## 2 Materials and Methods
### Viability MTT Assay  
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolzolium bromide) viability assay was performed according to the tested procedures and was modified see Figure 2 for the purposes of this study [37]. A 0.5 mL volume of unspecified soil microorganisms (the same microorganisms used in the respiration test) + 0.15 mL of phosphate buffer + 0.25 mL of sample (eluate) or buffer (control) were mixed in one glass tube. For the suspension, 100 mg of waste tire particles was added to the mixture. Triplicates were performed for the control, eluate and suspension. The samples were incubated in the thermostat (20 \(\pm\) 2 \({}^{\circ}\)C) in the dark for 168 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-difenyltetrazolzolium bromide (TTB) was added as a substrate (0.1 mL/tube) and the samples were incubated for next hour in the dark. A 7 mL volume of ethanol was then added into each tube. The blue product  
Figure 1: Scheme of experimental designs (respiration test)—preparation of samples.  
formazan from the microorganisms was extracted for the next 24 h. The formazan is a product of microorganismal metabolism. Dead microorganisms are not able to produce it and its production is thus an indicator of microorganismal fitness and viability. The levels of formazan were indirectly measured spectrophotometrically at 485 nm. The recommended duration for measurement is 1 h for cultivation of microorganisms at 37 \({}^{\circ}\)C. In this experiment, the temperature that was set for the entire experiment, i.e., 25 \({}^{\circ}\)C, was used. The formazan was incubated on Tuesdays for 24 h, as it was necessary for sufficient mixing of all suspension components. Some organisms can also live on the surface of the particles, and it would not be possible to achieve the maximum effect and yield of formazan with rapid incubation for 1 h. The incubation times of the formazan with the sample (24 h) were chosen based on previous measurements and experience with this test and solid samples of some natural soils and various building materials (unpublished, own preliminary data).  
The microbial data were evaluated using MS Excel. The oxygen loss (consumption by microorganisms or chemical degradation) during the time period (0 days versus 28 day) was calculated according to Formula (1):  
\[\mathrm{L-oxygen}(\%)=\frac{(\mathrm{T0-Ta})}{\mathrm{T0}}*100 \tag{1}\]  
where L--oxygen = oxygen loss in (%), T0 = oxygen level in time 0 and Ta = oxygen level in subsequent time.  
The toxicity of the waste tire eluate was calculated according to Formula (2):  
\[\mathrm{T(\%)}=\frac{(\mathrm{meanC(timeA)-meanS(timeA))}}{\mathrm{meanC( timeA)}}*100 \tag{2}\]  
where T (%) = toxicity (in%s), mean C(time A) = mean L--oxygen value of control in time A, and mean S(time A) = mean value of the tire sample (the eluate or the suspension) in time A.  
The inhibition of toxicity (in%s) was calculated according to Formula (3):  
\[\mathrm{R-T(\%)}=\frac{\mathrm{Tcontrol(TA)-Tsample(TA)}}{\mathrm{Tcontrol(TA)}}*100 \tag{3}\]  
where R - T = rate of toxicity (%), T control (TA) = toxicity value of the control in the time A and T sample (TA) = toxicity value of the sample in the time A.  
Figure 2: Scheme of experimental designs (MTT viability assay)—preparation of samples.  
The oxygen values were evaluated using the one-way analysis of variance (ANOVA) with the GraphPad InStat software (InStat version 3, San Diego, CA, USA). The multiple-comparison Tukey-Kramer test was performed at a 0.05 significance level.