A Method for the Extraction and Analysis of Microplastics from Tropical Agricultural Soils in Southeastern Brazil

Abstract

Microplastics (MP) are widespread pollutants that pose a risk to soil ecosystems globally, especially in agricultural soils. This study introduces a method to extract and identify MP in Brazilian tropical soils, targeting debris of low-density polyethylene (LDPE) and polyvinyl chloride (PVC) polymers, commonly present in agricultural settings. The method involves removing organic matter and extracting MP using density separation with three flotation solutions: distilled water, NaCl, and ZnCl₂. Extracted MP are then analyzed through optical microscopy and Fourier transform infrared spectroscopy. The organic matter removal efficiency ranged from 46% to 89%, depending on the initial organic matter content in the soil. Recovery rates for LDPE ranged from 81.0% to 98.8%, while PVC samples showed a range of 59.7% to 75.2%. Finally, this methodology was tested in four agricultural raw soil samples (i.e., without any polymer enrichment) The values found in the soil samples were 2517.5, 2245.0, 3867.5, and 1725.0 items kg⁻¹, for ferralsol, nitisol, gleysol, and cambisol samples, respectively, with MP having diverse shapes including fragments, granules, films, and fibers. This approach lays the groundwork for future studies on MP behavior in Brazilian tropical agricultural soils.

1. Introduction

Since the advent of synthetic plastics in the early twentieth century, their production and widespread utilization have experienced significant growth. Plastics play an integral role in nearly every facet of human activity, including agriculture, civil construction, medical equipment, industrial manufacturing, automotive, and electronics. This extensive application owes to their inherent plasticity, stable chemical properties, and remarkable impact resistance [1,2].

In agriculture, plastics find diverse uses, ranging from protective systems and coverings to irrigation, transportation, and storage of agricultural products. They serve in storing pesticides (herbicides and insecticides) and agricultural inputs (seeds and fertilizers) and contribute to irrigation systems and the controlled release of nitrogen, often in the form of nanocomposites [3,4,5,6]. However, despite their manifold advantages, the environmental repercussions of plastic usage are undeniable. Although many thermoplastic polymers are theoretically recyclable, the current recovery rate remains dismally low, hovering below 6% [7,8].

Over time, improperly disposed plastics undergo weathering and degradation, transforming into fragile and brittle fragments, thus forming microplastics (MP) or nanoplastics (NP). MP constitute solid plastic particles ranging in size from 1 µm to 5 mm, while NP are particles smaller than 100 nm [8,9,10]. Because of their diminutive size, these particles can be easily dispersed by wind and water currents over extensive distances, thereby infiltrating marine [11] and terrestrial ecosystems [4]. This wide dissemination poses a significant threat to ecosystems, as these particles can be ingested by marine organisms, potentially entering the food chain and impacting human health [12,13].

In agricultural soil, the primary sources of MP are polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), and polystyrene (PS) [14,15]. Particularly, LDPE and PVC MP were found in large quantities in agricultural soils [16], which is not surprising, as these plastics are widely used in modern agriculture. For instance, LDPE mulch film, which is used as a covering material in agriculture [17], accounts for approximately 2% of global plastic production each year [18]. PVC is also commonly used in irrigation devices [19].

In recent decades, numerous studies have focused on identifying and quantifying MP across various ecosystems [20,21], with research indicating that most plastic waste originates from terrestrial sources [22,23,24,25]. While research on MP in soil has expanded significantly, particularly in agricultural soils in China [26], driven by increasing global awareness of microplastic pollution [27], data on this issue remain remarkably scarce in regions such as South America [28]. In Brazil, studies have primarily targeted aquatic environments, such as lagoon sediments, rivers, recreational areas, and aquatic microfauna [29,30].

Adding to this challenge is the complexity of identifying and quantifying plastic materials in agricultural soils, which are highly heterogeneous matrices composed of mineral solids, organic matter, and other constituents arranged in a complex structure [31]. This makes it essential to assess and monitor the presence of MP in agricultural soils and to understand their potential impact on ecosystem quality and the well-being of both biota and humans.

This study aims to develop and evaluate a method for extracting and analyzing LDPE and PVC MP in four agricultural tropical soil types that are representative of Southeastern Brazil: Gleissolo Melânico Tb Distrófico típico (GM), Latossolo Vermelho-Amarelo Distrófico típico (LVA), Nitossolo Háplico Distrófico típico (NX), and Cambissolo Háplico Tb distrófico típico (CX), according to the Brazilian System of Soil Classification Brazil [32]. The MP extraction was performed using the density separation method, and their identification was performed through optical microscopy and Fourier transform infrared spectroscopy.

