*3.2. Abundance of Selected Taxa*

In the presence of the IL, most of the taxa showed a statistically significant reduction in their abundance. Only for two taxa, i.e., *N. perminuta* and *Navicula ramosissima*, did the abundance in the [BMIM]Cl solutions increase compared to the control solution (Figure 6a,b). Hence, they were identified as tolerant species to this IL. The abundance

of *N. perminuta* in the control solution on the seventh day decreased by 91% relative to the start of the experiment, while in both concentrations of the ionic liquid abundances of about 89% relative to the initial abundance were recorded. On the third day, in the lower [BMIM]Cl concentration cultures (1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−3), the cell abundance was 142% of the number in the control solution, while at the concentration of 1.75 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> it was as high as 289%. On day seven, as much as a tenfold rise in the cell abundance was observed for this taxon as compared to the control solution. A similar growth stimulation was noted on day three for *N. ramosissima*, but on day seven, the cell number was only 45% and 64% of that in the control depending on the [BMIM]Cl concentration.

**Figure 6.** Number of cells of selected microalgae during experiment: K—control solution; 0.00113—the concentration of 1.13 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl; 0.0175—the concentration of 1.75 <sup>×</sup> <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl. Statistically significant differences between the control solution and [BMIM]Cl treatments are marked with the asterisk. (**a**,**b**)—tolerant species positively affected by the ionic liquid; (**c**–**f**)—sensitive species negatively affected by the ionic liquid.

Figure 6c–f presents changes in the abundance of selected taxa considered most important in the communities in relation to the abundance based on the SIMPER analysis. The reduction in cell numbers in the [BMIM]Cl solutions tested indicated statistically significant (*p* < 0.05) growth inhibition. Such a response was characteristic of almost all the organisms observed in the microphytobenthic communities. However, depending on the taxon and concentration used, either a gradual reduction in abundance over time (e.g., *B. paxillifera* and *M. nummuloides* in the solution of 1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl) or a reduction in abundance followed by an initial increase (e.g., *B. paxillifera* and *M. nummuloides* in the solution of 1.75 × <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl or *T. fasciculata* in the solution of 1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl) was observed. The most dramatic changes in the abundance of *C. closterium* were noted. In the solution of IL of 1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−3, no living representatives of this species were observed on day seven, and in the 1.75 × <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm−3, only 14% of the abundance in the control solution was noted.

#### *3.3. Cell Condition in Selected Taxa*

Analysis of the chloroplast state in the cells provided complementary information on the differences in the response of the various taxa to [BMIM]Cl. In species considered tolerant, i.e., *N. perminuta* and *N. ramosissima*, a small number of cells with abnormally shaped chloroplasts were observed (Figure 7a,b). Interestingly, in the case of *N. perminuta*, cells with abnormally shaped chloroplasts were mainly observed in the control solution (e.g., up to 38% of all cells on the third day) and were not present or were only in small proportions in the IL solutions (up to 20% in the solution of 1.13 × <sup>100</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl). Similarly, in *N. ramosissima*, abnormally shaped chloroplasts were not observed in the cells at the beginning of the experiment. Deformed chloroplasts were present in the cells on the third day in the solution of 1.75 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl (15% of cells) and on the seventh day in the control solution (18% of cells) and in the solution of 1.13 × <sup>100</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> (50% of cells).

**Figure 7.** Condition of selected species shown as the percentage of cells with normal and abnormal chloroplasts. K indicates control cultures; 0.00113 and 0.0175 indicate cultures of lower (1.13 <sup>×</sup> <sup>100</sup>−<sup>3</sup> <sup>g</sup>·dm<sup>−</sup>3) and higher (1.75 <sup>×</sup> <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−3) tested [BMIM]Cl concentrations, respectively. (**a**,**b**)—tolerant species positively affected by the ionic liquid; (**c**–**f**)—sensitive species negatively affected by the ionic liquid.

For the taxa considered sensitive, cells with deformed chloroplasts were observed irrespective of the solution and day of the experiment (Figure 7c–f). For example, in *B. paxillifera* less than 15% of cells were characterized by deformed chloroplasts. Only in the solution of 1.75 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl on the third day was chloroplast degradation observed in 27% of the living cells. In *T. fasciculata*, except for at the beginning of the experiment, cells with degraded chloroplasts accounted for about 30–40% of all the cells. However, the highest number of cells with degraded chloroplasts was observed on the last day of testing in both IL solutions (about 50% of all cells). Similarly, for *M. nummuloides* in the control solution and at the beginning of the experiment, cells with abnormally

formed chloroplasts made up a small proportion of the population (0–16%). In contrast, in the solution of 1.13 × <sup>100</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl, up to 40% of the cells with damaged chloroplasts were observed on the seventh day and in the solution of 1.75 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm<sup>−</sup>3, 31% and 32% of cells on the third and seventh day, respectively. A significantly worse cell condition was observed in *C. closterium* as compared to the previously described species. In most of the solutions tested, cells with abnormally shaped chloroplasts accounted for about 40–85% of all the cells. Only at the solution of 1.75 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl did all of the cells have chloroplasts of normal shape, but the population size was low.

