Abstract-The androgenic potential of a New Zealand pulp and paper mill effluent was measured by applying a combinatioll of in vitro and in vivo bioassays with mosquitofish (Gambusia a!finis) and goldfish (Carassius aurarus). The in vivo method assessed the rate of gonopodial development (masculinization) and alterations from normal reproductive behavior in adult female mosqllitofish exposed for 21 d to lIntreated 01' secondary-treated pulp mill effluent. A second in vivo mosquitofish exposure tested the effect of gl ass-fiber (type C) fi Itration of secondary-treated effIlient on rates of expression of the same endpoints. Extractable organics analyses of effluents and extracts thereof were conducted. Mosqllitofish demonstrated significant masculinization on exposure to either treated 01' untreated effluent; the frequency of gonopodial development was reduced with efiluent secondary-treatment. Male mating behavior was observed in the masculinized adult females. Glass-fiber (type F) filtration of the treated effluent eliminated the masclilinizing effect, suggesting that the bioactive compounds were associated with the suspended solids. The in vitra method measlired the binding of compounds within a treated thermomechanicallbleached kraft cffluent extract to androgen receptors contained in goldfish testis cytosol. Exposure to extracts of either the particulate (glass-fiber filtered) 01' the dissolved organic fraction of the efflllent produced significant binding (as indicated by the displacement of radiolabeled testosterone) to the androgen reeeptor in goldfish gonadal tissue. Thus, the dissolved organics extract of the treated effluent contained compounds androgenic to goldfish in vitro but not to mosquitofish in vivo. The combined in vitra and in vivo data suggest that the effluent in question could exert effects on the reproductive physiology of fishes through an androgenic mechanism. The androgenic compounds androstenedione and testosterone were not detected in the extracts used far the in vitro component of this study.

The androgenic potential of a New Zealand pulp and paper mill effluent was measured by applying a combination of in vitro and in vivo bioassays with mosquitofish (Gambusia affinis) and goldfish (Carassius auratus). The in vivo method assessed the rate of gonopodial development (masculinization) and alterations from normal reproductive behavior in adult female mosquitofish exposed for 21 d to untreated or secondary-treated pulp mill effluent. A second in vivo mosquitofish exposure tested the effect of glass-fiber (type C) filtration of secondary-treated effluent on rates of expression of the same endpoints. Extractable organics analyses of effluents and extracts thereof were conducted. Mosquitofish demonstrated significant masculinization on exposure to either treated or untreated effluent; the frequency of gonopodial development was reduced with effluent secondary-treatment. Male mating behavior was observed in the masculinized adult females. Glass-fiber (type F) filtration of the treated effluent eliminated the masculinizing effect, suggesting that the bioactive compounds were associated with the suspended solids. The in vitro method measured the binding of compounds within a treated thermomechanical/bleached kraft effluent extract to androgen receptors contained in goldfish testis cytosol. Exposure to extracts of either the particulate (glass-fiber filtered) or the dissolved organic fraction of the effluent produced significant binding (as indicated by the displacement of radiolabeled testosterone) to the androgen receptor in goldfish gonadal tissue. Thus, the dissolved organics extract of the treated effluent contained compounds androgenic to goldfish in vitro but not to mosquitofish in vivo. The combined in vitro and in vivo data suggest that the effluent in question could exert effects on the reproductive physiology of fishes through an androgenic mechanism. The androgenic compounds androstenedione and testosterone were not detected in the extracts used for the in vitro component of this study.

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Rainbow trout were exposed to a secondary treated, thermomechanical/bleached kraft pulp and paper effluent in 12,000-L, flow-through exposure tanks at an environmental research facility located at a pulp and paper mill in Kawerau, New Zealand. Trout (age, 2+ years) were obtained from a local hatchery and exposed either to upstream river water or a nominal concentration of 12% (v/v) effluent diluted in upstream river water. Three treatment groups were used: Effluent exposure that started approximately three months before gonadal growth (eight-month total exposure), effluent exposure that started approximately halfway through gonadal development (two-month total exposure), and trout exposed to reference water alone for the total duration of the experiment. Trout were sacrificed just before spawning; exposure, growth, and reproductive endpoints were assessed during and at the termination of the experiment. Reduction in growth was observed in both sexes in the eight-month treatment group relative to the river water reference treatment group. No differences were observed in condition factor or liver size in either treatment. Females in the eight-month exposure group also had significantly lower ovary weight. The two-month exposure group showed no differences from the reference group in growth or somatic indices. Estradiol and testosterone were reduced in blood samples taken from the eight-month exposure group by four months into the experiment as compared to the reference treatment. Steroid and vitellogenin levels in individual female trout from this treatment were significantly correlated with gonadosomatic indices (GSI) measured at the termination of the experiment. The GSI was not correlated strongly or consistently with pregnenolone, nor were any treatment-related pregnenolone differences observed, indicating that the steroid hormone reductions likely were not related to cholesterol side-chain cleavage. Male trout showed significant induction of vitellogenin and lower 11-ketotestosterone during the experiment (only the eight-month group was examined), but this did not result in any significant differences in testes development. Thus, this study has shown an impact of pulp mill effluent exposure on the reproductive physiology of female trout that appeared to be hormonally mediated. Furthermore, the effect could only be manifest when the exposure was initiated before the start of gonad development.

Rainbow trout were exposed to a secondary treated, thermomechanical/bleached kraft pulp and paper effluent in 12,000-L, flow-through exposure tanks at an environmental research facility located at a pulp and paper mill in Kawerau, New Zealand. Trout (age, 2+ years) were obtained from a local hatchery and exposed either to upstream river water or a nominal concentration of 12% (v/v) effluent diluted in upstream river water. Three treatment groups were used: Effluent exposure that started approximately three months before gonadal growth (eight-month total exposure), effluent exposure that started approximately halfway through gonadal development (two-month total exposure), and trout exposed to reference water alone for the total duration of the experiment. Trout were sacrificed just before spawning; exposure, growth, and reproductive endpoints were assessed during and at the termination of the experiment. Reduction in growth was observed in both sexes in the eight-month treatment group relative to the river water reference treatment group. No differences were observed in condition factor or liver size in either treatment. Females in the eight-month exposure group also had significantly lower ovary weight. The two-month exposure group showed no differences from the reference group in growth or somatic indices. Estradiol and testosterone were reduced in blood samples taken from the eight-month exposure group by four months into the experiment as compared to the reference treatment. Steroid and vitellogenin levels in individual female trout from this treatment were significantly correlated with gonadosomatic indices (GSI) measured at the termination of the experiment. The GSI was not correlated strongly or consistently with pregnenolone, nor were any treatment-related pregnenolone differences observed, indicating that the steroid hormone reductions likely were not related to cholesterol side-chain cleavage. Male trout showed significant induction of vitellogenin and lower 11-ketotestosterone during the experiment (only the eight-month group was examined), but this did not result in any significant differences in testes development. Thus, this study has shown an impact of pulp mill effluent exposure on the reproductive physiology of female trout that appeared to be hormonally mediated. Furthermore, the effect could only be manifest when the exposure was initiated before the start of gonad development.