# Accumulation of Spherical Microplastics in Earthworms Tissues-Mapping Using Raman Microscopy
## 3 Results
### Test Results of Research Trials  
Detecting microplastics when measuring the entire tissue was difficult due to its thickness (several tens to several hundred micrometers) compared to the laser beam penetration depth of approximately several micrometers, which is why the signal was collected only from a selected focusing plane. Another limitation turned out to be the small size of the tested microplastics. The use of a sampling density of 40 \(\upmu\)m (spatial resolution 80-120 \(\upmu\)m) enabled the detection of VioletPEM microplastic particles in tissues (sizes up to 63 \(\upmu\)m); however, it turned out to be insufficient to detect GreenPEM microplastics, which are characterized to be smaller (up to 45 \(\upmu\)m). Figure 7A shows the result of Raman mapping of the tissue in which VioletPEM was identified based on TCA (marked with a red circle) (Figure 7B). Figure 5: (**A–C**). Natural tissue fluorescence and spectral bands for individual classes. Figure 6: (**A–E**). Measurement areas for control trials. The red color indicates the location of the exact scan. A summary of the visible image with the measurement area marked is shown in Figure 8A, and the Raman image created based on Raman mapping using the 633 nm excitation line and a 10\(\times\) objective for the tissue is shown in Figure 8B. The place where the spectrum corresponding to the spectral profile of the VioletPEM microplastic particle was identified is marked. The size of the measured area is 20,000 \(\times\) 4600 \(\upmu\)m, with a sampling density of 40 \(\upmu\)m.  
Figure 8: **(A,B)** Size of the measured area of 20,000 \(\times\) 4600 \(\upmu\)m, sampling of density 40 \(\upmu\)m (**A**) and more precise measurement of 9700 \(\times\) 3880 \(\upmu\)m, sampling density of 10 \(\upmu\)m (**B**). The red color indicates the location of the exact scan. Figure 7: **(A,B)** Preliminary mapping results with VioletPEM identification. The red color indicates the location of the exact scan. Mapping carried out for this tissue, performed with a higher sampling density--10 \(\upmu\)m--enabled the detection of a VioletPEM particle at a given focal plane. The microplastic particle found is marked with a red arrow (Figure 8B). The measurement area size is 9700 \(\times\) 3880 \(\upmu\)m, with a sampling density of 10 \(\upmu\)m.  
In the next step, smaller areas of 500 \(\times\) 500 \(\upmu\)m were measured for the tissue in selected places with a step of 5 \(\upmu\)m (spatial resolution of approx. 15 \(\upmu\)m). Visible images with the measurement area were marked, and the results of the TCA analysis were collected. Better resolution enabled the detection of both VioletPEM (Figure 9A-C) and GreenPEM (Figure 9D-F) microplastics. In turn, the areas shown in Figure 9G-I did not observe spectra corresponding to the spectral profiles of the tested microplastics. Similar results were obtained for another tissue; the analysis results are presented in Figure 10. VioletPEM microplastics were detected in the areas marked in Figure 10A,B,E, while GreenPEM microplastics were detected in the areas marked in Figure 10C,D. The places where no microplastics were detected are included in Figure 10F-H. All tissue areas for which Raman measurements were performed are presented. The results of the microscopic observations and Raman analyses confirmed the presence of plastic particles in all tested individuals, which may suggest the ability of the particles to break through tissue barriers. Microparticles were found in transverse muscles, coelomic fluid channels, and sepia constrictions. It is also possible that coelomic cells moving freely in the coelomic sac contributed to intratissue transmission. Observations in various light spectra also demonstrated natural tissue fluorescence, which is not related to the presence of plastic. This study highlighted the potential of Raman spectroscopy for the detection of microplastics in earthworm tissues but indicated significant limitations and requirements as to how samples should be prepared.