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The Santa Ana River (SAR) is the primary source of groundwater recharge for the Orange County Groundwater Basin in coastal southern California. Approximately 85% base flow in the SAR originates from wastewater treatment plants operated by three dischargers. An on-line, flow-through bioassay using Japanese medaka (Oryzias latipes) as a means of judging potential public health impacts was employed to evaluate the water quality of the surface water and shallow groundwater originating from the SAR. Three chronic (3-4.5 mo) exposures using orange-red (outbred, OR) and see-through (color mutant, ST-II) Japanese medaka as bioindicators were conducted to evaluate endocrinologic, reproductive, and morphologic endpoints. No statistically significant differences in gross morphological endpoints, mortality, gender ratios, and vitellogenin induction were observed in fish from SAR groundwater treatment compared to the group tested in solute reconstituted reverse osmosis-treated or granular activated carbon (GAC)-treated control waters. Significant differences were observed in egg reproduction and the time to hatch in SAR groundwater; however, total hatchability was not significantly lower. To evaluate the estrogenic activity of the surfacewater source of the groundwater, SAR surface water was evaluated for vitellogenin and gonadal histopathology in juvenile medaka with no effects observed. These results demonstrate that OR Japanese medaka may be a sensitive strain as an on-line monitor to predict potential impacts of water quality, but further studies are needed to elicit causative agents within the water mixture. [ABSTRACT FROM AUTHOR]