This paper presents a methodology for extracting and identifying microplastics in tropical soils. While based on previously reported approaches, the extraction method has been adapted explicitly for weathered soils. No prior study has applied this methodology to such soils, highlighting the novelty of this research. Additionally, research on MP extraction and identification in tropical soils, particularly in southeastern Brazil, remains limited. Thus, this study contributes to the knowledge about plastic pollution in this region of Brazil.

2. Materials and Methods

2.1. Sample Preparing

2.1.1. Microplastic Samples

The microplastics used in the experimental procedures were specifically created for this study. The polymers selected were chosen as they represent common sources of MP found in agricultural environments [33,34]. Pellets of low-density polyethylene (LDPE) and powder from polyvinyl chloride (PVC) were purchased from the polymers laboratory at the Federal University of Lavras, and pieces of PVC irrigation tubes were found in the study area. The plastic polymers had densities ranging from 0.91 to 0.925 g cm⁻³ for LDPE and from 1.25 to 1.50 g cm⁻³ for PVC, respectively. To obtain plastic particles with micrometric dimensions, the plastics were grated using a metal file. Subsequently, the particles were sieved through meshes of 2000 µm, 1000 µm, 500 µm, 210 µm, and 105 µm to obtain MP particles with sizes ranging from 105 µm to 2000 µm. This size range was selected because of the significant prevalence of MP in soil within this range [12]. The same mass proportion of the sieved MP was employed to produce the MP samples. Consequently, MP particles of varied sizes and shapes were generated through this process. Finally, the MP groups were prepared as follows: Group 1—LDPE microplastics, Group 2—PVC microplastics, and Group 3—samples with equal mass proportion of Groups 1 and 2.

2.1.2. Soil Samples

Raw soil samples used comprised four tropical soil types: Gleissolo Melânico Tb Distrófico típico (GM), Latossolo Vermelho-Amarelo Distrófico típico (LVA), Nitossolo Háplico Distrófico típico (NX), and Cambissolo Háplico Tb distrófico típico (CX), according to the Brazilian System of Soil Classification [32]. These correspond to gleysols (GM), ferralsols (LVA), nitisols (NX), and cambisols (CX), respectively, according to the WRB [35]. The samples were collected from the 0–20 cm soil layer at Muquem Farm and the Federal University of Lavras campus in the municipality of Lavras, Minas Gerais state, Brazil, in an area destined for agricultural use in August 2022. This depth was selected because the topsoil layer is critical for plant health. Moreover, most agricultural activities and MP deposition typically occur within these upper soil layers, making this zone essential for understanding MP contamination [28,36,37]. The soil samples were collected from a cultivated area, with the specific crop types detailed in

Table 1. Landscape, location, land use, physical, and chemical properties of the studied soils.

Characteristics	Soil Class
	LVA	NX	GM	CX
Location	21°12′16.62″ S 44°58′55.14″ W	21°11′56.09″ S 44°58′51.02″ W	21°12′4.23″ S 44°58′44.48″ W	21°13′49.59″ S 44°59′0.44″ W
Altitude (m)	996	978	960	918
Slope (%)	8–20	3–8	3–8	20–45
Current crop	Soybean	Soybean	Rice-Beans	Olive trees
Clay (g kg−1)	490	530	310	550
Silt (g kg−1)	100	60	220	170
Sand (g kg−1)	410	410	470	280
Bulk density (g cm−3)	1.36	1.29	0.75	0.92
Macroporosity (cm3 cm−3)	0.13	0.15	0.27	0.13
Microporosity (cm3 cm−3)	0.39	0.38	0.46	0.27
Total Porosity (cm3 cm−3)	0.52	0.52	0.73	0.40
pH (H2O)	5.6	6.9	5.0	5.0
Al3+ (cmolc dm−3)	0.10	0.10	0.20	0.00
H +Al (cmolc dm−3)	2.70	1.20	4.00	1.70
SB (cmolc dm−3)	5.68	8.69	9.15	1.86
eCEC (cmolc dm−3)	5.78	8.79	9.35	1.86
tCEC (cmolc dm−3)	8.38	9.89	13.15	3.56
BS (%)	67.78	87.90	69.55	52.28
m (%)	1.73	1.14	2.14	0.00
SOM (g kg−1)	30.8	26.3	172.9	4.70

LVA: ferralsol; NX: nitisol; GM: gleysol; CX: cambisol; Al³⁺: exchangeable aluminum; SB: sum of bases; eCEC: effective cation exchange capacity; tCEC: Cation exchange capacity at pH 7; BS: base saturation; m: aluminum saturation; SOM: soil organic matter.

