Ecotoxicity of Mixtures of IL and Lithium Salt †

: The applicability of ionic liquids (IL) has been increased during the last years and even new opportunities are becoming a reality, i.e., mixtures of pure IL and inorganic salt as electrolytes for smart electrochemical devices. In this work, the ecotoxicity of two protic ILs (Ethylammonium nitrate and Ethylimidazolium nitrate) and one aprotic IL (butylmethylpyrrolidinium bis(trifluoro omethylsulfonyl)imide) doped with the corresponding Lithium salt was tested towards changes on the bioluminescence of the bacteria Alii v ibrio fischeri , using the Microtox ® standard toxicity test. Half maximal effective concentration (EC 50 ) of these mixtures was determined over three standard periods of time and compared with the corresponding values to pure ILs. J.S.; methodology data, J.J.P., P.V., L.F.-M.; software, J.J.P., P.V., L.F.-M.; writing—original preparation, J.J.P., J.S., M.V.; funding acquisition, J.S.


Introduction
The applicability of Ionic liquids (IL) seems never-ending, since they are still not fully studied and mixtures of pure IL and inorganic salt burst in as electrolytes for smart electrochemical devices [1,2]. ILs can be divided into two different subclasses depending in their structural characteristics: protic (PIL) and aprotic (AIL) ionic liquids. PILs are formed by the transfer of proton from acid to base, and hence, they consist of proton-donor and -acceptor sites which are responsible for building extended three-dimensional hydrogen bond networks as in the case of water and AILs are mainly based on bulky organic cations (i.e., pyrrolidinium, imidazolium…) with long alkyl chain substituents and huge variety of anions (i.e., bis trifluoromethylsulfonyl imide (TFSI), tris(pentafluoroethyl)trifluorophosphate (FAP), halides). Despite having many useful properties (arising from the protic nature) and potential applicability, the literature on PILs is still scarce compared to their AIL [3,4].
In recent years, significant growth in the structure−property relationships of ILs has been achieved with a better understanding of the intermolecular forces [5,6]. As mentioned above, when IL properties are not still fully known, mixtures of ILs and inorganic salts increases gives new possibilities improving, in some particular cases, some of their properties [1,7]. Salgado et al. [1] have stated how melting and glass transition temperatures present lower values when salt concentration increases and thermal stability remains equal on mixture than pure IL. Kim et al. [8] have studied the mixture n-butyl-n-methylpyrrolidinium TFSI with Li TFSI salt showing slight decrease on ionic conductivity when salt concentration increases.
However, apart from good physico-chemical properties, current European Union environmental legislation including REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) [9] makes firm demands for safety materials, that some-  times, unfortunately ecotoxicity test are skipped since they are usually expensive and time-consuming [10]. Therefore, it is urgent to establish evaluation procedures to estimate the toxicity of ILs that can provide the needed information without taking too long and reducing the costs. Aliivibrio fischeri (A. fischeri) is a well-known marine luminescent bacterium with short reproductive cycle, and the toxicity inference for A. fischeri may be extrapolated for a wide variety of aquatic organisms and thus can be effectively applied for toxicological risk assessment [11,12]. In this work, the ecotoxicity of two protic ILs (Ethylammonium nitrate (EAN) and Ethylimidazolium nitrate (EIm NO3)) and one aprotic IL (butylmethylpyrrolidinium bis(trifluoromethylsulfonyl)imide (C4C1pyrr TFSI)) doped with the corresponding Lithium salt (Li NO3, for the protic ILs and Li TFSI for the aprotic IL) was tested towards changes on the bioluminescence of the bacteria Aliivibrio fischeri, using the Microtox ® standard toxicity test. The effective concentration (EC50) of these mixtures was determined over three standard periods of time, namely 5, 15, and 30 min and compared with the corresponding values to pure ILs.

