Kelly R. Munkittrick

Exposure to bleached kraft pulp mill effluent disrupts the pituitary-gonadal axis of white sucker at multiple sites

Our recent studies have demonstrated reproductive problems in white sucker (Catostomus commersoni) exposed to bleached kraft pulp mill effluent (BKME) at Jackfish Bay on Lake Superior. These fish exhibit delayed sexual maturity, reduced gonadal size, reduced secondary sexual characteristics, and circulating steroid levels depressed relative to those of reference populations. The present studies were designed to evaluate sites in the pituitary-gonadal axis of prespawning white sucker affected by BKME exposure. At the time of entry to the spawning stream, plasma levels of immunoreactive gonadotropin (GtH)-II (LH-type GtH) in male and female white sucker were 30- and 50-fold lower, respectively, than the levels in fish from a reference site. A single intraperitoneal injection of D-Arg6, Pro9N-Et sGnRH (sGnRH-A, 0.1 mg/kg) increased plasma GtH levels in male and female fish at both sites, although the magnitude of the response was greatly reduced in BKME-exposed fish. Fish at the BKME site did not ovulate in response to sGnRH-A, while 10 of 10 fish from the reference site ovulated within 6 hr. Plasma 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) levels were depressed in BKME-exposed fish and unlike fish at the reference site, failed to increase in response to sGnRH-A. Testosterone levels in both sexes and 11-ketostestosterone levels in males were elevated in fish from the reference site but were not further increased by GnRH treatment. In contrast, BKME-exposed fish exhibit a transitory increase in testosterone levels in response to the GnRH analog. In vitro incubations of ovarian follicles obtained from fish at the BKME site revealed depressed basal secretion of testosterone and 17,20 beta-P and reduced responsiveness to the GtH analog human chorionic gonadotropin and to forskolin, a direct activator of adenylate cyclase. By comparison, ovarian follicles from fish collected at BKME and reference sites produced similar levels of prostaglandin E basally and in response to a phorbol ester and calcium ionophore A23187, suggesting that BKME effects on ovarian function are selective and do not reflect a general impairment of ovarian function. BKME-exposed fish had plasma levels of testosterone glucuronide proportionately lower than those of reference fish, suggesting that there are site differences in the peripheral metabolism of steroids. These studies demonstrate that BKME exposure affects reproduction by acting at multiple sites in the pituitary-gonadal axis.

Changes in hepatic mixed-function oxygenase (MFO) activity, plasma steroid levels and age at maturity of a white sucker (Catostomus commersoni) population exposed to bleached kraft pulp mill effluent

Abstract The impacts of bleached kraft mill effluent (BKME) on a white sucker (Catostomus commersoni) population were examined during May, July and August 1989, and compared with two reference sites. At the time of this study, the effluent received only primary treatment. BKME-exposed white sucker exhibited increased liversomatic indices and elevated mixed-function oxygenase (MFO) activity in both July and August. They also showed lower gonadosomatic indices and an increased age to maturity. The females contained fewer eggs at maturity, while the males had reduced development of secondary sexual characteristics. These fish also had severe reductions in plasma steroid levels throughout the year, including testosterone, and 17α,20β-dihydroxyprogesterone in both sexes, as well as 11-ketotestosterone in males and 17β-estradiol in females. BKME-exposed white sucker were shorter, older and had decreased growth rates compared to those at the reference sites. These fish also exhibited an increased condition factor, yet showed decreased visceral lipid stores. Relative to those at the reference sites, the stomach contents of the BKME-exposed fish revealed reduced numbers of organisms per gut, reduced taxa per gut and an increased number of empty stomachs. The decreased energetic commitment to reproduction, along with the increased condition factor, suggested a disruption in metabolic capability and altered energy allocation in fish exposed to BKME.

An overview of recent studies on the potential of pulp‐mill effluents to alter reproductive parameters in fish

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

Exposure to bleached kraft pulp mill effluent reduces the steroid biosynthetic capacity of white sucker ovarian follicles

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.

Endocrine Toxicants and Reproductive Success in Fish

Abstract White sucker exposed to bleached kraft mill effluent (BKME) show a number of reproductive alterations correlated to reduced circulating reproductive steroid levels. The purpose of this experiment was to examine the steroid biosynthetic capacity of white sucker ovarian follicles undergoing vitellogenesis to determine if reductions in steroid production contribute to the reduced circulating steroid levels found during this stage of reproduction. We also examined the mechanisms responsible for steroid production within the ovary to determine where BKME exerts its impacts. Follicles collected from BKME-exposed fish during early and late vitellogenesis had reduced testosterone and 17β-extradiol production under basal and human chorionic gonadotropin stimulated conditions. Production of cyclic 3′,5′ adenosine monophosphate (cAMP) by ovarian follicles was similar between sites suggesting impacts occurred post-cAMP formation. Reduced conversion of exogenous testosterone to 17β-estradiol during August collections demonstrates reduced levels of the aromatase enzyme in BKME-exposed follicles during early vitellogenic stages. Measurement of pregnenolone production in October indicates similar P450 side chain cleavage enzyme activity between sites, suggesting disruptions downstream of pregnenolone formation were responsible for the reduced steroid productivity in ovarian follicles from BKME-exposed fish during this stage of the reproductive cycle.

