A lot of these evaluations have been practiced on Danio rerio (zebrafish) or Caenorhabditis elegans with concentrations between 1 and 100 mg. L-1. Before proceeding, ILs must be dried (water quantity is currently determined by a Karl — Fischer titration in ppm) and highly pure (determination by HPLC, NMR,…) [144].

Here are enunciated some microorganisms used to evaluate ILs toxicity: Danio rerio (zebrafish), Scenedesmus vacuolatus (green algae), Lemna minor (marine plant), Caenorhabditis elegans (earthworm), Physa acuta (aquatic snail), Oocystis submarine (Baltic algae), Cyclotella meneghiniana (Baltic algae), Daphnia magna (marine plant), Lactobacillus (bacteria), L. rhamnosus (bacteria), Vibrio fischeri (bacteria), E. Coli (bacteria), Pichia pastoris (bacteria), Bacillus cereus (bacteria) [51, 52, 140, 144, 145].

A high quantity of tests has been practiced on Vibrio fischeri, a marine bacteria [146—148]. In general, ILs are more toxic than acetonitrile, an organic solvent known for its toxicity [149]. They also have observed that pyridiniums are among the most toxic ILs and morpholiniums seems to be the less toxic [150]. Ammoniums are toxic towards Zebrafish [145] and other structures as choline-based ILs, morpholiniums, tropiniums, quinuclidiniums or alkylpyrrolidiniums were devel­oped to improve biocompatibility (in association with bromide anion) [149]. Alkyl chain length and the number of carbon atoms (>4 carbons) play an important role in toxicity. Longer chains exhibit higher toxicities [107, 108, 140, 144, 151]. The chain’s position on the ring does not influence the toxicity (e. g. methyl’s position on dimethylimidazoliums) [146].

Other evaluations on aquatic microorganism Dreissena polymorpha, a mollusc [152, 153], and Daphnia magna, a crustacean [154], showed similar tendencies (long alkyl chains increase lipophilicity and intensify toxicity).

Some functional groups like esters increase toxicity [141] whereas other func­tional groups like nitriles, hydroxyles or ethers reduce it [119, 149, 155]. Some 1-methyl-3-alkoxyalkylimidazoliums have been evaluated on bacterial targets have been found harmless, except if the cation wears long alkyl chains (>6-7 carbons) [106]. Moreover, aromatic groups like phenols show higher toxicity than a butyl chain [149].

Alvarez-Guerra et al. established a model comparing 30 anions and 64 cations on Vibrio fischeri [147].

Perfluoroanions as PF6~ in water at 50 °C can produce HF, via hydrolysis, and this acid can be very harmfull towards microorganisms [18, 119, 156—158]. The hydrolysis of PF6~’s hydrolysis was studied by Swatloski et al. and these authors proposed anions in principle non toxics but they confer an hydrophilic profile [159]. It was shown that RfBF3~ anions were very stable towards hydrolysis and produce less quantity of HF [160]. Ignat’ev et al. reported the synthesis of Rf3PF3~ anions (Rf is perfluoroalkyl chain containing 2 or 3 carbon atoms) less sensitive towards hydrolysis [156] and these ILs exhibit lower viscosities but higher toxicities [161].

All these studies demonstrate that the lipophilicity of the anion is a preponderant criteria [115]. It can be expected that the association with an appropriate cation could modify the global toxicity of the IL and balance the intrinsic anion toxicity [150].

Metabolites produced by degradation of ILs could be toxic too and a metabolisation route by cytochrome P450 of C4Mim cation has been proposed in the literature [44].

To conclude, all these studies show that lipophilicity of ILs is the determinant criteria related to toxicity and a hypothesis is that lipophilic cations can interact with lipidic membranes of microorganisms [150]. Considering this fact, measuring the Log POW parameter could be a relevant indicator to estimate on ILs toxicity [51, 52,162]. As we have evocated earlier, toxicity also depends on the biological target [119], so some results in appearance contradictory were reported in literature. As an example, NTf2~ is not toxic towards some bacteria whereas it is toxic towards some microorganisms (Scenedesmus vacuolatus and Vibrio fischeri) [150].

The environmental parameters must be considered because it was established that they can influence toxicity values. Indeed, it was published that NaCl content in water has a protective effect in high concentrations. Microorganism’s size and the cell membranes’ composition also play an important role since they are implicated in interactions between the IL and the biological target [142].

In aquatic environment, interactions with DOM (Dissolved Organic Mattercould) influence toxicity of ILs. Evaluations on Lemna minor demonstrated that association with high concentrations of DOM increase ILs toxicity [163].