The application of Schweizer's reagent with FTIR imaging spectroscopic solutions for microplastics advanced analysis of feces samples

The application of Schweizer's reagent with FTIR imaging spectroscopic solutions for microplastics advanced analysis of feces samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The application of Schweizer's reagent with FTIR imaging spectroscopic solutions for microplastics advanced analysis of feces samples Miloš Ilić, Tamara Mutić, Dragana Stanić-Vučinić, Mirjana Turkalj, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6427909/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The content, characteristics, and distribution of microplastics (MP) in feces samples are crucial for the investigation of human exposure and health risks. Feces, being rich in cellulose, is a particularly complex matrix for MP analysis. Numerous studies of microplastics in different matrices show how to remove organic matter from samples, but there are very few studies on the removal of cellulose. In this study, an efficient protocol for the digestion of children’s feces was developed and optimized as a combination of the innovative cellulose removal treatment of samples with previously known alkaline/oxidation treatments. To remove the cellulose, 40 mL of Schweizer's reagent was added for every 600 mg of dry sample for 40 mins. After that, the samples were passed through a 20 µm mesh sieve and washed with ultra-pure water. Samples were then subjected to alkaline digestion using 10% KOH for 24 hours at 40°C, followed by oxidative digestion using 30 mL of 15% hydrogen peroxide for 16 hours. MP content was determined in 14 feces samples from the Croatian region of Dalmatia, of which 7 samples contained MP. The number of MP ranged from 0 to 5, corresponding to concentrations between 1.18 and 7.25 particles per gram of sample. Among the detected polymers, polyethylene was the most prevalent (56% of particles) and the most dominant particle shape was fragment (68.75%). In comparison to alternative methods used for MP analysis in human feces, tour method efficiently remove cellulose and allow digestion of the matrix in a cost-effective and time efficient manner, allowing subsequent analysis by microFTIR. Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Due to its purpose, wide usability, low cost, and chemical inertness, plastic is widely spread in industry and people's everyday lives (Ho et al., 2022 ). Plastic pollution has drastically increased globally in the last two decades, with projections indicating an exponential rise of plastic waste to over 350 million tons annually (Ho et al., 2022 ; Schwabl et al., 2019 ). The most significant expansion and exponential increase in human exposure to microplastics was recorded during the COVID-19 pandemic due to increased consumption of single-use plastics, such as face masks, gloves, and disposable coats for virus prevention (Ricciardi et al., 2021 ). In most cases, microplastics (MPs) are formed through the degradation and fragmentation of macroplastics, influenced by natural environmental conditions and anthropogenic factors (Jenner et al., 2022 ; Ricciardi et al., 2021 ). Microplastics are not solely produced through fragmentation and degradation (secondary sources), but also leak into the environment by production in various industrial sectors (primary sources) where they have broad applications (Ragusa et al., 2021 ). According to existing data, microplastics have already entered the food chain. They are found in most marine animals, including the gastrointestinal tracts of fish, shellfish, and crabs, which humans often consume (Ibrahim et al., 2021 ; Jenner et al., 2022 ; Luqman et al., 2021 ; Mutić et al., 2024 ; Pérez-Guevara et al., 2021 ). Human exposure to microplastic particles most commonly occurs through the consumption of food and drinks contaminated with microplastics and inhaling these particles from the air (Abbasi & Turner, 2021 ). Approximately 74,000 microplastic particles enter the human body annually through ingestion and inhalation (Pérez-Guevara et al., 2021 ). Microplastics have been detected in saliva, hair, blood, skin surfaces, the human placenta, and lung tissue (Abbasi & Turner, 2021 ). One of the earliest signs