New guidelines for testing "Deep eutectic solvents" toxicity and their effects on the environment and living beings.

Deep eutectic solvents (DESs) were described at the beginning of this century as an alternative to ionic liquids (ILs) in green chemistry. Despite their obvious sustainable advantages as reaction media, there is still controversy about their potential toxicity. Most of the ecotoxicity assays done up to now involving DESs are based on antibiograms. This is not a good approach due to the high density and viscosity of most DESs already described. Additionally, antibiograms do not allow continuous monitoring of neither cellular growth nor changes on physicochemical parameters like culture acidification due to cellular growth or DESs metabolization. This work starts by displaying advantages and disadvantages of the DESs toxicity assays already reported. Then, using a new DES recently described and Escherichia coli as a model microorganism, liquid cultures with continuous monitoring of pH, temperature, shaking and optical density have been used, for the first time, to quantify potential toxicity of the DES as well as the degree of the cellular tolerance (in preadapted and non-preadapted cells). The results obtained show that this new DES is not toxic for E. coli at concentrations up to 300 mM and cellular preadaptation was crucial for the cells to grow. At concentrations between 300 mM and 450 mM, cells can tolerate this DES. Above 600 mM, the DES is toxic causing complete inhibition of growth. This toxicity is not only due to the chemical composition of the DES, but also due to the high acidification of the media caused by the DES hydrolysis during cellular growth. The consequences of sterilization procedures on the DES stability are also analysed into detail, finding that sterilization by autoclave promotes DES hydrolysis. From these results, new guidelines are proposed for furthers studies aiming to characterize and quantify DESs toxicity.