# Comprehensive Analysis of the Acute Toxicity of Ionic Liquids Using Microtax(r) Bioassays
## 1 Introduction  
The term \"ionic liquid\" (IL) encompasses a broad category, which generally consists of the combination of an organic cation and organic or inorganic anion, with a low melting point, i.e., lower than 100 \({}^{\circ}\)C, being one of its main characteristics. A wide variety of ions can be found, ranging from inorganic to organic, chiral or achiral, and including fully or partially ionized acids or bases, charged bridging ligands, metalate coordination polymers, and organic polymeric metal ions, among others [1, 2, 3]. The high anisotropic character of these compounds is mainly due to their characteristic electrostatic and dispersive interactions. These compounds are commonly labelled as green solvents, which has resulted in an increase in their potential applications in recent decades. Since the first IL, ethylammonium nitrate (EAN), was synthesized in 1914 by [PERSON], ILs have been used in a wide variety of fields, including synthesis, coordination chemistry, nanotechnology, polymer materials, and electrolytes [4, 5, 6, 7]. ILs can be classified in two different groups based on their chemical behavior: aprotic and protic ionic liquids. Protic ionic liquids (PILs) can be easily synthesized by combining a Bronsted acid and a Bronsted base, and they are characterized by the presence of a labile proton. Aprotic ionic liquids (APILs) are typically formed through Menshutkin-type reactions [8]. These fascinating compounds possess unique attributes, including the widely acknowledged low vapor pressure, high thermal and chemical stability, a broad electrochemical window, and low toxicity. However, their most noteworthy characteristic is their flexibilityof design or tunability, which allows the modification of physicochemical properties by adjusting anions and cations or introducing functional groups into their alkyl chains. This tunable nature has significantly increased the number of scientific studies analyzing their properties and the number of applications in recent decades. According to the Web of Knowledge (WOS) database, more than 9 thousand review papers had been published, which nearly 1000 corresponding to last past year, evidencing their interest and applicability. Three generations of ILs have been considered by some authors [9; 10]. The first generation is linked to halogenated anions, featuring high thermal stability and large liquid ranges but sensitivity to air and water. The second generation is stable to air and water but exhibits higher toxicity and reduced biodegradability, primarily associated with phosphonium and nitrogen-based anions. The third generation displays lower toxicity, high biodegradability, and biological activity, but is less suitable as a solvent. In addition to its optimal physico-chemical properties, the use of ILs has also been highlighted by the introduction of European Union environmental laws, specifically REACH (Regulation concerning Registration, Evaluation, Authorization, and Restriction of Chemicals), that emphasize the importance of using safe materials in the industrial applications. This underscores the principles of Green Chemistry, encompassing prevention, economic considerations, less hazardous chemical synthesis, efficient energy use, utilization of renewable raw and biodegradable materials, and the assurance of an adequate level of chemical safety. However, one of the characteristics of ILs most repeated in the papers is its low toxicity, as mentioned above; this recurrent statement is based on the low volatility of these compounds, without rigorous studies of the effects of these compounds on the different trophic levels. Unlike other physical and thermophysical properties, the influence of the two moieties of IL and substituents on toxicity is not yet clarified. Thus, the effect of the anion on the overall IL toxicity is the subject of debate in the literature, with different reports ranging from it having no or weak influence, to having a very important role in toxicity.