*2.3. Microscopic Analysis*

Qualitative and quantitative changes in assemblage composition and structure, i.e., changes in taxonomic composition and taxa abundance, were the primary parameters used to assess the changes in microphytobenthos. Microscopic analysis was conducted on microalgal material preserved in Lugol solution. The observations were conducted in all cultures on the third and seventh experiment day. Fifty fields of view were checked in Utermöhl chambers (2 mL) using a Nikon Eclipse TS100 inverted light microscope (magnifications of ×200 and ×400). All cells were counted and identified as laid out in the Utermöhl method [34] and Helcom [35] guidelines (cells or threads of 100 μm length are treated as units). Species were identified using appropriate keys and floras [36–43].

The analysis of the microalgal cell condition was also performed. For this purpose, the state of chloroplasts was observed and classified in one of three classes of cells: (1) live cells with normal chloroplasts, (2) live cells with abnormal chloroplasts, and (3) dead cells. Here, the results obtained for the two first cell classes are reported (Figure 2). The microalgal cell condition was evaluated in all cells counted in 50 fields of vision under a Nikon Eclipse 80i microscope fitted with a Nikon DSU2 camera at a magnification of ×400.

#### *2.4. Statistical Analysis*

Differences between means were verified with the Student's *t*-test using STATISTICA version 10 (StatSoft Polska Sp. z o.o., Kraków, Poland). Principal component analysis (PCA) was carried out with the Canoco 5 (Microcomputer Power, Ithaca, NY, USA) [44,45] and similarity percentage (SIMPER) with the PRIMER-e (PRIMER-e, Auckland, New Zealand).

**Figure 2.** Examples of cells of *Bacillaria paxilifera* (**a**) and *Navicula perminuta* (**b**) with normal (A) and abnormal chloroplasts (B). Chloroplasts treated as abnormal have a deformed shape compared to the ones correctly formed. The shape of the chloroplast itself and thus its alternations are species- and/or genus-specific.

#### **3. Results**

#### *3.1. Analysis of Taxonomic Composition and Structure*

A total of 46 microalgae species were identified, including 35 diatoms, 6 cyanobacteria, and 2 green algae taxa, as well as representatives of dinoflagellates (*Peridinium* sp.) and haptophytes (*Prymnesium* sp.) (the list of all identified taxa is in the Appendix A (Table A1).

At the beginning of the experiment, the highest number of cells, 38,800 ± 700 cells/mL, was found (Figure 3). On the third day, a 47% decrease in the number of microalgae cells was observed in the concentration of 1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−3, while in the concentration of 1.75 × <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm−<sup>3</sup> [BMIM]Cl, a drop by only 21% was observed. On the seventh day of testing, a similar abundance of microalgae was observed in both concentrations, 23–26% less than in the control solution. All differences in abundance between the concentrations tested and the control solution were statistically significant (*p* < 0.05).

The microphytobenthic communities were heavily dominated by diatoms, constituting from 70% to 92% of all the observed photosynthetic microorganisms. In the control cultures, the abundance of cyanobacteria did not exceed 10% (the highest number was observed on the third day of the experiment). On the seventh day, at both [BMIM]Cl concentrations, i.e., 1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> and 1.75 × <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm−3, they represented 23% and 28% of the total abundance, respectively. The abundance of *Prymnesium* sp. (Haptophyta) did not exceed 0.3% during the whole experiment, while the abundance of *Peridinium* sp. (Dinophyceae) in the control solution at the start of the tests was only 0.6%.

SIMPER similarity analysis showed a high similarity in community composition and structure at the control and both ionic liquid concentrations during the experiment. The average similarity was calculated as high as 71.92%. Based on the PCA analysis performed on the quantitative data, it was found that in addition to the concentration of the ionic liquid, the duration of the experiment also influenced the transformation of communities (Figure 4). At the start of the experiment (point K\_0, right part of the graph), the community was the richest (i.e., it was characterized by the highest number of species). On the third day of testing (upper left part of the graph), an increased proportion of cyanobacteria was observed (e.g., marked in graph as *cya\_sp*., *mic\_sp*., *spi\_mai*, *wor\_sp*.). The group of organisms dominating on the seventh day of testing (lower left part of the graph) included, among others, a diatom characterized by a relatively high resistance to ionic liquid— *Navicula perminuta* (marked as *nav\_per*). However, based on the PCA analysis, it was not possible to delineate groups of organisms that were unequivocally sensitive or tolerant to the IL tested.

**Figure 3.** Abundance of microalgae. K indicates control cultures; the numbers 0.00113 and 0.0175 indicate cultures of lower (1.13 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm<sup>−</sup>3) and higher (1.75 <sup>×</sup> <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm<sup>−</sup>3) tested [BMIM]Cl concentrations, respectively. The number after the underline denotes the day of the experiment. Statistically significant differences were marked with the asterisk. In each case, the value from the experimental variant was compared with the value for the control sample on the same day (*p* = 0.000002 for 0.00113\_3 vs. K\_3, *p* = 0.005617 for 0.0175\_3 vs. K\_3, *p* = 0.005178 for 0.00113\_7 vs. K\_7, *p* = 0.001357 for 0.0175\_7 vs. K\_7).

Based on SIMPER analysis, the most important species in the communities were distinguished in terms of abundance (Appendix A, Table A2). The most abundant species was *Bacillaria paxilifera* (up to 36% on the third day in the control solution) (Figure 5). The second most abundant species was *Tabularia fasciculata* (up to 23% of all cells on the initial day of the experiment). The abundance of *Diatoma vulgaris* ranged from 9% to 17% throughout the experiment. For *Melosira nummuloides*, the highest number of cells was observed on the seventh day of testing in the control solution (20% of all cells). Interesting changes were observed in the case of *N. perminuta*; the proportion of this species in the initial community did not exceed 8%, but on the seventh day at both [BMIM]Cl concentrations, i.e., 1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> and 1.75 × <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm−3, the share of this taxon increased to 15% and 16%, respectively. The highest share of *Cylindrotheca closterium* in the community was observed at the start of the experiment (13%), but on subsequent days the share did not exceed 8%. In the case of *Navicula gregaria*, a maximum share of 6% was observed at a concentration of 1.13 × <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−<sup>3</sup> on the seventh day of testing. While the share of the only representative of cyanobacteria, *Spirulina major*, did not exceed 1% during the whole experiment.

**Figure 4.** PCA (principal component analysis) of microphytobenthos species composition (based on the abundance data) from control and [BMIM]Cl treatments cultures. Percentage explained variation λ1 = 26%, λ2 = 22.4% λ3 = 17.9%. K indicates control cultures, 0.00113 and 0.0175 indicate cultures of lower (1.13 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>g</sup>·dm−3) and higher (1.75 <sup>×</sup> <sup>10</sup>−<sup>2</sup> <sup>g</sup>·dm−3) tested [BMIM]Cl concentrations, respectively. The number after the underline denotes the day of the experiment. Taxon codes are included in Table A1 in Appendix A.

**Figure 5.** Percentage abundance of the 8 most important species distinguished in the tested communities based on SIMPER analysis. Labels used: spi\_maj—*Spirulina major*, nav\_gre—*Navicula gregaria*, cyl\_clo—*Cylindrotheca closterium*, nav\_per—*Navicula perminuta*, mel\_num—*Melosira nummuloides*, dia\_vul—*Diatoma vulgaris*, tab\_fas—*Tabularia fasciculata*, bac\_pax—*Bacillaria paxilifera*.