Isolation of MFO inducers from tissues of white suckers caged in bleached kraft mill effluent

There is compelling evidence on a global scale for compromised growth and reproduction, altered development, and abnormal behaviour in feral fish that can be correlated or in some cases causally linked with exposure to endocrine disrupting chemicals (EDCs). Attributing cause and effect relationships for EDCs is a specific challenge for studies with feral fish as many factors including food availability, disease, competition and loss of habitat also affect reproduction and development. Even in cases where there are physiological responses of fish exposed to EDCs (e.g., changes in reproductive hormone titres, vitellogenin levels), the utility of these measures in extrapolating to whole animal reproductive or developmental outcomes is often limited. Although fish differ from other vertebrates in certain aspects of their endocrinology, there is little evidence that fish are more sensitive to the effects of EDCs. Therefore, to address why endocrine disruption seems so widespread in fish, it is necessary to consider aspects of fish physiology and their environment that may increase their exposure to EDCs. Dependence on aquatic respiration, strategies for iono-osmotic regulation, and maternal transfer of contaminants to eggs creates additional avenues by which fish are exposed to EDCs. This paper provides an overview of responses observed in feral fish populations that have been attributed to EDCs and illustrates many of the factors that need consideration in evaluating the risks posed by these chemicals.

Fish health in the Peace, Athabasca and Slave river systems

White sucker (Catostomus commersoni) caged for 3 days in a bleached kraft mill effluent (BKME) stream had elevated mixed function oxygenase (MFO) activities 15-90 fold those of fish caged in a reference stream. Liver composites of male and female fish were ground and extracted with dichloromethane (DCM), methanol or 50% DCM/methanol, and tested for MFO activity in rat hepatoma cells (H4IIE). There was no difference in the potency of H4IIE EROD induction among the three solvents, so DCM extracts were split into 31 fractions using reverse phase high pressure liquid chromatography (HPLC). H4IIE MFO activity was elevated in several fractions, with three early peaks and several later peaks of induction, indicating several classes or compounds causing MFO induction were present in the fish livers. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and chlorinated diphenylethers (CDPEs) were detected in several late-eluting fractions, but concentrations were not high enough for these compounds to be solely responsible for the observed induction. Induction by liver extracts decreased as cell exposure times increased (24, 48 or 72 h), suggesting that some inducers were more easily metabolized and eliminated from the H4IIE cells. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378-TCDD) had similar potency over 24, 48 and 72 h, as it was relatively resistant to metabolism.

Impact of bleached kraft mill effluent on population characteristics, liver MFO activity, and serum steroid levels of a lake superior white sucker (Catostomus commersoni) population

The response of large river systems to human development isoften hard to predict. The spatial scale of these systems makes themdifficult to study and their ecology (particularly in the case of largenorthern rivers) is often poorly understood. To provide an ecologicalassessment of three large river basins in northern Canada, the NorthernRiver Basins Study (NRBS) undertook a multi-disciplinary approach toassess environmental and socio-economic impacts of development. Resultspresented here focus on key findings relating to studies of fish healthwithin the basins. It was known at the outset of the NRBS that dioxins,furans and other organic contaminants would be present in the system.However, NRBS research indicated low levels of environmentalcontamination, particularly compared to other systems in Canada andelsewhere in the world. In addition, contaminant loads in fish generallyconform to Canadian guidelines for both aquatic and human health;although, levels of dioxins, furans, PCBs and mercury in biota didexceed guidelines at certain times and in certain locations. The weightof evidence indicates that many of the fish in these basins exhibitsigns of physiological stress. Of particular concern was the findingthat sex hormone levels in burbot and longnose sucker collected fromnear-field pulp-mill locations were significantly depressed, and thatnumbers of immature fish in these same locations were unexpectedly highand more likely to show external abnormalities. In addition, there is aperception that fish in the lower reaches of the Peace-Athabasca basinsand in the deltas are of lower quality. Studies of fish health have beenintegrated with other technical studies in a cumulative effectassessment and will provide a basis for future research and managementdecisions within these basins.