Accumulation of Spherical Microplastics in Earthworms Tissues-Mapping Using Raman Microscopy

Abstract

The presence of microplastics in the environment is now becoming a challenge for many scientific disciplines. Molecular diversity and spatial migration make it difficult to find plastic-free areas. Their negative, often toxic, effects affect plants and animals to varying degrees, causing many biochemical disorders, species degradation, and population changes. This study aimed to determine the possibility of accumulation of spherical low-density polyethylene particles of 38–63 µm (38–45 µm 1.00 g/cm³, and 53–63 µm 1.00 g/cm³) with fluorescent properties in muscle tissues of the cosmopolitan earthworm species Lumbricus terrestris, exposed to plastic contained in the soil at a concentration of 0.1% dry weight for 3 months. Analysis of the tissues by Raman microscopy included the estimation of mapping area size, sampling density, accumulation time, spectra, laser line, and laser power to detect plastic in the samples effectively. Our results demonstrate the ability of low-density polyethylene microparticles to accumulate in earthworm tissues and are presented graphically for the mapping area and images with plastic detection sites marked. In addition, this article highlights the potential of using Raman microscopy for research in the field of tissue analysis.

1. Introduction

Economic development and the constant demand for new materials have made plastics production one of the main branches of the manufacturing industry [1] Modifications and enrichment with stabilizers and dyes create many new types of plastics every day, more resistant to mechanical and biotic factors. According to various sources, a small part of used or damaged polymers is recycled (6–14%), and the rest goes to landfills or directly to ecosystems in the form of illegal dumps [2]. Despite their strength and resistance, plastics—as they are commonly called—gradually degrade, creating smaller and smaller particles [3]. According to the current nomenclature, these particles are called microplastics. There are many different types of microplastics, according to their chemical composition, origin, size, shape, and even color, but they all have a common denominator, which is the threat to the environment when left uncontrolled [4].

Numerous studies have documented the negative, often toxic, effects of microplastics on organisms. In the case of plants, germination, i.e., metabolic processes related to germination [5], adventitious root development, a decrease in biomass or the efficiency of the photosynthetic process [6], at the biochemical level, exposure to microplastics may be associated with increased levels of oxidative stress markers such as glutathione transferase (GST) and catalase (CAT), disruption of water-electrolyte and endocrine balance, which is associated with generative plant development [7]. There are reports of positive effects of plastic particles on plants, such as the longitudinal growth of roots or the stimulation of carotenoid synthesis [8], but these should be considered undesirable effects due to the presence of contamination. In the case of animals, the scale of research conducted appears extremely extensive, covering marine and terrestrial organisms of mammals, birds, fish, and invertebrates, with the smallest number of publications on reptiles. Relevant data are contained in an article by Porcino et al. [9], in which the authors emphasize the significant differences between studies of organisms in laboratory conditions and animals in the wild, with the implication that studies conducted using a single material under control conditions do not reflect both the variability of contaminants in the environment and the complexity of processes and mechanisms in an aquatic ecosystem, for example. Thus, some results may be overestimated relative to their actual values. Plastic particles can induce several changes once they enter the body, especially when they carry contaminants and toxins on their surface. In most species, the presence of microplastics can cause similar effects, such as decreased individual weight, gastrointestinal damage, accumulation in the liver or intestines, and effects on enzymatic activity and oxidative stress. In addition, many detailed and specific studies have been published on muscle dysplasia in birds, gill inflammation in fishes, impaired homeostasis in cephalopods, or basophilic cell volume in mollusks [10].

The greatest scientific challenge related to plastics is the possibility of their safe biodegradation, quick detection in various environments, and unification of analytical methodology. From the point of view of industrial development and the need for cheap materials, these challenges turn out to be extremely difficult to implement.

Currently, equipment is used that, based on the introduced database, can determine the plastic content in the sample with high accuracy, such as Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, Attenuated Total Reflection FTIR (ATR FTIR), Pyrolysis-gas Chromatography-Mass Spectrometry (Pyro-GC MS), Thermoextraction and Desorption Coupled With Gas Chromatography-Mass Spectroscopy (TED-GC-MS). Additionally, there are many methods of collecting, separating, cleaning, and coloring materials, but all these methods are not validated, usually require a specialist in their use, and describe a small and therefore unreliable sample, and the marking itself requires advanced equipment and specialists, making these markings not often available for commercial use [11].

