Assessment of the Presence of Microplastics in Stabilized Sewage Sludge: Analysis Methods and Environmental Impact

Abstract

Wastewater treatment processes can fragment microplastics (MPs), which may subsequently enter fertilizers applied in agricultural settings. This study aimed to quantify the occurrence of MPs in stabilized sewage sludge intended for fertilizer production. Matrix elimination was performed using an oxidative method to isolate MPs, followed by MPs separation with a saturated salt solution of appropriate density to enhance the accuracy of identification. The resulting samples were analyzed using spectroscopic and microscopic techniques to provide the detailed characterization of MPs content. The highest concentrations of MPs were recorded during the months of June, July, and May, with average values of 2942, 2341, and 1746 fragments per 100 g of dry weight, respectively. The analysis revealed that fragments were the dominant morphological form, and low-density polyethylene was the most common polymer type detected. These findings underscore a significant risk of MPs re-emission into the environment through the agricultural application of fertilizers derived from sewage sludge. Such practices may lead to the introduction of between 6110 and 13,889 MPs per square meter of soil, depending on the application rates, thereby posing potential risks to soil health and the broader ecosystem. This study highlights the importance of monitoring MPs content in fertilizers derived from wastewater treatment by-products.

1. Introduction

Micropollutants are natural and anthropogenic substances, including but not limited to microplastics (MPs), pharmaceuticals, industrial chemicals, and pesticides. They are mainly associated with human anthropogenic activity. Plastic production has been growing steadily since the end of the 1940s [1,2]. Since the beginning of the 1970s, plastic particles have been known to escape and accumulate in the environment [3]. Despite this, a dynamic increase in their production is observed due to their wide range of applications. In 2018, the world generated 360 million tons of plastic [4,5]. The main concern is that there has been a steady increase in plastic production between 1950 and 2000 [6]. MPs are defined as plastic pieces with sizes between 1 µm and 5 mm [4]. They are diverse in appearance (shape, color, structure) and composition. They originate from a wide variety of polymers and contain various chemical additives. There are two sources of MPs (see Figure 1). Primary MPs are produced in the form of tiny particles designed for commercial use, while secondary MPs come from larger fragments that have been partially degraded in the environment [7].

MPs are a serious problem for wastewater treatment plants. The removal of its fractions via wastewater treatment processes is complex. Moreover, the methods applied for oxidation, especially those for organic pollutants, may affect the comminution of MPs. The treated wastewater in the environment contains not only micropollutants [8,9] but can also contain a significant number of MPs that will accumulate in lakes, rivers, seas, and oceans [10,11,12,13].

Sewage sludge is another source of secondary re-emission of MPs into the environment. It is also used to make fertilizers [14]. Microplastic fractions accumulate in sewage sludge in large numbers [15,16]. The processes to which sludge is subjected during fertilizer production do not eliminate MPs from it [17,18]. There are also no legal standards regulating the content of MPs in such products.

Water is considered the largest carrier of microplastics [19]. There are few studies on the content of microplastics in sewage sludge samples, which is where they are retained due to their hydrophobic nature. Scientists warn that 90% of microplastics are retained in sewage sludge [20]. Studies also show that sewage sludge is the largest source of the emission of microplastics from fertilization, and it is more significant than the release of microplastics from other sources into the oceans or other water bodies [21,22,23,24]. In the case of Europe and North America, approximately 50% of the sludge is used to produce fertilizer [23]. Similarly, scientists determined levels from 87.6 to 545.9 MPs/kg of soil after annual fertilization with sludge-based products [20].

The purpose of this study was to analyze the forms and sizes of MPs included in stabilized sewage sludge collected in one of the wastewater treatment plants that uses it to produce organic-mineral fertilizers [9]. It should be noted that stabilized sewage sludge may also contain pharmaceuticals such as ibuprofen or diclofenac, as well as heavy metals. There is a risk of co-emission of pollutants because MPs can absorb the mentioned micropollutants [14,25].

