Ecotoxicological assessment of different choline chloride-based natural deep eutectic solvents: in vitro and in vivo approaches

Natural Deep Eutectic Solvents (NADES) have emerged over the last decade as a promising alternative to organic solvents. NADES are mixtures of two or more natural components interacting each other by hydrogen bonds to form a supramolecular mixture. Efforts have focused on demonstrating their lack of toxicity by studying the responses of different taxonomic groups. In this study, fish cells (RTG-2 line) and freshwater invertebrate embryos (Physa acuta) were used for the ecotoxicological assessment of seven choline chloride (ChCl)-based NADES. Three organic acids and four alcohols were used for the preparation of the NADES investigated. In a first step, the in vitro cytotoxicity of a single exposure to each NADES component, binary mixtures of their corresponding components and the prepared NADES were evaluated. The rank order of in vitro cytotoxicity of the individual components was: organic acids > ChCl > alcohols. NADES produced equal or lower cytotoxic effects than the corresponding mixtures, suggesting that NADES have their own toxicological behaviour, possibly supported by the maintenance of their supramolecular structure up to certain aqueous dilutions. Furthermore, the in vitro assessment showed that NADES prepared with acids were more cytotoxic than NADES prepared with alcohols, which was confirmed by the in vivo assessment. NADES prepared with acids produced embryo lethal effects from 0.025 % at short exposures (48h). For NADES prepared with alcohols embryo toxicity was found from 0.2 %. From our results, it can be concluded that ecotoxicological assessment can help in the selection of component combinations for the design of safe NADES.