Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3, and 42.7 micrograms/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1, and 18.4 micrograms/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590, and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 microliters/L) and 17 beta-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 35, and 42 of exposure, and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p-nonylphenol at concentrations of 5.4 micrograms/L or greater and to methoxychlor at concentrations of 2.5 micrograms/L or greater. Exposure to 0.64 microgram/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.