2. Materials and Methods

2.1. Materials

Zinc chloride (ZnCl₂, 99% purity) was purchased from ChemLab, Piekoszów, Poland. Hydrogen peroxide (H₂O₂, 30 wt % purity) was purchased from POCH, Gliwice, Poland. The HLP5 pure water system (Hydrolab, Straszyn, Poland) supplied the deionized water.

2.2. Sampling

In the experiment, stabilized wastewater sludge samples were tested. The samples were collected at the beginning of May, June, and July 2021 in a wastewater treatment plant (WWTP) located in the south of Poland. It treats around 7000 m³ of wastewater per day obtained from 62,000 of its inhabitants. The sediment was collected between 7:00 a.m. and 7:00 p.m. during press opening hours. Four samples of approximately 1.5 kg were taken at intervals of approximately 3 h, and all samples were mixed. The average amount of stabilized sewage sludge obtained in the WWTP is 458 Mg per month. Its mean calorific value is 13 ± 2 MJ/kg DM. The mean values of the concentrations of selected components in stabilized sewage sludge are presented in

Table 1. Characterization of stabilized sewage sludge.

Component	Content/Determined Value
Total moisture, wt % (% m/m)	78.2 ± 2.3
Dry matter, wt % (% m/m)	21.8 ± 2.3
Organic substance, wt % DM	64.7 ± 12.8
Nitrogen, wt % DM	5.3 ± 1.6
Phosphorus, wt % DM	2.7 ± 0.2
Magnesium, wt % DM	0.6 ± 0.1
Calcium, wt % DM	2.6 ± 0.5
Copper, wt % (mg/kg)	321.2 ± 68.0
Cadmium, wt % (mg/kg)	0.7 ± 0.2
Lead, wt % (mg/kg)	20.6 ± 7.9
Zinc, wt % (mg/kg)	674.1 ± 66.2
Chrome, wt % (mg/kg)	24.9 ± 3.8
Mercury, wt % (mg/kg)	0.3 ± 0.1

. These values were obtained based on the results obtained for six samples acquired during one year (values obtained from the WWTP). Furthermore, no live eggs from the intestinal parasites Ascaris sp., Trichuris sp., Toxocara sp., nor from bacteria of the genus Salmonella, were found in the tested stabilized sewage sludge. The samples were tested in an accredited testing laboratory, No. AB 213, according to standard PN-EN ISO 5667-13; 2011 [26].

2.3. Sample Processing

To remove moisture from the sewage sludge, the samples were subjected to the lyophilization process. The samples were lyophilized for 5 days at −50 °C. After this stage, the specimens were subjected to the oxidative etching and density separation process.

2.4. Separation and Digestion Methods

Each sample weight was 5 g of dry-weight stabilized sewage sludge. The samples were each converted to 100 g for comparative analysis. First, a density separation was performed using a saturated solution of zinc chloride (ZnCl₂), with 100 mL of saturated solution per 5 g of residue. The samples were placed in a stirrer and mixed for 30 min at 140 rpm. After mixing, the samples were left for 24 h to achieve density separation. The top layer was poured onto a filter and washed with deionized water. The filters were allowed to dry under natural conditions. The solid phase separation process was repeated twice to recover the most significant possible fraction of MPs. The next step was to subject the sample to a digestion process. For this purpose, a 15% hydrogen peroxide solution was prepared. The samples dried on the filter were washed with the prepared solution and transferred to a beaker. As in the case of density separation, 100 mL of hydrogen peroxide solution per 5 g of stabilized sewage sludge was used. The digestion process took 48 h. After this operation, the residue was filtered and flooded with 15% hydrogen peroxide solution for 48 h. The samples were quantitatively transferred to a filter and washed with deionized water, allowing for the removal of the unnecessary sample matrix. Then, the MPs analysis process was performed.

