# Accumulation of Spherical Microplastics in Earthworms Tissues-Mapping Using Raman Microscopy
## 4 Discussion  
The use of Raman microscopy to identify microparticles contained in tissues, despite many difficulties related to the appropriate setting of the laser power, turned out to be extremely effective. The mapped areas in various ranges of accumulation time and number of measurements were examined with a detection accuracy of up to 0.1 \(\upmu\)m, which gives high quantitative reliability. Raman spectroscopy's potential in detecting microplastics within biological tissues was also studied by [PERSON] et al. [19]. [PERSON] et al. [19] demonstrated the technique's capability to identify various types of microplastics in complex environmental samples through visual pseudo-color imaging, underlining the importance of precise detection methods for assessing ecological risks. Their approach, which combines cloud-point extraction with membrane filtration, highlights the advanced potential of Raman spectroscopy for accurately analyzing the category, quantity, location, and differentiation of microplastics [19]. Our detection of both VioletPEM and GreenPEM particles with high spatial resolution sampling corroborates the necessity for precise detection methods highlighted by [PERSON] et al. [21]) for assessing ecological risks associated with microplastics. Moreover, the specificity of Raman spectroscopy in differentiating microplastic types within biological matrices, as demonstrated in our study, is pivotal for environmental monitoring. This specificity aids in understanding the distribution and fate of different microplastics, as outlined by [PERSON] et al. [13], who explored the transformation and environmental risks of microplastics under UV irradiation. This specificity aids in understanding the distribution and fate of different microplastics, as outlined by [PERSON] et al. [22], who explored the transformation and environmental risks of microplastics under UV irradiation. The challenge of detecting smaller microplastics, such as GreenPEM, due to their size and the technique's spatial resolution limitations, underscores the need for advancements in sampling and analytical techniques. This challenge is consistent with the observations of [PERSON] et al. (2019) [11], who criququed current methodologies for their limited reliability in capturing the full spectrum of microplastic pollution. The observed ease of polyethylene absorption and accumulation in earthworm tissues at a concentration of 0.1% dry mass basis raises concerns about environmental and physiological impacts, resonating with [PERSON] et al. [12], who documented microplastic incorporation into _Lumbricus terrestris_ burrows. The presence in the soil and muscle accumulation may be related to disturbances in biochemical (enzymatic) processes, including the activity of glutathione s-transferase or lipid peroxidase, general oxidative stress, growth inhibition, or external and internal injuries resulting from contact with sharp edges of materials. It has been proven that particles are often catalysts for many pollutants and heavy metals, which affects their toxicity to living organisms. According to [PERSON] and [PERSON] [23], polyethylene damages male reproductive organs, which may contribute to population disorders within a microplastic-contaminated habitat. [PERSON] et al. [24] also describe immunological stress and the general weakening of individual immunity among _Eisenia fetida_; on the other hand, the authors of the article suggest that there is a chance to use the ability to accumulate microplastics in tissues for bioindication purposes. Our study's suggestion to leverage the accumulation capability of microplastics within earthworm tissues for bioindication  
Figure 11: (**A–D**) Plastic particles and natural tissue color under UV light.  
purposes finds a parallel in the work of [PERSON] and [PERSON] [18], who explored the role of earthworms in ecosystem functions and their potential as bioindicators.