Uncovering the cytotoxicity and growth inhibition of 14 Microcystis extracts on freshwater fish cell lines.

Freshwater cyanobacteria raise health and ecotoxicological concerns due to their production of secondary metabolites. The Microcystis genus synthesizes the well-known cyanotoxins, microcystins (MCs), but also various metabolites with documented sublethal and lethal effects. The toxic potential of such mixtures remains poorly characterized, although biota, including fish, are exposed to those metabolite cocktails through direct water uptake and/or trophic transfer. To provide preliminary answers to this complex issue, this study investigated the toxicity of 14 Microcystis extracts with distinct metabolite profiles using in vitro fish cell-based bioassays. Two freshwater fish cell lines, Rainbow Trout Liver-Waterloo 1 (RTL-W1) and Epithelioma Papulosum Cyprini (EPC), were selected as complementary models for ecotoxicity assessment. We quantified cell mortality and growth inhibition after 24 h and 48 h using flow cytometry. Toxicity data were integrated using a computed toxicity index based on the 0-100%-normalization of each endpoint regarding negative and positive controls, allowing ranking of extracts by overall toxic effects. Microcystis extracts induced heterogeneous toxicity, causing cell mortality and/or growth inhibition, with overall effects ranging from slight to moderate, and increasing by 48 h. Although MC occurrence significantly correlated with the toxicity index, their abundance levels were not directly linked to observed toxicity, suggesting that other emerging metabolites (e.g., aeruginosins, cyanopeptolins, cyclamides) may participate in the toxic effects. This study brought new insights into the toxic effects of cyanobacterial metabolite mixtures. These results advocate the interest of integrated metabolite profiling to fully evaluate cyanobacterial strain toxicity, which could use the developed data integration process to prioritize key metabolic compositions.