# Comprehensive Analysis of the Acute Toxicity of Ionic Liquids Using Microtax(r) Bioassays
## 2 Materials and Methods
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Name** & **Abbreviation** & **Purity** \\
**Molecular Mass (g/mol)** & **CAS Number** & **Molecular Mass (g/mol)** \\ \hline
1-(2-methoxyethyl)-1- & C\({}_{1}\)OC\({}_{2}\)C\({}_{1}\)PyrrFAP & \(>\)0.98 \({}^{2}\) \\ methylpyrrolidinium & 1195983-48-2 & 589.24 \\ tris(pentafluoroethyl)trifluorophosphate & & \\ \hline
1-hexyl-1-methylpyrrolidinium & C\({}_{6}\)C\({}_{1}\)Pyrr TFSI & \(>\)0.99 \({}^{1}\) \\ bis((trifluoromethyl)sulfonyl)imide & 380497-19-8 & 450.5 \\ \hline
1-octyl-1-methylpyrrolidinium & C\({}_{8}\)C\({}_{1}\)Pyrr TFSI & \(>\)0.99 \({}^{1}\) \\ bis((trifluoromethyl)sulfonyl)imide & 927021-43-0 & 478.5 \\ \hline
1-butyl-1-methylpyrrolidinium & C\({}_{4}\)C\({}_{1}\)Pyrr FAP & \(>\)0.98 \({}^{2}\) \\ tris(pentafluoroethyl) & 851856-47-8 & 587.28 \\ trifluorophosphate & & \\ \hline
1-butyl-1-methylpyrrolidinium & C\({}_{4}\)C\({}_{1}\)Pyrr C\({}_{4}\)FAP & \(>\)0.99 \({}^{2}\) \\ tris(nonafluorobutyl) trifluorophosphate & 851856-47-8 & 830.32 \\ \hline
1-butyl-1-methylpyrrolidinium & C\({}_{4}\)C\({}_{1}\)Pyrr C\({}_{8}\)FAP & \(>\)0.98 \({}^{2}\) \\ tris(perfluorooctyl) trifluorophosphate & -- & 1430.41 \\ \hline Tetrabutylphosphonium & Ph\({}_{3}\)t FAP & \(>\)0.98 \({}^{2}\) \\ tris(pentafluoroethyl) & 482635-81-4 & 704.36 \\ trifluorophosphate & \multicolumn{1}{c}{} & \\ \hline Propylammonium & EAN & \(>\)0.97 \({}^{1}\) \\ Nitrate & 22113-86-6 & 108.10 \\ \hline Butylammonium & PAN & \(>\)0.97 \({}^{1}\) \\ Nitrate & 22113-88-8 & 122.12 \\ \hline Ethylammonium & BAN & \(>\)0.97 \({}^{1}\) \\ Nitrate & 58888-50-9 & 136.15 \\ \hline Penthylammonium & PEAN & \(\geq\)99 \({}^{3}\) \\ nitrate & -- & 150.18 \\ \hline Hexylammonium & HEAN & \(\geq\)99 \({}^{3}\) \\ nitrate & -- & 164.20 \\ \hline Octylammonium & OAN & \(\geq\)99 \({}^{3}\) \\ nitrate & -- & 192.25 \\ \hline Ethylimidazolium & C\({}_{2}\)Im NO\({}_{3}\) & \(>\)0.98 \({}^{1}\) \\ nitrate & 501693-38-5 & 159.14 \\ \hline \({}^{1}\) Iolite; \({}^{2}\) Merck KGaA; \({}^{3}\) Synthesized [23]. & \\ \end{tabular}
\end{table} 
Table 1: _Cont._  
Figure 1: Ionic liquids selected for this work. Standard Microtok(r) liquid phase assays (M500 Analyzer--Modern water, Rema Tek LLC, Bonsall, USA) were employed for the evaluation of the acute toxicity by measuring the luminescence inhibition of the rod-shaped Gram-negative marine bacteria _Aliivibrio fischeri_ (_A. fischeri_), the bioluminescence of which, through a population-dependent mechanism known as quorum sensing, is sensitive to various toxic substances [24,25]. The light output was measured after being exposed to different IL aqueous serial solutions (ranging from 0 to 81.9%); 100% of the IL corresponds to the known concentration of a stock solution previously prepared, at 15 \({}^{\circ}\)C, and by comparing it with a blank control sample. EC\({}_{50}\) is commonly used as the primary parameter, representing the concentration causing a 50% reduction in bacterial luminescence. In addition, EC\({}_{10}\) and EC\({}_{20}\) (concentrations resulting in 10% and 20% reduction in initial luminescence, respectively) also offer valuable intermediate toxicity references. These concentrations are calculated, along with the corresponding 95% confidence intervals, through a non-linear regression using the least-squares method to fit the data to the logistic equation. The decrease in bioluminescence with increasing sample concentration serves as an integrated measure of physiological impairment of the bacteria, thereby demonstrating the toxic effect of the studied compound [12]. Two different classifications were employed in this study to discern the toxicity of the compounds. The first classification, widely used and proposed by [PERSON] and [PERSON] [26], is based on the values of EC\({}_{50}\) at 30 min.