## 3 Ecotoxicity
### Aquatic Biomodels
#### 3.1.1 Bacteria Biomedical
investigated the ecotoxicity of different mixtures of choline chloride (HBA) and carboxylic acids (acetic, lactic, glycolic, and citric acids) as HBD. In this case, all the of EC\({}_{50}\) results showed the same ascending pattern of toxicity--ChCl << ChCl:acid (2:1) << ChCl:acid (1:2) << acid--indicating that the toxicity increases with the acid content. These results in _A. fischeri_ indicate that the HBD composition plays an important role in the toxicity of the eutectic mixture. The mechanism of action of these compounds in _A. fischeri_ seems to be related to their ability to cross the cell membrane. It appears that the toxic effect occurs in the cytoplasm, altering the pH and affecting cell viability (hence, the toxic effect when increasing the pH of the acid) [73,92]. Even if the physicochemical properties of the eutectic mixtures were different from those of the starting materials, the toxicities between DESs and the reference organic acids were still similar. However, in this case, the predominant effect of the acids showed similar toxicities between DESs and their reference organic acids. The DESs tested were also found to be more toxic than the respective ionic liquids. This could be related to the presence of the delocalized charge of choline chloride because, as mentioned before, compounds with delocalized charges seem to be more toxic [87]. In the study carried out by [PERSON] et al., the toxicity of some DESs was tested in _A. fischeri_. These compounds were made up of combinations of ethylene glycol and 1-propanol as HBDs, and ammonium quaternary salts as HABs, at different molar ratios [89]. In this study, DESs were tested as aqueous solutions, so the components of the mixture were dissociated. Thus, the positively charged quaternary ammonium salt could have interacted with polysaccharides or peptide chains of the bacterial membrane thanks to the formation of hydrogen bonds or electrostatic interactions, producing a cellular disruption and generating the toxic effect. In this experiment, the trend already reported in other trials was also observed: the length of the alkyl chain affects toxicity. If the number of carbons in the alkyl chains increases, the lipophilicity leads to a higher permeability of the compound in the membrane [93,94]. In this case, the change in HBA structure caused the variation in toxicity. Other tests also considered the role of HBDs as a determinant of the toxicity of the compounds [59,61,85]. In another study conducted by the same authors, an estimation of the toxicity of some DESs was performed by obtaining the EC\({}_{50}\) values of their raw materials [95]. The objective of the test was to observe the synergistic or antagonistic behavior of choline chloride and ethylene glycol, glycerol, propionic acid, 1,2-propanediol, and urea mixtures at different molar ratios in _A. fischeri_. It was seen that most of the compounds showed an antagonistic effect, being less toxic in combination than their components separately. However, in some cases, a synergistic effect was observed, showing that sometimes the starting compounds had less toxicity than the DES. Even so, this was a predictive approach, and it is always preferable to physically carry out the tests on the corresponding biomodel [89,95]. Nevertheless, it was found that the toxicity of DESs cannot be based on predictions of the behavior of their starting materials.