Assessment of Reproductive Effects in Largemouth Bass (Micropterus salmoides) Exposed to Bleached/Unbleached Kraft Mill Effluents M. S. Sepu ´lveda, 1,2 D. S. Ruessler, 2 N. D. Denslow, 3 S. E. Holm, 4 T. R. Schoeb, 5 * T. S. Gross 1,2 1 Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA 2 USGS-BRD Florida Caribbean Science Center, 7920 NW 71st Street, Gainesville, Florida 32653, USA 3 Department of Biochemistry and Molecular Biology, College of Medicine, and Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida, Gainesville, Florida 32610, USA 4 Georgia-Pacific Corporation, 133 Peachtree Street NE, Atlanta, Georgia 03030, USA 5 Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA Received: 3 January 2001/Accepted: 4 June 2001 Abstract. This study evaluated the potential effects of different concentrations of bleached/unbleached kraft mill effluent (B/ UKME) on several reproductive endpoints in adult largemouth bass (Micropterus salmoides). The kraft mill studied produces a 50/50 mix of bleached/unbleached market pulp with an estimated release of 36 million gal of effluent/day. Bleaching sequences were C 90 d 10 EopHDp and CEHD for softwood (pines) and hardwoods (mainly tupelo, gums, magnolia, and water oaks), respectively. Bass were exposed to different ef- fluent concentrations (0 [controls, exposed to well water], 10, 20, 40, or 80%) for either 28 or 56 days. At the end of each exposure period, fish were euthanized, gonads collected for histological evaluation and determination of gonadosomatic index (GSI), and plasma was analyzed for 17-estradiol, 11- ketotestosterone, and vitellogenin (VTG). Largemouth bass exposed to B/UKME responded with changes at the biochem- ical level (decline in sex steroids in both sexes and VTG in females) that were usually translated into tissue/organ-level responses (declines in GSI in both sexes and in ovarian devel- opment in females). Although most of these responses occurred after exposing fish to 40% B/UKME concentrations or greater, some were observed after exposures to 20% B/UKME. These threshold concentrations fall within the 60% average yearly concentration of effluent that exists in the stream near the point of discharge (Rice Creek), but are above the 10% effluent concentration present in the St. Johns River. The chemical(s) responsible for such changes as well as their mode(s) of action remain unknown at this time. Results from our field studies have indicated altered reproduc- tive biomarkers for largemouth bass (Micropterus salmoides) sampled in the St. Johns River, downstream from a paper mill plant (Palatka, Florida) (Sepu ´lveda 2000; Sepu ´lveda et al. 2001). Fish inhabiting effluent-dominated streams had lower circulating levels of vitellogenin (VTG), decreased concentra- tions of plasma 11-ketotestosterone (11-KT) and 17-estradiol (E 2 ), and elevated mixed-function oxygenase (MFO) activity. These biochemical changes, however, were not necessarily related to impacts at higher and more resilient levels of bio- logical organization (organ, organism, and population). Georgia-Pacific’s Palatka plant has been in operation since 1947. This kraft mill produces a 50/50 mix of bleached/un- bleached market pulp with an estimated release of 36 million gal of effluent/day. During the time this study was conducted, production averaged 1,452 air-dried metric tons (ADMT)/pulp/ day from a furnish that consisted of 80% softwood (pines) and 20% hardwoods (mainly tupelo, gums, magnolia, and water oaks). The bleaching lines manufacture paper towels and tissue paper, whereas the unbleached line produces kraft bag and linerboard. Bleaching sequences were C 90 d 10 EopHDp and CEHD for the softwoods and hardwoods, respectively, where Cd = mixture of chlorine (C) and chlorine dioxide (d) in proportions designated by subscripts; Eop = extraction with alkali and the addition of elemental oxygen (o) and hydrogen peroxide (p); H = hypochlorite; and Dp = 100% d substitution with the addition of p. At the time of this study, effluents received secondary treatment, which consisted of both anaer- obic (500-acre basin) followed by aerobic (500-acre basin) biological degradation for a retention period of 40 days. Treated effluents are discharged into Rice Creek, a small trib- utary that runs for about 5 km prior to its confluence with the St. Johns River. Because Rice Creek is a small tributary, effluents can account for a large portion of its total flow (yearly average effluent concentration is estimated to be around 60%, with a range of 50% to 97%), but by the time effluents reach the St. Johns River, concentrations have fallen below 10% (Geor- gia-Pacific, personal communication). It should be noted, how- * Present address: University of Alabama, 402 Volker Hall, 1670 University Blvd., Birmingham, Alabama 35294, USA Correspondence to: M. S. Sepu ´lveda; email: marisol_sepulveda@ usgs.gov Arch. Environ. Contam. Toxicol. 41, 475– 482 (2001) DOI: 10.1007/s002440010274 ARCHIVES OF Environmental Contamination and T oxicology © 2001 Springer-Verlag New York Inc.