Aquatic Toxicology 148 (2014) 211–220 Contents lists available at ScienceDirect Aquatic Toxicology j ourna l ho me pa ge: www.elsevier.com/locate/aquatox Molecular signatures in rainbow darter (Etheostoma caeruleum) inhabiting an urbanized river reach receiving wastewater effluents P.A. Bahamonde a,∗ , G.R. Tetreault b,c , M.E. McMaster b , M.R. Servos c , C.J. Martyniuk a , K.R. Munkittrick a a Canadian Rivers Institute and Department of Biology, University of New Brunswick, Saint John, NB, Canada E2L 4L5 b Emerging Methods Branch, Aquatic Contaminant Research Division, Water Science and Technology Directorate, Environment Canada, Burlington, ON, Canada L7R 4A6 c University of Waterloo, Department of Biology, Waterloo, ON, Canada N2L 3G1 a r t i c l e i n f o Article history: Received 3 September 2013 Received in revised form 7 January 2014 Accepted 10 January 2014 Keywords: Rainbow darter Intersex Municipal wastewater effluents Endocrine disruption Sex differentiation genes Sexual development genes a b s t r a c t Rainbow darter (Etheostoma caeruleum) is a small benthic fish species found in North America that are abundant and distributed throughout the Grand River watershed, ON, Canada. Rainbow darter exhibit intersex in males at sites adjacent to municipal wastewater effluents (MWWE). In October 2010, female and male rainbow darter were collected at 3 sites (1 upstream reference and 2 downstream exposed sites) in the Grand River near the cities of Kitchener and Waterloo. The primary objectives of this research were (1) to characterize the responses of whole organism endpoints (i.e. condition factor (K), liversomatic (LSI) and gonadosomatic index (GSI), histopathology) to MWWEs and (2) to identify transcripts show- ing altered steady state abundance with exposure to MWWE in fish inhabiting municipal wastewater effluent-exposed areas. Genes measured in this study included vitellogenin, Sry-box containing protein 9 (sox9), forkhead box L2 (foxl2), doublesex and mab-3 related transcription factor 1 (dmrt1), cytochrome P450, family 11, subfamily A, polypeptide 1 (cyp11a) as well as estrogen (esr1, esrb) and androgen (ar) receptors. There were no changes in condition factor; however, there was a significant increase in LSI and a decrease in GSI in fish inhabiting downstream environments when compared with fish collected from the reference site. Males had a high incidence (∼70%) of intersex in downstream sites; characterized by the presence of oocytes within the testis. In the gonad, there were sex specific differences for genes related to sexual differentiation; dmrt1 was only expressed in males whereas foxl2 and sox9 were highly expressed in females compared to males. Expression levels of ar and esr1 were higher in females than males. Conversely, esrb was not differentially expressed between sexes or among sites. There were no differences detected for the genes investigated within sex among sites. This study is the first to report on gene expression changes in the rainbow darter, with emphasis on the differences in transcript abundance between sexes and how these changes relate to exposures to MWWEs. Molecular approaches are being investigated for their potential application to field ecotoxicology, and molecular bioassays for relevant, sentinel species in environmental monitoring programs are required to better understand the impact of anthropogenic impacts on species at risk in river systems. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Municipal wastewater effluents (MWWEs) are a complex mix- ture of residential, institutional, and industrial effluents that have been associated with eutrophication, low dissolved oxygen, high ammonia toxicity, and endocrine disruption in aquatic organisms (Shon et al., 2006). Endocrine disrupting compounds (EDCs) are exogenous substances that cause adverse health effects in an ∗ Corresponding author. Tel.: +1 506 648 5985; fax: +1 506 648 5811. E-mail addresses: paulina.bahamonde@unb.ca, p.bahamondec@gmail.com (P.A. Bahamonde). organism, or its progeny, as a result of changes in endocrine function (OECD, 1996). Sewages effluents contain ≥100,000 anthro- pogenic substances (Jobling et al., 2009), and many of these substances have the potential to be endocrine system modula- tors, including some pharmaceuticals and personal care products (PPCPs) (Carballa et al., 2004; Lishman et al., 2006), household and industrial chemicals (Stevens et al., 2003), organic materials (Shon et al., 2006), and natural or synthetic hormones (Esperanza et al., 2007; Servos et al., 2007; Ying et al., 2009). MWWEs have been associated with a variety of impacts in fish, from more mechanistic levels of biological organization (e.g. biochemical and molecular) (Albertsson et al., 2010; Robinson et al., 2003) to more ecologi- cally relevant levels of biological organization (e.g. population and 0166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2014.01.010