, where regular plowing, along with the application of fertilizers, pesticides, and other agricultural inputs that may serve as potential sources of MP contamination, are conducted. To ensure representative data, the sampling was performed randomly across the area. The soil samples were collected using a steel auger. Finally, samples were obtained with masses of 500 g; these were stored in glass jars to prevent contamination. Chemical and physical analyses were performed according to [38] (2017). The landscape, location, land use, physical, and chemical characteristics of the studied soils are detailed in Table 1.

The samples were air-dried, sieved through a 2000 µm mesh, and subjected to a 200 °C heat treatment for 24 h. This process aimed to degrade the target MP particles in the soil while minimally affecting the soil organic matter (SOM) [39,40]. By avoiding the combustion of SOM, we prevented any interference in the subsequent stages of the study.

Furthermore, research indicates that the presence of organic matter in soil can affect the extraction of MP, leading to inaccurate estimations of their concentration in the soil [6,41,42]. Therefore, before preparing the MP/soil samples, organic matter was removed using alkaline digestion, following the method described by [43] (2017). Briefly, ten grams of each soil sample were measured and transferred into a 200 mL beaker. Subsequently, 20 mL of a 30% KOH–NaClO solution was added to the soil and manually mixed for 2 min to ensure even distribution. The mixture underwent ultrasonic treatment in a water bath at room temperature for 5 min to facilitate further dispersion and breakdown of soil aggregates. Following this, the mixture was transferred to an oven set at 50 °C with forced air circulation to facilitate the evaporation of the alkaline solution. To ensure uniform digestion, the sample was manually shaken for 30 s every 4 h during its incubation in the oven. This digestion procedure was performed in triplicate to ensure the consistency and reliability of the results. Moreover, control soil samples were also prepared using the same protocol without adding the 30% KOH–NaClO solution. This control protocol was repeated in triplicate to serve as a baseline for comparison. The reagents were purchased from Neon and Alphatec Co. Ltd., São Paulo, Brazil.

2.1.3. Microplastic/Soil Samples

After the soil digestion procedure, the mixtures of MP and soil samples were prepared using 1 mg of MP per gram of soil (1 g kg⁻¹ dry weight). Each soil sample was mixed with the corresponding groups of MP (Groups 1, 2, or 3) in triplicate. These samples were named SOIL/LDPE, SOIL/PVC, and SOIL/LDPE–PVC (where SOIL is LVA, GM, CX, or NX).

The chemicals were purchased from different Brazilian suppliers in São Paulo city: sodium hypochlorite (NaClO) from Neon Co. Ltd., potassium hydroxide (KOH) from Alphatec, sodium chloride from Vetec, and zinc chloride (ZnCl₂) from Dinâmica Co., Ltd. All chemicals were of analytical grade, and purified distilled water was used throughout the experiments.

2.2. Extraction of Microplastics: Density Separation, Centrifugation, and Filtration

The method of density separation is widely employed to isolate MP particles from soil samples [8,14,16,17,22,44,45,46]. In this work, the extraction of MP in soil samples followed the methodology outlined by [4,47], incorporating modifications. We applied the technique using sequentially three different liquids to isolate MP from soil: purified distilled water (DW) (ρ = 1.00 g cm⁻³), a saturated solution of sodium chloride (NaCl) (S1) (ρ = 1.19 g cm⁻³), and zinc chloride (ZnCl₂) (S2) (ρ = 1.5 g cm⁻³).

Distilled water and NaCl solutions are affordable and noncorrosive alternatives, making them practical for laboratory use, as well as easy to prepare and handle compared to other methods [47]. The sequential use of these solutions with ZnCl₂ provides a more comprehensive approach to extracting microplastics of various densities, outperforming the efficiency of using high-density solutions such as calcium chloride (CaCl₂) alone [48]. Furthermore, denser solutions, such as calcium chloride, tend to be more expensive and may present handling challenges due to their corrosive potential [37].

