Evaluation of Short-Term Fish Reproductive Bioassays for Predicting Effects of a Canadian Bleached Kraft Mill Effluent

Abstract 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.