of human exposure to microplastic particles was their discovery in feces samples, indicating contamination of the gastrointestinal tract (Schwabl et al., 2019 ). Therefore, examining feces is a crucial step in figuring out whether or not ingested microplastics have been completely or almost entirely ingested and, consequently, whether or not microplastic exposure has taken place. Only a few studies have far analyzed microplastic content in human feces (Abbasi & Turner, 2021 ; Ho et al., 2022 ; Ibrahim et al., 2021 ; Luqman et al., 2021 ; Schwabl et al., 2019 ; N. Zhang et al., 2021 ). The study of microplastics in human feces is still in its early stages, primarily because of the challenges associated with quantifying microplastics and validating reliable methods. Digesting and isolating microplastics from feces represents a highly complex and challenging procedure. Due to this complexity, several methods have been developed for extracting microplastics from feces, including various chemical and enzymatic treatments (Toto et al., 2023 ; Yan et al., 2020 ). However, the validation and harmonization of microplastic quantification methods have not yet been developed. A methodology for isolating microplastics from human feces samples, involving a series of steps oxidizing agents and acids, was developed. This methodology includes using Fenton's reagent and digestion with 65% nitric acid at 50°C and 70°C (Yan et al., 2020 ). Toto et al. ( 2023 ) reported a method for digesting rat feces samples. This procedure involved acidic digestion using nitric acid and hydrogen peroxide, alkaline digestion using 10% potassium hydroxide, and enzymatic digestion using Viscozyme-L and cellulase. However, a drawback of these procedures lies in the use of nitric acid, which may potentially damage microplastics, as well as in the duration of the process (Toto et al., 2023 ). It is well known that human feces contain cellulose in a certain amount, as the human diet is largely based on plants that contain plant fibers (Danjo et al., 2008 ;). Cellulose is a polysaccharide dominant in the plant world, forming the structural foundation of plant cells (Taylor, 2008 ). The human body cannot break down cellulose because it lacks the enzymes and commensal microbiota necessary for its degradation. As a result, cellulose is excreted in feces in significant amounts (Chesterman et al., 2020 ). Cellulose represents a significant challenge in microplastic analysis using spectroscopic methods (Olsen et al., 2020 ), and it has become crucial to remove cellulose to ensure accurate results efficiently. Traditionally, a urea, thiourea, and KOH mixture has been widely studied as a method for cellulose removal. The effectiveness of this mixture is closely linked to its concentration, with an optimal mixture concentration of 8% typically yielding an efficiency range of 80–94%, which is considered theoretically satisfactory (Budtova & Navard, 2016 ). S. Zhang et al. ( 2010 ) report that this mixture achieves approximately 91% efficiency in removing cellulose fibers. However, a much more efficient method for cellulose removal is the use of Schweizer reagent, which has demonstrated 100% efficiency in removing cellulose, as shown in studies where cellulose was removed from toilet paper (Gupta et al., 2018 ). To date, Schweizer reagent has not been applied for cellulose removal from feces samples in order to analyze and isolate microplastics from these samples. This study focuses on the development of a novel, fast, and efficient method for extracting microplastics from feces samples and MP counting with their complete chemical characterization by microFTIR spectroscopy (µFTIR). A comprehensive digestion protocol was established to achieve this, combining cellulose removal, alkaline digestion, and oxidative digestion. Initially, we tested four different digestion protocols. The best protocol was further optimized and tested using in-house and commercial microplastic polymer standards. The objectives of our study were: (i) to optimize a highly efficient digestion protocol suitable for complex feces samples, introducing Schweizer’s reagent for superior cellulose removal, which has not been previously applied in this type of research; (ii) to evaluate a digestion