**1. Introduction**

The concept of sustainable development was introduced at the end of the 20th century, and it is based on the idea of social and economic development, which assumes that while meeting the needs of contemporary societies it will not limit the development opportunities for future generations. Sustainable development assumes a parallel development of the economy, society, and the environment. In line with these assumptions, more and more environmentally friendly substances, such as ionic liquids (ILs), have started to be used in industry. These are substances that are gaining increasing recognition and researchers in many scientific fields are interested in them as they are characterized by a set of specific properties, such as low vapor pressure, non-flammability, thermal and electrochemical stability, good conductivity, and catalytic properties [1,2]. They are used in various chemical processes, where they represent a new alternative to traditional organic solvents [3]. ILs are often called 'designer solvents'; the appropriate selection of anions and cations allows for the creation of a suitable chemical compound, depending on the future application [4,5]. However, with regard to ILs' specific features (e.g., high solubility, thermal stability, and/or poor biodegradability in water), they may potentially pollute the aquatic environment [6]. Science has known of ILs as solvents since 1914 [7]. However, the first stable ILs were described in 1995 [5]. Since then, there has been a rapid increase in interest in these substances, especially in terms of their effects on human health and the environment, as in, e.g., [6–15]. A major threat from ionic liquids is their low degradation rate. For example, a 28-day experiment showed a complete lack of biodegradation of [BMIM]Cl [7]. As a result, after years of research, their "green" status has been questioned [8–10]. Inadequate

**Citation:** Sylwestrzak, Z.; Zgrundo, A.; Pniewski, F. Effects of the Ionic Liquid [BMIM]Cl on the Baltic Microphytobenthic Communities. *J. Mar. Sci. Eng.* **2022**, *10*, 1223. https:// doi.org/10.3390/jmse10091223

Academic Editors: François Gagné, Stefano Magni and Valerio Matozzo

Received: 15 July 2022 Accepted: 28 August 2022 Published: 1 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

wastewater treatment, accidental spillage, or improper storage of waste contaminated with ILs can lead to the release of these substances into the environment where they subsequently cause negative effects in the ecosystem [1]. Numerous studies have proven the deleterious effects of ILs towards microalgae [16–21]. The toxicity of ILs depends on temperature and pH. Under conditions described as moderate, i.e., room temperature and pH close to neutral, these substances are stable. During industrial processes, the physicochemical conditions can change, and it has been shown that an acidic condition (pH about 3) and high temperature (of the range between 60 ◦C and 100 ◦C) accelerate the hydrolysis of the ILs, causing an increase in their toxicity. The IL used in this study was 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), which is characterized by a relatively short alkyl chain [9]. The toxicity of ILs increases with the elongation of the alkyl chain [2]; hence, the IL used here is not considered to be a highly toxic substance [22]. However, its toxicity is comparable to that of chlorinated organic substances, such as dichloromethane and chloroform; thus, it is more dangerous to the environment than common solvents of organic origin (e.g., acetone, methanol, and ethanol). Due to its properties, [BMIM]Cl is used in cellulose processing, among other areas. Although the ionic liquid itself can be recovered to a very high degree during industrial applications (such as the one mentioned above), the very process of creating the imidazole cation involves the use of large amounts of natural organic materials, energy, and solvents, causing harmful emissions to both air and water as well [7].

The photosynthetic organisms that form the microphytobenthos are extremely valuable elements of aquatic ecosystems due to their role as primary producers, combined with oxygen production and CO2 reduction. Understanding the joint response of photosynthetic organisms forming a microphytobenthic community is extremely important in order to reliably estimate the changes that may occur following the introduction of this increasingly common substance into the environment and to assess the associated risks.

