Jack Salole

Across the world, there is an increasing desire for the replacement of whole-organism toxicity tests with new approach methods (NAMs) that refine, reduce, and replace animal use. These principles, collectively known as the 3Rs, are critical to consider when discussing NAM development and implementation. Despite the frequency at which NAMs are discussed as replacements to existing toxicity test methods, the application to whole-effluent toxicity (WET) monitoring is largely ignored in favour of their use for chemical assessments. These WET test methods are an ideal receptor for NAMs due to their volume of use and highly repetitive nature as legislative tools used in many areas of the world. In this paper, we investigate this with a focus on Canadian effluent regulation, first discussing the current tools used to regulate effluent toxicity in Canada and how NAMs can aim to fill these roles rapidly to address the changing landscape of Canada’s policy on vertebrate use in toxicity testing. We discuss three NAMs being developed with potential to be applied to whole-effluent toxicity testing, summarizing current trends in the literature, and identify areas where they may struggle to may struggle to meet the needs of environmental regulations. Further research needs are highlighted. Finally, we propose ways to integrate these NAMs into environmental regulations to robustly evaluate effluent toxicity should future data should future data collected for effluents follow existing trends.

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 used 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 three separate mills with a total of five unique sampling locations; an additional eight 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 three 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. Salt stratification and predator cues: Impacts on freshwater species depend on habitat choice (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 un-ionized ammonia 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.

The RTgill-W1 in vitro assay is a new approach method designed as an alternative to one of the most widely used toxicity tests globally, the fish acute lethality test. The RTgill-W1 assay is standardized (Organisation for Economic Co-operation and Development; International Organisation of Standardization) but the test conditions could be optimized to allow for higher throughput, better replication, and lower costs. This study explores potential optimizations regarding the culturing conditions, plate format, and reference toxicant testing to make the RTgill-W1 assay more practical for widespread implementation. We demonstrate that the RTgill-W1 culture can be routinely split 1:3 without impacting test sensitivity (p = 0.207 to 0.612), which allows all work to be contained to a standard 5-day work week and 1.3x more tests over the current test methods. The test can adopt a 96-well plate format without impacting sensitivity (p = 0.672 to 0.889), dramatically improving the replication of the exposure wells and test controls and generating test data from a single plate. The fluorescent signal does not bleed across the smaller wells in the 96-well format to impact test endpoints (p = 0.465 to > 0.999). The reference toxicity test concentrations can be modified to increase confidence in the point estimate (EC50), allowing for more effective monitoring of assay performance. These optimizations improve the practicality and decrease the costs of the RTgill-W1 assay, which is particularly desirable for implementation in commercial and government laboratories that conduct regulatory toxicity testing.