Initially, we mixed 20 mL of distilled water with soil/MP sample (at a soil-to-MP ratio of 1 g:1 mg). The mixture was manually stirred for 20 min, then sonicated for 5 min and centrifuged for 5 min. Subsequently, it was left to settle for 48 h, allowing the supernatant to be carefully decanted and filtered through a cellulose ester membrane (MCE) filter with a diameter of 47 mm and a pore size of 0.22 µm. To the solid mixture remaining in the beaker, we added 20 mL of S1. Again, this mixture was manually stirred for 20 min, sonicated for 5 min, and then centrifuged to remove the supernatant. The supernatant underwent two rounds of filtration using both DW and Solution S1. Finally, 20 mL of S2 was added to the solid mixture in the beaker, and the previous procedure was repeated. In this instance, the supernatant was filtered through a membrane filter. All the membrane filters containing the MP were placed into Petri dishes, dried at room temperature, and then subjected to further analyses. The method of extracting MP from the soil samples is illustrated in Figure 1. Lastly, the potential contamination during the process was evaluated by conducting each extraction step on a soil-free solution.

2.3. Evaluation of Organic Matter Removal from the Soil Samples

The efficiency of removing organic matter using the KOH–NaClO solution was determined by comparing the organic matter content in soil samples with and without digestion. For this proposal, initially, we determined the organic matter content in the soil samples using the loss-on-ignition (LOI) method [49]. In brief, 10 g of each soil sample was dried in an oven at 105 °C for 2 h, until a constant dry weight was achieved. Then, it was combusted at 550 °C for 2 h and cooled to room temperature. Then, the organic matter content in the soil samples was calculated through the following equation:

$$\mathrm{L}\mathrm{O}\mathrm{I}={\displaystyle \frac{\mathrm{W}\left(105 °\mathrm{C}\right)-\mathrm{W}\left(550 °\mathrm{C}\right)}{\mathrm{W}\left(105 °\mathrm{C}\right)}}\times 100\%$$

(1)

where W(105 °C) represents the dry weight of the sample before combustion and W(550 °C) is the dry weight of the sample after heating to 550 °C (both in g).

The efficiency of removing organic matter from the soil samples was calculated using the following equation:

$$\mathrm{R}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{d}\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{c}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\left(\%\right)\mathrm{}=\mathrm{}{\displaystyle \frac{{\mathrm{S}\mathrm{O}\mathrm{M}}_{\mathrm{a}}}{{\mathrm{S}\mathrm{O}\mathrm{M}}_{\mathrm{i}}}}\times 100$$

(2)

where SOMi represents the mass percent of organic matter in the soil samples that did not undergo the digestion step and SOMa represents the mass percent of organic matter of soil samples after digestion.

2.4. Microplastic Recovery Rate

To assess the extraction efficiency of MP using the sequential application of three solutions, DW, S1, and S2, in SOIL/LDPE, SOIL/PVC, and SOIL/LDPE–PVC mixed samples, a mass difference calculation was performed, following the method described by [50] (2016). Once dried, the MP were transferred to a preweighed membrane filter and weighed using an analytical balance, and the recovery percentage was calculated using Equation (3):

$$\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{y}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left[\%\right]\mathrm{}=\mathrm{}{\displaystyle \frac{{\mathrm{W}}_3}{{\mathrm{W}}_2-{\mathrm{W}}_1}}\times 100$$

(3)

where W₁ is the weight (g) of the dry filters before extraction of MP, W₂ is the weight (g) of the dry filters with MP, and W₃ is the weight of MP mixed with soil samples. Using Equation (3), the MP recovery rate was evaluated in two steps: (i) after the density separation with distilled water and a sodium chloride solution (DW + S1) and (ii) after the density separation with a zinc chloride solution (S2).

2.5. Identification and Quantification of Microplastics

The MP were characterized and identified using optical microscopy and Fourier transform infrared spectroscopy (FTIR) in two stages: (i) before mixing with the soil samples and (ii) after the extraction method. The morphological characteristics of the particle surface and the size distribution of the MP were assessed using a Nikon Eclipse Ni 10× optical microscope and Imagin software version 4.6.0. Particles identified as potential plastics were identified using a Shimadzu IRAffinity 1S spectrometer. Measurements were made using the attenuated total reflectance (ATR) method, and spectra were recorded in the spectral range of 400–4000 cm⁻¹ with a scan rate of 16 and a spectral resolution of 4 cm⁻¹. The spectra were processed using the OriginPro 2023b software [51]. The spectroscopic analysis was performed by comparing the FTIR spectra of the MP samples with reference polymer spectra reported in the literature. Variations in band positions and intensities between the sample and reference spectra enabled a highly reliable identification of the polymer types. Based on this analysis, the FTIR spectra were identified as corresponding to low-density polyethylene (LDPE) and polyvinyl chloride (PVC), with no other foreign materials detected beyond these two polymers during the study.