Monitoring the response of organisms to potentially toxic substances introduced into the ecosystem is an important part of environmental quality control. To date, investigations into the toxicity of ionic liquids have provided information on the response of single algal strains under laboratory conditions, e.g., [21–23]. Previous ecotoxicological studies conducted on marine microphytobenthic communities in the Baltic Sea have tested substances such as irgarol 1051, Sea-Nine™211 (DCOIT), and TBT (trin-butyltin) [24,25]. However, most of the studies used only communities developed at salinities typical of marine waters, i.e., 32–36 PSU. Only studies conducted to determine the impact of glyphosate and copper ions on microphytobenthic communities were performed on organisms collected from environments with salinities around 8 [26,27]. Further ecotoxicological tests on other potentially toxic substances carried out on microphytobenthic communities typical of brackish waters, which are considered species-minimum waters for macrozoobenthos and macroalgae and aquatic higher plants, but which support an abundance and diversity of planktonic microorganisms [28], are an interesting contribution to the existing knowledge. In the case of ILs, the salinity aspect is extremely important because the toxicity of ILs increases in inverse proportion to the salinity [16–21]. At high salinity values, the toxicity of ILs decreases, probably due to the reduced permeability of the microalgal cell membranes which limits the migration of harmful cations [29]. Our study was designed to provide a general picture of the response of multispecies microalgal communities to an IL considered to be of relatively low toxicity, i.e., 1-butyl-3-methylimidazolium chloride—[BMIM]Cl, under brackish water conditions, and to complement the existing knowledge on the potential risks arising from the widespread use of this substance. For that purpose, observations were made at the population level, i.e., the change in species composition and the community dominance structure were determined, and at the cell level, i.e., the condition of the chloroplasts was analyzed.

#### **2. Materials and Methods**

#### *2.1. Study Area and Field Works*

The experiment investigating effects of the IL [BMIM]Cl on microphytobenthic communities is one of a series of tests based on the identical methodology described in detail in [26]. The experiments were conducted in parallel on communities with identical species composition to allow for the comparison of the results. In brief, the study material was collected from glass slides mounted on a dedicated culture panel (Figure 1b) exposed in the Gulf of Gda ´nsk waters at a distance of 300 m from the shore (54◦26 49 N, 8◦34 24 E) (Figure 1a) for two weeks. During this time, the temperature and salinity changed within limited ranges, i.e., 17–19 ◦C and 7.9–8.4 PSU, respectively. The 2-week incubation period allowed for the acquisition of a relatively rich and diverse microphytobenthic community but was still devoid of organisms such as fouling macroalgae and fauna that will eventually dominate the surface of any substrate in marine waters in the long term.

**Figure 1.** (**a**) Sampling site. The black dot indicates the location of the culture panel during exposure in the Gulf of Gda ´nsk (54◦26 51 N 18◦34 33 E). (**b**) The design of the culture panel used in this study.

#### *2.2. Microalgal Material Preparation Procedure and Experimental Design*

In the laboratory, the microphytobenthic communities were removed from the microscope slides by scraping them off with a scalpel. Subsequently, the microalgal cells were re-suspended in the seawater collected at the sampling site, which was first filtered through a glass filter (Whatman GF/C) and then autoclaved. The obtained microphytobenthos suspension was then sonicated, which allowed for the disruption and removal of cell aggregates. The sonication power was carefully chosen in order not to weaken or damage the cells [26].

The experiment was carried out in 250 mL flasks filled with 100 mL of microalgal suspension. Each microphytobenthos culture was insufflated with nitrogen for 30 s to remove heterotrophic microorganisms [30,31]. At the beginning of the experiment, the mean microalgal cell abundance was 38,800 cells/mL ± 700. Before the experiment, flasks with microalgal suspension were maintained in a thermostatic chamber for 72 h at constant light, temperature, and salinity conditions (i.e., 60 <sup>μ</sup>mol photons·m−2·s−<sup>1</sup> with a photoperiod L:D 16:8 h, 18 ◦C ± 1 ◦C, and 8 PSU, respectively) to let the communities acclimate to the

experimental conditions. The natural concentrations of the nutrient compounds in the sea water were: N-NH4 9.4 mg·m−3, N-NO3 102 mg·m−3, P-PO4 36 mg·m−3, and Si-SiO4 600 mg·m−3. As the nutrient concentrations in the natural Baltic water were sufficiently high to maintain the microphytobenthos community during the experiment, any kind of culture medium was not added. This was also dictated by the fact that the high nutrient content could facilitate the growth of random species rapidly responding to the increase in nutrient concentrations.

After the acclimation phase, the [BMIM]Cl toxicity tests were performed according to the following design: control—microphytobenthic assemblages kept in filtered sea water without the addition of the tested IL and test solutions—microphytobenthic assemblages treated with two [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<sup>−</sup>3. The lower [BMIM]Cl concentration selected for the experiment was previously proven to have significant effects on the species composition of the Baltic microphytobenthic communities [32]. The higher concentration of [BMIM]Cl was inferred from previously published values indicated as having inhibitory effects on algae [18,33]. All experimental treatments were carried out in triplicates.
