New Insight Regarding the Relationship Between Enantioselective Toxicity Difference and Enantiomeric Toxicity Interaction from Chiral Ionic Liquids

Chirality is an important property of molecules. The study of biological activity and toxicity of chiral molecules has important theoretical and practical significance for toxicology, pharmacology, and environmental science. The toxicological significance of chiral ionic liquids (ILs) has not been well revealed. In the present study, the enantiomeric joint toxicities of four pairs of chiral ILs 1-alkyl-3-methylimidazolium lactate to Allivibrio fischeri were systematically investigated by using a comprehensive approach including the co-toxicity coefficient (CTC) integrated with confidence interval (CI) method (CTCICI), concentration-response curve (CRC), and isobole analysis. The direct equipartition ray (EquRay) design was used to design five binary mixtures of enantiomers according to molar ratios of 1:5, 2:4, 3:3, 4:2, and 5:1. The toxicities of chiral ILs and their mixtures were determined using the microplate toxicity analysis (MTA) method. Concentration addition (CA) and independent action (IA) were used as the additive reference models to construct the predicted CRC and isobole of mixtures. On the whole, there was an enantioselective toxicity difference between [BMIM]D-Lac and [BMIM]L-Lac, and [HMIM]D-Lac and [HMIM]L-Lac, while no enantioselective toxicity difference was observed for [EMIM]D-Lac and [EMIM]L-Lac, and [OMIM]D-Lac and [OMIM]L-Lac. Thereinto, the enantiomer mixtures of [BMIM]D-Lac and [BMIM]L-Lac, and [HMIM]D-Lac and [HMIM]L-Lac presented antagonistic action, and the enantiomer mixtures of [EMIM]D-Lac and [EMIM]L-Lac, and [OMIM]D-Lac and [OMIM]L-Lac overall presented additive action. Moreover, the greatest antagonistic toxicity interaction occurred at the equimolar ratio of enantiomers. Based on these results, we proposed two hypotheses, (1) chiral molecules with enantioselective toxicity difference tended to produce toxicity interactions, (2) the highest or lowest toxicity was usually at the equimolar ratio and its adjacent ratio for the enantiomer mixture. These hypotheses will need to be further validated by other enantiomer mixtures.


Introduction
Chirality is the geometric characteristic of a molecule being not superimposable on its mirror image formed by inversion through a point by pure rotation and translation. Chiral molecules with a single chiral center have two stereochemical arrangements, which have identical physicochemical properties, imidazolium tartrate can cause enantioselective oxidative stress to Scenedesmus obliquus, and the toxicity of [RMIM]L-(+)-tartrate treatment was greater than [RMIM]D-(-)-tartrate with enantioselectivity [32]. However, the toxicity interaction of chiral ILs enantiomers was still not entirely clear and has not been systematically studied up to now.
In the present study, we selected four pairs of chiral ILs composed of imidazolium cation and lactate anion as the enantiomer components. They were [ Their specific information is shown in the section of Materials and Methods. Among them, the imidazole cations were not chiral. While lactate anion has two enantiomers of L-lactate and D-lactate, and the human body can only metabolize the L-lactic acid [33]. We want to know how coexisting chiral ILs enantiomers interact with each other in the toxicity. Such considerations prompt us to initiate this study, which aims (1) to investigate the enantioselective toxicity difference for chiral ILs, (2) to investigate the enantiomeric toxicity interaction of chiral ILs, and (3) to determine whether there was some relationship between enantioselective toxicity difference and enantiomeric toxicity interaction.