Only glass and metal laboratory equipment was used during the analysis. The samples were tightly protected against the migration of MPs from the environment. Only outerwear made of non-synthetic materials was worn by the researchers. A blank test was performed without an environmental sample, which showed no evidence of the presence of microplastics.

2.5. Sample Processing

2.5.1. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

The morphology of microplastic (MPs) particles isolated from stabilized sewage sludge was initially examined using a PerkinElmer Frontier Spotlight 400 FTIR microscope. For this analysis, all residues on the filter were transferred onto a microscope slide. During examination, the MPs were counted and categorized based on their color and shape, distinguishing between fibers and fragments [13]. Fourier transform infrared (FTIR) spectroscopy was employed to record the IR transmittance spectra of the MPs and to identify the characteristic functional groups. A PerkinElmer Frontier attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectrometer, equipped with a mid-infrared (MIR) range, a DTGS detector, and an additional wide-range MCT detector, was used. The FTIR spectra were recorded within the wavenumber range of 530–4000 cm⁻¹, averaging 64 scans per spectrum at room temperature [27].

2.5.2. Confocal Microscope

To complement the IR spectroscopy results, a Raman confocal microscope (Ulm, Germany) was employed. Imaging was conducted using a WiTec Alpha 300 R microscope with a 10× air lens (Zeiss EC Epiplan-Neofluar Dic 10/0.25, Ulm, Germany) objective, coupled with a 532 nm excitation laser and a UHRS 300 spectrometer (600 g/mm) equipped with a highly efficient, thermoelectrically cooled CCD camera. During imaging, specimens were placed on CaF₂ glass slides [13].

2.5.3. FIB/SEM (Focused Ion Beam/Scanning Electron Microscope)

Microstructural characterization was carried out using FEI Versa 3D SEM (Waltham, MA, USA). Through the use of the low vacuum (LV) mode in SEM, surface imaging was performed on the sample surface, which was not coated with a conductive layer. SEM measurements were performed at a water vapor pressure of 70 Pa and an accelerating voltage of 20 kV using a secondary electron (SE) detector.

2.5.4. Statistical Analysis

For the analysis, a measure of the relative dispersion of the data set was used to determine the variability.

The coefficient of variation (CV) and relative standard deviation (RSD) were used to analyze the measure of relative dispersion of the data set to determine variability.

$$\mathrm{RSD}={\displaystyle \frac{s}{\mu }},$$

(1)

s—standard deviation.

µ—average.

CV—coefficient of variation.

CV = RSD × 100%

(2)

RSD—relative standard deviation.

3. Results and Discussions

Using the FTIR microscope (Figure 2) and the confocal microscope (Figure 3), photos of the separated plastics were captured for later statistical analysis. Based on of the photos acquired, a quantitative analysis of the separated MPs was carried out. The MPs were counted and divided according to color, shape, size, and quality.

The largest number of MPs was found in the June samples, with about 2942 fragments per 100 g of dry-weight stabilized sewage sludge. The smallest number of MPs was found in the samples from May, which contained an average of 1746 particles per 100 g from dry matter of stabilized sewage sludge. This may be related to the increased use of disposable utensils, straws, cutlery, and packaging during these months (Figure 4). Increased rates have also been found in dry sludge during the warmest months (May–September). Based on the arithmetic mean, standard deviation, and relative standard deviation, the coefficient of variation was estimated for samples from May, June, and July.

Based on the results obtained regarding the amount of microplastics in the samples, arithmetic means and standard deviations were calculated.

For the May samples, the mean was approximately 1746 fragments, with a standard deviation of 364. The coefficient of variation (CV) was 20.87%, indicating very little variability in the data in this group.

For the June samples, the average was 2942 fractions, and the standard deviation was 407. The coefficient of variation (CV) in this case was 13.83%, which also indicates a very low variability of the results.

Average variability was observed in the July samples. The arithmetic mean was approximately 2341 fractions, and the standard deviation was 871. The coefficient of variation (CV) was 37.22%.

All calculations indicate that for the May and June samples, the variability of the data was very low, while for the July samples, it was moderate.