protocol for isolating MPs from feces samples suitable for the counting of MPs and their complete chemical characterization by microFTIR; and (iii) to validate the method efficiency, recovery rate, and the preservation of polymer integrity. At the end, the optimized protocol was applied to 14 feces samples from the Dalmatian coastal region to determine microplastic content. The analyzed microplastic-related parameters included polymer type, particle size and size distribution, particle shape, total number of particles per sample, and particle count per gram of sample. The digestion protocol presented here has proven to be suitable for children’s feces samples, which is essential as fecal analysis has become an important tool for monitoring the extent of microplastic exposure of live organisms and humans. 2. Materials and methods 2.1 Sample Collection and Preparation To investigate human exposure to microplastics, frozen feces samples from children were obtained from the Srebrnjak Children's Hospital in Croatia. The samples were collected from the population living in Dalmatia, a coastal region of Croatia stretching along the eastern shore of the Adriatic Sea. Each sample was carefully packaged in a 50 mL glass jar with a metal lid and labelled with a unique numeric code on an attached paper strip. Each sample weighed between 4 to 7 grams. These samples were then packed in a cardboard box and stored in a freezer at -20°C until further processing for digestion. 2.2 Materials and methods Chemicals: Viscozyme from Aspergillus sp, solution, ≥ 100 FBGU/g, (Sigma-Aldrich, St. Louis, USA); Cellulase from Trichoderma reesei, aqueous solution, ≥ 700 units/g, (Sigma-Aldrich, St. Louis, USA); Glacial acetic acid 100%, p.a. (Zorka pharma, Šabac, Serbia), Sodium acetate, anhydrous, p.a. (Centrohem, Belgrade, Serbia), KOH (Merck, Darmstadt, Germany); Nitric acid (65%), analytical reagent grade (Merck, Darmstadt, Germany) Hydrogen peroxide 30%, analytical reagent grade (99.9%), (Fisher Scientific, USA); abs EtOH (HPLC grade, Merck, Darmstadt, Germany), Copper(II) sulphate pentahydrate (Merck, Darmstadt, Germany), Ammonia solution, 30%, (Honeywell; North Carolina), NaOH (Merck, Darmstadt, Germany), Urea, reagent grade (99%), (Merck, Darmstadt, Germany), Thiourea, reagent grade (99%), (Merck, Darmstadt, Germany), NaHCO 3 (Merck, Darmstadt, Germany). Schweizer reagent : Schweizer's reagent is a metal ammine complex with the formula [Cu(NH 3 ) 4 (H 2 O) 2 ](OH) 2 . The reagent was prepared according to Reaction 1 by dissolving 6 g of copper(II)-sulphate pentahydrate (CuSO 4 ∙5H 2 O) in 75 mL of water under constant stirring until complete dissolution was achieved. A volume of 48 mL of 1 M sodium hydroxide (NaOH) solution was then carefully added dropwise while stirring, forming a light blue gelatinous precipitate. The precipitate was allowed to settle, separated from the solution by filter paper filtration, and washed thoroughly with 20–30 mL of water to remove residual ions. The washed precipitate was transferred to a clean beaker, and concentrated ammonia solution was added dropwise with continuous stirring until the precipitate was dissolved entirely, forming a clear, deep blue solution. The resulting solution was filtered through a PVDF membrane filter (pore size: 0.22 µm; diameter: 47 mm) to remove any remaining impurities. The prepared Schweizer's reagent was stored in an airtight container and protected from light to ensure stability. Standards : Seven in-house produced MP standards (50–500 µm) were used: polypropylene (PP), high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyvinylchloride (PVC), polystyrene (PS), polyethylene terephthalate (PET), polycaprolactam (PA-6), were obtained from Ghent University Polymer Department and characterized as described previously (Mutić et al., 2024 ). Monodisperse microparticle size PS standard dispersion (Supelco, Product No 59336, approximately 3.6x10 4 particles/ml, of uniform size 100 µm) was purchased from Sigma-Aldrich (Sigma Aldrich, St. Louis, USA). Filters: GF/A glass microfiber filters (Whatman, 1.2 µm, Sigma Aldrich, St. Louis, USA), PTFE membrane filter (0.45 µm, (Hyundai Micro, Seoul, Korea) and stainless-steel filters pore size 20 and 10 µm (Xinmingde