Single Enantiomer Toxicity
All of the studied enantiomers inhibited Allivibrio fischeri (AVF) in a concentration-dependent manner, with log-sigmoidal CRC for the four pairs of chiral ILs shown in Figure 1. The concentration of the stock solutions (C 0 ) and following diluted solutions (C 1 -C 11 ) of chiral ILs and their mixtures were shown in Table S1. The regression models and the estimated parameters of the toxicity of single enantiomer to AVF are summarized in Table 1. The CRCs can be fitted by the two-parameter Weibull function with RMSE < 0.09 and R 2 > 0.91. The variability of the blank control in the test was controlled within ±20%. The indicators of effect concentration EC 80 , EC 50 , and EC 30 are shown in Table 1. According to these indicators, the toxicity order of single enantiomers was OL ≥ OD > HL > HD > BD > BL > EL ≥ ED. With the increase of the number of carbon atoms in the alkyl chains of the imidazolium cations, the toxicity of chiral ILs increased gradually. The EC 50 of EL was 143 times that of OL, and the EC 50 of ED was 137 times that of OD. Stock et al. also reported that ILs with long alkyl chains showed higher AVF inhibitive toxicity [34].   Previous studies have demonstrated that the enantiomers of chiral pesticides have different biological toxicities [9]. In the present study, the EC 50 of BL was 1.7 times that of BD, and the EC 50 of HD was 2.1 times that of HL. BL and BD, and HD HL showed enantioselective toxicity to AVF. While enantioselective toxicity difference was not observed for ED and EL, and OD and OL to AVF as shown in Figure 2A. Chen et al. reported the enantioselective toxicity of these four pairs of chiral lactate ILs to Scenedesmus obliquus, the EC 50 value of EL was twice that of ED to algae, while no enantioselective toxicity difference was found for L-lactic acid and D-lactic acid [31]. Therefore, the enantioselective toxicity difference of the chiral ILs enantiomers with lactate anion should be attributed to the interaction between anions and cations. Furthermore, the mechanisms of enantioselective toxicity difference of chiral ILs at the molecular and cellular levels will need further study to be elucidated [32]. ILs were different from ordinary neutral molecules. ILs can be regarded as a mixture of anions and cations. Stolte et al. proposed that the toxicity of single ILs can be calculated based on their anion and cation toxicity using the CA model [37]. Previous studies showed that the strong electrostatic interactions between an achiral cation and a chiral anion can result in the transfer, induction, and amplification of chiral information [38,39]. Such ion-pairing effects between achiral imidazolium cation and chiral lactate anion may induce the enantioselective toxicity difference. The n-octanol/water partition coefficient (logP o/w ) was an important parameter that can be used to simulate ILs diffusion from the aqueous phase to the bacterial cell membrane, and was also associated with the chemical toxicity [35]. We determined the logP o/w of these chiral ILs as shown in Figure 2B and Table S2. It can be seen that basically the logP o/w of L-lactate was greater than the logP o/w of D-lactate, except for the octyl IL in the opposite order. Meanwhile, compared with ethyl, hexyl, and octyl ILs, the logP o/w of butyl IL was the smallest, indicating that the trend of butyl IL participating in water was the largest relative to n-octanol. Normally, with the increase of the length of alkyl chain, the toxicity and lipophilicity of ILs were increased as shown in Figure 2A,B. However, the relationship between lipophilicity (logP o/w ) and toxicity (e.g., pEC 50 ) was generally an inverted U-shaped, as shown in Figure 2C and the reference [36]. Therefore, there may be some balance, resulting in enantioselective toxicity difference in the imidazole lactate ILs with an intermediate number of carbon atoms. Chen et al. also observed that chiral ILs with greater carbon chain lengths no longer exhibited enantioselectivity, due to changes in the toxicity weightings of the cations [31]. Furthermore, the mechanisms of enantioselective toxicity difference of chiral ILs at the molecular and cellular levels will need further study to be elucidated [32].
ILs were different from ordinary neutral molecules. ILs can be regarded as a mixture of anions and cations. Stolte et al. proposed that the toxicity of single ILs can be calculated based on their anion and cation toxicity using the CA model [37]. Previous studies showed that the strong electrostatic interactions between an achiral cation and a chiral anion can result in the transfer, induction, and amplification of chiral information [38,39]. Such ion-pairing effects between achiral imidazolium cation and chiral lactate anion may induce the enantioselective toxicity difference.