Spatial and temporal variations in microplastics (MPs) abundance in sludge are associated with urban indicators, including population density, level of urbanization, economic income, and climate factors [28].

Black was the dominant color in the samples tested in May, June, and July (Figure 5). In May, black fragments constituted an average of 968 fractions, with fibers comprising 234 per 100 g from dry matter of stabilized sewage sludge. In June, the number of black fragments was 1306, and the number of fibers was 293. In July, black fragments constituted 816 and fibers 282. The number of colored fragments in May was on average 296, and the number of fibers was 247. The number of colored pieces in June was 894 and 449 fibers. Meanwhile, in July, the average number of colored pieces was 670, with 553 fibers. It can be seen that the colored fragments and fibers had a similar value, except for in June, when the colored fragments were almost twice as dominant. However, a trend is visible in the division order, i.e., black fragments > colored fragments > colored fibers > black fibers, in relation to each month.

Color provides a qualitative indication of the age of the MPs present in the samples, as well as the origin of the plastic [29]. Regarding color, some authors note the fact that the majority of MPs are colorful, which shows that they are broken from colored plastic products [28]. On the contrary, others indicate that the sludge was dominated by both clear (white or transparent) and colored fibers [30]. Hatinoglu e Dilek Sanin (2021) [20] found that most MPs in sludge are white (80%), clear and black (70%), and red and blue (40%). Similar results are obtained by Franco et al. (2023) [31], who observed white, black, brown, blue, red, green, and other colored fibers. Other colors include violet, yellow, gray, and orange.

In terms of morphology, MPs are primarily categorized into fibers, shafts, films, flakes, and spheres. The results of the conducted research also indicate that the largest percentage of MPs are fragments in the samples from May, June, and July (Figure 5). Similarly, in this work (Figure 5), fibers and fragments represented the highest proportion of MPs [20,30,31], unlike the work of Li et al., which found that MPs are mainly composed of polyolefin (fibers and rollers), acrylic fibers (fibers), polyethylene (films), polyamide (films), alkyd resin (flakes), and polystyrene (balls) [22]. However, completely different results were obtained by Lusher et al. (2017). They found that the types of MPs in sediments included beads (37.6%), flakes (31.8%), and fibers (28.9%) [32].

The size of MPs were fractions greater than 300 µm, which were identified in July (an average of 2351 fractions per 100 g from dry matter of stabilized sewage sludge) and in June (an average of 2157 fractions) (Figure 6). Samples from May exhibited a size of up to 1500 µm (average 1376 fractions). Fractions up to 300 µm constituted a significant minority. In this case, these were the most common in June (an average of 418 fractions) and May (an average of 184 fractions) (Figure 6). A certain trend can be noticed. Larger microplastic fractions (>300 µm) dominated in samples from July (2351 fractions per 100 g of dry weight of sediment) and June (2157 fractions), while samples from May contained fewer of these larger fractions (1376). In turn, smaller fractions (<300 µm) were the minority in each sample, but their number was the highest in June (418 fractions) and May (184 fractions).

It can be seen that larger fractions dominated, especially in the summer period (June–July), and the number of smaller fractions varied more over time.

In wastewater, in the sewage sludge from which MPs originate, dimensions of 25 µm, 100 µm, and 500 µm were used most often for size classification [33,34,35]. Analysis of the literature shows that, on average, more than 90% of MPs in wastewater are less than 500 µm in size, and in some samples, approximately 60% of MPs were smaller than 100 µm [36,37].

