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Since 1995, most mills in Canada have biotreatment in order to meet effluent regulatory limits for toxicity, biochemical oxygen demand, and total suspended solids. With occasional exceptions, the limits have been met. However, questions remain about effluent biotreatment regarding environmental protection, such as the reproductive capacity of fish. To address these concerns, a series of before-after studies were undertaken during the past decade. These included i) comparisons of effluents before and after biotreatment by means of fish (vitellogenin activity and egg production) and Ceriodaphnia (young production) tests done in the laboratory and ii) comparisons of fish communities in a river before and after the installation of effluent biotreatment at two mill sites. In all laboratory tests and with respect to all endpoints examined in these tests, the effects of the effluents after biotreatment were less or nonexistent when compared with the effects of the effluents before biotreatment. The assessment of the fish communities based on various metrics (e.g., percent piscivores, percent fish with anomalies) indicated improved conditions after the installation of biotreatment. Taken overall, the results indicated that biotreatment has improved effluent quality and this has resulted in clear improvements for the receiving environment.

To evaluate currently available bioassays for their use in investigating the causes of pulp and paper mill effluent effects on fish reproduction, the responses of wild white sucker (Catostomus commersoni) collected from the receiving environment at the bleached kraft mill at La Tuque, Quebec, were compared with responses of fathead minnow (Pimephales promelas) exposed to effluent in a laboratory lifecycle test. White sucker collected at effluent exposed sites had increased liver size but none of the reproductive effects that had been documented in earlier field studies at this site. Exposure to 1, 3, 10, 30, and 100% bleached kraft mill effluent (BKME) in the lab led to significantly decreased length, but increased weight and liver size in male fathead minnow. Female length was also decreased and liver size was increased at high effluent exposures. Most effluent concentrations (1 to 30%) significantly increased egg production compared with controls. The fathead minnow lifecycle assay mirrored the effects seen in wild fish captured downstream of the BKME discharge. These results will be used to select short-term fish tests for investigating the causes of and solutions to the effects of mill effluents on fish reproduction.

Under the Canadian Environmental Effects Monitoring (EEM) program for pulp and paper effluents, the observation of a national response pattern of decreased gonad size and increased fish condition and liver size has triggered a centralized multiagency investigation of cause (IOC) of reproductive impacts in fishes. The purpose of the component of the IOC study presented here is to compare a number of fish bioassays for determining reproductive and reproductive-endocrine effects of a bleached kraft mill effluent. The bleached kraft mill chosen for this study had demonstrated the national response pattern in previous EEM cycles. The bioassays employed to examine reproduction were fathead minnow (Pimephales promelas) 5- and 21-d, mummichog (Fundulus heteroclitus) 25-d, and zebrafish (Danio rerio) 7-d tests, all of which had egg production as the primary reproductive endpoint. Additional bioassays examining reproductive-endocrine endpoints included a 7-d mummichog test, a 7- and a 21-d threespine stickleback (Gasterosteus aculeatus) test, a rainbow trout (Oncorhynchus mykiss) 7-d test, and in vitro sex steroid receptor and plasma protein binding bioassays. The zebrafish and fathead minnow reproductive tests showed significant suppression of egg production at the 100% effluent concentration. Endocrine data derived from the tests showed that this effluent did not impact steroidogenic endpoints at any concentration. Bioassays showed that this effluent i) was capable of eliciting cytochrome P4501A induction at as low as 10% vol/vol effluent, ii) was weakly androgenic at 10% vol/vol, and iii) showed no evidence of in vivo estrogenicity. These results were consistent with in vitro receptor binding assays showing a highly variable level of androgenic equivalents over six months of effluent testing, with little evidence of estrogenic activity. Bioassay results were consistent in that the overall conclusion was that this effluent has only a weak potential to cause reproductive impairment and would likely not do so at environmentally relevant concentrations. Field studies and a fathead minnow lifecycle study conducted concurrently were in agreement with reproductive bioassay results as white sucker exposed in the receiving environment no longer had significantly reduced gonadal development. Overall, this study provided evidence that the laboratory assays evaluated for various reproductive endpoints have potential application for future IOC work.

Pulp and paper mill effluents have been reported to cause changes in reproductive indicators of fish in laboratory and field studies. These changes include reduced egg production and gonad size, and altered hormone levels and expression of secondary sex characteristics. We examined the performance of biotreatment plants for their potential in abating effects of pulp and paper mill effluents on fish reproduction under laboratory conditions. A bleached kraft mill effluent (BKME) treated in an aerated lagoon and a thermomechanical pulp mill effluent (TMPE) treated by aerobic sludge in a sequential batch reactor were selected for study. Mature fathead minnows (Pimephales promelas) were exposed to effluents before and after biotreatment under continuous renewal conditions for 21 days. Egg production was monitored daily, while morphometric parameters (length, weight, gonad size), secondary sexual characteristics, and steroid hormone and vitellogenin levels were measured at the end of the effluent exposure. The effluent from both mills before biotreatment impaired the reproductive capacity of minnows (egg production) at concentrations of 10 and 20% vol/vol, but not at 2% vol/vol. Exposure to biotreated effluents from both mills at concentrations of 2, 10, 20, and 40% vol/vol caused no significant differences in overall reproductive capacity of minnows as compared with controls. These results indicate that biotreatment can significantly improve the quality of a BKME and an effluent from a TMP mill with respect to the reproductive capacity of fish as determined in laboratory tests.