Microplastics are present not only in marine ecosystems but also in the land, forests, and agricultural areas, and although many studies focus on their impact in the places mentioned above, it should be remembered that the main source of microplastics is cities [12]. Building renovation works, urban dust, degradation of paint particles, tread rubber particles, and clothing fibers constitute most of the fractions described. Additionally, particles of plastics intentionally produced in small sizes, such as those for abrasive materials or cosmetics, have been detected. Plastics of various sizes and shapes generated in this way are carried by wind, rain, or artificially generated gusts over considerable distances, often reaching rural areas.

Detecting plastics in agricultural soils is complicated for many reasons, ranging from the diversity of the plastics themselves to the diversity of the soil and the variety of terrain and natural barriers to particles. Therefore, a precise estimation of the concentration in a specific site is difficult [13].

Earthworms belong to the subclass Oligochaeta and are widely distributed mainly in Central, Western, and partly Eastern Europe. Often called cosmopolitan organisms, they have adapted well to live in various ecosystems, including those that are subject to strong urbanization by humans. Species diversity allows us to identify typical soil-dwelling species that dig tunnels up to 3 m deep into the ground, species that prefer to feed above the soil surface, mainly in composts, and species that prefer a mixed environment. Importantly, earthworms are also common in large urban agglomerations despite theoretically unfavorable conditions such as vibrations and large surfaces covered with asphalt or paving stones [14].

There are many ways for microplastics to enter the soil, as well as many opportunities for further degradation. As Hou et al. [15] points out, one of the main sources is foil used for mulching, giving the example of China, where the areas covered with foil in 2015 amounted to 18 million hectares; the scale in Europe and the USA is similar, although China is still a leader in both the production and use of plastics [15]. Foil residues contribute to increased water evaporation, reduced biomass, and the overall condition of plants [16]. Another source is groundwater purified from sewage treatment plants and rainwater runoff. Water is an excellent carrier of microplastic particles, and, as research shows, it significantly contributes to the pollution of agricultural and forest soils by rinsing plastic particles from facades, roads, sidewalks, and road signs [13]. Sewage treatment plants, despite being well adapted to the absorption of plastics reaching a level of 99%, also contribute to the spread mainly of clothing fibers and cosmetic items containing plastic particles. The scale of soil pollution is significantly influenced by uncontrolled waste dumps, which are often located close to green areas, forests, or water reservoirs [17]. In this case, susceptibility to UV radiation, changes in temperature and humidity, and wind-promoting friction between plastics cause the release of significant amounts of microplastics, the concentration of which can range from 0.01 to 0.7% in the surface soil. It can also be assumed that wind plays a role in the migration of plastics in most cases, mainly by spreading urban particles outside the city zone, as in the case of car tread rubber particles, which are easily carried over up to 8 km. On a global scale, it has been observed that wind currents from sub-Saharan Africa carrying heated air over Europe contribute to the intercontinental drift of particles, which results in plastic reaching ice caps and areas uninhabited by humans [18].

The accumulation is related to the permanent presence of plastics in the tissues of living organisms, documented based on microscopic analysis [19] or molecular staining [20]. So far, accumulation has been described in many species of saltwater fish, birds, and humans. In the case of fish, as many as 47.8% of the tested individuals showed the presence of plastics at a concentration of 4.11–2.85 pcs/individual, and the particle size ranged from 0.054 μm to 0.765 μm. The presence of plastics has many negative effects, such as a sense of false satiety, internal injuries to the intestines and digestive tract, physiological stress, reduced fertility and survival of offspring, growth inhibition, and the dominance of organisms better adapted to the plastic-laden environment. Scientists indicate that, in addition, particles are often vectors of toxins that lead to the inflammation and death of organisms [12]. The problem of accumulation seems to be extremely difficult to combat because the multitude of colors, shapes, and particles increases the chances of mistaken consumption of plastics as natural food. Additionally, microplastics float at different depths in the water, which also favors their accidental ingestion by fish or crustaceans.

The aim of this study was to determine the molecular accumulation of low-density spherical polyethylene in a selected species of earthworms (Lumbricus terrestris). Taking into account the individual anatomy, previous publications, and the nature of this research, the precise determination of the route of accumulation, the impact on biochemical processes, and the amount of the material turn out to be extremely important. Earthworms play a key role in the soil ecosystem, regardless of whether we are discussing cosmopolitan species adapted to urban soils or bioindicators of clean, rural soil [18]. Earthworms, often called soil engineers, contribute to the better circulation of organic matter, increase the absorption of mineral compounds by plants, produce soil aggregates, and increase carbon circulation in the soil.

