In the early 1990s, many Canadian pulp and paper mills implemented process changes to comply with new regulations that came into effect in 1993. These regulations placed stricter guidelines on a number of parameters in effluent discharges, including limits on acute toxicity, on the discharges of suspended solids, and on biochemical oxygen demand. To meet these new regulations, many of the older Canadian pulp and paper mills had to install secondary treatment systems. The investment by the Canadian pulp and paper industry was in excess of $5 billion, and the implementation of the new regulations and the process changes took several years. The new regulations were an extension of regulations designed in the early 1970s and were not designed specifically to address the reproductive responses recently reported in fish collected downstream of mills in Scandinavia and North America. This report describes a series of projects conducted between 1991 and 1996 to evaluate the effectiveness of the new regulations to address the issue of reproductive responses in fish associated with exposure to pulp-mill effluents. These studies have shown that the existing short-term bioassays do not adequately predict the potential of effluents to affect reproduction in wild fish. Laboratory testing using fathead minnows exposed over a full life cycle confirmed depression in sex steroid production, delay in sexual maturity, reduced egg production, and changes in secondary sex characteristics documented at some sites. Our studies demonstrated that both steroid hormone changes and induction of liver detoxification enzymes take place quickly. While short-term exposures can predict the potential of some effluents to impact steroid hormone production, there is no readily available assay that can be widely applied. In the absence of a usable and transferable laboratory bioassay, field collections were conducted at a number of sites. Generalizations are not possible at this time, but impacts have been seen at a variety of sites, and partial recovery has been documented at five sites in North America following various process and waste treatment changes. Data gaps and critical research areas are identified.

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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.