A bleached-kraft mill effluent fraction causing induction of a fish mixed-function oxygenase enzyme

Pulp mill effluents contain a myriad of chemicals that have the potential to cause deleterious effects on aquatic biota in receiving waters. Some of these chemicals evoke an acute lethal response of exposed biota while others evoke sublethal responses. One such sublethal response is the induction of mixed-function oxygenases (MFO) in fish, specifically the CYPlA1 enzyme ethoxy-resorufin-o-deethylase (EROD). We followed the partitioning of the inducing chemicals in pulp mill effluent fractions by Toxicity Identification Evaluation (TIE), or bioassay-driven chemical analysis. This procedure was eventually modified to a more direct technique involving centrifugation, filtration, cleanup procedures, and C-18 solid-phase adsorption. The extracts from the fractionation of two pulp mill effluents after secondary treatment were tested for EROD-inducing activity in a 4-d rainbow trout bioassay. The methanol extracts of particulates/colloids showed significant inducing capacity in Mill A effluent but not in Mill B effluent. The C-18 methanol extracts induced activity from both effluents, with extracts from Mill A causing the greatest response. The particulate/colloidal extract (Mill A) was used as the source material for chemicals which caused EROD induction. The fraction was purified by solid-phase extraction techniques and reverse-phase high-performance liquid chromatography. The majority of the EROD activity was found in the moderately nonpolar region of the chromatogram (K-ow = 4.6 to 5.1).