Reproductive Responses of Common Carp (Cyprinus carpio) Exposed in Cages to Influent of the Las Vegas Wash in Lake Mead, Nevada, from Late Winter to Early Spring ERIN M. SNYDER,* , SHANE A. SNYDER, ,‡ KEVIN L. KELLY, § TIMOTHY S. GROSS, O DANIEL L. VILLENEUVE, , | SCOTT D. FITZGERALD, ⊥ SERGIO A. VILLALOBOS, ,# AND JOHN P. GIESY Department of Zoology, National Food Safety and Toxicology Center, and Center for Integrative Toxicology, Michigan State University, 218C Food Safety and Toxicology Building, East Lansing, Michigan 48824, U.S. Bureau of Reclamation, Ecological Research and Investigations (D-8220), Denver Federal Center, P.O. Box 25007, Denver, Colorado 80225, U.S. Geological SurveysBiological Resources Division, Florida Caribbean Science Center, 7920 NW 71st Street, Gainesville, Florida 32653, and Department of Pathobiology and Diagnostic Investigation and Diagnostic Center for Population and Animal Health, P.O. Box 30076, Lansing, Michigan 48909-7576 The Las Vegas Wash (LW) delivers tertiary-treated municipal wastewater effluent, nonpotable shallow groundwater seepage, and runoff from the urbanized Las Vegas Valley to Las Vegas Bay (LX) of Lake Mead. To investigate the potential for contaminants in LW influent to produce effects indicative of endocrine disruption in vivo, adult male and female common carp (Cyprinus carpio) were exposed in cages for 42-48 d at four sites in Lake Mead: LW, LX, and two reference locations in the lake. End points examined included gonadosomatic index; gonad histology; concentrations of plasma vitellogenin (VTG) and plasma sex steroids (17-estradiol (E2), testosterone (T), 11- ketotestosterone (11-KT)); plasma estrogen:androgen ratios (E2:T, E2:11-KT), in vitro production of T by gonad tissue, and hepatopancreas ethoxyresorufin O-deethylase activity. Few differences among fish caged at different sites were potentially attributable to exposure to contaminants in LW influent. Male carp caged at LW had slightly greater concentrations of plasma VTG than those at other sites, and a modest elevation in plasma E2 was observed in male carp at LX and LW, but causes other than contaminant exposure cannot be ruled out. Water temperature differences among sites complicated interpretation of the results. Variation in some end points among carp at two different reference sites supports the use of multiple reference sites in field studies of the effects of endocrine- disrupting chemicals. Introduction Lake Mead is a large reservoir formed by impoundment of the Colorado River behind the Hoover Dam. The reservoir serves as a source of domestic and agricultural water for more than 22 million users (1) and is a popular recreation area. The Las Vegas Wash (LW) delivers tertiary-treated municipal wastewater effluent (87-88%), nonpotable shallow groundwater (6%), and urban runoff (6%) from the urbanized Las Vegas Valley (1, 2) to the Boulder Basin of Lake Mead via the Las Vegas Bay (LX) (Figure 1). LW serves as the sole drainage of the Las Vegas Valley hydrographic basin and contributes approximately 1.5% of the flow into Lake Mead, while the Colorado River provides approximately 97% of the flow, and the Virgin and Muddy Rivers contribute an additional 1.5%. In 1996, the U. S. Geological Survey (USGS) reported that endocrine disruption was occurring in feral adult male and female common carp (Cyprinus carpio) collected in the vicinity of LW and LX (3). Evidence included alterations in plasma sex steroid concentrations in male and female carp relative to those in carp at a reference site and increased concentrations of the estrogen-inducible plasma protein vitellogenin (VTG) in the blood of male carp in the absence of a concomitant increase in plasma 17-estradiol (E2). The latter indicates that the fish were exposed to an unidentified exogenous estrogenic chemical (4). Previous research has revealed increased concentrations of VTG in the blood of carp (4-10) and other cyprinids (11, 12) exposed to municipal wastewater treatment plant (WWTP) effluent in enclosures or captured from riverine sites influenced by WWTP effluent. Estrogenic chemicals found in municipal WWTP effluent and implicated as potential causative agents for VTG induction in male fish exposed to these effluents include the animal steroids E2 and estrone, the oral contraceptive medication component ethinylestradiol (EE2), and the alkylphenol polyethoxylates (APE) and their degradation products, the alkylphenols (AP) (5, 13-15). Extracts from water samples collected from LW and LX in April 1997 were subjected to a bioassay-directed fraction- ation and analysis scheme designed to identify the bioactive estrogenic constituents of the LW influent (16). Results indicated that the relatively polar compounds E2 and EE2 were largely responsible for the observed in vitro bioactivity, while relatively nonpolar compounds (polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, organo- chlorine pesticides) and moderately polar compounds (oc- tylphenol, nonylphenol) were not likely to have contributed significantly to the estrogenic activity (16). The current study was conducted in mid-February through late March to early April of 1999 to determine whether exposure to LW influent, and particularly to estrogenic contaminants found therein, causes reproductive endocrine disruption in adult carp. * Corresponding author present address: Black & Veatch Cor- poration, 4040 S. Eastern Ave., Suite 330, Las Vegas, NV 89119; phone: (702)732-0448; fax: (702)732-7578; e-mail: SnyderEM@ bv.com. Michigan State University. ‡ Present address: Southern Nevada Water Authority, 1350 Richard Bunker Ave., Henderson, NV 89015. § U.S. Bureau of Reclamation. O U.S. Geological Survey. | Present address: U.S. EPA Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804-2595. ⊥ Department of Pathobiology and Diagnostic Investigation and Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI 48824. # Present address: Syngenta Crop Protection, Inc., Ecological Toxicology & Environmental Safety, P.O. Box 18300, Greensboro, NC 27419. Environ. Sci. Technol. 2004, 38, 6385-6395 10.1021/es049690n CCC: $27.50  2004 American Chemical Society VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 6385 Published on Web 11/02/2004