2.6. Application of Microplastic Extraction Method in Raw Agricultural Soils

The extraction method was evaluated on raw samples of the soils collected previously by performing the same procedure outlined earlier, without the pretreatment heat step in the soil and the addition of MP. For each tested sample, the quantification of the number of particles of MP was performed, accompanied by characterizing and quantifying of the extracted polymers. The abundance of MP was recorded as pieces kg⁻¹ of dry soil by counting the pieces of MP in the total area of the filter. The shapes were classified as fragments (hard angular pieces), granules (circular or semicircular particles), fibers (elongated filaments), and films (soft, transparent flakes). In addition, the longest part of the particle length was measured and recorded in microns (µm) [52,53].

2.7. Statistical Analyses

Statistical analyses were performed to assess potential disparities in the soil digestion process with the alkaline solution in the SOM content measured by the LOI method in the soils, the differences between the studied polymers in the recovery rate of MP, and the differences in the quantity of MP particles.

The normality of the variables under study was verified using the Shapiro–Wilk test, the analysis of variance (ANOVA) was performed between treatments, and the Tukey test assessed the differences of means at a significance level of 5%. The data were transformed using the Box–Cox transformation. Statistical analyses and models were conducted using the R Software (R Core Team, Vienna, Austria, 2022) version 4.2.2 [54] with the Tidyverse and ggplot2 libraries.

3. Results and Discussion

3.1. Efficiency of Digestion Method for Organic Matter Removal in Soil Samples

The digestion of soil organic matter (SOM) is essential to minimize errors in the visualization, quantification, and spectroscopic identification of MP [37]. Furthermore, this process helps release MP trapped within the soil’s organic matrix [3,55], making extraction easier by reducing sample complexity. This ensures that the resulting data more accurately reflect the true presence and concentration of MP in the soil [47]. Therefore, it is necessary to remove organic matter from soil samples without damaging the MP [56,57]. The SOM content of samples without undergoing the digestion process, using the loss-on-ignition (LOI) method, was 17.9% for LVA, 22.5% for NX, 45.0% for GM, and 9.2% for CX. Similarly, the SOM content was determined in soil samples subjected to digestion. Consequently, on average, the SOM removal, expressed as percentages, was as follows: 70.9 ± 2.9% for LVA soil, 82.7 ± 17.9% for NX soil, 89.6 ± 1.3% for GM soil, and 46.7 ± 2.8% for CX soil (see Figure 2). Statistical analysis showed significant differences among the rates of organic matter removal in the four types of soil (p < 0.05). All estimates showed coefficients of variation of less than 23%.

According to these results, the CX sample had the lowest SOM value, while the GM soil had the highest. This last result was expected, since, because of its redoximorphic characteristics [58], the GM soil type accumulates a large amount of SOM, thus confirming the higher removal found. When comparing the rate at which SOM was removed from the GM, LVA, and NX samples, the removal rate was lower in the latter two. This can be explained by the higher abundance of microaggregates in the LVA and NX soil types compared to the GX soil [58]. These microaggregates in soil could hinder the SOM from the samples. On the other hand, the efficiency of SOM removal is also influenced by the organic matter content present in the soil samples. The soil samples with the lower SOM content (e.g., CX soil) exhibited correspondingly diminished rates of SOM removal. Radford et al. [45] observed that samples with low organic content displayed variations in the quantity of SOM removed depending on the employed digestion methods. In contrast, samples characterized by high SOM content did not exhibit notable differences in the amount of SOM removed under various treatments, including alkaline digestion. Alkaline digestion preserves the integrity of MP analysis in soil, unlike acid digestion [59], as will be elucidated later.

3.2. Characterization of the Microplastics Before and After the Extraction Method

As previously mentioned, MP were created in the laboratory and sorted by size before being grouped for soil incorporation. Optical microscopy was employed to assess the size distribution of MP within each group [60,61], while FTIR spectroscopy was utilized for the characterization of MP before soil insertion [62]. The findings revealed that MP ranging from 105 to 1000 μm constituted 91.9% of Group 1, 81.5% of Group 2, and 89.4% of Group 3. MP sized between 1000 and 2000 μm accounted for 6.8% in Group 1, 9.6% in Group 2, and 2.9% in Group 3. Samples from Groups 2 and 3 contained particles that were smaller than 105 µm. This was expected, because PVC powder typically includes particles below 100 μm [8], which could pass through the sieve used in this study. Figure 3 shows an optical microscopic image of two samples of MP in different shapes and sizes and the spectra recorded using FTIR.

In Figure 3a, the characteristic FTIR bands of PVC are presented. The bands between 2958 and 2890 cm⁻¹ correspond to different stretching vibration modes of the CH and CH₂ bonds. At 1426 cm⁻¹, –CH₂–deformation vibrations (wagging of the methylene groups) were observed [63]. The 1254 and 1330 cm⁻¹ bands corresponded to C–H rocking deformation and C–H deformation of CHCl, respectively. The 1095 cm⁻¹ band related to C–C stretching vibrations, while the 959 cm⁻¹ band was assigned to trans-CH wagging vibrations. The stretching vibration of the –C–Cl bond was associated with the 834 cm⁻¹ band. Finally, the 610 and 695 cm⁻¹ bands were attributed to the stretching of the C–Cl bond. The FTIR spectrum for LDPE MP is presented in Figure 3b, where the characteristic LDPE bands can be readily identified. Specifically, the spectrum reveals the 2915 and 2848 cm⁻¹ bands, corresponding to the asymmetric and symmetric C–H stretch of the methylene groups (CH₂), respectively. Additionally, the 1454 and 720 cm⁻¹ bands could be attributed to the scissoring and rocking vibrations of the methylene groups [62,64].

After the extraction, a total of 191 MP particles were analyzed using optical microscopy, and their sizes were determined by calculating the maximum Feret diameter. Figure 4 shows the frequency distribution graph of MP recovered by size in the different soils studied.

These results showed that the MP particles recovered from all soil types mostly ranged from 105 to 1000 µm, with a small number of particles were detected between 1000 and 2000 µm (as shown in Figure 4), similarly to the sizes of MP observed before extraction. However, the proportion of MP removed by size distribution changed depending on soil type. The method utilized in this study demonstrates limited efficiency in extracting MP larger than 800 µm across all soil types, particularly in CX soils. These results suggest that MP of this size range were easily trapped in the soil, probably because of various factors involved in the separation process, including centrifugation time and speed. Similar results were observed in [4], which reported that the recovery of certain plastic particles may be hindered during centrifugation. During soil decantation, some plastic particles would be prevented from floating, thus reducing their recovery. Another hypothesis is related to the granular form of the PVC powder particles, which could not be completely separated from the fine soil particles during the isolation process, especially in clayey soils such as those studied in CX, NX, and LVA (Table 1). A similar situation was reported in [65] (2018).

According to the FTIR results (see Figure 5), after the separation density, the FTIR spectra did not show significant deviations from the initial spectra, making the identification of the polymer possible. This means that the characteristic band polymers kept their intensity, allowing their identification after their extraction step.

3.3. Recovery Efficiency of Microplastics Through Extraction Method

To ensure an accurate quantification of MP concentrations in soil samples, it is essential to evaluate the extraction efficiency. Figure 6 shows the recovery rates for MP extracted in two steps with distilled water and sodium chloride and with the zinc chloride solution. It also shows the total MP recovery rates for all soil samples. The results showed a significant difference in the recovery rates of LDPE compared with PVC MP (Figure 6) when using the density separation method with DW and S1 solution across all soil types.

Specifically, the DW + S1 solution recovered over 50% of the mass of LDPE MP, whereas only about 25% of PVC was recovered. The average recovery rates of MP in LDPE–PVC/soil samples using DW and S1 solutions varied between different soil types. Specifically, the rates were 28.2% for LVA soil, 55.0% for NX soil, 43.9% for GM soil, and 47.9% for CX soil. In all cases, the recovery rates of MP were lower than those obtained from LDPE/soil and PVC/soil samples. Therefore, the separation method using a saturated NaCl solution did not result in high recovery rates of MP.

The NaCl-saturated solution is commonly used to extract low-density MP, such as LDPE, from different environments because of its low cost and environmental benefits [17]. However, our findings indicate that it is not effective when used in soils with high clay content, which was the case with our samples (Table 1). Ref. [50] (2016) reported a similar situation, in which water and NaCl were tested for extracting MP from PVC and PET polymers in sediment-type soil samples, obtaining a recovery rate of less than 60%, particularly for sizes between 800 and 1000 µm. Ref. [15] (2022) obtained low recovery rates of LDPE, reporting a rate of around 66% for particles smaller than 1 mm in agricultural soil, especially clay loam soil, while using a saturated NaCl solution [66]. According to the literature [45], the extraction of MP with sizes ranging from 0.25 to 1 mm from clayey soils depends mainly on the time process of density separation. This is because the clay content makes it difficult to separate the MP that adhere to the clay. In the case of our samples, the time used to mix the MP in the soil was probably not enough to separate the MP, allowing their flotation.

