## 5 Discussion of the Results
### Particle Concentration in Tissues  
This study clearly shows significant differences in the tissue accumulation of earthworms that were reared under laboratory conditions and in outdoor containers with large soil volumes. The worms who were forced to forage in 3L containers showed a linear increase in microplastic particle concentration, which was triggered by the lack of mobility and stability of the plastics throughout the soil and food. These results further demonstrate the ability to accumulate microplastic in earthworm bodies to a high degree and the potential for further analysis for other polymer groups. Tests conducted on macro-cultured earthworms were variable with respect to the concentration, and it seems that earthwormsavoided areas where microplastic accumulated at a concentration of \(\sim\)0.6% and that the particle size also had a significant impact on the results. The authors draw the bold conclusion that earthworms are unable to sense plastic below 40 um, meaning that foraging in an area with a concentration of less than 0.6% and a particle size of less than 40 um occurred without the appearance of signs of stress or a desire to relocate. The size and concentration of microplastics in the soil have a significant impact on their accumulation in earthworms. Higher concentrations and smaller particle sizes increase the likelihood of their ingestion and accumulation in tissues [49]. In addition, particles with spherical shapes were most common in the tissue analysis, followed closely by irregular fragments of a similar size. The tissue images and optical analysis did not clearly prove signs of inflammation in the sites where the material was labeled, nor damage to the tissues themselves, as is often reported in articles. Therefore, it can be concluded that mechanical damage to the gastrointestinal tract or intestines can only be caused by larger fragments that damage tissues with their sharp edges during intestinal peristalsis. These conclusions are consistent with previous studies, although some aspects require further verification. In [50], which used Raman microscopy, the authors showed that spherical microplastics accumulate more often in earthworm tissues, which coincides with the results presented in the current study. However, some previous studies [43] have suggested that even small microplastic particles can cause subtle inflammatory reactions. Additionally, these results fit into the broader context of research on the ecotoxicity of microplastics in terrestrial ecosystems. [PERSON] et al. [51] suggest that the ability of earthworms to avoid areas with high concentrations of microplastics may be a behavioral strategy to minimize its negative impact. Nevertheless, the lack of visible reactions to particles that are smaller than 40 um requires further investigation, especially with regard to potential sublethal effects such as oxidative stress or metabolic disorders. The described particles are two orders of magnitude smaller, so there is a chance of internal tissue penetration. It is also possible that the smallest fragments of microplastic enter the body through the respiratory cavities along the body via the mucus produced. The available literature indicates that there are many problems associated with the detection of microplastic in soil and the whole procedure for its detection [52; 53], pointing to the variability of the matrix that is soil, the number of samples taken, their size and spatial distribution, or the consideration of soil hydrodynamics.