High-throughput toxicity screening of novel azepanium and 3-methylpiperidinium ionic liquids

Ionic liquids (ILs) have been employed as potentially environmentally friendly replacements for harmful organic solvents, but have also been studied for their use in bioelectrochemical applications, such as in microbial electrochemistry for bioenergy production, or in industrial biocatalysis. For these processes, low microbial toxicity is important and there is a growing need for microbial toxicology studies for novel ILs. In this study, we report initial toxicity data for novel ILs, based on azepanium and 3-methylpiperidinium cations. Agar disc diffusion assays are used, along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations, to obtain rapid and inexpensive initial toxicity data for these novel ILs against Escherichia coli and Staphylococcus epidermidis. Many of the novel ILs characterised possess low microbial toxicity relative to well-studied ILs, highlighting their potential for further study in applications where this is a desirable property.


Introduction
The emergence of ionic liquids (ILs) as environmentally friendly replacements for harmful organic solvents and electrolytes continues to gain in popularity. Some early generation ILs, such as those containing some cations combined with [BF 4 ] À , [PF 6 ] À , and [NTf 2 ] À , were initially touted as being 'green' due to properties like low volatility. Further studies however, showed them to be quite toxic to a number of organisms. 1 This highlights the importance of early toxicological studies conducted alongside the synthesis of novel ILs. This is of paramount importance, whether they are to be used in industrial applications or in consumer products. The number of antimicrobial susceptibility studies involving ILs continues to increase, with ILs based on a number of functional groups, including imidazolium, 2-5 alkylimidazolium lactates, 6 oxygen-functionalised imidazolium esters, 7 pyrrolidinium and piperidinium, 5 quaternary ammonium ILs, [8][9][10][11] morpholinium-based ILs, 12 and phosphonium ILs. 13 Novel ILs have evolved through various generations 14 in order to improve their original use, 15,16 as well as to reduce their toxicity. For example, rst generation alkylimidazolium/ alkylpyridinium chloroaluminate/halide ILs were developed for electrochemical applications, 17,18 with [C 4 pyr][Al 2 Cl 7 ] À exhibiting both cyto-and ecotoxicity. Later generation ILs, including quaternary ammonium ILs, have been studied as electrolytes for batteries, 19 with cholinium-based quaternary ammonium ILs possessing lower toxicity their predecessors. 8,20,21 More recently, pyrrolidinium, piperidinium and azepanium-based ILs have been proposed as green electrolytes for battery systems, fuel cells and high voltage supercapacitors. [22][23][24] ILs, which play important roles in electrochemical applications as highlighted above, can also be applied in microbial electrochemistry, such as in microbial fuel cells and supercapacitor manufacture. [25][26][27][28] Ammonium-based polymer IL membranes have also been employed for wastewater treatment and bioenergy production using microbial fuel cells. 29,30 Unlike for IL use as biocides, where high microbial toxicity is benecial, low toxicity is desirable in microbial electrochemical and biocatalysis applications, so as not to inhibit the microorganisms involved in these biotransformations.
Similarly, low microbial toxicity is a desirable property for ILs involved in whole cell biocatalysis, in order to maintain the viability of the microorganism carrying out the biotransformation. ILs have been used to improve a number of biocatalytic processes, with their versatility allowing them to act as substitutes for both organic solvents and in aqueous two-phase systems, as well as possessing low ammability and negligible vapour pressure. 31,32 ILs have been employed in a range of other applications involving microbes and their enzymes, such as biomass treatment and biodiesel production. 33,34 In order to further much needed emphasis on early stage toxicity testing of novel ILs, this paper reports initial toxicity data for a new family of ILs based on azepanium and 3-methylpiperidinium cations. These are combined with the per-uorinated anions bis(triuoromethane) sulfonimide ([NTf 2 ] À ), triuoromethanesulfonate ([CF 3 SO 3 ] À ), and triuoroacetate ([TFA] À ), methylsulfate ([CH 3 SO 4 ] À ) and halide (I À and Cl À ) anions. Previously published data show moderate viscosities and remarkably wide electrochemical windows for these types of ILs, and suggests their potential for use as electrolytes, battery materials, or in synthetic media. 35,36 In this study, agar disc diffusion assays are used, along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations, to obtain rapid and inexpensive initial toxicity data for these novel azepanium and 3-methylpiperidinium ILs. Twenty-ve different ILs were tested in all, including some halogenated ILs commonly used as starting materials for synthesis. Toxicity to bacterial strains Escherichia coli and Staphylococcus epidermidis were assessed, representing well studied examples of both Gram negative and Gram positive bacteria respectively.

IL synthesis, disc preparation and sample quantication
The ILs used in this study were synthesized as described previously. 36,37 The cations and anions used in this study are shown, along with their structures, in Table 1 and 2 respectively. IL purity was assessed by NMR spectroscopy, accurate mass ESI-MS, and CHNS elemental analysis. 36,37 CHNS analysis is summarised in ESI (Table S1 †). Grade 1 Whatman lter paper discs (6 mm diameter) were prepared using a metal hole punch and sterilised using a dry air oven. Sterile 1.5 mL microcentrifuge tubes were weighed both empty and with a sterile lter paper disc inside. 5 mL IL was added and a new weight recorded, before transferring the disc to the surface of an agar plate. The tube, along with residual IL was reweighed, and the amount of IL on the disc was determined from these measurements.