Enantiomer Mixture CRC and CTC
The mixture CRCs predicted by CA and IA together with the experimental data and the fitted curves were integrated and displayed in Figure 1. These observed CRCs can also be depicted by the Weibull function. In all cases, the R 2 s were greater than 0.85 and the RMSEs less than 0.13.
The EC 80 , EC 50 , and EC 30 values for enantiomer mixtures are listed in Table 1. Using the EC x,i , EC x,mix , and P i , the CTC of IL enantiomer mixtures can be obtained as shown in Table 2. According to the CTCICI method [18], at 30%, 50%, and 80% effect levels, the mixtures of BD and BL, HL, and HD, and OD and OL overall presented antagonistic action, except for the additive action of H2, H3 at 80% effect, H1 at 50% effect, B4, B5, H1, O1, O2, and O4 at 30% effect. While ED and EL mixtures presented additive action at 30% and 50% effect levels, and presented synergistic action at 80% effect level except for the additive action of the E3 mixture. Theoretically, CTC [40] was the deformation expression of CA, and the reciprocal form of the combination index [41]. Therefore, CTC only reflected the judgment result of CA on toxic interaction [42]. The advantages of CTC were simple, intuitive, quantitative, and widely used. Although in most cases, the difference between the CA and IA predictions were small for an assessment of mixture toxicity [43]. However, sometimes this difference can be too big to ignore [44,45]. The next step was to further study the toxic interaction of these four pairs of enantiomer mixtures from the perspective of CRC based on CA and IA in combination with CI. Comprehensively speaking, the predicted curves of CA and IA were above the observed CRC CI for BD and BL, and HL and HD mixtures, these two pairs of enantiomers were antagonistic action. While the predicted curves of CA and IA were within the observed CRC CI for ED and EL, and OD and OL mixtures, so these two pairs of enantiomers were additive action, except that the mixtures of ED and EL were synergistic action at the 80% effect level. Single enantiomer toxicity analysis indicated that enantioselective toxicity differences were observed for BD and BL, and HL and HD mixtures, and that were not observed for ED and EL, and OD and OL mixtures. This generated a hypothesis that chiral molecules with enantioselective toxicity difference tended to produce toxicity interaction. Figure 3 showed the isoboles of four pairs of chiral ILs mixtures at 30%, 50%, and 80% effect levels. In all situations, the CA isoboles were below the IA isoboles, which was the reflection of CA CRC above IA CRC. In general, the observed isoboles of the mixtures of ED and EL, and OD and OL were relatively close to the predicted isoboles of CA and IA, and the CI of the observed isoboles can basically contain the CA or IA isobole, so the two pairs of enantiomers were additive action. The only exception was ED and EL mixture presenting synergistic action at 80% effect level. In the Section 2.2, mixtures of OD and OL were judged to show antagonistic action. However, based on IA, these mixtures were determined to be additive. Therefore, CA and IA should be used in combination to comprehensively judge the toxicity interaction of mixtures to avoid qualitative error [18]. On the other hand, the observed isoboles of the mixtures of BD and BL, and HD and HL deviated from the predicted isoboles of CA and IA far upward, and the CI of the observed isoboles basically cannot contain the CA or IA isobole, so these two pairs of enantiomers showed antagonistic action, and the mixture with molar ratio 1:1 generally had the largest deviation trend indicating the greatest toxic interaction. It was generally accepted that CA was applicable to similar acting chemicals and IA was applicable to dissimilar acting chemicals [46]. It should be more likely that enantiomers were applicable to CA, but our results indicated that IA was closer to mixture observed isobole than CA. Therefore, the question was aroused as to whether CA and IA were related to the mechanism of action (MoA). Our results supported that CA and IA were both only the additive reference models, which were not applicable to associate with the MoA. The additivity assumption was merely a working concept and did not necessarily reflect the reality [47].