Machinery, Henan, China) were used for filtration. Silicone filters for µFTIR, 1 µm pore size (Thermo Fisher Scientific, Waltham, USA). Equipments: Fourier transform infrared spectrometer, which couples a microscope (micro-FTIR, iN10 Nicolet, Thermo Fisher Scientific, Waltham, USA), was used for MPs identification. The instrument has an ultra-fast motorized stage and a liquid nitrogen-cooled mercury cadmium telluride detector (MCT detector). Olympus Microscope BX51M Metallurgical (Olympus Corporation, Tokyo, Japan) with Polarization and Differential Interference Contrast (DIC) was used for sample observation and analysis, Laminar flow cabinet (ALPINA, LVAC-K1300). 2.3 Quality assurance and quality control (QA/QC) Given the ubiquitous presence of MP, there is a risk of contamination in the laboratory. Due to this, a cotton lab coat and nitrile gloves were always used during sample processing, digestion, and analysis. Glass containers, metallic spoons, spatula, and tweezers were used in every step and carefully cleaned with Milli-Q water between each sample. The equipment in contact with the samples or the chemical solutions was washed before use and then covered with aluminum foil to avoid microplastic airborne contamination. All the chemicals used in the protocol were pre-filtered with WhatmanTM GF/C (1.2 µm pore size) and kept in glass containers with glass lids. Also, to minimize the risk of contamination, the samples in glass containers were covered with aluminum foil during all the procedures. All the steps in the protocol were performed in a laminar flow cabinet (ALPINA, LVAC-K1300). Positive and blank controls were carried out in triplicate. The feces samples were spiked with a known amount of microplastic, and recovery rates were analyzed. Blank controls, without feces, were carried out in duplicate to evaluate the procedure and background contamination. 2.4 Development of digestion protocol for analyzing microplastics in feces samples 2.4.1 Protocol A: acidic, alkaline, and enzymatic digestion The initial protocol, protocol A, combines digestion with acidic, alkaline, and enzymatic digestion (Toto et al., 2023 ). The digestion begins with an acidic step using a 30 mL mixture of 15% H 2 O 2 and 5% HNO 3 for 24 hours at 40°C. Following this, the samples were filtered using 20 µm stainless steel filters. The next step was alkaline digestion with 30 mL of 10% KOH under the same conditions (24 hours at 40°C). After alkaline digestion, the samples were filtered using 20 µm stainless steel filters. The next step involved enzymatic digestion using a 25 mL mixture of Viscozyme L and Cellulase enzymes in an acetate buffer at pH 5.1 for 24 hours at 40°C. The samples were filtered using 20 µm stainless steel filters, and oxidative digestion was performed using 30 mL of 15% hydrogen peroxide for 16 hours at room temperature. Finally, the samples were transferred to silicone filters for µFTIR analysis and stored in glass Petri dishes until further analysis using µFTIR. 2.4.2 Protocol B: Protocol with pre-step removing cellulose with 8% urea, 8% thiourea and 8% KOH Protocol B included a pre-treatment step to remove cellulose from samples, as described by (Egea-Corbacho et al., 2022 ). This pre-treatment involved treating the sample with 40 mL of mixture solution containing 8% urea, 8% thiourea, and 8% KOH per 0.1 g of the sample. Feces samples, ranging from 0.3 to 0.6 g, were treated with 120 mL of an 8% mixture solution containing 8% urea, 8% thiourea, and 8% KOH. The mixture was covered with a watch glass and incubated at -20°C for 40 minutes. Following this, the samples were allowed to equilibrate to room temperature. The samples were then filtered through 20 µm stainless steel filters and rinsed with 200 mL of MiliQ water to ensure the quantitative transfer of all material onto the filter. A mixture of 30 mL of 5% HNO 3 and 15% H 2 O 2 was added directly in situ on the filter, and digestion was performed for 24 hours at 40°C with constant stirring at 200 rpm. Subsequently, the samples were neutralized to pH 7 using NaHCO 3 , and a 25 mL mixture of Viscozyme L (3.19 units/mL) and Cellulase (32.31 units/mL) enzymes in acetate buffer (pH = 5) was introduced. The mixture was incubated for 4 days at 40°C with constant