Alternatives to Fish Acute Whole Effluent Toxicity (WET) Testing: Predictability of RTgill-W1 Cells and Fathead Minnow Embryos with Actual Wastewater Samples

Toxicity assays using fish cells and embryos continue to gain momentum as a more ethical and informative alternative to fish acute toxicity testing. The goal of our study was to test the accuracy of RTgill-W1 cells and the fathead minnow (Pimephales promelas) embryos to predict actual whole effluent toxicity (WET) in the fathead minnow larvae. The three models were compared concurrently using samples of various origins and treatment types. Additionally, the toxicity of reference toxicants (Cd, Cu, NH3-N, 3,4-dichloraniline, and benzalkonium chloride) spiked into a nontoxic wastewater was compared. The toxicity of reference toxicants was tested in isosmotic and hypoosmotic exposure media in RTgill-W1 cells. Of the 28 wastewater samples, 14 induced a toxic response in fish larvae. Embryos predicted 11 of the 14 wastewater samples toxic to the larvae, whereas RTgill-W1 cells predicted the toxicity of all 14 toxic samples to the larvae. In addition, embryos and RTgill-W1 cells predicted toxicity in two and six additional samples, respectively, that were nontoxic to larvae. Exposures in hypoosmotic medium significantly increased sensitivity of RTgill-W1 cells to all reference toxicants, excluding benzalkonium chloride, compared to exposures in isosmotic medium and showed toxicity levels similar to that in larvae. Thus, hypoosmotic exposure medium should be considered for aquatic toxicity testing applications. Overall, both gill cell and embryo models predicted toxicity in the majority of wastewater samples toxic to larvae and demonstrated their applicability for regulatory WET testing.

Automated summary

Toxicity assays using fish cells and embryos are gaining popularity as an ethical and informative alternative to traditional toxicity testing methods. This study confirmed the accuracy of RTgill-W1 cells and fathead minnow embryos in predicting whole effluent toxicity in fathead minnow larvae, and demonstrated their applicability for regulatory testing. The results also highlighted the potential benefits of using hypoosmotic exposure media in aquatic toxicity testing.