## 4 Conclusions  
* Waste fires from transport were tested in biodegradation and toxicological experiments with a nonspecific soil microbial community in the present study. The results showed various toxicities but no biodegradation of the waste tires during 28 days of exposure.
* Higher toxicity was found during the respiration inhibition test as well as for the viability assay in the case of suspensions with tire particles than for pure eluates. Such results raise the question of whether such an experimental design should always be used when tests are performed using solid sample leachates, or if both variants should be used together in one experimental design for a more ecologically relevant and realistic comparison.
* The viability tests represented an acute toxic effect and the respiration inhibition test a chronic effect. However, we compared the results from the period of 168 h, and we found that the microorganisms were more sensitive in the short viability test than in the chronic test during the course of 168 h. A similar comparison has never been studied in ecotoxicological papers and as such we are not able to review and discuss these types of results.
* The non-degradability of compounds in the waste eluates indicated that the leachates from waste tires were not subject to degradation. These results were also strongly influenced by a short-term experiment (a duration of only 28 days). Unfortunately, this confirmed the problematic removal of waste tires, which do not undergo environmental degradation. Therefore, the negative ecotoxicological results on soil and aquatic organisms, together with the large leaching of heavy metals or organic pollutants and limited biodegradation, lead to the conclusion that waste tire particles are not a harmless material and their possible use and processing with various techniques needs to be further investigated.
* Other microorganismal or sub-cellular assays should be used for the assessment of toxicity and biodegradability of rubber tires in the future. Another possibility is the use of multi-generation or multi-species tests or in microcosms or mesocosms in aquatic or terrestrial environments. Different species or types of microorganisms should be used as model organisms--mainly yeasts, bacteria, green or blue algae, molds, fungi and their mixtures.
* The practical significance of the conducted research lies in the knowledge that both types of methodologies (eluates + suspension of solid tested particles) are supposed to be used simultaneously. This finding should be reflected in the methodologies used for legislative purposes in the determination of ecotoxicity for microbial communities.  
Conceptualization, K.K.; methodology, K.K.; investigation, K.K.; writing--original draft preparation, K.K., J.F. and R.C.; writing--review and editing, K.K. and R.C.; supervision, R.C. All authors have read and agreed to the published version of the manuscript.  
This research has been supported by the Czech Science Foundation under Project No. FV40054 and the Czech Technical University project No. SGS22/137/OHKI/3T/11.  
Not applicable.  
Data Availability Statement: Data supporting the reported results can be obtained from the authors.  
The authors declare no conflict of interest.