Enantiomer Mixture Toxicity Assessment Based on Isobole
In the present study, we used three methods of CTCICI, CRC, and isobole to evaluate the toxic interaction of enantiomers of chiral ILs. To date, there were few examples of evaluating enantiomeric mixture effects based on isobole [16]. Most of studies regarding enantiomer mixture toxicity evaluation were based on statistical judgments by comparing with the toxicity of single enantiomers It was generally accepted that CA was applicable to similar acting chemicals and IA was applicable to dissimilar acting chemicals [46]. It should be more likely that enantiomers were applicable to CA, but our results indicated that IA was closer to mixture observed isobole than CA. Therefore, the question was aroused as to whether CA and IA were related to the mechanism of action (MoA). Our results supported that CA and IA were both only the additive reference models, which were not applicable to associate with the MoA. The additivity assumption was merely a working concept and did not necessarily reflect the reality [47].
In the present study, we used three methods of CTCICI, CRC, and isobole to evaluate the toxic interaction of enantiomers of chiral ILs. To date, there were few examples of evaluating enantiomeric mixture effects based on isobole [16]. Most of studies regarding enantiomer mixture toxicity evaluation were based on statistical judgments by comparing with the toxicity of single enantiomers [48]. The advantage of this approach was that it was simple to operate and did not require the introduction of an additive reference model. In particular, when the effect concentration of enantiomer mixture was greater than or less than the corresponding effect concentration of enantiomer components, the mixture would show antagonistic or synergistic action [49]. In theory, this was still based on the CA principle. As shown in Table 1, all the mixtures of BD and BL, HD and HL, and OD and OL at 30%, 50%, and 80% effect for all molar ratios, the enantiomer mixture effect concentrations (EC x,mix ) were all greater than single enantiomer effect concentrations (EC x,i ), which indicated that these mixtures were theoretically antagonistic based on CA. Only the mixture of ED and EL at all molar ratios, the mixture 80%-effect concentrations (EC 80,mix ) were all smaller than the enantiomer 80%-effect concentrations (EC 80,i ), which indicated that these mixtures were theoretically synergistic based on CA. In these cases, toxic interactions can be determined even without the use of statistical judgments, additive models, or isobole. However, when the mixture effect concentration was within or not far from the enantiomer effect concentration, it was necessary to use the additive model or isobole in combination with the CI. Comparatively speaking, isobole had the natural applicability, comprehensiveness, and accuracy in judging enantiomeric toxic interactions.

Relationship between Mixture Toxicity and Enantiomer Concentration Proportions
Previous studies indicated that there was biphasic relationship between the binary mixture toxicity and the concentration proportion (P i ) of components [18]. A pair of enantiomers of chiral molecules can form the natural binary mixtures. It can be seen that in Figure 4, there were two pairs of relatively obvious U-shaped relationship between the P i of components and the toxicities (pEC 30 , pEC 50 , pEC 80 ) of mixtures of BD and BL, and HL and HD. There were two pairs of inverted U-shaped relationships between the P i of components and the toxicities (pEC 30 , pEC 50 , pEC 80 ) of mixtures of EL and ED, and OD and OL. This phenomenon basically conformed to the climax hypothesis proposed by Lin et al. [50]. The climax hypothesis concluded that there was a climax at the equitoxic ratio when plotting the toxic ratios of individual chemicals in mixtures versus their joint effects [51].
In the present study, we deliberately designed the enantiomer mixtures according to their molar ratio, and found that the highest or lowest toxicity point was usually at the equimolar ratio, especially for the enantiomers with differential toxicity. Therefore, for the enantiomer mixture, the climax hypothesis was reduced to a new form that the highest or lowest toxicity was usually at the equimolar ratio. To show the difference, we called this type of Climax hypothesis the Crown hypothesis according to the shape of the isobole. This new improved hypothesis needed to be tested by other enantiomeric binary mixtures. 10

Implications
The equimolar ratio corresponding to the greatest antagonism may reflect a certain mechanism in itself. When the ratio of enantiomers was greatly different, the one with a large proportion played a dominant role, the antagonism was also small accordingly. However, when the proportion of the two enantiomers was the same, the two had even influence and produced the greatest antagonism. More quantitative and accurate interpretation may need to apply the molecular simulation to a specific protein molecular target, such as the photobacterium luciferase.
Our results showed that the antagonistic toxicity interaction was strongest when the enantiomeric mixture was at the equimolar ratio. Raffa et al. also observed that the mixture of the tramadol enantiomers with the ratio (−)/(+) = 1/1 produced the strongest synergistic action to mice for the antinociception effect expressed as inhibition of acetylcholine-induced abdominal constriction [16]. When mixtures were presented in equimolar ratio and its adjacent ratio, the maximum medicinal interaction can also be observed in drug synergies. In fact, the ratio-dependent synergy had also been