There are many reports documenting the ingestion of microplastic particles by earthworms and their ability to forage and recognize contaminants selectively, but there are few publications describing the subsequent fate of the particles in the digestive tract. The purpose of this study was to determine the potential for permanent microplastic accumulation in L. terrestris muscle tissues exposed to 38–63 µm low-density polyethylene (38–45 µm 1.00 g/cm³ and 53–63 µm 1.00 g/cm³) contained in the soil at a concentration of 0.1% dry weight for an incubation period of 3 months. An additional goal was to use Raman microscopy as an innovative method for measuring plastics of very small sizes.

2. Materials and Methods

2.1. Research Materials Used

All earthworms were tested for species purity and reproduced under controlled conditions. Adult specimens were used for this study and placed in containers with soil and horse manure in a 3/1 ratio. The manure was frozen for 48 h before mixing to avoid the presence of other individuals. Microplastics were used in the form of spherical particles with sizes of 38–45 µm (GreenPEM 38–45 µm 1.00 g/cc, Fluorescent Green Response: Peak emission of 515 nm when excited at 414 nm), (Figure 1A) and VioletPEM 53–63 µm 1.00 g/cc, Fluorescent Violet Response: Peak emission 584 of nm when excited at 636 nm) (Figure 1B), originated from Cospheric LLC, Somis, CA, USA, with fluorescent properties which, on a dry weight basis, constituted 0.1% of the total mixture. The exposure took place in a controlled room with a temperature of 18 °C and lighting 12/12 h for 3 months. After this time, the individuals collected for testing were placed on an agar medium for 12 h and then into containers with clean soil that did not contain any plastic. After 21 days, the individuals were transferred back to the agar medium for 24 h, washed with distilled water, and frozen by thermal shock at -80 °C. Then, the individuals were deprived of their intestines and digestive tract. After thorough washing again, they were subjected to microscopic analysis using a fluorescence microscope and Raman microscopy.

2.2. Study of Microplastics in Earthworm Tissues

Raman spectroscopy was performed on WITec Alpha 300R and WITec Alpha 300RSA+ spectrometers. Both spectrometers are equipped with a confocal microscope and a TrueSurface attachment. In order to detect the analyzed microplastics in earthworm tissues, three excitation lines were tested: 532, 633, and 785 nm. Due to the strong fluorescence of microplastics during measurement using the 532 nm line, it was not possible to record Raman spectra. In the case of the 785 nm line, a spectrum was obtained for GreenPEM microplastics, while for VioletPEM, mainly fluorescence was observed, which was difficult to distinguish from the fluorescence of the own tissue. Therefore, the measurements were performed using a laser excited at a wavelength of 633 nm.

Using this laser line, it was possible to obtain a spectrum for GreenPEM microplastics. For the VioletPEM microplastics, strong fluorescence with a characteristic spectral profile was observed, allowing the spectra to be distinguished from the intrinsic fluorescence of the examined tissues with high efficiency. In the first stage, single spectra of microplastic samples were made with the following parameters: accumulation number of 10, single spectrum accumulation time of 0.5 s, objective lens with 20× magnification. In the case of GreenPEM microplastics, the radiation power was 12 mW, while for VioletPEM, it was set to 1 mW. Raman mapping involved the sequential collection of individual spectra from a specific surface. Each time, the spectrum was measured over the entire spectral range, i.e., 0–2800 cm⁻¹. Images were obtained using a 10× (NA 0.25) or 20× (NA 0.5) objective.

Measurements were carried out with the following settings:

(a) Mapping large tissue fragments: sampling density of 40–10 μm, map size depending on the tissue size, spectrum accumulation time of 0.1 s, radiation power on the sample of 9–12 mW.

(b) Mapping of smaller tissue fragments: sampling density of 5 μm, map size of 500 × 500 μm, spectrum accumulation time of 0.1 s, radiation power of 9–12 mW. The sampling density was experimentally adjusted to the size of the microplastics in the tissues. Raman images were prepared based on the True Component Analysis (TCA). This analysis makes it possible to identify different components in the sample based on Raman spectra and create an average spectrum for them. The result also includes images showing the distribution of the accumulation of components identified in the sample. The TCA image is constructed in such a way that intense yellow pixels indicate high spectral intensity for the identified component. TCA analysis made it possible to isolate classes containing spectra corresponding to the spectral profile of microplastic standards (control samples).

The GreenPEM spectrum has a set of characteristic bands at 687, 1215, 1296, 1446, and 1538 cm⁻¹ (Figure 2A). In turn, for the VioletPEM microplastics, very strong but characteristic fluorescence is observed, present even at low laser powers (1 mW) (Figure 2B). It is important that the fluorescence spectrum profile is different and more intense than the fluorescence of the tissue itself, making it possible to distinguish this microplastic from the tissue.

