*2.2. Bioaccumulation*

To evaluate bioaccumulation of the tested antidepressants in protozoa, *S. ambiguum* was incubated with the antidepressants at three concentrations: low (10 μg/L), medium (25 μg/L), and high (100 μg/L) for six days uptake phase, followed by six days depuration phase.

Whole-body internal concentrations based on the parent compound were measured. The concentrations of the compounds inside the protozoa and in the medium were determined four times in each research phase. The results of the concentration of the tested antidepressants in *S. ambiguum* cells and in the medium are shown in Figure 2 and Table A2, while the BCF values are presented in Figure 3 and Table A3. From the internal concentration data, it can be concluded that uptake and elimination kinetics vary greatly between the tested pharmaceuticals. *S. ambiguum* accumulated significant amounts of sertraline and mianserin, but di fferent bioaccumulation scenarios were observed in each case and for each drug concentration. The concentration of sertraline in the protozoan cells increased gradually during the uptake phase for low and medium drug concentration. For the highest level tested, the highest sertraline concentration was determined after 24 h, followed by a gradual decrease in its concentration. In the depuration phase, the sertraline intracellular concentration remained at a high level, falling by only 40% of the highest concentration (all tested concentrations). Mianserin reached its highest concentration in *S. ambiguum* cells after two days of incubation. After six days, its level dropped to 60–70% and then gradually decreased in the depuration phase. Fluoxetine and paroxetine were not accumulated inside the protozoan cells, and their BCF values during the uptake phase never exceeded 1000 L/kg, while for mianserin and sertraline, the BCF values reached much higher at 4939 and 34,092 L/kg, respectively. The U.S. Environmental Protection Agency has established a BCF ranging from 100 to 1000 L/kg to indicate a medium concern for bioaccumulation [13]; compounds with BCF > 1000 L/kg are considered to be highly bioaccumulating.

The bioaccumulation of SSRIs has been reported in invertebrates and fish by many authors [17,32–34], and the results varied depending on the species. The BCF for sertraline calculated by Grabicova et al. [17] for *E. octoculata* and *Hydropsyche* sp. was higher than 2000 L/kg, while Du et al. [32] found that the BCF value for *Planorbid* sp. was only 990 L/kg. These values were an order of magnitude lower than our results obtained for *S. ambiguum*. The largest spread of results was published for fluoxetine. The value close to our value was obtained by Franzellitti et al. [33] in the marine mussel *Mytilus galloprovincialis*; after seven days of treatment at the concentrations of 30 and 300 ng/L, the BCF ranged from 200 to 800 L/kg. A higher value of 3000 L/kg was reported by Du et al. [32] for *Planorbid* sp. In contrast, Meredith-Williams et al. [34] obtained quite di fferent BCF values of 185,900 L/kg and 1387 L/kg in freshwater shrimp (*Gammarus pulex*) and the water boatman (*Notonecta glauca*), respectively. According to these authors, the 2–3 orders of magnitude higher BCF values for fluoxetine in *G. pulex* resulted from the limited depuration in these animals. Our results (Figures 2 and 3) also indicate low depuration of the tested pharmaceuticals from *S. ambiguum*. In the most cases, after transferring the protozoa to a fresh medium, the intracellular concentration decreased only 2–3 times. The di fferences in the degree of uptake across the di fferent organisms may be due to di fferences in the mode of respiration, behavior, and pH of the test system. Moreover, the BCF values are reduced as organism size increases and increase with increasing lipid content [34,35]. However, Rubach et al. [36] found no relationship between lipid content and chlorpyrifos uptake in all 15 species of fish they tested. Lipophilicity is the most often used criterion for predicting the bioaccumulation potential. According to European guidelines on environmental risk assessment of medicinal products for human use [37], all drug substances with log P > 4.5 should be considered to be potentially persistent and should be screened for bioaccumulation; however, OECD uses lower criteria of only log P > 3 [38]. Based on the calculated log P values, Howard and Muir [13] classified sertraline, fluoxetine, and paroxetine as potentially bioaccumulative. However, at neutral pH, the log D values are much lower than log P values (Table 1), and this can explain such low BCF values for fluoxetine (log D: 1.23–1.81) and paroxetine (log D: 0.01–0.61). Grabicova et al. [17] showed that the antidepressive drug citalopram tended to accumulate in organisms, and the extent of accumulation was equivalent to the extent of metabolic transformation and removal from the body.

**Figure 2.** Concentration of tested antidepressants in *S. ambiguum* cells [μg/g].

**Figure 3.** Bioconcentration factor expressed as the ratio of concentration of tested antidepressants in *S. ambiguum* cells to the concentration in water.

After transferring *S. ambiguum* to a clean solution, very slow elimination was observed, and the drugs were detected inside the cells at concentrations up to 11,000 higher than that in the water phase (Figure 3 and Table A3). This indicates that the protozoa were unable to excrete the accumulated antidepressants. The bioaccumulation of drugs in subcellular organelles may eventually result in phospholipidosis and alkalinization of the lysosomes [39]. Two mechanisms are responsible for the accumulation of the basic amphiphilic compounds in cells: binding to phospholipids and lysosomal trapping [40]. The cell membrane and membranes of cellular organelles are permeable to non-ionized compounds [39]. The most acidic pH of protozoa food vacuoles ranges between 3.5 and 4.0. In these conditions, all the tested antidepressants became protonated and cannot pass through the membrane back to the cytosol, which may result in their accumulation within the lysosomes [39]. This phenomenon is called lysosomotropism and has been found in different mammalian cells [39–41]. However, to the best of our knowledge, it has not ye<sup>t</sup> been studied in protozoa. The degree of ion trapping depends on membrane permeability, the pH gradient between the cytosol and lysosome, and physicochemical properties of the compound such as pKa [41]. In our present study, vacuolization of the protozoan cells was observed after six days of incubation with the highest tested concentration of sertraline (100 μg/L) (date not presented). This suggests an effect of the drug on vacuole membrane; however, this hypothesis needs to be confirmed in future research.
