Determining the effects of a mixture of an endocrine disrupting compound, 17a-ethinylestradiol, and ammonia on fathead minnow (Pimephales promelas) reproduction Brandon M. Armstrong a , James M. Lazorchak b,⇑ , Cheryl A. Murphy a,c , Herman J. Haring d , Kathleen M. Jensen e , Mark E. Smith d a Department of Fisheries and Wildlife, Michigan State University, 13 Natural Resources, East Lansing, MI 48824, United States b U.S. Environmental Protection Agency, National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268, United States c Lyman Briggs College, Michigan State University, Holmes Hall, East Lansing, MI 48824, United States d The McConnell Group c/o U.S. Environmental Protection Agency, 26 W. Martin Luther King Dr., Cincinnati, OH 45268, United States e U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804, United States highlights  We utilized a 21D reproduction protocol to determine the effects of EE2 and NH 3 .  Exposure to a mixture of EE2 and NH 3 at their respective NOAEC induced mortality.  Exposure to EE2, NH 3 and their mixture at their NOAEC did not affect reproduction. article info Article history: Received 20 February 2014 Received in revised form 26 May 2014 Accepted 14 June 2014 Handling Editor: S. Jobling Keywords: Ammonia Ethinylestradiol Mixture Mortality Fecundity Promelas abstract Aquatic organisms are exposed to a multitude of contaminants and to fully understand the impact of multiple stressors on fish populations, we must first understand the mechanism of action for each toxicant and how the combined effects manifest at the level of the individual. 17a-ethinylestradiol (EE2) has been known to cause adverse reproductive effects including reduced fecundity and fertility, intersex and skewed sex ratios in fish by mimicking naturally produced estrogen at low concentrations. Ammonia can cause adverse reproductive and mortality effects in individual fish through effects or dam- age to the central nervous system. Both EE2 and ammonia are found in most municipal effluents in var- ious concentrations. A flow-through diluter system was used to test the individual effects of these two contaminants at their respective no observable adverse effect concentration (NOAEC) as well as their combined effects on fathead minnow, (Pimephales promelas) reproduction in a mixture exposure. While neither contaminant nor their mixture altered reproduction in terms of fecundity, their mixture resulted in significant fathead minnow mortality during a 21 d exposure. This study demonstrated the need to consider mixture effects when assessing risk for toxicity testing with multiple stressors. Ó 2014 Published by Elsevier Ltd. 1. Introduction Concern over the harmful effects of trace chemicals found in wastewater treatment plant (WWTP) effluent has risen over the past two decades with the discovery that such chemicals often act as endocrine disruptors (Lange et al., 2001; Parrott and Blunt 2005; Kidd et al., 2007; Bergman et al., 2013). Wastewater treatment plant effluent is often comprised of a mixture of chemicals from household and industrial sources and little is known about either the effects of individual endocrine disrupting chemicals (EDCs) on fish and wildlife or their combinations. A mix- ture of chemicals can have unexplained interactive effects on the endocrine system of fish and other aquatic organisms (Jobling and Tyler, 2003). One group of EDCs found in effluents are xenoestrogens, chemicals that mimic naturally produced estrogen and have the potential to disrupt the endocrine system during the critical sexual development period (Lange et al., 2001). These estrogen mimics http://dx.doi.org/10.1016/j.chemosphere.2014.06.049 0045-6535/Ó 2014 Published by Elsevier Ltd. ⇑ Corresponding author. Tel.: +1 513 569 7076. E-mail address: lazorchak.jim@epa.gov (J.M. Lazorchak). Chemosphere 120 (2015) 108–114 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere