Prediction of acute fish toxicity (AFT) and fish embryo toxicity (FET) tests by cytotoxicity assays using liver and embryo zebrafish cell lines (ZFL and ZEM2S)

Abstract

Fish cell-based assays represent potential alternative methods to vertebrates' use in ecotoxicology. In this study, we evaluated the cytotoxicity of thirteen chemicals, chosen from OECD guidelines 236 and 249, in two zebrafish cell lines (ZEM2S and ZFL). We aimed to investigate whether the IC50 values obtained by viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict the LC50 values of Acute Fish Toxicity (AFT) test and Fish Embryo Toxicity (FET) test. There was no significant difference between the values obtained by the different viability assays. ZFL strongly correlated with AFT and FET tests (R²_AFT = 0.73-0.90; R²_FET48h = 0.79-0.90; R²_FET96h = 0.76-0.87), while ZEM2S correlated better with the FET test (48h) (R² = 0.70-0.86) and weakly with AFT and FET tests (96h) (R²_AFT = 0.68-0.74 and R²_FET96h = 0.62-0.64). The predicted LC50 values allowed the correct categorization of the chemicals in 76.9% (AFT test) - 90.9% (FET test) using ZFL and in 30.7% (AFT test) - 63.6% (FET test) using ZEM2S considering the US EPA criterion for classifying acute aquatic toxicity. ZFL is a promising cell line to be used in alternative methods to adult fish and fish embryos in ecotoxicity assessments, and the method performed in 96-well plates is advantageous in promoting high-throughput cytotoxicity assessment.

Keywords: Alternative methods; Aquatic toxicology; In vitro-in vivo correlation; Viability assays; Zebrafish cell lines.