The regulatory Environmental Effects Monitoring (EEM) program in Canada has found that pulp and paper mill effluents reduce the gonad size of wild fish. In such cases, there is a need for Investigation of Cause (IOC) and Investigation of Solution (IOS) studies to find the cause(s) for these responses as well as cost-effective mitigation strategies. IOC and IOS work requires the availability of practical (in terms of time and effluent volume needs) and biologically meaningful tests for assessing fish reproduction in the laboratory. One available fish reproduction test uses adult fathead minnow (Pimephales promelas) and involves a two- to three-week pre-exposure period and a three-week exposure to a test substance. Work was undertaken to determine if both the pre-exposure and effluent-exposure phases could be reduced, leading to a shorter test with a lower effluent volume requirement. For this purpose, effluent samples were taken from two thermomechanical (newsprint) pulp mills and used in tests with ≤7 days of pre-exposure and 5 to 6 days of effluent exposure. Egg production was monitored in each test, and in tests with effluents from one mill, other endpoints - such as gonad somatic indices and whole-body sex steroid levels, as well as vitellogenin activity in males - were measured. The shortened fish reproduction test in this study provided results similar to tests of longer duration, specifically in terms of egg production, and was able to distinguish between pulp and paper mill effluents of different potencies. The test was also found to be directly applicable for toxicity identification evaluation work as it was clearly able to distinguish between the solids and soluble fractions of a biotreated newsprint mill effluent. As such, the test is a promising tool for EEM-related IOC and IOS work.

In Canada, effluents from pulp and paper mills are regulated for toxicity. The regulation requires ≥50% survival of rainbow trout (Oncorhynchus mykiss) exposed to full-strength (i.e., 100%) effluent for 96 h in tests that must be conducted monthly. The regulation also calls for ≥50% survival of Daphnia magna exposed to 100% effluent for 48 h in weekly monitoring tests. Every year, about 10 to 25% of the mills exceed the regulatory limit at least once in tests with either rainbow trout or Daphnia magna. Between 1996 and 2003, we investigated 84 such cases from 32 mills. Of the 84 investigations, 49 involved only trout, 29 involved only Daphnia, and six involved both species. We identified the actual cause of toxicity in 70% of the cases and partially or tentatively identified the toxicant(s) in 17% more. In the cases involving only trout, the most frequent causes of toxicity were related to biotreatment performance (e.g., ammonia). In the cases involving only Daphnia, the most frequent cause of toxicity seemed to be related to polymeric formulations. For those cases involving both trout and Daphnia, the cause of toxicity was traced to more than one compound or to excess concentrations of a specific compound resulting from a spill or biotreatment-related problems.

A questionnaire was sent to 75 mills in Canada requesting information on compliance in 1999 with respect to the toxicity regulation for liquid discharges. This regulation requires that at least 50% of rainbow trout and Daphnia magna survive in the discharge from the mill in toxicity tests. Of the 74 mills that responded, 81% and 74% reported no toxicity failures for process effluents in rainbow trout and Daphnia magna tests, respectively. For mills with infrequent or no toxicity episodes during the year (1 or 0 for trout; 1–4 or 0 for Daphnia magna), 89% and 92% of the mills met this criterion for trout and Daphnia magna tests, respectively. In addition to process effluents, 28 mills had separate cooling water discharges and, of these, ten experienced at least one episode of cooling water toxicity. Ammonia and accidental spills were most frequently cited by the mills as likely causes of process effluent toxicity. The most frequent causes cited for cooling water toxicity were chlorine and accidental contamination from other sources.

Rainbow trout exposed to effluent from a UNOX (oxygen activated sludge) treatment plant were found to exhibit signs of distress during the first hours of exposure. While the final effluent was not acutely lethal at full strength, it was still important to determine the cause of the distress to ensure that the mill's effluents remain in regulatory compliance. The use of a series of effluent manipulations involving pH adjustment and aeration as well as observations of symptoms exhibited by the fish were successful in identifying carbon dioxide as the cause of fish distress for the UNOX-treated effluent. For rainbow trout, the symptoms of exposure to elevated levels of carbon dioxide of 100 mg/L or greater included loss of equilibrium, erratic swimming, gasping at the surface, sinking to the bottom of the test container and paralysis. The fish were found to eventually recover as the carbon dioxide was stripped from the effluent due to aeration during the test. A carbon dioxide concentration of 250 mg/L, however, was found to be lethal to trout A survey of mills using oxygen activated sludge treatment systems indicated that the carbon dioxide levels in effluents from such operations can range from 48 to 251 mg/L. Solutions for eliminating the fish distress and possible mortality associated with such effluents would be to lower the carbon dioxide level to below 100 mg/L by aeration or pH adjustment.