Implications
The equimolar ratio corresponding to the greatest antagonism may reflect a certain mechanism in itself. When the ratio of enantiomers was greatly different, the one with a large proportion played a dominant role, the antagonism was also small accordingly. However, when the proportion of the two enantiomers was the same, the two had even influence and produced the greatest antagonism. More quantitative and accurate interpretation may need to apply the molecular simulation to a specific protein molecular target, such as the photobacterium luciferase.
Our results showed that the antagonistic toxicity interaction was strongest when the enantiomeric mixture was at the equimolar ratio. Raffa et al. also observed that the mixture of the tramadol enantiomers with the ratio (−)/(+) = 1/1 produced the strongest synergistic action to mice for the antinociception effect expressed as inhibition of acetylcholine-induced abdominal constriction [16]. When mixtures were presented in equimolar ratio and its adjacent ratio, the maximum medicinal interaction can also be observed in drug synergies. In fact, the ratio-dependent synergy had also been used in pharmacology. Ribavirin and disulfiram in molar ratio 2:1 presented the maximal antibacterial synergy against methicillin-resistant Staphylococcus aureus proliferation [52]. Indacrinone was a diuretic, the (−) isomer had diuretic effect with increasing the uric acid levels in blood, while the (+) isomer can promote the excretion of uric acid. It was possible to improve the therapeutic effects of indacrinone by manipulation of the enantiomer ratio, such as (−)/(+) = 1/4 [53]. However, whether the equimolar ratio and its adjacent ratio presenting the maximum toxic interaction was universal required further verification.
For the ILs toxicity, it was generally accepted that the cations played a major role and were more important than anions. Our results suggested that the interaction between cations and anions may actually be more important than single ions. Our results also showed that the toxicity of octyl ILs was two orders of magnitude greater than that of ethyl ILs, while different configurations of lactate ILs had no fixed order of toxicity. To minimize the environmental risk, the chiral ILs with short alkyl chains and enantioselective toxicities should be taken into consideration [31].
The stock solutions of ILs were separately prepared through dissolving them in the deionized water and stored in a 4 • C refrigerator. The stock solutions of IL mixtures were prepared through mixing the stock solutions of individual ILs according to their concentration ratios assigned. The concentration of the stock solutions (C 0 ) and following diluted solutions (C 1 -C 11 ) of chiral ILs and their mixtures were shown in the Supplementary Materials. used in pharmacology. Ribavirin and disulfiram in molar ratio 2:1 presented the maximal antibacterial synergy against methicillin-resistant Staphylococcus aureus proliferation [52]. Indacrinone was a diuretic, the (−) isomer had diuretic effect with increasing the uric acid levels in blood, while the (+) isomer can promote the excretion of uric acid. It was possible to improve the therapeutic effects of indacrinone by manipulation of the enantiomer ratio, such as (−)/(+) = 1/4 [53]. However, whether the equimolar ratio and its adjacent ratio presenting the maximum toxic interaction was universal required further verification. For the ILs toxicity, it was generally accepted that the cations played a major role and were more important than anions. Our results suggested that the interaction between cations and anions may actually be more important than single ions. Our results also showed that the toxicity of octyl ILs was two orders of magnitude greater than that of ethyl ILs, while different configurations of lactate ILs had no fixed order of toxicity. To minimize the environmental risk, the chiral ILs with short alkyl chains and enantioselective toxicities should be taken into consideration [31].

Chemicals
The IL components  Table 3.
The stock solutions of ILs were separately prepared through dissolving them in the deionized water and stored in a 4 °C refrigerator. The stock solutions of IL mixtures were prepared through mixing the stock solutions of individual ILs according to their concentration ratios assigned. The concentration of the stock solutions (C0) and following diluted solutions (C1-C11) of chiral ILs and their mixtures were shown in the Supplementary materials. used in pharmacology. Ribavirin and disulfiram in molar ratio 2:1 presented the maximal antibacterial synergy against methicillin-resistant Staphylococcus aureus proliferation [52]. Indacrinone was a diuretic, the (−) isomer had diuretic effect with increasing the uric acid levels in blood, while the (+) isomer can promote the excretion of uric acid. It was possible to improve the therapeutic effects of indacrinone by manipulation of the enantiomer ratio, such as (−)/(+) = 1/4 [53]. However, whether the equimolar ratio and its adjacent ratio presenting the maximum toxic interaction was universal required further verification. For the ILs toxicity, it was generally accepted that the cations played a major role and were more important than anions. Our results suggested that the interaction between cations and anions may actually be more important than single ions. Our results also showed that the toxicity of octyl ILs was two orders of magnitude greater than that of ethyl ILs, while different configurations of lactate ILs had no fixed order of toxicity. To minimize the environmental risk, the chiral ILs with short alkyl chains and enantioselective toxicities should be taken into consideration [31].