Chemicals
The main characteristics of selected ILs (EAN, EIm NO3, and C4C1pyrr TFSI) with the corresponding Lithium salt (Li NO3, and Li TFSI) are indicated in Table 1. Nitrate ionic liquids were dried into high vacuum. Water content for all the samples was below 100 ppm. The different solutions of IL+salt mixtures were prepared by mixing both components with the help of an ultrasound bath and a magnetic stirrer during, at least, 48 h. Saturated solutions have been reached using the hydrated salts by increasing molality in intervals of 0.5 mol·kg −1 until saturation point at room temperature [1]. The molar fraction (χ) of metal salts and pure IL and the final molecular mass (Mm) for every saturated mixture are indicated in Table 2.

Experimental Section
Acute toxicity was assessed by determining the luminescence inhibition of the marine bacteria Aliivibrio fischeri (A. fischeri). A Microtox ® M500 Analyzer (Modern water) was used. After exposing the bacteria to each different IL or IL+salt solutions (from 0 to 81.9%) at 5, 15, and 30 min, the light output was measured and compared with a blank control sample. The concentration of the sample (mg L −1 ) which produces a 50, 20, and 10% luminescence inhibition after exposure at the three selected times (5, 15, and 30 min) is designated as the Effective Concentration (EC50, EC20, and EC10, respectively) and is calculated, together with the corresponding 95% confidence intervals, through a non-linear regression, using the least-squares method to fit the data to the logistic equation [11]. Figure 1 shows the behavior of % of luminescence relative to control Aliivibrio fischeri bacteria versus concentration, of C4C1pyrr TFSI and C4C1pyrr TFSI + TFSI 1.5 m mixture. The luminescence strongly decreases with the concentration of the toxic, following a logistic equation. Furthermore, it can be seen how the salt affects, negatively in this case, to the toxicity of the final solution mixture. From this curves the effective concentration values (EC10, EC20, and EC50) of the different compounds at different times have been calculated and exposed in Table 3. Results found in Table 3 exhibit the well-known effect of time in the toxicity results, which typically describes the lowest EC values for the highest time of exposure. Some authors [13][14][15] have reported that aromatic cations (imidazolium in this case) are more toxic than non-aromatic based ILs (pyrrolidinium and ammonium); this statement perfectly fits with our results that present the following trend: EAN < < C4C1pyrr TFSI < EIm NO3.

Results and Discussion
According to Passino & Smith classification [16], pure EAN can be considered as harmless (>1000 mg L −1 ), and pure C4C1pyrr TFSI and EIm NO3 practically harmless (10 mg L −1 < EC50 < 100 mg L −1 ). Scarce values of the toxicity of these compounds can be found in literature, to the best of our knowledge, except for pure EAN [17] and C4C1pyrr TFSI [15], who found similar values than the present study.
It can be seen how lithium salt induces different effects on these pure ILs: whereas LiNO3 seems that does not induce changes on EC values for pure EAN, EIm NO3 + Li NO3 mixture presents higher EC values than the corresponding to pure one, indicating slightly lower toxicity in mixture than in pure IL [16]. Nevertheless Li TFSI clearly decrease EC values for the pure Aprotic ILs, being the mixture more toxic than pure IL, considered as moderately toxic on EC20 and EC10 indicators [16]. No previous bibliographic data have been found for the mixtures of IL with salt. The differences between these results can be explained in terms of the saturation concentration of the different salts on the ILs as it is exposed in Table 2. Since EAN + LiNO3 is the mixture with the lowest salt molar fraction and the lowest toxicity effect, and C4C1pyrr TFSI + Li TFSI is the mixture with the highest salt molar fraction and, consequently, highest toxicity change.

Conclusions
The main conclusion of this work is that there are some differences between PIL and APIL toxicity, and even among the two selected PILs. Salt addition clearly affects to the toxicity and is highly depending on the molar fraction, as expected. Ethylammonium nitrate and its mixture can be considered as non-toxic, EIm NO3 pure is slightly toxic meanwhile its mixture is non-toxic, and finally, C4C1pyrr TFSI goes from practically harmless to moderately toxic when lithium salt is added.