**4. Conclusions**

We successfully performed a laboratory experiment designed to obtain comprehensive results for acute toxicity, bioconcentration, and biotransformation by determining the biological activity of four antidepressants on the protozoan *S. ambiguum*. The tested compounds were acutely toxic to *S. ambiguum*, and moreover, sublethal effects quickly became lethal ones. Sertraline was the most toxic among the studied antidepressants. However, the toxic effects occur at concentrations at least two orders of magnitude higher than those determined in effluents and freshwaters. Thus, it can be concluded that the tested antidepressants are unlikely to represent a risk to the aquatic protozoa. The results also showed the relationship between pH and toxicity, which has two consequences. First, the pH of the water should be more strictly defined in the aquatic toxicity guidelines to prevent high inter- and intra-laboratory variability of the results. Second, pH of the water and effluent should be considered in the environmental risk assessment, especially for ionizable compounds.

On the basis on the bioconcentration tests, it can be concluded that uptake and elimination kinetics vary greatly between the tested pharmaceuticals. The highest BCF value was obtained for sertraline and mianserin, but different bioaccumulation scenarios can be observed for each pharmaceutical and for each concentration. Our results also indicate that the protozoan cells were unable to excrete the accumulated antidepressants. We suspect that the main reason of the toxic effects and high bioaccumulation ratio were the interactions between the tested drugs and lysosomal membrane phospholipids, which lead to vacuolization. Thus, future research should focus on analyzing the transmission of antidepressants accumulated in vacuoles and/or their effects on the next generations of organisms.

For the first time, the research for the biotransformation products of antidepressants were conducted in the protozoa. However, because of the low abundance of possible biotransformation products, their structure could not be elucidated. This part of the present work revealed a potential for further investigation of pharmaceutical metabolism in protozoa exposed to drugs under natural conditions.

**Author Contributions:** Conceptualization: A.D., G.N.-J. and M.W.; methodology: J.G., G.N.-J., A.D. and M.W.; formal analysis: J.G., G.N.-J., A.D. and M.W.; investigation: J.G., G.N.-J., A.D., M.W. and A.O.; resources: J.G., G.N.-J., A.D., M.W. and A.O.; data curation: J.G., G.N.-J., A.D. and M.W.; supervision: J.G., G.N.-J. and A.D.; writing—original draft preparation: J.G., G.N.-J., A.O.; A.D. and M.W.; writing—review and editing: J.G., G.N.-J., A.D., M.W. and A.O.; funding acquisition: G.N.-J.; project administration: G.N.-J.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by the Medical University of Warsaw (Grant number: FW14/N/2017). LC–MS/MS analysis on QTRAP was carried out using the CePT infrastructure financed by the European Union—the European Regional Development Fund within the Operational Program "Innovative economy" for 2007–2013.

**Acknowledgments:** The authors wish to thank Ryszard Marszałek for technical assistance during LC–MS/MS analysis.

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