Chemicals
The IL components  Table 3.
The stock solutions of ILs were separately prepared through dissolving them in the deionized water and stored in a 4 °C refrigerator. The stock solutions of IL mixtures were prepared through mixing the stock solutions of individual ILs according to their concentration ratios assigned. The concentration of the stock solutions (C0) and following diluted solutions (C1-C11) of chiral ILs and their mixtures were shown in the Supplementary materials. used in pharmacology. Ribavirin and disulfiram in molar ratio 2:1 presented the maximal antibacterial synergy against methicillin-resistant Staphylococcus aureus proliferation [52]. Indacrinone was a diuretic, the (−) isomer had diuretic effect with increasing the uric acid levels in blood, while the (+) isomer can promote the excretion of uric acid. It was possible to improve the therapeutic effects of indacrinone by manipulation of the enantiomer ratio, such as (−)/(+) = 1/4 [53]. However, whether the equimolar ratio and its adjacent ratio presenting the maximum toxic interaction was universal required further verification. For the ILs toxicity, it was generally accepted that the cations played a major role and were more important than anions. Our results suggested that the interaction between cations and anions may actually be more important than single ions. Our results also showed that the toxicity of octyl ILs was two orders of magnitude greater than that of ethyl ILs, while different configurations of lactate ILs had no fixed order of toxicity. To minimize the environmental risk, the chiral ILs with short alkyl chains and enantioselective toxicities should be taken into consideration [31].

Chemicals
The IL components  Table 3.
The stock solutions of ILs were separately prepared through dissolving them in the deionized water and stored in a 4 °C refrigerator. The stock solutions of IL mixtures were prepared through mixing the stock solutions of individual ILs according to their concentration ratios assigned. The concentration of the stock solutions (C0) and following diluted solutions (C1-C11) of chiral ILs and their mixtures were shown in the Supplementary materials. used in pharmacology. Ribavirin and disulfiram in molar ratio 2:1 presented the maximal antibacterial synergy against methicillin-resistant Staphylococcus aureus proliferation [52]. Indacrinone was a diuretic, the (−) isomer had diuretic effect with increasing the uric acid levels in blood, while the (+) isomer can promote the excretion of uric acid. It was possible to improve the therapeutic effects of indacrinone by manipulation of the enantiomer ratio, such as (−)/(+) = 1/4 [53]. However, whether the equimolar ratio and its adjacent ratio presenting the maximum toxic interaction was universal required further verification. For the ILs toxicity, it was generally accepted that the cations played a major role and were more important than anions. Our results suggested that the interaction between cations and anions may actually be more important than single ions. Our results also showed that the toxicity of octyl ILs was two orders of magnitude greater than that of ethyl ILs, while different configurations of lactate ILs had no fixed order of toxicity. To minimize the environmental risk, the chiral ILs with short alkyl chains and enantioselective toxicities should be taken into consideration [31].

Chemicals
The IL components  Table 3.
The stock solutions of ILs were separately prepared through dissolving them in the deionized water and stored in a 4 °C refrigerator. The stock solutions of IL mixtures were prepared through mixing the stock solutions of individual ILs according to their concentration ratios assigned. The concentration of the stock solutions (C0) and following diluted solutions (C1-C11) of chiral ILs and their mixtures were shown in the Supplementary materials.

Photobacterium Toxicity Test
The photobacterium Aliivibrio fischeri (Strain number 1H00019) was purchased from Marine Culture Collection of China (MCCC). The culture medium consisted of 1 g KH2PO4, 4.7 g Na2HPO4·12H2O, 0.3 g MgSO4·7H2O, 0.5 g (NH4)2HPO4, 30 g NaCl, 5.0 g yeast extract powder, 5.0 g tryptone, 3.0 g glycerin, and 1000 mL water, and was adjusted to pH 6.7 ± 0.3. The AVF was grown in the culture medium at 22 ± 1 °C by shaking (120 r/min) for 8-12 h during the logarithmic growth phase until the relative light unit reached 1×10 6 for the toxicity test.
The toxicities of single ILs and their mixtures were expressed as an inhibition of the AVF luminescence. According to the methods of MTA [54], IL chemicals and their mixtures with 11 concentration series in eight repeats and eight controls were arranged in a microplate. First, 100 µL water was added to eight wells in the twelfth column as blank controls, 100 µL of the solutions of IL chemicals, and their mixtures with 11 gradient concentrations according to a geometric dilution factors of 0.5 were added to the wells from the first to the eleventh column. Then, 100 µL AVF suspension was added into each well to reach the final volume of 200 µL. The relative light units (RLUs) of the AVF system exposed to single ILs and their mixtures were determined on Synergy 2 Multi-Mode Microplate Readers (BioTek Instruments, Winooski, VT, USA) with a 96-well white flat bottom microplate (Corning 3917) after 30 min of exposure at 26 ± 1 °C.
The inhibitive effect (E of x%) of individual ILs and their mixtures was calculated using Equation (1). The CRCs were fitted by Weibull function shown in Equation (2) using least squares method [55]. The goodness of fit of statistical models was evaluated by R 2 and RMSE. As a quantitative measure of the uncertainty, the observation-based 95% CI was determined [56].
where L0 is the average of RLUs of controls, L is the average of RLUs of treatments, E is inhibitive effect of AVF luminescence, C is chemical concentration, a is location parameter, and b is slope parameter.