The results indicated that after using the S2 solution for extraction, the recovery rate of MP in the soil and LDPE samples ranged from 81.0% to 99.0%. In contrast, the recovery rate in the soil and PVC samples ranged from 59.7% to 75.2%. Notably, a high recovery rate was achieved for low-density MP, while the extraction of PVC from all soil types yielded lower recovery rates.

The substantial challenge encountered in extracting PVC MP from the soil, particularly those smaller than 300 μm, is worth noting, as emphasized in previous studies [50]. Our observations underscored this difficulty, which was particularly evident in PVC/CX soil. This soil type’s propensity for compaction increases soil density, posing hurdles in extracting smaller and denser MP, which tend to adhere to soil particles.

Finally, the study findings revealed that the average recovery rates of MP in soil/LDPE and soil/PVC–LDPE samples were 89.6% ± 11.9% and 86.4% ± 8.4%, respectively. However, the average recovery rate for PVC polymers was 66.0% ± 22.9%.

Several studies have highlighted the challenge of extracting PVC MP from soil, which is primarily due to their high density (ρ = 1.30–1.58 g/cm³) [28], which reduces the efficiency of density-based separation methods [67], resulting in low recovery rates. In our study, a zinc chloride (ZnCl₂) solution with a density of 1.5 g/cm³ was used slightly below the maximum density of PVC. This density difference prevented small PVC fragments and granules from floating [68], making their extraction via the flotation method applied in this study unfeasible. Additionally, the heterogeneous composition of the soil further complicated the separation and specific characterization of PVC MP [69].

3.4. Testing the Recovery of Microplastics from Raw Agricultural Soils

The method of digestion of organic matter and density separation described above was used to analyze MP in raw agricultural soil samples (LVA, NX, GM, and CX). This procedure enabled the determination of MP abundance per unit mass of soil. The values found in the soil samples were 2517.5 ± 481 items kg⁻¹, 2245 ± 501 items kg⁻¹, 3867.5 ± 1120 items kg⁻¹, and 1725 ± 95 items kg⁻¹ for the LVA, NX, GM, and CX samples, respectively (Figure 7c). The LVA, NX, and GM samples exhibited higher abundances of MP in the soil. These soils stemmed from an area characterized by intensive agricultural practices, including semiannual crops such as rice, soybeans, and beans (Table 1). In contrast, the CX samples, which demonstrated a lower presence of MP in the soil, pertained to an area predominantly occupied by perennial olive trees, thus experiencing minimal agricultural activity. Consequently, the reduced occurrence of MP in the CX samples can be attributed to their land use, as this soil remained unaffected by major sources of soil MP, such as biosolids, organic fertilizers, mulch, and atmospheric deposition [34,70]. For instance, ref. [71] (2023) conducted a study on soil MP in Coimbra, Portugal, comparing sites with and without agricultural use. An average of 104 × 10³ items kg⁻¹ MP was reported in agricultural soils, while forested sites exhibited a lower average of 55 × 10³ items kg⁻¹, higher than those reported in this study. In contrast, [13] (2024) reported that the abundance of MP ranged from 21 to 210 particles per 100 g of dry soil sample collected from critical MP pollution areas in Surat in Gujarat, India; these MP concentrations were close to those found in this study.

Moreover, agricultural soils are susceptible to erosion, facilitating the transport of MP through surface runoff [72]. Consequently, soils at lower elevations tend to accumulate a higher abundance of MP. In the case of our samples, the GM, NX, and LVA soils were from regions with slopes ranging from 3 to 8%, whereas the CX soil featured a steeper slope, ranging from 20 to 45%. This difference in topography suggests that the lower-altitude location of GM soils enables them to receive MP through surface runoff, leading to a greater accumulation of MP in these areas [73].

The abundance of MP found in soil samples differed from those reported in other surveys carried out on agricultural soils. For instance, on Hainan Island in China, the concentration of MP in the study area ranged from 2.8 × 10³ to 82.5 × 10³ particles kg⁻¹, with an average concentration of 15.461 × 10³ particles kg⁻¹ and sizes between 20 and 200 µm [74]. Thirty-two soil samples were collected from four agricultural farms in Bangladesh, and their mean MP concentration was found to be 21.3 x 10³ particles kg⁻¹; the size range of the MP was between 1.0 and 1.5 mm [75]. In a typical agricultural township near the southeast coast of China, the concentration of MP ranged from 70.2 to 851.3 particles kg⁻¹, with an average of 314 particles kg⁻¹ in coastal plain soils [76].