#### **4. Discussion**

ILs as solvents with salt structures have been known since 1914 [7]. However, the first stable ILs were described in 1995 [5]. The beginning of the 21st century brought the possibility of designing chemical compounds combining required biological properties with preferred physicochemical characteristics. Currently, these substances are being studied on a massive scale, as evidenced by the number of peer-reviewed publications e.g., [6,9,10,12–14]. Furthermore, the list of their potential applications as reaction media in many industrial fields is growing [9,12]. ILs have also found applications in medicine due to their antibacterial, antifungal, anticholinergic, and local anesthetic activities and agrochemistry as bactericides, fungicides, herbicides, plant growth stimulants, or wood preservatives [9,46]. Although ILs are popular in research and economics, this does not necessarily correspond to the amount of research related to the monitoring of these compounds in the environment and the subsequent risk assessment; the number of papers focusing on the presence of ILs or their compounds in the environment remains small [10,47–50]. In one of such studies, 1310 pollutants were identified in riverine waters in Germany, among which ca. 20 different compounds belonging to ILs were detected in concentrations of up to <sup>μ</sup>g·dm−<sup>3</sup> [48]. In addition, in the United States, based on analyses of sediments from lakes located within the state of Minnesota, it was shown that the concentration of the IL C4-PYR was 0.053 <sup>μ</sup>g·dm−<sup>3</sup> [51]. In this context, one of the ILs, i.e., 1-octyl-3-methyl imidazolium, is of particular significance as it was identified not only in environmental samples [52] but also in human blood [53].

The picture is completed by the fact that imadazolium-based ILs, such as [BMIM]Cl tested here, have a low rate of biodegradation and are resistant to photodegradation [54–56]. Previous studies have shown that ILs, after eventual emission into the environment, may behave similarly to some persistent organic pollutants [57]. An extremely important aspect is also the fact that the technology to effectively remove ILs from wastewater is still being developed [4]. Hence, it is to be expected that, due to the increasing popularity of ILs, they will be used widely and, consequently, will be uncontrollably introduced into the aquatic environment, remaining there for a long time due to the difficulties associated with water treatment and their poor biodegradability. Therefore, it is of paramount importance to investigate the ILs' toxicity under environmental conditions and not only under controlled laboratory conditions [58]. Our tests on the effects of the IL [BMIM]Cl, considered to be relatively harmless [32,33,57], on the whole communities of microphytobenthos collected from the environment, allowed us to determine the response of a wide spectrum of microorganisms and not just single strains as in standard ecotoxicological tests. Changes in the tested communities at the population and cellular level show in a more reliable way the direction of the changes to which the microphytobenthos, an important component of the marine ecosystem, is subjected.

In a study using cumulative impact assessment, it was found that the [BMIM]Cl turned out to be the least hazardous among the imidazolium chloride ionic liquids with the Safe Environmental Concentration (SEC) as high as 750 × <sup>100</sup>−<sup>3</sup> mmol/L, which corresponds to the concentration of 1.31 × <sup>100</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> [59]. In this study, however, it was shown that the concentration of 1.13 × <sup>100</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> already reduced the abundance of dominant species by 20% to 60% within 3 days and up to 75% within 7 days. Only two diatom species of higher abundances showed resistance and gained a quantitative advantage in the studied communities. The number of cells of *N. perminuta*, for example, increased, reaching almost 290% of the abundance in the control solution on the third and 1000% on the seventh day of the experiment. As a result, the total abundance of cells comprising the communities decreased by only 25%. Although the composition of the communities remained similar, the abundance structure changed due to the strong dominance of tolerant taxa—apart from diatoms, cyanobacteria also showed some resistance. Similar observations regarding the substitution of sensitive taxa by tolerant or indifferent taxa were found during experiments on the same Baltic microalgal communities, testing the effects of copper chloride [27] and glyphosate [26]. However, effects induced by the aforementioned substances were also manifested by a drastic shift in species composition, i.e., the increased contribution of cyanobacteria to the total community abundance.

Interesting changes were observed for the second taxon selected as resistant to the presence of the IL [BMIM]Cl—*N. ramosissima*. On the third day of the experiment, its cell numbers increased significantly, but on the seventh day, the cell abundance again decreased in the IL concentrations tested. Such a reaction may indicate depletion of the IL molecules, the presence of which has a stimulating effect on the test organisms. For example, in toxicological tests conducted by [20] on *Scenedesmus obliquus*, low concentrations of ILs have been shown to stimulate cells for biological activity (e.g., by changing the activity of catalase and superoxide dismutase). However, this may also be a response related to the competitive activity of other taxa, such as the predominant *N. perminuta*. Similarly, [21] selected from their study several species for which the IL [BMIM]Cl was practically harmless, i.e., the cyanobacterium *Anabaeana cylindrica* and the green alga *Chlorella pyrenoidosa*, and in the case of the green alga *Dunaliella salina*, they concluded that it was relatively harmless.