MPs separated from stabilized sewage sludge displayed the highest LDPE content. Figure 7 shows the most frequently obtained spectrum during ATR-FTIR analysis (Figure 7). The intensity at 3340 cm⁻¹ is responsible for CH stretching vibration =C–H [38]. The bands around 2918 cm⁻¹ are responsible for C–H stretching vibrations, typical for pure polyethylene. Peaks between 2840–2950 cm⁻¹ indicate both symmetric and asymmetric CH stretching vibrations [39]. The intensity at 1744 cm⁻¹ indicates the presence of carbonyl group stretching C=O [40]. The same is true for the peak at 1622 cm⁻¹, which indicates the C=O group coupled to the C=C group derived from the vinyl group [41,42]. The band at 1562 cm⁻¹ comes from the vibrations of the C=C group. Vibrations at 1460 cm⁻¹ are C–H deformation vibrations and demonstrate the crystallinity of polyethylene. In the case of high-density polyethylene (HDPE), there would be a multiplicity of peaks. Peaks at 1385 and 1353 cm⁻¹ are indicative of polymer branching, which is typically found in LDPE [43]. The B-Bands at 1244 1243 are visible, which indicates the content of oxygen compounds. They can also be derived from phthalates and other fillers and plasticizers. The peak at 1028 cm⁻¹ can be responsible for oxygen bonds (C–O, C–OH) [38].

During the analysis of the literature sources, it was discovered that more than 30 types of microplastic polymers are detected in wastewater and wastewater treatment plants. The most common polymers were polyester, polyethylene, polyamide (PA), polyolefin, acrylic, and others. These types of plastic are representative of the types of plastic products used in people’s daily lives [44], e.g., personal care products, shampoo bottles, pipes, etc. Polypropylene (PP) is also very common and is used in food packaging, snack wrappers, auto parts, etc. [20]. Polyethylene was dominant in the samples analyzed. It is used in personal care products, including body and facial scrubs, food packaging films, and water bottles [34,36,45,46].

The microstructure of the separated microplastics was examined using SEM. The structures differed from each other within the same polymer, i.e., a smooth structure with various defect sizes (Figure 8A) and a honeycomb-shaped structure typical of foam materials (Figure 8B) [47]. The SEM micrographs show numerous microcracks and cracks, which indicate mechanical damage to the surface (Figure 8). Additionally, there are noticeable signs of surface peeling. Due to resistance to biodegradation and the action of physical and chemical factors, MPs accumulate in sewage sludge and, along with it, are introduced into the soil (for example, when used in agriculture) [48]. In the soil environment, other organic micropollutants, such as PAHs, are released into the environmental morphology of the MPs [48].

A review of the literature indicates that wastewater treatment processes may lead to the fragmentation of microplastics (MPs), which then end up in fertilizers used in agriculture, which is confirmed by the results of this study. MPs concentrations in stabilized sewage sludge intended for fertilizer production were significant, especially in the summer months. Microplastics act as both carriers and sources of organic and inorganic pollutants. They can adsorb toxic substances such as polychlorinated biphenyls (PCBs), dioxins, DDT or polycyclic aromatic hydrocarbons (PAHs) and transfer them between various ecosystem components [49]. Additionally, chemical compounds used as additives to plastics, e.g., polybrominated diphenyl ethers, may penetrate the tissues of organisms, disturbing their digestive activity [50].

The results of this work confirm that microplastics present in fertilizers may increase environmental risk, not only by introducing them into the soil, but also by the possibility of them transporting and accumulating harmful chemical compounds. In the context of a growing population and the reduction of agricultural regions in favor of urbanized and industrial areas, the results emphasize the need to monitor the MPs content in fertilizer products obtained from wastewater treatment.

As reported by Daisy Harley-Nyang et al., the application of biosolids to agricultural land as fertilizers can potentially release 1.61 × 10¹⁰ and 1.02 × 10¹⁰ MPs in anaerobically digested and lime-stabilized sludge, respectively, every month. This illustrates the extent to which MPs can enter the terrestrial environment by this route [16].