Experimental Design and Toxicity Evaluation of Mixtures
The five binary mixtures of each pair of enantiomers were designed using the EquRay design [57], every two enantiomers were mixed according to molar ratios of 1:5, 2:4, 3:3, 4:2, and 5:1. The molar ratio was chosen instead of the toxic unit ratio to construct the racemic mixture and to increase the generality of the experimental design.
The models of CA shown in Equation (3) and IA shown in Equation (4) were used to predict the mixture effect concentration (ECx,mix) corresponding to the mixture x% effect, and the predicted CRC of the mixture was also presented [58]. The predicted ECx,mix was multiplied by the enantiomer concentration fraction (Pi) to obtain the two partial concentrations which formed a point in the twodimensional Cartesian coordinates. These points were connected to form the mixture predicted

Photobacterium Toxicity Test
The photobacterium Aliivibrio fischeri (Strain number 1H00019) was purchased from Marine Culture Collection of China (MCCC). The culture medium consisted of 1 g KH2PO4, 4.7 g Na2HPO4·12H2O, 0.3 g MgSO4·7H2O, 0.5 g (NH4)2HPO4, 30 g NaCl, 5.0 g yeast extract powder, 5.0 g tryptone, 3.0 g glycerin, and 1000 mL water, and was adjusted to pH 6.7 ± 0.3. The AVF was grown in the culture medium at 22 ± 1 °C by shaking (120 r/min) for 8-12 h during the logarithmic growth phase until the relative light unit reached 1×10 6 for the toxicity test.
The toxicities of single ILs and their mixtures were expressed as an inhibition of the AVF luminescence. According to the methods of MTA [54], IL chemicals and their mixtures with 11 concentration series in eight repeats and eight controls were arranged in a microplate. First, 100 µL water was added to eight wells in the twelfth column as blank controls, 100 µL of the solutions of IL chemicals, and their mixtures with 11 gradient concentrations according to a geometric dilution factors of 0.5 were added to the wells from the first to the eleventh column. Then, 100 µL AVF suspension was added into each well to reach the final volume of 200 µL. The relative light units (RLUs) of the AVF system exposed to single ILs and their mixtures were determined on Synergy 2 Multi-Mode Microplate Readers (BioTek Instruments, Winooski, VT, USA) with a 96-well white flat bottom microplate (Corning 3917) after 30 min of exposure at 26 ± 1 °C.
The inhibitive effect (E of x%) of individual ILs and their mixtures was calculated using Equation (1). The CRCs were fitted by Weibull function shown in Equation (2) using least squares method [55]. The goodness of fit of statistical models was evaluated by R 2 and RMSE. As a quantitative measure of the uncertainty, the observation-based 95% CI was determined [56].
where L0 is the average of RLUs of controls, L is the average of RLUs of treatments, E is inhibitive effect of AVF luminescence, C is chemical concentration, a is location parameter, and b is slope parameter.

