## 2 Topical Research Areas Related to Ionic Liquids and the Contribution of Spiroammonium Compounds
### Green Chemistry and the IL Concepts
#### 2.2.3 The Toxic Properties of ILs; Their Screening with Organisms of Different Trophic Levels  
A one-sided perspective of ionic liquids as \"green\" solvents based solely on their non-volatility and non-flammability does not guarantee their overall non-toxicity. Like all chemical products, ionic liquids require a rigorous, comprehensive approach in terms of their impact on human health and the environment. Although ILs can lessen the risk of air pollution due to their low vapor pressure, they have significant solubility [57] as well as high stability [58] in water. Additionally, the expanding range of IL applications in many fields of the chemical industry has led to a growing threat of contamination to aquatic and terrestrial environments by these compounds [59; 60; 61]. In addition, the toxicity of ILs should be a focus not only in applications related to biomedical research but also in all other cases where people have to interact with them directly in production processes, from the laboratory to industrial scales. There is a limited number of studies on the toxic properties of ILs compared to the number of studies on their numerous applications. Thus, out of the 8000 publications discovered (in the Web of Science database) devoted to the applied issues of ILs, only 250 (or 3%) studied the toxic properties of ILs [62]. In the last 20 years, the toxicity of ILs towards cells and microorganisms has been heavily investigated, with the main aim of assessing the risks associated with their potential use in industrial applications and developing strategies to design greener ILs [63]. Another important issue is related to the effect of ILs on different organisms. For example, an IL can be toxic to one organism but practically harmless to another; therefore, it is important to analyze the toxic effect of ILs on different organisms, from simpler ones (bacteria or fungi) to more complex ones (plants and animals) [64]. The impact of aromaticity on the toxicity of different cations (pyridinium, piperidinium, pyrrolinium, and imidazolium) and hydrophobic anions (bis(trifluoromethylsulfonyl)imide [NTf\({}_{2}\)] and hexafluorophosphate [PF\({}_{6}\)]) has been measured [65]. Standard assays that use organisms of different trophic levels, such as bacteria _Vibriofscheri_ (Microto\({}^{\text{\textregistered}}\)), green microalgae _P. subcapitata_, and cladoceran _D. magna_, enable evaluations of the consistency of structure-activity relationships across different biological targets. The results clearly indicate that the studied ILs can be divided into two groups that present very different dependencies of toxicity in relation to water solubility. One of the groups comprises non-aromatic ILs, which are based on piperidinium and pyrrolinium. This group presents much lower water solubility and toxicity than aromatic ILs that are based on the imidazolium and pyridinium cations; however, both [PF\({}_{6}\)] and [NTf\({}_{2}\)] anions have little impact on toxicity: [C\({}_{3}\)C\({}_{1}\)pyrrol] \(<\) [C\({}_{3}\)C\({}_{1}\)pip] \(<\) [C\({}_{3}\)C\({}_{1}\)] \(<\) [C\({}_{3}\)C\({}_{1}\)pyr]. When testing imidazolium ILs [C\({}_{n}\)C\({}_{1}\)im][NTf\({}_{2}\)] (n = 1-8), after 15 min of exposure to the luminescent marine bacteria _V. fscheri_, general regularity was demonstrated which, with an increase in the chain length of alkyl radicals C and a subsequent increase in hydrophobicity, increased the toxicity of the ILs [66; 67; 68; 69; 70]. Thus, in a row [C\({}_{1-8}\)C\({}_{1}\)im][NTf\({}_{2}\)], the EC\({}_{50}\) value consistently decreases when moving from n = 1, \(\ldots\) to n = 8 from 2362.78 mg\(\cdot\)L\({}^{-1}\) to 6.44 mg\(\cdot\)L\({}^{-1}\). The reported results suggest the possibility of an IL design with an enhanced hydrophobic character and lower toxicity by eliminating their aromatic nature. The studies described in the Toxicity Report under the National Toxicology Program [71] evaluate the toxicological potential of selected substances in laboratory animals (two species: rats and mice).