Agar disc diffusion tests
Agar disc diffusion tests were adapted from previously described reports. 20

Effect of anion selection
Characterization of these novel ILs shows a low toxicity prole for the majority of ILs tested, against both the Gram negative E. coli and Gram positive S. epidermidis. Many of the ILs tested produced very low or no inhibition of growth for either E. coli or S.   Table 3). Incorporation of the bistriimide anion [NTf 2 ] À resulted in a profound increase in toxicity, producing the greatest zones of inhibition across all cations it was combined with, with the exception of [C 6 mm b pip] + . Comparisons of toxicity against E. coli for different anion/cation combinations are shown in Fig. 1, with similar trends observed against S. epidermidis. These ndings agree with previously published studies, which also reported high toxicity when [NTf 2 ] À was employed as an anion, particularly in combination with oxygen-functionalised cations. 44

Effect of side chain oxygenation
The potential to tune IL toxicity through side chain oxygenation is interesting and could provide a route to non-toxic ILs with the desired physicochemical biological properties. Previous literature suggests oxygenation of side chains leads to a reduction in IL toxicity.  (Table 3).
This opposite effect is observed however when [NTf 2 ] À is selected as anion. In disc diffusion assays involving E. coli, the zone of inhibition, and therefore toxicity, increased from 5. Again, a similar trend is observed in the S. epidermidis experiments (Table 3). These results may be attributed to the combination of the hydrophobic bistriimide anion with a more hydrophilic cation. As has been previously reported, it appears the more hydrophilic the cation, the more the anion will be "pulled" into aqueous solution, effectively increasing the concentration of the bistriimide anion in the agar and making the IL more toxic. 39

Other effects
Despite previous reports, 45,48 an increase in the number of carbon atoms in the ring structure did not result in increased  Physical properties of ILs, such as their solubilities in media, can also inuence their toxicity. The water solubility of ILs is largely determined by the hydrophobicity of their constituent cations and anions, 49 and this will also have had an effect on the ability of the ILs tested in this study to dissolve in water-based media and produce zones of growth inhibition.
Similar trends were observed between the two microorganisms used in this study, E. coli and S. epidermidis. ILs containing the [NTf 2 ] À anion appeared to be more toxic towards E. coli than S. epidermidis, with the exception of [C 4  These results are hardly surprising, given the high toxicity observed with bistriimide anions in the disc diffusion assays. Indeed, there is a strong inverse correlation between zone of inhibition from the disc diffusion assay and MIC value, as shown in Fig. 2.
In comparison to many previously reported MIC values, the novel ILs characterised in this study possess relatively low microbial toxicity, making them ideal for microbiological applications. For example, propargyl-functionalised piperidinium ILs, although designed for their toxicity, have been shown to have much lower MIC values against E. coli of 50 mg mL À1 , 5 whilst the ILs tested in this study have MICs many times that value. MIC values were also determined for a range of phosphonium bromide and chloride ILs against E. coli by Cieniecka-Rosłonkiewicz and co-workers, with values ranging from 0.01 to 0.02 mM for the bromides and 0.0025 to 0.113 mM for the chlorides. 13 Work by the Gathergood group reported an MIC value of 16 mg mL À1 (0.0618 mM) for imidazolium-based IL 1- Fig. 1 Effect of anion selection on toxicity towards E. coli, as measured by zone of inhibition using the agar disc diffusion test. methyl-3-decylimidazolium against E. coli, 7 and Pernak and coworkers reported MIC values of between 8 and 62.5 mg mL À1 for ammonium-based ILs against E. coli. 10 These examples, across a range of IL classes, serve to highlight the relatively low toxicity of the ILs characterized in this study. Whilst low microbial toxicity is obviously undesirable for ILs if they are to employed as biocides, it is benecial for their use in microbiological applications, such as biocatalysis, where it is critical for microbial function to be maintained.

Conclusions
In this study we have characterised the microbial toxicity of a number of novel ILs towards the well studied bacteria E. coli and S. epidermidis. Choice of anion had a particularly sizeable effect on toxicity, with [NTf 2 ] À in particular engendering high levels of toxicity across all ILs tested. Azepanium and piperidinium-based ILs had comparable toxicity proles, and cations with oxygenated side chains exhibited reduced microbial toxicity with most anions. Low microbial toxicity towards both E. coli and S. epidermidis was observed for ILs with [MeOC 2 mpip] + and [MeOC 2 OC 2 mm b pip] + cations, highlighting in particular their potential for use in microbe-associated applications. Early characterisation of microbial toxicity is vital in determining the applicability of novel ILs for various uses. Comparisons of MIC values to previously reported ILs suggest those tested in this study have low overall toxicity, making them potential candidates for use in microbiological applications.

Conflicts of interest
There are no conicts to declare.