RTgill-W1 assay detects pulp and paper mill effluent toxicity

Globally, there has been a push for the development of new approach methods that refine, reduce, and replace animal use in toxicity testing. A large source of this animal use is whole-effluent toxicity testing, which is widely employed in regulation to determine the toxicity of effluents. The rainbow trout acute lethality test is one of the most widely used in North America for this purpose, as it empirically and irrefutably demonstrates effluent toxicity. Despite its strengths, this test is an ideal candidate for replacement due to its frequent use. One replacement is the RTgill-W1 assay, which uses a rainbow trout gill cell line to assess the toxicity of effluent samples. Despite its promise, the RTgill-W1 assay has rarely been investigated for its potential in replacing the rainbow trout acute lethality test. This study assessed the toxicity of pulp and paper mill effluents using the RTgill-W1 assay. Sixty-two effluents were sourced from 3 separate mills with a total of 5 unique sampling locations; an additional 8 effluents were created by mixing effluents together. Of the 70 effluents, 50 were assessed with both the rainbow trout acute lethality test and the RTgill-W1 assay. Twenty of these caused >50% mortality to the rainbow trout and >50% decline in cell viability. An additional 3 effluents caused a >50% decline in cell viability. In effluents causing >50% mortality and a decline in cell viability, there was a strong correlation in toxicity (r = 0.786, p <0.001). The alamarBlue endpoint was more sensitive in filtered effluents (p <0.001). The effluent matrix did not influence the test endpoints, but NH3 was observed to cause significant vacuolation of the cells. 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.