A solution of silver nanoparticles with a concentration of 20 mg/L and particle size of 10 - 20 nm was purchased from Dr. Juice (Miskolc, Hungary). Silver nitrate (AgNO₃) was obtained from Reanal Laborvegyszer. (Budapest, Hungary). Sodium hydrogen selenite (NaHSeO₃) was obtained from VWR International (Lutterworth, Leics, UK) and sodium selenate decahydrate (Na₂SeO₄∙10H₂O) were obtained from Scharlau Chemie (Barcelona, Spain).

Stock solutions of 20 mg/L of silver nanoparticles and silver nitrate, 80.0 mg/L of sodium hydrogen selenite, and sodium selenate decahydrate were prepared and then diluted in serial dilutions (until 10⁻¹⁰). 20 µL fresh culture of P. caudatum was exposed with 20 µL solutions on the glass slide in control groups. LC95 values of every toxicant were determined. Surviving time and the effect of concentration are applied to calculate the LC50 concentration values. The NOEL (No Observed Effect Level) concentration was defined as the concentration when the surviving is longer than 20 min.

500 µL fresh culture of P. caudatum was treated with 500 µL selenium nanoparticles (800 mg/L) at room temperature for 2 h in experimental groups. At the end of the treatment, 20 µL of the treated culture was placed on the middle of

the glass slide, and while observing under a microscope 20 µL toxicants were added. Then, the survival time, locomotor behavior, and morphological changes of P. caudatum are continuously observed under the microscope for 20 min. After more than 20 min, the solution on the glass slide became dry out and the cells were started to die. The location of selenium nanoparticles inside the cell is described by light microscopy attached to a CCD camera, and scanning electron microscope (Hitachi S-4300).

The concentration of selenium nanoparticles higher than 800 mg/L was shown to exert no effects on locomotion and morphology of P. caudatum. Within 5 min of treatment, selenium nanoparticles started to be accumulated inside the cells. Figure 2 shows the black spots are inside P. caudatum which are red with a proper illumination (white light from the side). Also, scanning electron microscopic image with X-ray mapping shows the location of selenium nanoparticles in the cell (Figure 3). These results indicated no morphological changes in P. caudatum.

For toxicants, results showed that ≥1.25 mg/L of silver nanoparticles, ≥2.5 mg/L of silver nitrate, ≥10.0 mg/L of sodium hydrogen selenite, and ≥20.0 mg/L of sodium selenate have a lethal effect on P. caudatum in the control groups as presented in Figure 4. The survival time vs. concentrations has a special shape, which is similar to the pH titration curve. Its inflection point gives the LC50 value. All P. caudatum died within 18 min due to cell lysis. In the present experiments, we observed that these toxicants affected the test organism, P. caudatum in a concentration-dependent manner.

Silver especially AgNPs are known for their antibacterial effect and are used in various nanomedicine and biomedical applications. However, there is increasing concern related to the biological impact of the use of AgNPs on a large scale, and the possible toxicity to the environment and health [20]. Several works show that silver nanoparticles exert an inhibitory effect in all biological systems such as the virus [24], bacteria [25], protozoa [26], algae [27], and fungi [28] regardless of their structural or physiological characteristics, at the cellular level. For instance, 30 - 50 µg/L of silver nanoparticles have a rapid toxic effect on Paramecium within 15 min [29]. In other words, AgNPs have also the capacity to kill cells with different levels of complexity, both prokaryotic and eukaryotic. Most of the reports show an effective concentration near 0.1 mg/L of silver [27] [28]. It is classified that sodium selenite is highly toxic, and sodium selenate is moderately toxic to fingerlings of tilapia [30]. Generally, selenium bioavailability depends on their

size, chemical type, and dose in the source. The elemental selenium nanoparticles have very good bioavailability as indicated by enhancement of serum antioxidant status and selenium concentration in blood and tissues [31], and much lower toxicity as indicated by median lethal dose, acute liver injury, survival rate, and short-term toxicity [32] compared to other chemical types.

