Ex Vivo Gill Assays as an Alternative Approach to Fish Experimentation

DE LA TORRE, FERNANDO; LOZANO, ISMAEL; LO NOSTRO, F; SCARCIA, PAOLA

Montevideo

Congreso; SETAC Latin America 15th Biennial Meeting; 2023

SETAC Latin America

Fish acute toxicity studies are one of the most frequently conducted vertebrate ecotoxicology tests where death is the intended endpoint, therefore, opportunities to Replace, Reduce, and Refine Regulatory of those tests has become of increasing importance. Addressing the need for alternatives, sub-organismal responses proved to be useful to assess chemical stress and were referred to biochemical/physiological changes (biomarkers) measured in vivo or by in vitro bioassays using isolated cell lines exposed to extracted and enriched surface water. Interestingly the freshwater fish gills are a focal point for studies that seek to understand deleterious effects of various contaminants of emerging concern (EC). In this context, predicting fish acute toxicity of chemicals by means gill ex vivo assay is an alternative method to the conventional approach in line with the 3Rs ideas. The aim of this study was to assess the adverse response of selected oxidative stress biomarkers in different model fish species after ex vivo gill exposure to ECs (fipronil, Fp; triclosan, Tcs; nanosilver, AgNPs and ivermectin, Iv). Alternative studies were conductedin order to compare: Fp in vivo and ex vivo responses; the mitigation effect of humic acids on the toxicity of NPs and the photolytic degradation of TCS. These short-term assays were conducted with native (Prochilodus lineatus, Cyphocarax voga, Piaractus mesopotamicus, Corydoras paleatus) and standardized species (Cyprinus carpio) as a preliminary screening approach where environmental representative concentrations of ECs were assessed. Briefly, four fish were used and four pairs of gill arches were obtained that were randomly assigned to four treatment groups (n=5): freshwater fish saline solution (Ctrl); methanol in saline solution (CtrlSv); and two nominal concentrations of C1 and C2 prepared from a methanol stock solution of the EC. After incubating in glass containers for 1h at 25°C, branchial arches were removed and kept at −80 °C until biochemical analysis. Results indicated that biomarker responses were dependent on fish species and the assayed ECs and alternative assays gave rise to valuable information. The ex vivo model provided a versatile way of exposure with a reduced number of fish and generate rapid information from a key target organ of toxicity. Besides, native test species were able to generate responseswith several ECs showing response capacity even under environmentally relevant concentrations.