Research shows that 0.5 kg of fertilizer is applied per m² per year for agricultural soils [14]. With such a large number of MPs found in sewage sludge samples, we can determine the number of MPs that will be emitted per year per 1 m² of soil. Taking into account the smallest number of MPs found in the May samples, about 1746 fragments per 100 g of sewage sludge, and the information that 70–95% of sewage sludge is used for fertilizer production, the annual emission per m² from fertilizer can reach 6110 to 8295 fractions [51,52]. Taking into account the largest number of MPs in the June samples, 2924 fractions per 100 g of dry matter, the emission could range from 10,234 to 13,889 fractions. It should also be emphasized that, unlike micropollutants such as pharmaceuticals, they are present much longer, as their decomposition process is very slow, and they break down into smaller fragments, which increases their bioavailability [53]. In addition to the many toxic substances contained in MPs absorbed by plants and animals, there is another cause for concern, i.e., the danger of contamination by microorganisms that develop on the biological membrane (the so-called biofilm) formed on the microplastic surface [54]. So far, only a limited number of studies have been carried out on this topic in aquatic ecosystems, but it is an issue of concern for environmental microbiologists. The diverse group of microorganisms (making up the so-called plastisphere) discovered on the surface of the microplastics in the North Atlantic was initially described by Zettler [55].

4. Conclusions

The research focused on the analysis of stabilized sewage sludge for the presence of MPs. The statistical analysis was related to months. The tested samples contained huge amounts of plastic microparticles, and their highest content was recorded in June, July, and May, respectively. The MPs content in the stabilized sewage sludge from May amounted to an average of 1745 fragments and was the smallest in relation to the rest of the tested samples. The microplastic content in June averaged 2942 fragments, and samples from this period displayed the highest number of MPs. On the contrary, the July samples showed an average of 2340 microplastic fractions per 100 g of dry matter. The researchers noticed the effect of the number of MPs as a function of weather conditions. A greater number of plastics was recorded at higher temperatures. In each of the samples, fragments dominated, especially black ones, by about 44%. Colored pieces were the second most abundant. The most common polymer in the stabilized sewage sludge was low-density polyethylene (LDPE). The most likely sources of these substances are plastic bags, bottles, nets, and drinking straws. The highest number of fractions above 300 µm were found in samples from June and July and the lowest in May; the highest number of fractions below 300 µm were found in samples from May, followed by June and July. This may be due to the presence of plastics under conditions that have a greater impact on their degradation, such as temperature changes.

SEM images showed that the type of the separated microplastics varies, from smooth with defects and crevices to highly porous. In each case, there is a potential for the sorption of various types of pollutants, including heavy metals, pharmaceuticals, and pesticides. Studies have confirmed the risk of re-emission of MPs into the environment by stabilized sewage sludge and fertilizers produced from it. Converting the masses of the samples to 100 g of dry mass allows the number of plastic microfractions to be compared with each other. Attention should be paid, for example, to the most harmful fractions, which are characterized by the smallest size and negligible weight. The quantitative content of MPs should be a necessary precedent used in estimating the amount of this type of contamination and is used by many scientists dealing with this problem. From the research, it can be concluded that the use of sewage sludge to obtain organic mineral fertilizers enables their re-emission into the environment. Future research, which is in progress, focuses on the analysis of organic mineral fertilizers based on stabilized sewage sludge.

In this study, the authors analyzed the occurrence of microplastics (MPs) in stabilized sewage sludge from a specific wastewater treatment plant (WWTP), providing specific data that contribute to the understanding of MPs in the context of sewage sludge used for fertilizer production. Although issues related to MPs in wastewater treatment processes are documented, a novel aspect of this study is the detailed quantification of MPs in the specific wastewater treatment plant under consideration, as well as the identification of the dominant polymer types and morphological forms present. In addition, the study also focuses on the potential environmental risk posed by polymers, given their role as pollutant vectors, which has been less emphasized in previous work. Research on the presence of MPs in fertilizers and their potential impact on the environment has been combined. This paper makes a timely and important contribution to the growing body of research on microplastics in agricultural systems. The findings not only increase existing knowledge, but also provide new insights into the transport and re-emission of MPs into the environment, highlighting important issues such as the need to monitor and regulate MPs in agricultural products obtained from wastewater treatment.