*3.3. Bioaccumulation Test*

The experiments were carried out in 250 mL glass beakers filled with 200 mL of sample or control. As a diluent and control, an inorganic medium (Tyrod solution) was used, with pH adjusted to 7.4. The bioaccumulation experiments were performed for the individual drugs: fluoxetine, sertraline, paroxetine, and mianserin (10, 25 and 100 μg/L). The experimental scheme is presented in Table 3. A total of 1000 protozoan cells were added to each beaker, and the beakers were incubated for 6 days (144 h) at 25 ◦C in darkness. A total of 100 protozoan cells were subsampled from each test beaker after 2 h and after 1, 2, and 6 days of incubation. Simultaneously, 1 mL of water from each sample was taken for chemical analysis. After 6 days, the protozoa (approximately 500) were transferred to a new glass beaker with the fresh Tyrod solution for testing the depuration of the accumulated drugs. Next, 100 protozoan cells and 1 mL of water were subsampled after 1, 2, and 6 days of incubation. The test was performed in duplicate.

**Table 3.** Bioaccumulation experiment with protozoan *S. ambiguum*. During the sampling 1 mL of medium and 100 protozoans were collected for chemical analyses.


Quantitative analyses were performed using Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA), equipped with a degasser, a thermostated autosampler, a binary pump, and connected in series to a QTRAP®4000 (AB SCIEX, Framingham, MA, USA) equipped with a Turbo Ion Spray source operated in the positive mode. The curtain gas, ion source gas 1, ion source gas 2 and collision gas (all high purity nitrogen) were set at 35 psi, 60 psi, 40 psi and 'medium' instrument units, respectively, and the ion spray voltage and source temperature were set at 5000 V and 600 ◦C, respectively. Chromatographic separation was achieved with a Kinetex RP-18 column (100 mm, 4.6 mm, particle size 2.6 μm) supplied by Phenomenex (Torrance, CA, USA). The column was maintained at 40 ◦C at the flow rate of 0.5 mL/min. The mobile phases consisted of HPLC grade water with 0.2% formic acid as eluent A and acetonitrile with 0.2% formic acid as eluent B. The gradient (%B) was as follows: 0 min, 10%; 1 min, 10%; 8 min, 90%; 9 min, 90%. The volume of injection was 10 μL. The target compounds were analyzed in the multiple reaction monitoring (MRM) mode (Table A4) by monitoring two transitions between the precursor ion and the most abundant fragment ions for each compound.

Preparation of *S. ambiguum* samples for HPLC analysis involved mixing 50 μL of sample (100 protozoan cells + medium) with IS (50 μL) and acetonitrile (100 μL). The samples were vortexed (10 min), placed for 10 min in a freezer (at −20 ◦C) and then centrifuged (5 min at 10,000× *g*). The supernatant (150 μL) was mixed with 375 μL of water and transferred to the autosampler vial. The concentration of pharmaceuticals in organisms was calculated using the measured concentration of the pharmaceutical in the medium and the volume of *S. ambiguum*. An average volume of 100 cells of *S. ambiguum* was 0.50 μL. The preparation of medium samples for HPLC analysis involved centrifugation (10 min at 10,000× *g*), mixing with the IS (9:1) and transferring to vials. No clean up procedure was used.

The validation was performed according to the European Medicines Agency guideline [37]. For *S. ambiguum* extracts, two linearity ranges were selected: 1–100 μg/<sup>L</sup> and 50–10,000 μg/<sup>L</sup> of homogenate. For medium samples, the linearity was selected as 0.2–100 μg/L. The coefficients of determination for curves was above 0.99. All validation experiments (accuracy, precision, variation of the relative matrix effect and stability) met the European Medicines Agency (EMEA) acceptance criteria [51].

The concentration of the tested antidepressants in *S ambiguum* was expressed as μg/g assuming the density of the organism as 1 g/mL. The bioconcentration factor was calculated by dividing the substance concentration in organisms to the concentration in the medium and was expressed as L/kg.

#### *3.4. Analysis of Biotransformation of Drugs*

The biotransformation of the drugs by the protozoa was analyzed for the four antidepressants: fluoxetine, mianserin, paroxetine, and sertraline. The test beakers were prepared in a manner similar to that for the bioaccumulation experiment. However, only one concentration (100 μg/L) of the drug was tested, and no depuration phase was performed. Concomitant with the tested sample, two control samples were incubated: the abiotic degradation control containing only the same concentration of the tested pharmaceutical (described as "drug control") and the organism control containing only protozoa. After 2 days of incubation in darkness, 500 protozoan cells in 100 μL of medium were transferred to the Eppendorf tube, and 200 μL of acetonitrile was then added. Samples were vortexed (10 min), placed for 10 min in the freezer (at −20 ◦C) and centrifuged (5 min at 10,000× *g*). The supernatant (150 μL) was mixed with 375 μL of water and transferred to the autosampler vial. Furthermore, 100 mL of medium was sampled at the end of experiment and poured into the preconditioned Oasis HLB (Waters) *spe* cartridge (30 mg). The analytes were eluted with 2 × 3 mL of methanol. The methanol was evaporated under the stream of nitrogen, and the extract was reconstituted with 1 mL of acetonitrile:water (1:9, *v*/*v*). The analysis of transformation products was performed with Ultra High Performance Liquid Chromatography (UHPLC) Dionex Ultimate 3000 with a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer system. Heat electrospray ionization (HESI) was operated in the positive mode. Full MS scans were acquired over *m*/*z* 75–1100 range with the resolution of 70,000 (*m*/*z* 200). Standard mass spectrometric conditions for all experiments were as follows: spray voltage: 3.5 kV; sheath gas pressure: 60 arb; aux gas pressure: 20 arb; sweep gas pressure: 0 arb; heated capillary temperature: 320 ◦C; loop count: 3; isolation window: *m*/*z* 3.0; and dynamic exclusion: 6.0 s. Chromatographic separation was achieved using a Kinetex RP-18 column (100 mm × 4.6 mm, 2.6 μm) supplied by Phenomenex and equipped with a security guard. The column was maintained at 40 ◦C at the flow rate of 0.3 mL/min. The mobile phases consisted of HPLC grade water with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as eluent B. The gradient (%B) was as follows: 0 min–10%; 1.5 min–10%; 7.0 min–90%; 12 min–90%. The volume of injection was 10 μL.

All the chromatograms obtained in the biotransformation experiments were integrated with Compound Discoverer Software. The area of the peaks obtained for the sample (protozoa in the drug solution) was divided by the area of the corresponding peaks of the control (protozoa in the medium). Similarly, the area of the peaks obtained for the drug control was divided by the area of the corresponding peaks of the medium. Thus, three values were obtained: tested medium, extract from the protozoan cells, and control medium.
