# Comprehensive Analysis of the Acute Toxicity of Ionic Liquids Using Microtax(r) Bioassays
## 4 Conclusions  
In this study, the ecotoxicity of a set of ILs with different ionic natures was evaluated. The assessment was based on the inhibition of the bioluminescence of the bacteria _Aliivibrio fischeri_ with different concentrations of the ILs using the Microtox(r) standard toxicity test. The most remarkable findings of this study are the following:  
* This study found that 16 of the 30 compounds were either non-toxic or practically harmless, and none were at the highest levels of the two classifications considered. No preferential effect of cation or anion on toxicity have been found, although the effect on bacteria is determined by the combination of both. * Protic ILs exhibit lower toxicity compared to aprotic ones at the shorter alkyl chain length, and non-aromatic ILs generally demonstrate lower toxicity than aromatic ones. Additionally, water solubility plays a significant role, with lower toxicity associated with higher hydrophilicity within each group. * Ionic liquids with ammonium cations presented the lower toxicity, while the imidazolium-based ILs are more harmful for the shortest alkyl chain length ILs, although the toxicity increases with this alkyl chain, with OAN and C8C1 Im TESI being the most toxic ILs, both with C = 8, the longest chain considered in this work. * The toxicity of similar cations ranged from the TESI anion, the most toxic, to nitrate-based ILs, the least toxic. * FAP-based ILs, which present the opposite behavior regarding the chain length to the other anion ILs; i.e., the more toxic ILs correspond to the ILs with the shorter alkyl chain. \begin{table}
\begin{tabular}{l c c} \hline \hline
**IL** & **[PERSON] and [PERSON] [(26)]** & **[PERSON] et al. [(27)]** \\ \hline C\({}_{2}\)C\({}_{1}\)Im TESI & Practically harmless & Non-toxic \\ C\({}_{4}\)C\({}_{1}\)Im TESI & Toxic & Toxic \\ C\({}_{4}\)C\({}_{1}\)Im TESI [(12)] & Practically harmless & Non-toxic \\ C\({}_{6}\)C\({}_{1}\)Im TESI & Toxic & Toxic \\ C\({}_{5}\)C\({}_{1}\)Im TESI & Toxic & Very toxic \\ AC\({}_{1}\)Im TESI & Practically harmless & Non-toxic \\ AC\({}_{1}\)Im Cl & Relatively harmless & Non-toxic \\ AC\({}_{1}\)Im DCA & Relatively harmless & Non-toxic \\ C\({}_{4}\)C\({}_{1}\)Im FAP & Toxic & Toxic \\ C\({}_{1}\)C\({}_{1}\)Im DMP & Relatively harmless & Non-toxic \\ C\({}_{2}\)Py TESI & Practically harmless & Non-toxic \\ C\({}_{4}\)Py TESI & Practically harmless & Non-toxic \\ C\({}_{6}\)Py TESI & Toxic & Toxic \\ C\({}_{3}\)C\({}_{1}\)Pip TESI & Practically harmless & Non-toxic \\ C\({}_{4}\)C\({}_{1}\)Pip TFSI & Practically harmless & Non-toxic \\ C\({}_{4}\)C\({}_{1}\)Pyr TFSI [(12)] & Relatively harmless & Non-toxic \\ C\({}_{6}\)C\({}_{1}\)Pyr TFSI & Toxic & Toxic \\ C\({}_{6}\)C\({}_{1}\)Pyr TFSI & Toxic & Toxic \\ C\({}_{4}\)C\({}_{1}\)Pyr FAP & Practically harmless & Non-toxic \\ C\({}_{1}\)OC\({}_{2}\)C\({}_{1}\)Pyrr FAP & Toxic & Toxic \\ C\({}_{4}\)C\({}_{1}\)Pyrr C\({}_{4}\)FAP & Toxic & Toxic \\ C\({}_{4}\)C\({}_{1}\)Pyrr C\({}_{8}\)FAP & – & – \\ Ph\({}_{3}\)FAP & Relatively harmless & Non-toxic \\ EAN [(12)] & Relatively harmless & Non-toxic \\ PAN [(23)] & Practically harmless & Toxic \\ BAN [(23)] & Practically harmless & Toxic \\ PEAN [(23)] & Relatively harmless & Non-toxic \\ HEAN [(23)] & Toxic & Toxic \\ OAN [(23)] & Toxic & Very toxic \\ C\({}_{2}\)Im NO\({}_{3}\)[(12)] & Practically harmless & Non-toxic \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Toxicity level for the selected ILs obtained by [PERSON] and [PERSON] [(26)] and [PERSON] et al. [(27)] criteria. The identification of a critical alkyl size (CAS) at C = 6 was documented in this study, defining a tipping point in toxicity behavior related to the length of the chain. The presence of six or more carbons in the alkyl chain results in a significant increase in toxicity levels. Conceptualization, J.J.P. and J.S. ; methodology, J.J.P. ; formal analysis, A.S.-A., R.S.E., T.T. and P.V. ; writing--original draft preparation, J.J.P., M.V. and J.S. ; writing--review and editing, J.J.P., M.V., A.S.-A. and J.S.