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A Decade of Research on the Environmental Impacts of Pulp and Paper Mill Effluents in Canada (1992-2002)

3.3 Research Program to Identify the Causative Compounds, How to Eliminate Them, and Determine Their Short and Long-Term Environmental Effects

The results from the large survey of Ontario pulp and paper mills were completed and published. This study was designed to investigate the receiving areas below a large number of pulp and paper mills to determine whether discharges were associated with any of the physiological responses evident in fish collected in Jackfish Bay , Lake Superior . It was also designed to determine whether there was any correlation between waste treatment and the presence of biological responses in wild fish, and whether there was any association between the use of chlorine as a bleaching agent and these responses (Figure 1) (Munkittrick et al., 1994a).

Alterations were demonstrated in fish collected downstream of all effluent discharges, although responses were not consistent at all sites (Munkittrick et al., 1994a). Although white sucker collected near bleached kraft mills exhibited the highest EROD induction and dioxin levels (Servos et al., 1994), elevated enzyme activity was observed in fish from sites that did not use chlorine, and depressions in sex steroid levels were not correlated with the level of EROD activity (Munkittrick et al., 1994a). The absence of chlorine bleaching or the presence of secondary treatment did not eliminate responses in fish, including decreased circulating levels of sex steroids, decreased gonadal size and increased liver size (Munkittrick et al., 1994a). Although there was a positive correlation between EROD and dioxin equivalents overall, some discrepancies suggested that additional compounds may be involved (Servos et al., 1994). There were no relationships between reproductive responses in wild fish downstream of the mill sites and dioxin equivalents (Servos et al., 1994) or with any of the receiving water laboratory toxicity tests (Robinson et al., 1994). The surveys' main findings were that a) induction of hepatic EROD enzymes and depressions of some plasma sex steroids were found downstream of several pulp and paper mills; b) these changes were seen at some mills without chlorine bleaching and at mills that had secondary treatment; c) substantial dilutions of non toxic effluent did not appear to remove these responses; d) the dominant factor determining the presence or absence of responses appeared to be dilution level; and e) lab toxicity tests on invertebrates, rainbow trout, and fathead minnows could not predict the presence of these responses in wild fish (Munkittrick et al., 1994a). In conclusion, there was no correlation between reproductive effects in wild fish and pulping process, dioxin contamination, AOX production, AOX levels in the receiving water or the use of chlorine. The identity of the chemical(s) responsible for the reproductive effects remained unknown and it was not known whether the addition of chlorine to the chemical(s) altered their potencies.

Studies also continued at a number of the locations examined prior to the new effluent regulations. Servos et al., (1992) had previously documented minimal effects at a modernized bleached kraft mill on the Spanish River in Northeastern Ontario . This mill was also included in the larger survey of Ontario mills and although some steroid differences were demonstrated, impacts on gonadal development were not found (Munkittrick et al., 1994a). However, effluent from this mill was shown by Robinson (1994) to affect sexual maturation and hormone levels in fathead minnows during chronic laboratory exposures, although at concentrations above those found in the receiving environment (Figure 1). This information was important because it demonstrated that some modernized mills may produce or release compounds capable of reproductive impacts, but fish at a specific receiving environment may not show responses for any of a variety of reasons, including species sensitivity, effluent dilution or site-specific receiving environment conditions which may offer the fish refuge from exposures. It is important to determine whether the absence of responses in wild fish at any specific site, are because the mill does not produce the chemicals or the fish are protected by some other mechanism.

Follow-up studies were also conducted at the two sites on the Moose River basin that were included in the earlier 10 mill study (Table 1). Nickle et al. (1997) conducted more detailed studies at Smooth Rock Falls (BKME) and Kapuskasing (Thermo-mechanical pulp - TMP) prior to the installation of secondary treatment at these sites in order to provide detailed pre-secondary treatment data to potentially follow recovery. His studies confirmed both physical and biochemical changes in white sucker exposed to both primary treated BKME and TMP mill effluent. Both mill effluents contained compounds that induced MFO activity, increased peroxisomal fatty acyl-CoA oxidase activity and reduced circulating sex steroid levels. However, in this study, exposure to the BKME discharge was associated with reductions in gonadal development, whereas the TMP discharge had no effects on gonad size but led to increases in liver size. Earlier studies at the TMP site however, did demonstrate differences in ovarian development downstream of the TMP discharge (Munkittrick et al., 1994a). The reproductive alterations documented did not appear to be in response to estrogenic substances present in the effluent as no induction of vitellogenin was evident. In fact reductions in vitellogenin in females downstream of the primary treated BKME site corresponded to reductions in circulating 17ß-estradiol levels (Nickel et al., 1997). Increases in peroxisomal proliferation within the liver tissue may have been partially responsible for the increase in liver size in white sucker downstream of the primary treated TMP mill in Kapuskasing.