stirring at 200 rpm. The samples were again filtered using 20 µm stainless steel filters and rinsed with 200 mL of MiliQ water. A 30 mL of 10% KOH solution was added directly to the filters, and digestion was conducted for 24 hours at 40°C with constant stirring at 200 rpm. After the alkaline digestion, oxidative digestion was performed using 30 mL of 15% hydrogen peroxide for 16 hours at room temperature. The samples were filtered through 20 µm stainless steel filters, and the filters were sonicated in an ultrasonic bath with 20 mL of 50% ethanol. Finally, the samples were transferred to silicone filters for µFTIR analysis and stored in glass Petri dishes until further analysis using µFTIR. 2.4.3 Protocol C: Protocol with pre-step removing cellulose with Schweizer reagent This protocol includes a preliminary step for cellulose removal using Schweizer’s reagent, followed by four main digestion steps: acidic digestion, enzymatic digestion, alkaline digestion, and oxidative digestion. Feces samples were treated with 40 mL of Schweizer’s reagent for 40 minutes at room temperature under constant stirring at 200 rpm. After the treatment, the samples were filtered through 20 µm stainless steel filters to remove undigested residues and rinsed with 200 mL of Milli-Q water to ensure the quantitative transfer of all material onto the filter. Subsequently, 30 mL of a 5% HNO 3 and 15% H 2 O 2 mixture was added in situ directly onto the filter, and digestion was performed for 24 hours at 40°C with constant stirring at 200 rpm. The samples were then neutralized to pH 7 using NaHCO 3 , after which 25 mL of a Viscozyme L (3.19 units/mL) and Cellulase (32.31 units/mL) enzyme mixture in acetate buffer (pH 5) was introduced. The enzymatic digestion step was carried out for 4 days at 40°C with constant stirring at 200 rpm. After enzymatic digestion, the samples were filtered again using 20 µm stainless steel filters and rinsed with 200 mL of Milli-Q water. Then, 30 mL of a 10% KOH solution was added directly to the filters, and alkaline digestion was conducted for 24 hours at 40°C with constant stirring at 200 rpm. This was followed by oxidative digestion using 30 mL of 15% hydrogen peroxide, which was performed for 16 hours at room temperature. Finally, the samples were filtered through 20 µm stainless steel filters, and the filters were subjected to ultrasonic treatment in 20 mL of 50% ethanol. The processed samples were then transferred to silicone filters for µFTIR analysis and stored in glass Petri dishes until further analysis. 2.4.4 Protocol D: Final optimized protocol for cellulose removal and microplastic detection in human feces The final protocol D is based on three digestion steps: applying Schweizer's reagent and two main steps: alkaline digestion (with 10% KOH) and oxidative digestion (with 15% H 2 O 2 ). The feces samples were treated with 40 mL of Schweizer’s reagent for 40 minutes at room temperature under constant stirring at 200 rpm. After treatment, the samples were filtered through 20 µm stainless steel filters, rinsed with 200 mL of Milli-Q water, and subjected to alkaline digestion using 30 mL of 10% KOH for 24 hours at 40°C directly on the filter with constant stirring at 200 rpm. Subsequently, the samples were filtered again through 20 µm stainless steel filters, rinsed with 200 mL of Milli-Q water, and both filters were transferred to an Erlenmeyer flask for oxidative digestion. This step was carried out at room temperature using 30 mL of 15% hydrogen peroxide for 16 hours. After oxidative digestion, the samples were filtered again through 20 µm stainless steel filters. The filters were then sonicated in an ultrasonic bath with 20 mL of 50% ethanol to ensure optimal recovery. Finally, the processed samples were transferred to silicone filters, stored in glass Petri dishes, and prepared for µFTIR analysis. 