## 2 Topical Research Areas Related to Ionic Liquids and the Contribution of Spiroammonium Compounds
### Green Chemistry and the IL Concepts
#### 2.2.4 Creation of the Low-Toxic Ionic Liquids and the Variants of Their Use  
The history of ILs dates back to the first generation in 1914 with the synthesis of liquid salts of ethylammonium, which were unstable and sensitive to moisture [76]. The second generation of ILs was marked by the synthesis of a stable 1-ethyl-3-methylimidazolium cation IL, in which the anions were tetrafluoroborate and hexafluorophosphate ([BF\({}_{4}\)] and [PF\({}_{6}\)]) [77]. The enormous possibilities of combinations of organic cations with anions, both organic and inorganic, have led to a variety of IL structures with different physicochemical properties, which can be intelligently designed depending on important applications in fields such as chemistry, energy, pharmaceuticals, etc. Time demands on environmental protection and increased attention to human health are reflected in the development of third-generation ILs. Third-generation ILs are biodegradable cations and anions, and natural compounds containing choline, amino acids, or carbohydrates have been developed for IL production [78]. The main trend in the creation of non-toxic and biodegradable ILs is the use of cationic and anionic fragments from a number of components, which occur in nature and can be metabolized to form non-toxic residues. They are expected to constitute greener replacements for traditional ILs, and their use is associated with applications in the food and pharmaceutical industries [79,80]. Choline, a biologically widespread molecule and an essential micronutrient, is able to degrade completely under aerobic conditions [81]. 'Bio-ILs', composed wholly of biomaterials, have been developed in two stages from choline cations and carboxylic acid anionsfound in nature. In the first stage, a water solution of cholinium iodide was passed through an anion exchange resin (SUPELCO1 AMBERLITE IRA-78) to form [Ch][OH]. [Ch][Ac] was obtained via an ion exchange reaction of an aqueous solution of [Ch][OH] with a slight excess of acetic acid in water. The same method was used to synthesize a series of cholinium ILs with monobasic anions (acetate, glycolate, benzoate, propionate, and tiglate) and acidic anions of dibasic acids (H-succinate, H-malate, H-tartrate, H-maleate, and H-fumarate). After coupling with the choline cation, all new ILs, except for an IL with a H-tartrate anion, were room-temperature ILs (RTILs) with a m.p. below 100 \({}^{\circ}\)C. The authors suggested a possible use of these ILs in biomass pretreatment [82]. Cholinium and amino acid-based ionic liquids [AA ILs] occupy a place of their own due to their exceptional biocompatibility stemming from being entirely made of metabolic molecular components. AA is the building unit of proteins and an essential component of our food. Choline (once known as vitamin B4) is a metabolite directly synthesized by the human body and is also an essential dietary nutrient [83]. Eighteen promising new cholinium amino acid ionic liquids ([Ch][AA] ILs) have demonstrated biocompatibility and low toxicity toward enzymes and bacteria [84]. Their inhibitory potentials to acetylcholinesterase (EC\({}_{50}\) values of 2400-3800 \(\upmu\)M) are weaker by an approximate order of magnitude than the traditional IL 1-butyl-3-methylimidazolium tetrafluoroborate (EC\({}_{50}\) value of 330 \(\upmu\)M). The determination of the toxicity of [Ch][AA] ILs and [Ch][AcO] and [Ch][Cl] to representative bacteria (_Escherichia coli, Staphylococcus aureus, Salmonella enteritidis, and Listeria monocytogenes_) has demonstrated that [Ch][AcO] and [Ch][Cl] show lower toxicity towards the test bacteria than [Ch][AA] ILs. However, similar to enzyme inhibitory results, imidazolium-based [Bmim][BF\({}_{4}\)] has been much more toxic than these novel ILs. With relatively few exceptions, [Ch][AA] ILs with lower toxicity have shown higher degradability.