Although the abundance of MP in Brazilian agricultural soils has not been reported yet, the abundance of MP in mangroves has [77]. According to [48] (2022), there was a remarkable presence of MP in six Brazilian mangrove soils, registering a value of 10.782 x10³ particles kg⁻¹ (max. = 31.087 × 10³ particles kg⁻¹), the main MP being fibers with a dominant size between 150 and 200 µm.

The average size of MP particles found in agricultural soils exhibits variability based on their shape (Figure 7). The majority of particles have a size of less than 300 µm, while only fibers have an average size of 750 µm. Ref. [78] (2021) reported that the highest proportion of MP, around 54.29% of the total items in all soils, had a size of less than 200 m. A similar situation was reported in [79] (2021), where MP with a size of less than 500 µm in agricultural soils, specifically in a layer between 10 to 25 cm in depth, indicated that smaller MP tend to migrate to deeper layers of the soil. Ref. [80] (2021) noted that the predominant forms of MP were fragments (46.3%) and films (25.4%), similar proportions to those identified in this research.

The MP characterized here were influenced by soil class and landscape position, offering opportunities for investigating diffuse pollution and the development of environmental tracer studies [47,80,81,82,83].

The MP were further characterized under the microscope by measuring the size and shape of the particles. Figure 7a shows the percentage distribution of the different forms (fragment, film, fiber, and granule) in the four soil samples collected (LVA, NX, GM, and CX). Fragments were the form with the highest percentage of occurrence in all the soils, followed by granules > films > fibers. In general, the predominant form was a fragment, and, in particular, in the CX and NX soil samples, there were no particles in the form of fibers. According to Figure 7b, the MP in the shape of granules had an average size of 167.8 ± 55.7 µm; those in the shape of fibers, 759.4 ± 469 µm; those in the shape of films, 214.2 ± 140 µm; and those in the shape of fragments, 268.8 ± 215.8 µm. The fiber-shaped particles showed the greatest variation in size compared with the other shapes.

The results obtained regarding the abundance, size, and shapes of MP found in the four tropical soil types studied showed similarities with findings reported by other authors. Specifically, the MP abundance observed in Figure 7c falls within the global average concentration range of 2900 ± 7600 items kg⁻¹, derived from 89 studies [37].

Moreover, most of the detected MP were smaller than 1000 µm [68], with a higher frequency of fragments, films, and granules [67]. In contrast, fibers, though less abundant, exhibited an average size ranging from 970 µm to 2000 µm [47].

For future research on MP in tropical soils, it is crucial to evaluate their potential effects on soil health and the environment. Following the advances made by other researchers, it is essential to understand how MP can alter soil structure and water dynamics [84], affect microbial communities [85], the increased opportunity for MP to enter into the aggregate fractions [86] and migrate from soil to aquatic ecosystems, compromising both ecosystem health and water quality [72]. Additionally, it is important to investigate how MP act as reservoirs for pathogens and contaminants [87]. Moreover, exploring how MP may affect plant health and modify soil dynamics [88] is necessary, as this can directly impact soil fertility [53] and ultimately reduce agricultural production [26]. These research directions are essential to understanding the extent of MP impacts and developing strategies to mitigate their long-term effects on tropical ecosystems.

4. Conclusions

In this study, it a methodology was proposed for the extraction, characterization, and identification of two MP (PVC and LDPE) and their testing in four Brazilian tropical agricultural soils. The procedure encompassed four key steps: (i) removal of organic matter, (ii) density-based separation, (iii) identification, and (iv) characterization of MP. The recovery efficiency exceeded 88% for LDPE and 66% for PVC. Remarkably, the ZnCl2 extraction solution proved to be highly effective in recovering denser MP.

This methodology ensured optimal extraction conditions and characterization of MP in the four types of tropical soils studied. The extraction method revealed significant differences in MP extraction among agricultural soil samples, with the GM sample showing a notably higher abundance of MP than NX and CX.

The results obtained provide a valuable methodological and reference foundation that will support future researchers in the extraction, characterization, and analysis of MP in tropical soils. Additionally, these findings serve as a wake-up call for farmers regarding the potential impact of agricultural activities on MP accumulation in soils, emphasizing the importance of adopting more sustainable practices to reduce plastic waste generation and prevent soil quality degradation.