Typically, the reactions of taxa to toxicants tested in communities are milder than in laboratory tests conducted on monocultures [26,27,60]. In our studies, conducted on communities grown in nature, *B. paxillifera* cell counts decreased by 55% on the seventh day of the experiment in the solution of 1.75 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl. A very similar response was observed in toxicological tests conducted on a monoculture of *B. paxillifera* isolated from the Baltic Sea—the concentration of 1.75 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> decreased the growth of the strain by 58% on the seventh day of testing [61]. A similarly large reduction was shown for the same concentration in *T. fasciculata* (50% reduction on the seventh day). Moreover, [19] observed an inhibition of 50% cell growth (EC50) in the planktonic diatom *Skeletonema marinoi* at the concentration of 0.1 mM [BMIM]Cl (1.745 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−3). In turn, for the green alga *Chlorella pyrenoidosa*, 50% inhibition of growth (IC50) was shown for the concentration of 21.4 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [21]. The phenomenon of the same species reacting the same way to a substance regardless of how it is cultured and tested (individually or in communities) may indicate that the communities as a whole, but also the individual components of the community, do not necessarily have mechanisms to protect them from the toxic effects of the IL under testing.

Exposure time was also shown to be a variable having an effect on the action of the IL because, as reported by [62], excessive accumulation of the IL in microorganisms increases its effect. In the case of *M. nummuloides*, which was part of the tested community in our study, a significant increase in cell number was observed in the control solution during the experiment, while on the third day at the [BMIM]Cl solution of 1.75 × <sup>100</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> 50% fewer cells were observed, while on the seventh day the abundance decreased to 8% of the abundance in the control solution. A similarly rapid abundance reduction response to [BMIM]Cl was observed for the green alga *Dunaliella salina* [21]. This implies that there is a group of sensitive species in natural marine phytobenthic communities that may be rapidly eliminated from the environment while exposed to ILs, e.g., the aforementioned *M. nummuloides* or *C. closterium*.

The cell condition index used in our study, which consists of an assessment of the chloroplast state, confirmed the observations based on cell abundance. In the species considered sensitive, the percentage of cells with deformed chloroplasts was much higher than in the resistant species, e.g., up to a half of the observed cells of *T. fasciculata* on the seventh day of testing had degraded chloroplasts at both solutions of [BMIM]Cl. The negative effect on the chloroplast condition was confirmed by toxicity studies on five ILs ([Cnmim]Cl, *n* = 6, 8, 10, 12, 16). Ultrastructural morphology performed during the study revealed IL negative effects on various cellular structures, e.g., chloroplast grana became loose and mitochondria and their intermembranes swelled [22]. Other studies also confirmed that chloroplast damage can be considered as an indicator of microalgal degradation [27,63].

Tests carried out on the Baltic Sea microphytobenthic communities from the Gulf of Gda ´nsk made it possible to assess the effect of the IL [BMIM]Cl on the microorganisms comprising this formation. The study showed that 1-butyl-3-methylimidazolium chloride is a relatively harmful substance for the entire community, which contradicts the assessment carried out by reports based on cumulative impact assessment [47,59,64]. Thus, we have confirmed that even ILs considered to be of relatively low hazard have a significant impact on the aquatic environment. Hence, we are convinced that this group of compounds requires special attention in the context of testing its effects on different ecosystem components, its bioaccumulation, and its fate in the environment, as suggested recently by a growing group of authors [9,11,64].

#### **5. Conclusions**

During this study, the toxic influence of [BMIM]Cl on the marine microphytobenthic communities was demonstrated. The majority of species comprising the tested community reacted negatively to the presence of [BMIM]Cl at concentrations between 10−<sup>3</sup> and <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm<sup>−</sup>3, with a reduction in cell abundance and a deterioration in cell condition. Only in the case of two dominant diatom species, *N. perminuta* and *N. ramosissima*, was a stimulation of growth observed. In conclusion, the IL [BMIM]Cl on a short time scale contributes to a reduction in the abundance of species representing diverse taxonomic groups, which translates into the decrease in the total abundance and biomass of the microphytobenthic communities. However, despite the elimination of individual taxa, it does not lead to the degradation of entire communities but to their transformation into communities strongly dominated by a few resistant taxa.

**Author Contributions:** Conceptualization, Z.S., A.Z. and F.P.; methodology, Z.S., A.Z. and F.P.; software, Z.S.; validation, Z.S., A.Z. and F.P.; formal analysis, Z.S., A.Z. and F.P.; investigation, Z.S.; resources, Z.S. and A.Z.; writing—original draft preparation, Z.S., A.Z. and F.P.; writing—review and editing, Z.S., A.Z. and F.P.; visualization, Z.S.; supervision, A.Z.; project administration, Z.S.; funding acquisition, Z.S. and A.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a Research Project for Young Scientists from the Faculty of Oceanography and Geography, University of Gdansk (No. 538-G245-B209-16).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are available upon request to the corresponding author.

**Acknowledgments:** The authors thank Adam Latała for his support in developing the idea for the experiments.

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