**1. Introduction**

Protozoa play an important role in the aquatic food web as primary consumers. They are common in surface waters and activated sludge in waste-water treatment plants (WWTP), where they feed on bacteria and may ingest pollutants directly from water. *Spirostomum ambiguum* is one of the largest ciliated protozoa with long generation time of about 70 h. It tolerates pH changes from 5.5 to 8.0, can be cultured in laboratory, and stored in an inorganic medium for at least eight days [1]. Thus, it is a very convenient organism and has been used in ecotoxicological studies for more than 25 years [1–3].

Antidepressants are one of the major group of pharmaceuticals used worldwide. Sertraline, fluoxetine, and paroxetine, belonging to the most commonly used selective serotonin re-uptake inhibitors (SSRIs), were ranked 14, 31, and 68, respectively, on the top 300 best-selling drugs in 2020 with 38.3, 21.9, and 11.7 million prescriptions, respectively, in the U.S. in 2017 (www.clincalc.com, accessed: 7 February 2020). Mianserin is an atypical, tetracyclic antidepressant used for the treatment of major depressive disorders. Antidepressants, as with other pharmaceutically active compounds (PhACs), are released into freshwaters mainly with waste-water, and they have been detected in

effluents, freshwaters, and even drinking waters in many countries [4–9]. Mole and Brooks [10] wrote a comprehensive review of the occurrence of SSRIs in the environment. They found that fluoxetine, citalopram, paroxetine, and sertraline, and their main metabolites norfluoxetine and norsertraline were the most commonly detected antidepressants. Their concentrations in influents and e ffluents were up to several μg/L. SSRIs have been identified not only in water and sediments but also in fish caught in the e ffluent waste streams and in molluscs and fish tissues commonly consumed by humans [11]. Most of the SSRIs are slowly degraded under the influence of both biotic and abiotic factors, and due to the continuous discharge are called pseudo-persistent contaminants [11].

High biological activity of antidepressants, especially SSRIs, has been reported for algae [12] and crustaceans and fish [11]. In short-term acute toxicity tests, the EC50 values ranged from 0.2 to 10 mg/<sup>L</sup> [11,12]. However, in chronic toxicity assays, the lowest observed e ffect concentration (LOEC) values were much lower, as low as 0.0136 mg/<sup>L</sup> for *Pseudokirchneriella subcapitata* [12]. Moreover, SSRIs are considered to be potentially bioaccumulative [13]. They were detected in fish [14,15] and bivalve [16] tissues. Sertraline was the most bioaccumulating compound in the e ffluent reach stream [17]. Its bioaccumulation factor (BCF) in two benthic organisms *Hydropsyche* sp. and *Erpobdella octoculata* ranged from 920 to 2100 L/kg and was close to the predicted value. There is a lack of knowledge of the response of protozoa to PhACs, probably due to their small size that entails the use of more sophisticated research techniques.

The analysis of the occurrence of PhACs and their human metabolites in the environment has been restricted to compounds with standards available in the market [8,10,18]. With the development of new analytical mass spectrometers, i.e., Quadrupole time of flight (QTOF) working in all ion MS/MS modes and high-resolution Orbitrap ™, it has become possible to detect a huge amount of non-target PhAC metabolites and transformation products [18,19] not only of human origin but also of microbial origin. However, neither PhAC nor their metabolites have ye<sup>t</sup> been reported for their e ffect on protozoa. Some antidepressant metabolites, e.g., norfluoxetine, have even higher biological activity as parent compounds [20]. Thus, to recognize the complete risk of drugs occurring in the aquatic environment, it is important to know both biotic and abiotic transformation of these compounds.

The present study aimed to evaluate the biological activity of four antidepressants, namely fluoxetine, sertraline, paroxetine and mianserin, on the ciliated protozoan *S. ambiguum*. Our comprehensive study included analysis of acute toxicity, bioconcentration, and biotransformation. Acute toxicity was evaluated with the prolonged, seven-day Spirotox assay. As the tested compounds ionize in water solution, three pH levels from the range of the pH of natural freshwaters (6.0, 6.5 and 7.4) were tested, and the relationship between pH and toxicity was discussed. The bioconcentration of the tested pharmaceuticals was analyzed in the six days uptake followed by six days depuration phases, while the biotransformation products were identified after two days of incubation of the protozoa with the tested antidepressants. In the bioconcentration tests, high-performance liquid chromatography (HPLC) with the mass spectrometer detector QTRAP ™ was used, while in the biotransformation tests, UPLC with MS/MS Orbitrap ™ was applied with post facto analysis performed by Compound Discoverer Software (Thermo Fisher Scientific, Waltham, MA). To the best of our knowledge, this is the first study on the bioaccumulation and possible biotransformation of PhACs by protozoa.
