Inhibition Effect of Ionic Liquid [Hmim]Cl on Microcystis Growth and Toxin Production

Ionic liquids (ILs) are known as “green solvents” and widely used in industrial applications. However, little research has been conducted on cyanobacteria. This study was conducted to investigate the toxicity of ionic liquids ([Hmim]Cl) on Microcystis aeruginosa PCC7806. The EC50 (72 h) of [Hmim]Cl on the growth of Microcystis aeruginosa PCC 7806 was 10.624 ± 0.221 mg L−1. The possible mechanism of toxicity of [Hmim]Cl against M. aeruginosa PCC 7806 was evaluated by measuring cell growth, photosynthetic pigment contents, chlorophyll fluorescence transients, cell ultrastructure, and transcription of the microcystin-producing gene (mcyB). The concentrations of chlorophyll a and carotenoids were significantly reduced in treated M. aeruginosa cultures. The results of chlorophyll fluorescence transients showed that [Hmim]Cl could destruct the electron-accepting side of the photosystem II of M. aeruginosa PCC 7806. Transmission electron microscopy demonstrated cell damage including changes in the structure of the cell wall and cell membrane, thylakoid destruction, and nucleoid disassembly. The transcription of the mcyB gene was also inhibited under [Hmim]Cl stress. In summary, this study provides new insights into the toxicity of [Hmim]Cl on cyanobactreia.


Introduction
Demand for green technologies has increased rapidly in recent years. Ionic liquids (ILs) have attracted great attention as promising organic solvents. ILs are low-temperature molten salts, which possess many unique physiochemical characteristics and excellent solvent properties (negligible vapor pressure, high thermal, chemical, and electrochemical stability) [1]. Therefore, ILs have been considered to be "green" substitutes for conventional volatile organic compounds (VOCs) used in synthetic and analytical processes [2]. Although ILs can reduce the potential risk of air pollution due to their negligible vapor pressures, they may pose ecological risks to aquatic ecosystems, due to their high solubility in water. In addition, many studies have found that ILs show poor biodegradability [3].
Algae are crucial primary producers for the food chain in aquatic ecosystems and play an important role in nutrient cycling. Algae have been used for hazard assessment of aquatic contaminants [4,5], because of their rapid response to environmental changes. Several studies have been done on the toxicity of ILs on aquatic organisms, such as green algae [6], diatoms [7], cladocerans [8], mussels [9], and fish [10]. However, little research has been conducted on cyanobacteria [11].
Global warming and eutrophication have enhanced the proliferation of cyanobacteria in freshwater bodies [12,13]. Microcystis aeruginosa (Kützing) Kützing 1846 is one of the most widespread and dominant cyanobacterial species [14,15]. M. aeruginosa also has the capability of producing secondary metabolites such as microcystin, the synthesis of 2 of 9 which is sensitive to the surrounding environment [16,17]. Thus, the response of M. aeruginosa to IL stress can be used for assessing the impact of ILs on aquatic ecosystems. Oxygenic photosynthesis of cyanobacteria is easily affected by hazardous substances such as ILs [18]. The chlorophyll fluorescence measurement is an important index to evaluate the performance of photosynthesis processes and is usually measured via the redox states of photosystem II (PSII) and the electron-donating and -accepting sides of the active reaction centers. Krause et al. [19] and Strasser and Strasser [20] established the JIP-test, which conducts a kinetic analysis of the light-induced chlorophyll fluorescence rise curve based on the theory of biofilm energy flow. The JIP-test reflects the physiological state of cyanobacterial PSII by detecting the effects of different factors on its structure and function [18,21,22].
The compound 1-hexyl-3-methylimidazolium chloride, which is abbreviated as [Hmim]Cl, is one of the widely used ILs [23]. Until now, there has been no available study concerning the effects of [Hmim]Cl on cyanobacteria. The current study aims to illustrate the possible toxicological mechanism of [Hmim]Cl on cyanobacteria from the view of cell growth, photosynthesis, and gene expression.

Materials and Methods
The M. aeruginosa PCC 7806 was provided by the Institute of Hydrobiology, Chinese Academy of Sciences, and was cultured in a BG11 liquid medium [24]. The culture was maintained at a light-dark cycle of 12 h:12 h with a light intensity of 3000 lx under a controlled temperature (28 • C). The [Hmim]Cl (99% purity) was purchased from the Chengjie Chemical Company (Shanghai, China).

Measurement of Cell Growth and Photosynthetic Pigments Content
The [Hmim]Cl was prepared and added into the M. aeruginosa PCC 7806 culture with a cell density of 10 4 cells mL −1 . The final concentrations of [Hmim]Cl were set at 5, 10, 20, and 30 mg L −1 . All experiments were performed in triplicate. The cell density and pigment contents in the control and treated cultures were measured after 24 and 72 h of incubation. To measure the pigment contents, the cultures were centrifuged for 10 min at 8000 rpm, and the supernatants were discarded. The chlorophyll a(Chl a) and carotenoids were extracted from cell pellets using 80% acetone for 24 h in darkness at 4 • C. Absorbance of the extracts was measured using a spectrophotometer at 663, 645, and 450 nm. The following equations set by Richards et al. (1952) [25] were used for the calculation of the pigment contents.

Measurement of Chlorophyll Fluorescence Transients
The polyphasic rise of chlorophyll fluorescence transients was detected by PEA (AquaPen-C AP-C 100, Photon Systems Instruments, The Czech Republic). The JIP transients were measured after exposure to [Hmim]Cl stress for 24 h and 72 h, respectively. Cells from the control and treated cultures were placed on special plastic clips. The parameters of absorption (ABS/RC), trapping (TR 0 /RC), electron transport (ET 0 /RC), dissipation (DI 0 /RC), the probability that a trapped exciton moves an electron into the electron transport chain beyond QA (ψ 0 ), the maximum quantum yield of primary photochemistry (ϕP 0 ), the quantum yield of electron transport (ϕE 0 ), the probability that an absorbed photon is dissipated (ϕD 0 ), relative fluorescence values [(1 − V K /V J ) r ], and the performance indexes of photosystem II were calculated from the chlorophyll fluorescence transients [26,27]. The intensity of actinic light was set at 30 µmol photons/(m 2 ·s). The equations used for calculating the above parameters were as follows [27]: ET 0 /RC = (TR 0 /RC)ψ 0

Observation of Cell Ultrastructure
Changes in the ultrastructure of the treated M. aeruginosa cells at 10 and 30 mg L −1 of [Hmim]Cl were detected using transmission electron microscopy. After 48 h of exposure, culture cells were centrifuged for 15 min at 4000 rpm under 4 • C, and the supernatant was discarded. The cell pellets were fixed with 2.5% glutaraldehyde solution. The samples were sent to the Institute of Hydrobiology, Chinese Academy of Sciences, for electron microscopic analysis. Changes in the ultrastructure of the treated cells were identified by comparison with the control group.

RNA Extraction and Reverse Transcription
The cells of M. aeruginosa PCC 7806 were harvested after 24 h and 72 h of exposure to each concentration of [Hmim]Cl. The cultures were centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. Cell pellets were re-suspended in a Trizol reagent (Invitrogen, Waltham, MA, USA) and then frozen and thawed for three times. Total RNAs were extracted following the manufacturer's manual of Trizol reagent and dissolved with diethyl pyrocarbonate-treated water. Reverse transcription was conducted using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany).

Determination of mcyB Gene Transcription
The primers 30F and 108R were used for the detection of the mcyB gene [16], whereas 5 -GGACGGGTGAGTAACGCGTA-3 and 5 -CCCATTGCGGAAAATTCCCC-3 were used to amplify 16S rRNA gene as the internal control [28]. The transcription of mcyB in M. aeruginosa was determined by qPCR after 24 h and 72 h of exposure to each concentration of [Hmim]Cl. The qPCR reaction was prepared with final volumes of 10 µL containing 5 µL of Master Mix (SYBR Green, CWBIO, China), 0.2 µL of each primer (10 µmol L −1 ), and 0.4 µL cDNA and 4 µL of sterile water. The qPCR conditions were set as follows: 95 • C for 10 min; 40 cycles at 95 • C for 15 s, 59 • C for 30 s, and 72 • C for 30 s. The relative transcription level of the mcyB gene was calculated using 2 −∆∆Ct where ∆∆Ct = (Ct target gene − Ct 16S rrn ) stress − (Ct target gene − Ct 16Srrn ) control [29].

Statistical Analysis
The experimental data were analyzed by SPSS 20.0 (ANOVA, LSD). Differences were considered to be significant at p < 0.05.

