi-assay screening approach for assessment of endocrine-active contaminants in wastewater effluent samples Chris D. Metcalfe a, ⁎, Sonya Kleywegt b , Robert J. Letcher c , Edward Topp d , Purva Wagh e , Vance L. Trudeau e , Thomas W. Moon e a Environmental and Resource Studies, Trent University, Peterborough, ON, K9J 7B8, Canada b Standards Development Branch, Ontario Ministry of the Environment, 40 St. Clair Ave. West, Toronto, ON, M4V 1M2, Canada c Ecotoxicology and Wildlife Health Division, Science and Technology Branch, Environment Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON, K1A 0H3, Canada d Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON, N5V 7T3, Canada e Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, ON, K1N 6N5, Canada HIGHLIGHTS • Tested wastewater for in vitro activity for comparison to analytical measurements of EDCs. • Estrogenic activity was detected only in extracts that contained estradiol or estrone. • Positive relationship observed for binding to thyroid transport protein and 4-OH-PBDE17. • Wastewater showed response in peroxisome proliferator receptor assay, but no relationship observed with levels of contaminants. • Feasible to use in vitro testing as a screening tool before instrumental analysis of contaminants. abstract article info Article history: Received 2 December 2012 Received in revised form 24 February 2013 Accepted 24 February 2013 Available online xxxx Keywords: Estrogens Thyroid PPAR PBDEs Pharmaceuticals Personal care products Environmental agencies must monitor an ever increasing range of contaminants of emerging concern, including endocrine disrupting compounds (EDCs). An alternative to using ultra-trace chemical analysis of samples for EDCs is to test for biological activity using in vitro screening assays, then use these assay results to direct analytical chemistry approaches. In this study, we used both analytical approaches and in vitro bioassays to characterize the EDCs present in treated wastewater from four wastewater treatment plants (WWTPs) in Ontario, Canada. Estrogen-mediated activity was assessed using a yeast estrogenicity screening (YES) assay. An in vitro competitive binding assay was used to assess capacity to interfere with binding of the thyroid hormone, thyroxine (T4) to the recombinant human thyroid hormone transport protein, transthyretin (i.e. hTTR). An in vitro binding assay with a rat peroxisome proliferator responsive element transfected into a rainbow trout gill cell line was used to evaluate binding and subsequent gene expression via the peroxisome proliferator activated receptor (PPAR). Analyses of a suite of contaminants known to be EDCs in extracts from treated wastewater were conducted using either gas chromatography with mass spectrometry (GC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS). Estrogenic activity was detected in the YES assay only in those extracts that contained detectable amounts of estradiol (E2). There was a positive relationship between the degree of response in the T4-hTTR assay and the amounts of polybrominated diphenyl ether (PBDE) congeners 47 and 99, triclosan and the PBDE metabolite, 4-OH-BDE17. Several wastewater extracts gave a positive response in the PPAR assay, but these re- sponses were not correlated with the amounts of any of the EDCs analyzed by LC-MS/MS. Overall, these data in- dicate that a step-wise approach is feasible using a combination of in vitro testing and instrumental analysis to monitor for EDCs in wastewater and other environmental matrixes. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Modulation or disruption of the homeostatic function of endocrine systems by xenobiotic compounds continues to be a concern in humans and in wildlife (Hotchkiss et al., 2008). The aquatic environment is the ultimate sink for natural and man-made chemicals. One of the primary sources for the release of endocrine disrupting compounds (EDCs) into the aquatic environment is municipal wastewater. In Canada, there are a number of classes of contaminants that have been designated as EDCs that are discharged into surface waters from wastewater treatment plants (WWTPs), including natural estrogen hormones, alkylphenol Science of the Total Environment 454-455 (2013) 132–140 ⁎ Corresponding author. Tel.: +1 705 748 1011x7272; fax: +1 705 748 1569. E-mail address: cmetcalfe@trentu.ca (C.D. Metcalfe). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.02.074 Contents lists available at SciVerse ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv