## 3 Results and Discussion  
The authors referred to the levels of heavy metals in the tested tire eluates in one of the previous studies [20]. The concentration of metal leakage from rubber crumbs into water was determined using an inductively coupled plasma optical emission spectrometer Agilent 5110 SVDV device ICP-OES (Agilent technologies, Santa Clara, CA, USA), equipped with a SeaSpray glass concentric nebulizer. The eluate contained a barium (Ba) concentration of 0.021 mg/L and a zinc (Zn) concentration of 0.273 mg/L. The other commonly determined elements (Mn, B, Si, P, Cu, Pb) were under the detection limit [20]. On the other hand, the chemical analysis also confirmed the higher levels of pollutants in solutions more than 1-25 times when the tire particles were in the test solutions for 28 days [51]. The levels of boron, iron and silicon were also under the limit of quantification after 24 h. The barium level was 0.053 mg/L, the manganese level 0.031 mg/L and the zinc concentration 5.670 mg/L [51]. PAHs were detected in units of mg/kg. Only pyrene levels were around 30 mg/kg in the tire particles [20]. However, PAHs are generally volatile compounds and if we prepared the eluate and performed the microbial tests, these substances could enter the air above the leachate or suspension level with the microorganisms in the test vessels. Therefore, it can be assumed that during the test these substances did not have as large of an effect on the obtained toxicity results in the respiration test over 28 days. This means only the metals present could probably serve as a source of toxicity in addition to sugar as food, as they are not able to evaporate in the aquatic or soil environment. On the other hand, PAHs together with metals may have affected the toxicity and cell survival in the viability assay. It has been proven many times that both inorganic and organic pollutants have an effect on the cellular level and cause damage to membranes, oxidative stress or damage to genetic information [52; 53; 54; 55]. It should be emphasized that no similar study has been published to date. Therefore, it is not possible to compare our data with the values and conclusions of other studies on the same topic. The results of the biodegradation and toxicological respiration test indicate that significant effects were found (see Table 1 and Figure 3). The oxygen loss in the tire eluates without microorganisms showed the same values during the duration of the experiment--these results are expressed in Table 1, part \"D\". This indicates that oxygen was not chemically degraded, and the chemical composition of the sample was constant. On the other hand, samples of the eluate containing microorganisms showed decreasing levels of oxygen during the time period. This was caused by microorganisms and their metabolic processes (Table 1, part \"B\"). The same results were found for control samples with microorganisms but without the tested eluate (Table 1, part \"A\"). The consumption of oxygen between the control and the eluate sampled did not differ in the case of the eluates, contrary to the suspension of particles (cf. Table 1, parts \"B and C\"). The lower oxygen consumption indicates that microorganisms were less abundant in the presence of tire particles or that their metabolism was lower in comparison to the control and microorganisms in the clear eluates (cf. Table 1, parts \"A, B and C\"). The continual involvement of heavy metals and organic pollutants can lead to such conclusions. The toxicity of samples with the prolonged presence of tire particles was confirmed during the course of the experiment (Figures 3 and 4). The biodegradation potential was not recorded because the oxygen values were similar or less for both tire samples in comparison to the control (Figure 4). The statistical analysis confirmed statistically significant differences in the oxygen values between only some of the individual times at a 0.05 significance level.