Inhibition of [Hmim]Cl on M. aeruginosa PCC 7806 Growth
As displayed in Figure 1a, cell growth was not affected by [Hmim]Cl at the concentrations of 5 and 10 mg L −1 at 24 h. However, cell growth was significantly inhibited by higher concentrations of [Hmim]Cl with 25% decrease at 30 mg L −1 . After 72 h, the cell density of the control culture and the culture exposed to 5 mg L −1 of [Hmim]Cl increased by 118% and 61%, respectively. For cultures exposed to higher concentrations of [Hmim]Cl, cell density decreased after 72 h of exposure. No growth was observed at 10 mg L −1 of [Hmim]Cl, and cell density decreased by 23% in culture exposed to 30  trations of 5 and 10 mg L −1 at 24 h. However, cell growth was significantly inhibited higher concentrations of [Hmim]Cl with 25% decrease at 30 mg L −1 . After 72 h, the density of the control culture and the culture exposed to 5 mg L −1 of [Hmim]Cl increa by 118% and 61%, respectively. For cultures exposed to higher concentrations [Hmim]Cl, cell density decreased after 72 h of exposure. No growth was observed at mg L −1 of [Hmim]Cl, and cell density decreased by 23% in culture exposed to 30   . Different uppercase letters represent significant ferences (p < 0.05) between exposure times within the same treatment. Different lowercase let represent significant differences (p < 0.05) between treatments within the same exposure time.

Effects of [Hmim]Cl on the Pigment Contents
The effects of [Hmim]Cl on the pigment contents were similar to those on the growth ( Figure 2). The Chl a concentration in the control culture increased by 34.5% a 174.2% after 24 h and 72 h of incubation, respectively. However, little changes were served in the treated cultures at 24 h when compared with 0 time. Although the Ch concentrations in the treated cultures increased after 72 h of exposure, they remained s nificantly lower than in the control culture. The inhibition of [Hmim]Cl on caroteno production was more apparent than that on Chl a production. The increase of the caro noids' concentrations in the treated cultures was lower than 50% in comparison to th times in the control culture after 72 h of incubation. . Different uppercase letters represent significant differences (p < 0.05) between exposure times within the same treatment. Different lowercase letters represent significant differences (p < 0.05) between treatments within the same exposure time.

Effects of [Hmim]Cl on the Pigment Contents
The effects of [Hmim]Cl on the pigment contents were similar to those on the cell growth ( Figure 2). The Chl a concentration in the control culture increased by 34.5% and 174.2% after 24 h and 72 h of incubation, respectively. However, little changes were observed in the treated cultures at 24 h when compared with 0 time. Although the Chl a concentrations in the treated cultures increased after 72 h of exposure, they remained significantly lower than in the control culture. The inhibition of [Hmim]Cl on carotenoids production was more apparent than that on Chl a production. The increase of the carotenoids' concentrations in the treated cultures was lower than 50% in comparison to three times in the control culture after 72 h of incubation.

Inhibition of [Hmim]Cl on M. aeruginosa PCC 7806 Growth
As displayed in Figure 1a, cell growth was not affected by [Hmim]Cl at the concentrations of 5 and 10 mg L −1 at 24 h. However, cell growth was significantly inhibited by higher concentrations of [Hmim]Cl with 25% decrease at 30 mg L −1 . After 72 h, the cell density of the control culture and the culture exposed to 5 mg L −1 of [Hmim]Cl increased by 118% and 61%, respectively. For cultures exposed to higher concentrations of [Hmim]Cl, cell density decreased after 72 h of exposure. No growth was observed at 10 mg L −1 of [Hmim]Cl, and cell density decreased by 23% in culture exposed to 30   . Different uppercase letters represent significant differences (p < 0.05) between exposure times within the same treatment. Different lowercase letters represent significant differences (p < 0.05) between treatments within the same exposure time.

Effects of [Hmim]Cl on the Pigment Contents
The effects of [Hmim]Cl on the pigment contents were similar to those on the cell growth ( Figure 2). The Chl a concentration in the control culture increased by 34.5% and 174.2% after 24 h and 72 h of incubation, respectively. However, little changes were observed in the treated cultures at 24 h when compared with 0 time. Although the Chl a concentrations in the treated cultures increased after 72 h of exposure, they remained significantly lower than in the control culture. The inhibition of [Hmim]Cl on carotenoids production was more apparent than that on Chl a production. The increase of the carotenoids' concentrations in the treated cultures was lower than 50% in comparison to three times in the control culture after 72 h of incubation. (a) Chl a concentration; (b) carotenoids concentration. Different uppercase letters represent significant differences (p < 0.05) between exposure times within the same treatment. Different lowercase letters represent significant differences (p < 0.05) between treatments within the same exposure time.

Characteristics of Chlorophyll Fluorescence Transients
The parameters of the JIP test are illustrated as a radar plot (Figure 3). At 24 h, TR 0 /RC, ϕP 0 , ϕD 0 , and (1 − V K /V J ) r were not affected by [Hmim]Cl, whereas PI ABS was significantly inhibited at all concentrations of [Hmim]Cl. The parameters ABS/RC and DI 0 /RC at 5 mg L −1 were significantly larger than those of the control culture and ET 0 /RC and ψ 0 were significantly lower at 30 mg L −1 .
nificantly inhibited at all concentrations of [Hmim]Cl. The parameters ABS/RC and DI0/RC at 5 mg L −1 were significantly larger than those of the control culture and ET0/RC and ψ0 were significantly lower at 30 mg L −1 .
The effects of [Hmim]Cl on chlorophyll fluorescence transients were more apparent at 72 h compared with those at 24 h. The φP0 value was significantly lower than that of the control culture after 72 h of exposure. DI0/RC was doubled at 5 and 20 mg L −1 and increased 3 times at 10 mg L −1 when compared with the values at 24 h. The ABS/RC was significantly increased at all concentrations at 72 h, whereas PIABS was significantly inhibited.   The effects of [Hmim]Cl on chlorophyll fluorescence transients were more apparent at 72 h compared with those at 24 h. The ϕP 0 value was significantly lower than that of the control culture after 72 h of exposure. DI 0 /RC was doubled at 5 and 20 mg L −1 and increased 3 times at 10 mg L −1 when compared with the values at 24 h. The ABS/RC was significantly increased at all concentrations at 72 h, whereas PI ABS was significantly inhibited.

Transcription of mcyB Gene
The relative expression of mycB gene at different concentrations of [Hmim]Cl was significantly lower than that of the control culture ( Figure 5). With the exception of 5 mg L −1 , the gene expressions in treated cultures were increased significantly after 72 h of incubation.

Transcription of mcyB Gene
The relative expression of mycB gene at different concentrations of [Hmim]Cl was significantly lower than that of the control culture ( Figure 5). With the exception of

Transcription of mcyB Gene
The relative expression of mycB gene at different concentrations of [Hmim significantly lower than that of the control culture ( Figure 5). With the exception L −1 , the gene expressions in treated cultures were increased significantly after 72 cubation.

Figure 5. Effects of increasing [Hmim]
Cl concentrations on the transcription of mcyB gene. D uppercase letters represent significant differences (p < 0.05) between exposure times within t treatment. Different lowercase letters represent significant differences (p < 0.05) between tre within the same exposure time.

Discussion
Recently, several studies have been published regarding the structural char tics, toxicity, environmental fate, and application of ILs [30,31]. Although ILs ha validated as green solvents instead of the hazardous organic solvents, they can neg affect the aquatic environment [3].
A toxicological assessment of ILs in aquatic organisms involves bacteria (Vibr eri), green algae (Selenastrum capricornutum, Oocystis submarina), diatoms (C meneghiniana), mollusks (Physa acuta), vertebrates (Danio rerio, Rana nigromaculata nus carpio), and plants (Lemna minor) [7]. A few kinds of aquatic organisms are infer simple, and their cell membrane is the first target that can be affected by ILs [32, could cause the destruction of a membrane lipid structure and the change of comp due to their properties as ionic surfactants. The long alkyl chain of [Hmim]Cl in t onic part increases the lipophilicity and consequently interacts with the phosphol layer and hydrophobic part of membrane proteins. This leads to the leakage of

Discussion
Recently, several studies have been published regarding the structural characteristics, toxicity, environmental fate, and application of ILs [30,31]. Although ILs have been validated as green solvents instead of the hazardous organic solvents, they can negatively affect the aquatic environment [3].
A toxicological assessment of ILs in aquatic organisms involves bacteria (Vibrio fischeri), green algae (Selenastrum capricornutum, Oocystis submarina), diatoms (Cyclotella meneghiniana), mollusks (Physa acuta), vertebrates (Danio rerio, Rana nigromaculata, Cyprinus carpio), and plants (Lemna minor) [7]. A few kinds of aquatic organisms are inferior and simple, and their cell membrane is the first target that can be affected by ILs [32,33]. ILs could cause the destruction of a membrane lipid structure and the change of composition due to their properties as ionic surfactants. The long alkyl chain of [Hmim]Cl in the cationic part increases the lipophilicity and consequently interacts with the phospholipid bilayer and hydrophobic part of membrane proteins. This leads to the leakage of cellular contents and then cell death [34]. Such interpretation can explain the decrease of the M. aeruginosa cell density in the current study. The experimental results revealed that the EC 50 (72 h) of [Hmim]Cl for the growth of M. aeruginosa PCC 7806 was 10.624 ± 0.221 mg L −1 , referring to the low biodegradability as mentioned in the previous studies on imidazole or pyridine ILs [35][36][37].
Previous study revealed that ILs can inhibit algal photosynthesis by causing a decrease in the content of chlorophyll, thereby affecting the growth of algae [38,39]. Similarly, the contents of both Chlorophyll a and carotenoids were significantly decreased after 72 h of exposure to [Hmim]Cl. These results may be attributed to the long alkyl chain of [Hmim]Cl, which can lead to a leakage of cellular contents [30,37,40].
Chlorophyll fluorescence transients could be used to indicate the structure and function of PSII [27] and have become an important method to examine the response of plants and cyanobacteria to environmental stress [41]. According to the energy-flow model diagram of Strasser and Strasser (1995) [20], ϕP 0 can represent the electron-transfer performance of the electron donor side of the PSII reaction center. After 72 h of exposure, ϕP 0 was significantly inhibited, indicating that the electron donor side of the PSII reaction center of M. aeruginosa is the target site of [Hmim]Cl. ψ 0 and ϕE 0 can reflect the performance of the electron acceptor side of the PSII reaction center. Inhibition of ψ 0 and ϕE 0 by [Hmim]Cl indicated that the electron acceptor side of the PSII reaction center is also the target site of [Hmim]Cl. ABS/RC, TR 0 /RC, DI 0 /RC, and ϕD 0 values increased after extending the exposure time. These results indicate that although exposure to [Hmim]Cl leads to partial reaction center inactivation or cleavage, the efficiency of the active reaction center is improved. The values of (1 − V K /V J ) r can represent the state of the oxygen-evolving complex of PS II [26]. However, the relative value of (1 − V K /V J ) r showed no significant changes when compared with the control culture ( Figure 3). Therefore, the oxygen-evolving complex was not the target of [Hmim]Cl. On the other hand, PI ABS , which is the performance index of photosynthesis, was markedly decreased in all treatments. The results from this study show that [Hmim]Cl would cause serious damage to the photosynthetic system of M. aeruginosa. It also proves that PI ABS is more sensitive to reflect the changes of the photosynthetic system.
Previous investigation reported that ionic liquids can impair cells via lipid peroxidation [42] and DNA damage [43]. The results of ultrastructural observation by TEM indicate that both the thylakoid membrane and the cellular membrane were all damaged by [Hmim]Cl at 30 mg L −1 . These results suggest that the mechanism of the toxicity of [Hmim]Cl against M. aeruginosa PCC 7806 may also be involved in the damage of membrane. The experimental data show that [Hmim]Cl had a short-time strong inhibition on the transcription of mcyB, which recovered slowly after 72 h. Given that there is a positive relationship between the growth rate and microcystin production [44], the rapid inhibition of mcyB transcription provided further evidence for the toxic effects of [Hmim]Cl on M. aeruginosa at transcription level. It also reflects that the potential threat indicated by [Hmim]Cl caused the cells to allocate energy toward cell division instead of toxin production.
The results of this study found that an adverse effect was showed on M. aeruginosa under different concentrations of [Hmim]Cl, but this is just the indoor research. If many ILs flowed into the aquatic ecosystem, threats may be superimposed on the algae with other substances, such as heavy metals and pesticides. The combined toxicity of ILs requires further study.

Conclusions
The main conclusions may be summarized as follows.

Data Availability Statement:
The authors declare that all data and materials are available to be shared on a formal request.