Evidence of neuroendocrine disruption in freshwater mussels exposed to municipal wastewaters F. Gagné a, ⁎, C. André a , P. Cejka b , R. Hausler c , M. Fournier d a Fluvial Ecosystem Research, Environment Canada, Montréal, Que., Canada b Montreal Wastewater Treatment Plant, Montréal, Que., Canada c STEPPE, École de Technologie Supérieure, Montréal, Que., Canada d INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, Que., Canada abstract article info Article history: Received 14 March 2011 Received in revised form 15 April 2011 Accepted 22 April 2011 Available online 24 June 2011 Keywords: Neuroendocrine disruption Municipal wastewater effluents Freshwater mussels Oxidative stress The purpose of this study was to test the hypothesis that exposure to municipal effluents can disrupt the neuroendocrine system in Elliptio complanata freshwater mussels. The capacity of ozonation to mitigate these effects was also examined. Mussels were exposed for 14 days to a continuous flow of increasing concentrations of the effluent before and after ozonation. Neuroendocrinal effects were examined by tracking changes in acetylcholinesterase, glutamate, gamma-aminobutyrate, serotonin, dopamine and their respective adenylcyclase activities in synapse membranes, monoamine oxidase and vitellogenin-like proteins. Oxidative stress and damage were examined by superoxide dismutase and lipid peroxidation, respectively, in the visceral tissues. The results revealed that the exposure of freshwater mussels increased the levels of vitellogenin-like proteins in both the primary-treated and ozonated effluents, dopamine and glutamate, and decreased the turnover of the neurostimulant acetylcholine. Moreover, these endpoints were significantly correlated with oxidative stress and damage. A canonical analysis of the responses revealed that dopamine and the neuroexcitatory neuromediators-acetylcholesterase and glutamate-were the endpoints more strongly related with oxidative stress and damage. Mussel morphology and estrogenic biomarkers (vitellogenin-like proteins, gonad lipid stores) were also significantly related, albeit to a lesser extent, to oxidative stress and damage. In general, ozone treatment was not sufficient to mitigate the observed neuroendocrinal effects in freshwater mussels. We conclude that the continuous exposure of freshwater mussels to municipal wastewater effluents leads to neuroendrocinal alterations and to oxidative stress. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. 1. Introduction Urban effluents are major sources of a complex array of pollutants in the aquatic environment. Many of the chemicals identified as potent endocrine disruptors include 17α-ethynyl-estradiol-17β (used in birth control drugs), nonylphenol (a breakdown product of alkylphe- nol polyethoxylate surfactants), and estradiol-17β (Sumpter and Jobling, 1995; Sabik et al., 2003; Verthaak et al., 2005). Moreover, these wastewaters harbor many pharmaceutical and personal care products (PPCPs; Kummerer, 2001) which can not only act as potential endocrine disruptors but disrupt the neuroendocrine pathways in organisms. For example, final maturation of gametes and spawning depends on the balance of the neurotransmitters serotonin and dopamine, which can influence the contraction of smooth muscle to assist egg release in mussels and fish (Gibbons and Castagna, 1984; Fong et al., 1993). This process is further assisted by the production of prostaglandins such as cyclooxygenase, which is the rate-limiting enzyme for the production of prostaglandins (Matsumani and Nomura, 1987; Flippin et al., 2007). Hence, the presence of commonly consumed serotonergic drugs (fluoxetine, tricyclic antidepressants), non-steroidal anti-inflammatory drugs (cyclooxygenase inhibitors) and estrogens (ethynylestradiol) could act as neuroendocrine disruptors of reproduc- tion in aquatic invertebrates and fish. Indeed, tricyclic antidepressants were shown to suppress spawning and fertilization in zebra mussels (Hardedge et al., 1997). A recent study revealed that a physico-chemical treated effluent contains significant amounts of selective serotonin- reuptake inhibitors which were bioavailable to brook trout (Lajeunesse et al., 2011). This study also revealed that sertraline liver tissue level could reach concentrations up to 10 ng/g and was negatively correlated with serotonin-dependent Na/K-ATPase in synapse membrane preparations. Freshwater mussels are key representatives of the benthic commu- nity in many lakes and rivers in countries worldwide. These organisms are particularly at risk of contamination by neuroendocrine disruptors because they are sedentary, long-lived, and filter high volumes of water and suspended matter during respiration and feeding. In searching for markers of neuroendocrine disruption beyond estrogenic chemicals, Science of the Total Environment 409 (2011) 3711–3718 ⁎ Corresponding author. E-mail address: francois.gagne@ec.gc.a (F. Gagné). 0048-9697/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2011.04.037 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv