Extending the toxicity-testing paradigm for freshwater mussels: Assessing chronic reproductive effects of the synthetic estrogen 17α-ethinylestradiol on the unionid mussel Elliptio complanata Jeremy A. Leonard a, ⁎, W. Gregory Cope a , Edward J. Hammer b , M. Christopher Barnhart c , Robert B. Bringolf d a Department of Applied Ecology, Box 7617, North Carolina State University, Raleigh, NC 27695, USA b U.S. Environmental Protection Agency, 77 West Jackson Blvd., Chicago, IL 60604, USA c Department of Biology, 901 South Avenue, Missouri State University, Springfield, MO 65897, USA d Warnell School of Forestry and Natural Resources, University of Georgia, 180 East Green Street, Athens, GA 30602, USA abstract article info Article history: Received 22 June 2016 Received in revised form 30 August 2016 Accepted 1 September 2016 Available online 06 September 2016 Surface water concentrations of the synthetic estrogen 17α-ethinylestradiol (EE2) as low as 1 ng/L can cause adverse reproductive effects in fish under acute and chronic exposure conditions, whereas higher concentrations (N 5 ng/L) in acute studies are necessary to elicit adverse effects in freshwater mussels. Prolonged chronic expo- sures of freshwater mussels to EE2 remain un-evaluated. An extended duration testing paradigm was used to examine reproductive and biochemical (carbohydrate, lipid, protein) effects of EE2 on the unionid mussel, Elliptio complanata, throughout its reproductive cycle. Mussels were exposed to a control and EE2 concentrations (5 and 50 ng/L) in six discrete and sequential 28 d tests, and in one discrete and simultaneous 180 d test, from February through August. Foot protrusion and siphoning behavior were recorded daily, along with conglutinate releases and larval (glochidia) mortality. Gonad, hemolymph, and gonad fluid samples were taken for biochemical and vitellogenin-like protein (Vtg) analysis post-exposure. Female mussels released eggs and conglutinates during the months of April to June, indicating sexual maturation during this time. Conglutinates released in the 5 ng/L treatment in 28 d exposures contained fewer glochidia and more eggs, and increased concentrations of Vtg in hemolymph were observed from April to August in the 5 ng/L treatment during the 180 d exposure. Results in- dicate that the 180 d test approach, concurrent with the sequence of 28 d tests, enabled a more robust evaluation of mussel behavior and physiology than would have been possible with a single short-term (28 d) test. © 2016 Elsevier Inc. All rights reserved. Keywords: carbohydrate Elliptio complanata ethinylestradiol glochidia lipid protein Unionid vitellogenin 1. Introduction Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and can alter behavior, neurodevelopmental processes, primary and secondary sex characteristics, and reproductive success of exposed individuals. Studies of adverse effects in aquatic vertebrates caused by estrogenic or androgenic compounds are numerous (Guillette et al., 2000; Parks et al., 2001; Sumpter and Johnson, 2008). Evidence of endocrine disruption in bivalves exposed to individual estrogenic compounds and urban effluents has also been observed (Ortiz-Zarragoitia and Cajaraville, 2005). Much of the estrogenic activity of contaminants found in surface waters arises from their incomplete biodegradation in wastewater treatment processes prior to discharge (Desbrow et al., 1998). 17α-ethinylestradiol (EE2) is often used as a synthetic contraceptive agent or in estrogen replacement therapy. It is frequently excreted in the urine and feces of individuals taking the medication and retained in surface water after wastewater treatment (Hannah et al., 2009). EE2 is a lipophilic compound (log K ow = 3.67), and as such, easily diffuses across the lipid bilayer membrane of vertebrate cells to activate nuclear estrogen receptors, which in turn activate estrogen response elements of hormone-regulated genes (Filby et al., 2007). Induction of the hepatic egg-precursor protein vitellogenin (Vtg) is a reliable indica- tor of exposure to estrogenic EDCs like EE2 in aquatic vertebrates such as teleost fishes (Orn et al., 2003), and may also indicate such exposures in the marine mussel Mytilus edulis (Ciocan et al., 2010). Concentrations of EE2 in surface water have been found to range from below detection limits at 0.01 ng/L (Hinteman et al., 2006) to as high as 273 ng/L (Kolpin et al., 2002), though the latter value is likely orders of magnitude above concentrations that are commonly measured. It is estimated that concentrations are likely ~2–5 ng/L after sewage treatment and instream dilution and degradation (Hannah et al., 2009). Concentrations of EE2 in surface water as low as Comparative Biochemistry and Physiology, Part C 191 (2017) 14–25 ⁎ Corresponding author at: United States Environmental Protection Agency, 109 TW Alexander Dr., Mail Code E205-1, Research Triangle Park, NC 27709, USA. E-mail address: leonard.jeremy@epa.gov (J.A. Leonard). http://dx.doi.org/10.1016/j.cbpc.2016.09.002 1532-0456/© 2016 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc