THE APPLICATION OF THE RTGILL-W1 CELL LINE ASSAY TO DETECT PULP AND PAPER MILL EFFLUENT TOXICITY

Abstract

There is an increasing demand for new approach methods that refine, reduce, and replace vertebrate animal use in toxicity testing. A major source of vertebrate use is whole-effluent toxicity (WET) testing, which uses an estimated 3 million fish in North America each year. The rainbow trout acute lethality test is the most common WET test method and is widely applied in routine compliance testing making it an ideal candidate for replacement. The RTgill-W1 assay is a potential replacement that exposes rainbow trout gill cells to effluent and evaluates toxicity using fluorescent indicators. This thesis investigates if the RTgill-W1 assay is a viable alternative to the rainbow trout acute lethality test. Optimizations to the RTgill-W1 assay to improve performance monitoring and reduce assay cost by 40 to 75%. The ability of the RTgill-W1 assay to pulp and paper effluent toxicity was evaluated thoroughly using 70 effluent samples. One of three fluorescent indicators used in the RTgill-W1 assay is strong influenced by unionized ammonia in effluents. As ammonia is a common component of effluent samples, this indicator was suggested to be omitted in regulatory frameworks. Of the effluents evaluated, 20 caused acute lethality to fish and all 20 caused toxicity in the cell line. The calculated effect meaures had a strong correlation, suggesting that cell viability can predict fish lethality for pulp and paper mill effluents. An additional 3 samples were toxic to the cells but not the fish, suggesting the RTgill-W1 assay may be more sensitive to effluent toxicity. Regulatory frameworks for the implementation of the RTgill-W1 assay are presented estimated to reduce fish use by ~ 90% for pulp and paper mill effluents. Overall, these results are promising for the implementation of the RTgill-W1 assay as a replacement to the rainbow trout acute lethality test for pulp mill effluents.