Experimental Design and Toxicity Evaluation of Mixtures
The five binary mixtures of each pair of enantiomers were designed using the EquRay design [57], every two enantiomers were mixed according to molar ratios of 1:5, 2:4, 3:3, 4:2, and 5:1. The molar ratio was chosen instead of the toxic unit ratio to construct the racemic mixture and to increase the generality of the experimental design.
The models of CA shown in Equation (3) and IA shown in Equation (4) were used to predict the mixture effect concentration (ECx,mix) corresponding to the mixture x% effect, and the predicted CRC of the mixture was also presented [58]. The predicted ECx,mix was multiplied by the enantiomer concentration fraction (Pi) to obtain the two partial concentrations which formed a point in the twodimensional Cartesian coordinates. These points were connected to form the mixture predicted 98% 284.39
The toxicities of single ILs and their mixtures were expressed as an inhibition of the AVF luminescence. According to the methods of MTA [54], IL chemicals and their mixtures with 11 concentration series in eight repeats and eight controls were arranged in a microplate. First, 100 µL water was added to eight wells in the twelfth column as blank controls, 100 µL of the solutions of IL chemicals, and their mixtures with 11 gradient concentrations according to a geometric dilution factors of 0.5 were added to the wells from the first to the eleventh column. Then, 100 µL AVF suspension was added into each well to reach the final volume of 200 µL. The relative light units (RLUs) of the AVF system exposed to single ILs and their mixtures were determined on Synergy 2 Multi-Mode Microplate Readers (BioTek Instruments, Winooski, VT, USA) with a 96-well white flat bottom microplate (Corning 3917) after 30 min of exposure at 26 ± 1 • C.
The inhibitive effect (E of x%) of individual ILs and their mixtures was calculated using Equation (1). The CRCs were fitted by Weibull function shown in Equation (2) using least squares method [55]. The goodness of fit of statistical models was evaluated by R 2 and RMSE. As a quantitative measure of the uncertainty, the observation-based 95% CI was determined [56].
where L 0 is the average of RLUs of controls, L is the average of RLUs of treatments, E is inhibitive effect of AVF luminescence, C is chemical concentration, a is location parameter, and b is slope parameter.

Experimental Design and Toxicity Evaluation of Mixtures
The five binary mixtures of each pair of enantiomers were designed using the EquRay design [57], every two enantiomers were mixed according to molar ratios of 1:5, 2:4, 3:3, 4:2, and 5:1. The molar ratio was chosen instead of the toxic unit ratio to construct the racemic mixture and to increase the generality of the experimental design.
The models of CA shown in Equation (3) and IA shown in Equation (4) were used to predict the mixture effect concentration (EC x,mix ) corresponding to the mixture x% effect, and the predicted CRC of the mixture was also presented [58]. The predicted EC x,mix was multiplied by the enantiomer concentration fraction (P i ) to obtain the two partial concentrations which formed a point in the two-dimensional Cartesian coordinates. These points were connected to form the mixture predicted isobole. When the CIs of mixture observed isobole were containing, above, or below the mixture predicted isobole, the mixture was judged to present additive, antagonistic, or synergistic action, respectively.
For CRC, when the mixture predicted CRC was located within the CIs of mixture observed CRC, the mixture presented additive action. When the mixture predicted CRC was located above or below the mixture observed CRC CI, the mixture presented antagonistic or synergistic action, respectively. At the single effect point level, the toxic interactions of mixtures were evaluated using the components EC x,i , and mixture observed EC x,mix and its 95% CI according to the CTCICI method developed recently [18]. The CTC were computed using Equation (5). When 100 was included in the CI of mixture CTC, the mixture presented additive action. When the CI of mixture CTC was greater or smaller than 100, the mixture presented synergistic or antagonistic action, respectively.
where n is the number of mixture components, EC x,i is the concentration of ith component eliciting the x% effect, EC x,mix is the concentration of a mixture eliciting the x% effect, P i is the concentration proportion of ith component in a mixture, F i is individual concentration-response functions, CTC is co-toxicity coefficient.

Conclusions
The toxicities of the four pairs of chiral ionic liquids (ILs) to Allivibrio fischeri were explored. Interestingly, two pairs of chiral ILs showed enantioselective toxicity difference, while the other two pairs of chiral ILs showed no enantioselective toxicity difference. Thereinto, the enantiomer mixtures of two pairs of chiral ILs with enantioselective toxicity difference presented antagonistic action, that without enantioselective toxicity difference overall presented additive action. Moreover, the greatest toxic interaction occurred at the enantiomer molar ratio 1:1. These results may have important implications and practical applications for chiral molecules in toxicology, pharmacology, environmental science, pesticide science, and other research fields.

Conflicts of Interest:
The authors declare no conflict of interest.