2.4.5 Evaluation of a protocol for isolation MPs from feces samples for microFTIR analysis The protocol regarding digestion efficiency for MP analysis, recovery rates, and polymer integrity was evaluated by recording microFTIR spectra. The digestion efficiency was expressed in percentages (%), such as the ratio between matrix residue after digestion and sample weight before digestion. The weight of the matrix residue was determined by the weight difference of the dried filter (silicone filter used for µFTIR 1.0 µm). The dry weight of each filter, obtained by placing filters at 60 0 C for 24 h, was measured before and after digestion to assess the proportion of remaining organic matter on each filter after digestion. For each sample, digestion efficiency (E) was calculated according to the formula: \(\:\varvec{E}=\left(\frac{\varvec{W}-\varvec{D}}{\varvec{W}}\right)\:\varvec{x}100\varvec{\%}\) where: W= a mass of the feces sample, F = a mass of the filter before digestion, R = a mass of the filter after digestion, D=R−F– the difference in filter mass. To perform the recovery test, solid in-house standards were prepared by suspending microplastic particles in Milli-Q water. A few drops of Milli-Q water were added to a clean microscope slide containing the solid plastic standards, creating a suspension of particles. Individual microplastic particles were visually detected, carefully selected, and transferred into the sample using an inoculating loop. This method ensured precise sample spiking with a known number of microplastic particles, enabling accurate recovery efficiency evaluation. The recovery rate (%) was calculated as a ratio of the count of particles lying on filters after digestion and the number of particles subjected to digestion for each polymer type. In addition, a PS standard solution was used, containing approximately 3.6 x 10 4 spherical particles/mL, with an average particle diameter of 100 µm, as confirmed by the manufacturer’s specifications. The standard was diluted to 18 particles per 100 µL. The feces samples were spiked with 100 µL of this standard, and recovery rates were analyzed (positive control). Blank controls, without a feces sample, were carried out to evaluate the procedure and background contamination. To check the influence of the optimized digestion protocol on the integrity of MPs, chemical identification of standard particles (LDPE, HDPE, PP, PVC, PS, PA, PET) was performed before and after the complete digestion procedure by micro-FTIR. FTIR spectra of standard particles after digestion were compared with spectra libraries. 2.4.6. Microplastic identification Quantitative analysis and polymer identification with microFTIR spectroscopy Quantitative analysis and polymer identification were conducted using the ThermoFisher Nicolet iN10 FTIR microscope (ThermoFisher, USA). Fast mapping of the silicone filter was performed in reflection mode, covering an area of 1 cm × 1 cm and utilizing a cooled MCT detector. The resulting chemical maps were analyzed for each filter, with a focus on peak height intensity. Spectral acquisition was conducted at a resolution of 8 cm⁻¹, with 16 scans collected per measurement point. All particles were manually examined, irrespective of their composition. A particle was classified as plastic if its spectral match with the reference library exceeded 70%. The spectral database comprises over 185,000 spectra from more than a hundred different libraries, including commercially available and in-house collected datasets. Particle dimensions were determined using microscopy imaging and a digital ruler tool to measure the longest dimension (maximum Feret diameter). The aperture was set to 50 µm, with a detection limit of 10 µm. 3. Results and discussion 3.1 Initial tests In order to evaluate and optimize sample preparation methods for the isolation of MP from the feces samples, a literature study on extraction protocols was carried out. Different approaches were systematically evaluated and optimized to address the specific challenges associated with feces digestion. A comparison of tested methods regarding different steps is presented in Table 1 . Table 1 Tested protocols Step Protocol A (Yan 2020) Protocol B (Toto 2023) Protocol C Protocol D (new protocol) Pre-treatment None 120 mL of a mixture containing 8% urea, 8% thiourea, and 8% KOH 40 mL of Schweizer reagent; 40 minutes 40 mL of Schweizer reagent; 40 minutes Acidic digestion 30 mL of 5% HNO 3 and 15% H 2 O 2 for 24 hours at 40°C, stirred at 200 rpm. 30 mL of 5% HNO 3 and 15% H 2 O 2 for 24 hours at 40°C, stirred at 200 rpm. 30 mL of 5% HNO 3 and 15% H 2 O 2 for 24 hours at 40°C, stirred at 