Studies on the St. Maurice River found changes in liver glycogen levels, plasma steroids, and liver MFO levels (Hodson et al., 1992; Gagnon et al., 1994a,b). Continued study at this site examined in more detail the reproductive alterations demonstrated in the original studies. These follow-up studies concluded that the depressions in steroid levels were not related to changes in hormone biosynthesis, but rather to increased metabolism (Gagnon et al., 1994b). Some of the differences between the conclusions of the Jackfish Bay and Moose River studies and those of Gagnon et al., (1994a,b) could have been due to the effects of capture and handling stress on steroid hormone levels (Van Der Kraak et al., 1992). Follow-up studies at Jackfish Bay demonstrated that handling stress (McMaster et al., 1994) and confinement stress (Jardine et al., 1996) led to dramatic changes in physiological parameters, and that holding fish for a short time period to "recover" was not sufficient for overcoming these effects of capture stress in the white sucker species (Jardine et al., 1996).

The 2½ year, multidisciplinary study of the Wapiti/Smoky River ecosystem in northwestern Alberta was also completed. The Weyerhaeuser Canada Ltd. mill was one of the newest most modern mills in the country and during these studies upgraded their process from 25 to 70% chlorine dioxide substitution in the fall of 1990 and to 100% substitution in July of 1992. Overall findings were that AOX did not correlate with dioxins, furans, chlorinated phenolics or environmental impacts and that implementation of 70% chlorine dioxide substitution resulted in declines in dioxins/furans and chlorinated phenolics to near or below detection limits (Swanson et al., 1993). Although fish demonstrated induction of the liver detoxification system that paralleled dilution of the effluent, it was not correlated with health effects in mountain whitefish or longnose sucker. These fish did demonstrate a response related to nutrient enrichment which led to increased condition factor and increased internal fat stores (Swanson et al., 1993). The absence of significant changes in physiological parameters in the Wapiti River studies could have been related to a number of factors, including the pulping process and waste treatment employed by the mill, the dramatic seasonal changes in dilution, or the high mobility of the fish species present (Swanson et al., 1993, 1994; Kloepper-Sams & Benton, 1994; Owens et al., 1994a,b; Pryke et al., 1995).

Studies continued at the original Jackfish Bay location with a number of different objectives. Although numerous improve-ments had occurred in Jackfish Bay following the installation of secondary treatment (Karl, 1992), white sucker had not demonstrated similar improvements in reproductive function. For this reason, wild fish were continually monitored throughout the reproductive growing season for evidence of potential recovery following the installation of secondary treatment in the fall of 1989. The first signs of potential recovery in reproductive function were demonstrated in early September of 1993. Although gonadal size differences were still evident, no site differences in circulating steroid levels (Jardine, 1994) or in vitro production (McMaster unpubl. data) were found. However, circulating and in vitro differences were present in samples collected in late September and late October 1993, at this same site (McMaster et al., 1996a). The first signs of wild fish recovery corresponded well with caging studies conducted with goldfish, as studies conducted during the spring of 1993 demonstrated effluent potential to reduce steroid production, while studies conducted in the summer and fall of 1993 as well as the spring of 1994 failed to demonstrate reproductive effects using this short-term exposure protocol (McMaster et al., 1996a) (See Development and Application of Bioassays). This partial recovery in reproductive function at the time coincided with a series of mill process changes in May and June 1993 associated with increasing the level of chlorine dioxide substitution to 70% (Munkittrick et al., 1997a).

Early studies at Jackfish Bay concluded that one of the main factors responsible for lowering sex hormone levels during prespawning periods was occurring within the steroid biosynthetic pathway in a reduced ability to make steroid hormones (Van Der Kraak et al., 1992). McMaster et al. (1995a) continued these studies and determined that BKME exposed white sucker also have reduced steroid biosynthetic capacities during both early and later stages of vitellogenic growth. Further studies identified specific locations within the steroid biosynthetic pathway impacted, such as reduced aromatase activity during early vitellogenic stages and disruptions downstream of pregnenolone formation during later stages of vitellogenic growth (McMaster et al., 1995a). Continued studies into impacts in the biosynthetic pathway measured both the levels of the steroid precursor substrates and assessed the enzyme conversion rates within the steroid biosynthetic pathway. These studies identified specific lesions associated with this decreased steroid productivity and further showed that these particular sites of disruption within the pathway differed with the reproductive state of the fish. Although a number of locations within the steroid biosynthetic pathway were altered with BKME exposure, the major alteration appeared to be due to reductions in the availability of the steroid substrate cholesterol (McMaster, 1995; McMaster et al., 1996b).

Bezte et al. (1997) conducted a study on the Winnipeg River downstream of a primary treated groundwood/sulphite pulp and paper mill in Pine Falls , Manitoba (Table 1). Similar to some of the mills in the 10-mill survey, this mill also did not bleach pulp. White sucker collected downstream of the effluent discharge demonstrated induced EROD activity, altered fecundity, reduced hepatic vitamins, increased liver sizes, and reductions in circulating steroid levels (Bezte et al., 1997). Examination of the ability of ovarian follicles to produce reproductive steroids at this site also demonstrated a reduced capacity of the gonads to produce these reproductive steroids (McMaster et al., 1996b) similar to those documented at Jackfish Bay on Lake Superior (McMaster et al., 1995a).

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