*2.1. Toxicity*

The protozoan *S. ambiguum* could be stored in an inorganic medium for a long time without losing its viability; thus, the Spirotox test could be prolonged up to seven days. In all tests, the toxic e ffect percent in the negative control was less than 10%; thus, the results of the tests were valid.

Sertraline was the most toxic antidepressant in all the tested approaches, with EC50 in the range of 0.2–0.7 mg/<sup>L</sup> (Figure 1 and Table A1). Paroxetine and fluoxetine were three-fold, while mianserin was 10-fold less toxic than sertraline. The tested compounds were acutely toxic to *S. ambiguum*, as the

LC50 and EC50 values were close to each other. This implies that sublethal e ffects quickly became lethal ones. Moreover, in most cases, the EC20 values were less than two times lower than EC50 values (Table A1). Only for fluoxetine and mianserin tested at pH lower than seven, the EC50 to EC20 ratio was higher than two. EC20 is a threshold value that indicates the threat to the population of the tested organism. This implies that the EC50 value is a good predictive value that can be used to predict the e ffects of the substances on an entire population. As expected, the toxicity increased with the time of incubation, and the seven-day values were much lower than the one- and the two-day values. The toxicity also depended on the pH of the medium. *S. ambiguum* could be tested in a wide range of pH from 5.5 to 8.0. The toxicity was measured at three pH values 6.0; 6.5 and 7.4 to imitate natural freshwaters. For all tested antidepressants, an increase in toxicity was observed with increasing pH. For SSRIs, the step change can be seen between pH 6.0 and 6.5, while for mianserin, the toxicity increased gradually with the increase in pH, especially after one and two days of incubation. The relationship between toxicity and pH of the medium was previously reported for nitrophenols [21], and to the best of our knowledge, this relationship has not been tested for pharmaceuticals thus far. The toxicity-to-water pH relationship has two consequences. First, the pH of the water should be more strictly defined in the ecotoxicity guidelines to prevent high variability of the results. The present data indicate that the pH shift by only one unit may result in a significant change in toxicity. Second, pH of the water and e ffluent should be considered in the environmental risk assessment of the ionizable compounds. The tested antidepressants are cationic amphiphilic drugs that ionize in acidic solutions, and the bioavailability of the ionized form of the compound is lower than that of the non-ionized one. For many amphiphilic compounds, the biological activity may be predicted using the pH-dependent water/octanol partition coe fficient (log D) instead of log P. Taking into account the whole group of compounds tested there was no correlation between the toxicity of the antidepressants to *S. ambiguum* and lipophilicity expressed by both log P and log D coe fficients (Table 1). Thus, their biological activity cannot be explained by the simple non-polar and polar narcosis mechanism of action [22]. The tested drugs inhibit neurotransmitter's (serotonin) re-uptake in vertebrate's tissues. Minguez et al. [23] reported the correlation of SSRI toxicity towards *Daphnia* with the log P coe fficient. However, they also observed irreversible cell lysis in the abalone hemocytes, probably due to interactions between the drugs and lysosomal membrane phospholipids [23]. As vacuolization was the first symptom of toxicity of the tested compounds in *S. ambiguum*, we expected that such interactions also occur in protozoaandarethemainreasonoftoxice ffects.


**Table 1.** Physicochemical characteristics of the tested antidepressants.

pKa: acid dissociation constant obtained from www.drugbank.ca; Log P: Octanol-water partition coe fficient obtained from www.drugbank.ca; Log D: pH-dependent octanol-water partition coe fficient calculated with logD predictor (KLOP algorithm, MarvinSketch 15.2.23. software (https://disco.chemaxon.com/apps).

**Figure 1.** Toxicity of tested antidepressants in Spirotox test after 1, 2, and 7 days incubation (EC50 expressed in mg/L).

Antidepressants, especially sertraline, are very potent against parasitic protozoa with IC50 of 0.16 mg/<sup>L</sup> and 0.24 mg/<sup>L</sup> for *Plasmodium falciparum* and *Trypanosoma brucei rhodosiensis*, respectively, and are considered to be applicable in the treatment of relevant tropical diseases caused by these parasites [24]. Palit and Ali [25] reported high activity of sertraline against another parasite protozoan *Leishmania donovani*. They hypothesized that sertraline induces cell apoptosis by lowering adenosine triphosphate (ATP) levels, resulting in a reduction in oxygen consumption. However, more research is needed to prove this hypothesis and to determine the mode of action of antidepressants towards protozoa.

The protozoan *S. ambiguum* appeared to be comparably sensitive as other organisms used in acute toxicity bioassays. Similar to our results, sertraline was reported to be the most toxic antidepressant to crustaceans with 48-h LC50 of 0.12 mg/<sup>L</sup> for *Ceriodaphnia dubia* [26], 24-h LC50 of 0.6 mg/<sup>L</sup> for *Thamnocephalus platyurus* [27] and 48-h EC50 of 0.92 mg/<sup>L</sup> for *Daphnia magna* [28]. Slightly lower toxicity was reported for fluoxetine, ranging from 0.23 and 0.82 mg/<sup>L</sup> for *C. dubia* and *D. magna* [12] to 0.85 mg/<sup>L</sup> for *T. platyurus* [29]. Contrary to the previous two antidepressants, paroxetine was

10-fold less toxic to *D. magna* (6.3 mg/L) [28] than to *C. dubia* (0.58 mg/L) [26]. Very little information is available for mianserin. Wawryniuk et al. [30] reported 24-h LC50 of 1.8 mg/<sup>L</sup> for *T. platyurus*, while Minguez et al. [23] showed 48-h EC50 of 7.81 mg/<sup>L</sup> for *D. magna*. Similar acute toxicity data were reported for fish: 48-h LC50 of 0.198 mg/<sup>L</sup> for fluoxetine towards *Pimephales promelas* [31] and 96-h LC50 of 0.38 mg/<sup>L</sup> for sertraline towards *Oncorhynchus mykiss* [27]. These values are 2–3 orders of magnitude higher than the levels of antidepressants detected in municipal e ffluents and freshwaters, and therefore, the acute toxicity e ffect is not expected in the environmental samples.
