In Vivo and in Vitro Assessment of the Androgenic Potential of a Pulp and Paper Mill Effluent

Abstract

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.

Summary of a Decade of Research on the Effects of a New Zealand Pulp and Paper Mill on Reproduction in Fishes

“…In the fi rst mosquitofi sh experiment, signifi cant masculinization was demonstrated at all effl uent concentrations of treated and untreated effl uent (Ellis et al 2003). Treatment appeared to remove some of the androgenic potency of the effl uent, though masculinization was still present at environmentally relevant concentrations of 15% effl uent.…”

“…This bioassay takes advantage of the sexual dimorphism in male and female mosquitofi sh with the male possessing an elongated anal fi n which acts as a sexual organ. This organ, called a gonopodium, can be grown in a female exposed to endogenous androgenic compounds (Ellis et al 2003).…”

“…Mosquitofi sh exposures were initiated in late 1998. Mosquitofi sh were exposed to Tasman effl uent for 21 d with daily static renewal (Ellis et al 2003). Untreated effl uent at a 15% dilution and treated effl uent at 15 and 70% dilutions were utilized for those experiments.…”

“…During the mosquitofi sh exposures from 1998 to 2002, a measure the androgenic potency of the effl uents was conducted using in vitro receptor binding techniques (Ellis et al 2003;Bandelj et al 2006). These techniques involved chemical extraction of the effl uent, and concentration of the extract followed by a measure of the ability of the extract to displace radiolabelled steroid from a receptor preparation.…”

The Effects of Final Treated Effluent and In-Mill Waste Streams from a Canadian Thermomechanical Pulp and Paper Mill on Mummichog (Fundulus heteroclitus) Reproduction

“…Fish exposed to pulp mill effl uents in both fi eld and laboratory studies have shown alterations in their reproductive performance, including reduced gonad size (Munkittrick et al 1992;Parrott et al 2004), reduced egg production (Parrott et al 2004;Rickwood et al 2006), delays in maturation (Munkittrick et al 1992), male-biased sex ratios (Larsson et al 2000;Örn et al 2006), masculinization of females (Larsson and Förlin 2002;Ellis et al 2003), reduced development of secondary sexual characteristics (McMaster et al 1991), and depressed steroid hormone levels (Munkittrick et al 1992;Dubé and MacLatchy 2000).…”