3. Results

The Raman microscopy analysis takes into account natural tissue fluorescence, which is not related to the presence of plastics. The characteristic discolorations are clearly visible under the UV matrix and in the Raman microscopy laser spectrum. Determining the band for individual spectra makes it possible to distinguish individual parts in terms of chemical composition and density.

3.1. Results of Microscopic Analysis of the Spectrum, Control Sample

A comparison of the visible image of the entire tissue with the measurement site marked is shown in Figure 3A, and the Raman image, created based on Raman mapping using the 785 nm excitation line and a 20× objective, is shown in Figure 3B. The measured area is 20,400 × 4800 μm, with a sampling density of 40 μm. No areas containing spectra were identified in the tissue whose spectral profile could correspond to the spectra of microplastics.

Figure 4B and Figure 5A,B show the absence of bands characteristic of microplastic samples; only fluorescence coming from the tissue itself is visible. The area size is 23,000 × 4200 µm, the sampling density is 40 µm, the accumulation time is 0.2 s, the laser line is 532 nm, and the laser power is 10 mW.

Despite a higher sampling density (10 µm) and, therefore, better spatial resolution, it was not possible to obtain a signal indicating the presence of microplastics (Figure 6A–E).

3.2. 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 μm (spatial resolution 80–120 μm) enabled the detection of VioletPEM microplastic particles in tissues (sizes up to 63 μm); however, it turned out to be insufficient to detect GreenPEM microplastics, which are characterized to be smaller (up to 45 μm). 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).

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× 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 × 4600 μm, with a sampling density of 40 μm.

Mapping carried out for this tissue, performed with a higher sampling density—10 μm—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 × 3880 μm, with a sampling density of 10 μm.

In the next step, smaller areas of 500 × 500 μm were measured for the tissue in selected places with a step of 5 μm (spatial resolution of approx. 15 μm). 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. Accumulated microplastics, both VioletPEM and GreenPEM, were detected and identified in the measured earthworm tissues. Detection of both types of microplastics was only possible when Raman imaging was performed with good spatial resolution, i.e., a sampling density of 5 μm. In the case of measurements with lower resolution, only VioletPEM particles, which had a larger diameter, were detected.

3.3. An Image of Microplastics Under UV Light

The image of tissues under UV light containing fluorescent plastic particles with a characteristic band is not a method that is sufficiently effective due to the previously mentioned strong intrinsic fluorescence of the tissue. An effective solution seems to be confirming the presence and type of material based on Raman analysis. If the chemical composition of the material is known and the bands visible during analysis are described, testing with a specific density gives highly effective results. Figure 11 shows microscopic photos of GreenPEM 38–45 µm against the background of tissue structures with a similar range of colors seen. Figure 11A,B contain a plastic particle at different depths.

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 μm, which gives high quantitative reliability. Raman spectroscopy’s potential in detecting microplastics within biological tissues was also studied by Liu et al. [19]. Liu 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 Liu 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 Tian 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 Cheng 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 Prata et al. (2019) [11], who critiqued 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 Huerta Lwanga 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 Kwak and An [23], polyethylene damages male reproductive organs, which may contribute to population disorders within a microplastic-contaminated habitat. Rodríguez-Seijo 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 purposes finds a parallel in the work of Zaller and Saxler [18], who explored the role of earthworms in ecosystem functions and their potential as bioindicators.

5. Conclusions

The application of Raman microscopy for identifying microparticles in earthworm tissues, despite the challenges associated with properly adjusting the laser power, proved to be highly effective. This innovative study is the first to use this technique for the parameters being investigated. The method has shown great promise and can be integrated into research on the accumulation of microplastics in animals. In conclusion, while our findings affirm the potential of Raman spectroscopy in microplastic detection within earthworm tissues, they also highlight the complexities and challenges inherent in accurately assessing and mitigating the ecological impacts of microplastics. Our study’s proposal to utilize the accumulation of microplastics in earthworm tissues for bioindication purposes underscores the potential of earthworms as important bioindicators in monitoring environmental contamination. Their role in ecosystem functions, combined with their ability to accumulate pollutants, makes them valuable for assessing the presence and impact of microplastics in terrestrial environments. This approach could enhance our understanding of how microplastics affect biodiversity and ecosystem health, offering a novel method for tracking pollution in natural systems. Future research should focus on refining detection methodologies and exploring the ecological roles of bioindicator species in monitoring environmental health.