As part of the Long-Term Receiving Water Study, effluent from a bleached kraft mill (BKME) employing oxygen delignification (OD) and elemental chlorine free bleaching (ECF, 100% CLO2 substitution) was used to evaluate biological response using Ceriodaphnia dubia whole effluent toxicity (WET) acute and chronic tests, and a fathead minnow (Pimephales promelas) 7-d, 28-d, and 56-d survival and growth bioassays, and life cycle test. These bioassays were conducted to determine effluent dose-response patterns in survival, growth, and reproduction; and to relate laboratory findings to instream patterns of biota (fish, macroinvertebrates, and periphyton). Measured endpoints included C. dubia survival and reproduction, and fathead minnow survival, growth (biomass), egg production (total eggs and eggs/female/day), egg hatchability and size, gonad somatic index (GSI), liver somatic index (LSI), condition factor, vitellogenin (VTG), testosterone, 11-ketotestosterone (11-KT), 17β-estradiol, nuptial tubercles, and histopathological analyses.

Biological effects of effluent were among the lowest measured by NCASI, with nearly all endpoints showing no difference from the control in 100% effluent, or not showing a consistent dose-response pattern. Ceriodaphnia dubia 7-d survival and reproduction were unaffected by 100% v/v effluent. Fathead minnow 7-day, 28-day, and 56-day juvenile growth and survival tests were unaffected by effluent exposure, regardless of whether juveniles were hatched from exposed or unexposed parents. The IC25 for total eggs, numbers of spawns and eggs/female/day were all >100% effluent, with no effect of effluent exposure on egg hatchability or adult biomass or survival. Most biomarkers were unaffected by effluent exposure. Exceptions included lower testosterone concentrations in both males (IC25=76.9%) and females (IC25=35.7%), lower 11-ketotestosterone in males (IC25=19.8%), and lower 17β-estradiol in females (IC25=31.4%). Egg size showed substantial variability within test treatment and across effluent concentrations, suggesting that this endpoint might not be useful for identifying effluent toxicity. Histological examination of gonad tissue showed no abnormalities in either sex. Females had continuous oocyte recruitment and were capable of multiple spawns, while males had no change in the size or number of Sertoli cells and spermatozoa present in sperm ducts, indicating they were capable of spawning.

Based on the instream waste concentration of the receiving water (mean 33%), this study indicates that the margin of safety for instream biological communities is ~3X. This accurately predicts the observed general absence of effluent effects on receiving water biota observed in field studies in the effluent receiving water.