The lethal concentration of toxicants was significantly decreased by 2 times in the experimental groups which were pre-treated with selenium nanoparticles (800 mg/L). Namely, the lethal concentrations were found as ≥2.5 mg/L, ≥5.0 mg/L, ≥20.0 mg/L, and ≥40.0 mg/L for silver nanoparticles, silver nitrate, sodium hydrogen selenite, and sodium selenate, respectively (Figure 4). There were no changes in locomotion and morphology of P. caudatum in the experimental groups under the exposure of previously determined lethal concentrations of the toxicants. Particularly, P. caudatum did not exhibit any alteration in their shape, blebbing, lysis, and swimming. And their survival time was extended by the administration of selenium nanoparticles (Figure 4). The changes in the experimental group are compared with the control groups in Figure 4. These results are theoretically consistent with the results of other research methods. For instance, bulk selenium (selenite) treatment in a mouse model was more toxic than selenium nanoparticles in terms of deleterious effects on mice growth, liver functions, and hepatic lipid peroxidation [33], and the selenium nanoparticles dramatically reduce the death incurred by acute toxicity associated with bulk selenium up to 4 times in a rodent model [34]. Also, selenium nanoparticles protect against As(III)-induced cell death and DNA damage [21], and supplementation of selenium with lead reduced the lead level in serum [19]. Selenium antagonizes the toxicity of arsenic and cadmium mainly through sequestration of these elements into biologically inert complexes and/or through the action of selenium-dependent antioxidant enzymes [35]. Besides, Ansar et al., reported selenium supplementation reduces the AgNP-induced hepatotoxicity in rats [20]. Basically, the toxicity of AgNPs is explained due to the release of Ag ions in the system or extensive systemic distribution of Ag in tissues causing oxidative stress, protein or DNA damage, and apoptotic cell death [36] [37] [38]. Selenium restores the activity of important antioxidant enzymes such as glutathione peroxidase and thioredoxin reductase [39]. Therefore, the antidote effect of selenium nanoparticles can be explained by the restoration of target selenoprotein activity and restoring the intracellular redox environment. Basically, impairment of the thioredoxin and glutaredoxin systems allows for proliferation of cytosol and mitochondrial reactive oxygen and nitrogen species which lead to mitochondrial injury/loss, lipid peroxidation, calcium dyshomeostasis, impairment of protein repair, and apoptosis [40]. For instance, mercury binds to the selenocysteine binding site of thioredoxin reductase 1 in the cytosol and thioredoxin reductase 2 in the mitochondria dramatically inhibiting their function [41]. Basically, metals have a lower affinity for thiol groups and a higher affinity for selenium containing groups. In this case, selenium supplementation, with limitations, may have a beneficial role in restoring adequate selenium status and mitigating the toxicity of mercury [42].

Different changes in the locomotion and morphology of P. caudatum were observed for the exposure of the silver and bulk selenium during the experiment. P. caudatum affected by silver mainly showed alteration of their shape by developing irregular blebbing of the cell membrane before cell lysis. There were occurred single or multiple blebs in the cell membrane. The process of blebbing is a common phenomenon during apoptosis. At the same time, fragmentation and disintegration of macronucleus were also observed with increased time of exposure. These morphological changes in the experimental group lasted longer than the control group. Similar kind of changes on the cell membrane of P. caudatum was observed with xenobiotics, monocroptophos (>90 mg/L), and fenthion in P. caudatum. The complete cell lysis occurred within 17 min on average ranging from 5 to 20 min [6] [43] [44].

The effect of bulk selenium mainly showed the locomotion changes. Particularly, due to the high dose, the swimming of P. caudatum stopped completely, but his ciliates kept moving until cell lysis. As the concentrations decreased, sequential changes such as the swimming speed and changes (circular movement), immobile, and cell lysis were observed. In other words, P. caudatum died after they became immobile. Gradual decrease in the swimming speed, with the increased time of exposure, is due to the effect of the toxicants on the cellular metabolism and morphological changes. Similar changes like reduction in the swimming speeds, altered morphology, and generation times were observed when P. caudatum was exposed to a higher concentration (>350 mg/L) of acephate [7] [44].