Effects of pulp mill wastewater treatment on phytosterol biotransformation and genomic response in rainbow trout

Phytosterols are ubiquitous, naturally occurring plant steroids that are released into solution during pulping processes. These chemicals have formerly been identified as endocrine disrupting compounds. Phytosterols are known to be degraded by microorganisms during biological wastewater treatment, but the metabolites produced during this process and their subsequent effects on fish remain unclear. The objectives of the present study were to determine whether phytosterol biotransformation products can be identified in pulp mill effluents and to establish a relationship between wastewater treatment conditions, concentrations of phytosterols and their metabolites, and genomic response in fish. To accomplish this, a benchscale biological wastewater treatment system was operated using aerobic and microaerophilic treatment trains with a range of solids retention times (SRTs). By varying these operational parameters, four independently treated effluents were produced. Treated effluents were characterized for concentrations of phytosterols and their potential biotransformation products, including thirteen estrogenic and three androgenic compounds. Results showed microaerophilic effluents to contain sterols concentrations at levels two to three orders of magnitude higher than aerobic effluents. Sterols biotransformation products were identified in each effluent and included cholesterol, desmosterol, dihydrocholesterol and estrone. Estrone was the most ecologically relevant metabolite identified and was detected in final treated aerobic effluent at a maximum concentration of 623 ng/L. Following chemical characterization, underyearling rainbow trout were exposed to the effluents at 1% and 10% g/g for 96 hours. Liver tissues were dissected, persevered and analyzed by quantitative polymerase chain reaction. Genes selected for examination of molecular level responses were VTG1, VEPB, VEPG, NME, AR and THRa. Results demonstrated a strong estrogenic and thyroid response with a clear pattern among wastewater treatment conditions. Microaerophilic effluent generated with a short SRT induced the strongest response and was able to do so at both 10% and 1% concentrations. Aerobic effluents induced a significant response at 10% effluent concentration but only for NME, which proved the most sensitive of all genes tested. This study highlights the use of genomic analysis as a predictive indicator for endocrine disrupting effects and provides insight into the operation of wastewater treatment systems for reduction of biological effects.Applied Science, Faculty ofCivil Engineering, Department ofGraduat