200 rpm. None Alkaline digestion 30 mL of 10% KOH; 24 hours 30 mL of 10% KOH; 24 hours 30 mL of 10% KOH; 24 hours 30 mL of 10% KOH; 24 hours Enzymatic digestion Acetic buffer (pH = 5) with 25 mL Viscozyme and cellulase. Incubation at 40°C for 4 days, stirred at 200 rpm. Acetic buffer (pH = 5) with 25 mL Viscozyme and cellulase. Incubation at 40°C for 4 days, stirred at 200 rpm. Acetic buffer (pH = 5) with 25 mL Viscozyme and cellulase. Incubation at 40°C for 4 days, stirred at 200 rpm. None Oxidative digestion 30 mL of 15% H 2 O 2 ; 16 hours 30 mL of 15% H 2 O 2 ;16 hours 30 mL of 15% H 2 O 2 ;16 hours 30 mL of 15% H 2 O 2 ;16 hours Total time 7 days 7 days 4 days 2 days The initial protocol, protocol A, was adapted from the method described by Toto et al. ( 2023 ) for rat feces digestion and modified. The reason for starting with this protocol was that it involves steps that have shown promising results when applied to human feces samples, making it a suitable candidate for adaptation in our study. Protocol A was applied, which included acid, alkaline, enzymatic, and oxidative digestion. In most studies, acidic conditions and potent oxidizing agents were employed to digest feces samples and detect microplastics. However, numerous studies have examined the impact of strong acids and oxidizing agents on the integrity of microplastics (Adedapo et al., 2024 ; Liu et al., 2022 ; Ortiz et al., 2022 ; Schrank et al., 2022 ; Sipps et al., 2023 ). For instance, 65% nitric acid has been shown to be particularly harmful to polymers such as polyamide (PA), polyethylene terephthalate (PET), and polyurethane (PUR), leading to their complete dissolution at elevated temperatures (Schrank et al., 2022 ). Additionally, significant surface changes have been observed in polyethylene (PE) and polypropylene (PP) after exposure to nitric acid, which reacts with functional groups on the surface of microplastics, resulting in molecular-level alterations) (Schrank et al., 2022 ). Adedapo et al. ( 2024 ) emphasized that polypropylene (PP) is highly susceptible to degradation under the influence of nitric acid, with substantial degradation occurring at both room and elevated temperatures. However, filtration was impossible immediately after the first step of acid digestion with hydrogen peroxide and nitric acid due to the high cellulose content, which clogged the 20 µm stainless steel filter. Therefore, this protocol was not further optimized. Optimization efforts were directed toward removing cellulose from the samples before applying the combined digestion protocol. 3.2 Optimized Pre-Treatment as an Additional Step for Superior Cellulose Removal Efficient digestion is critical for ensuring accurate downstream analyses, such as polymer identification through µFTIR spectroscopy, and underscores the importance of selecting appropriate reagents and protocols in microplastic research. To achieve high digestion efficiency, pretreatment protocols were essential in overcoming challenges related to cellulose removal. To address this issue, an additional step was introduced to reduce the cellulose content in the samples. These pre-treatments aim to remove a significant amount of cellulose, which causes issues during digestion, further treatment, and the filtration of digest through a 20 µm stainless steel filter. Therefore, comparing these two methods and concluding which pretreatment method is more compelling is crucial. Protocol B included a pretreatment step for cellulose removal with a mixture of 8% urea, 8% thiourea, and 8% KOH, followed by acid, alkaline, enzymatic, and oxidative digestion. Protocol C included Schweizer’s reagent as the pretreatment step, followed by acid, alkaline, enzymatic, and oxidative digestion. In most studies examining the efficiency of the 8% urea, 8% thiourea, and 8% KOH mixture, the efficiency itself depends on the concentration of the mix. An optimal concentration of 8% usually provides an efficiency range of 80–94%, which is theoretically satisfactory (Budtova & Navard, 2016 ). According to Zhang et al., ( 2010 ), the efficiency of cellulose fiber removal with this mixture is approximately 91%. Schweizer's reagent consists of copper(II)-hydroxide dissolved in ammonia, forming the tetraamminediaquacopper(II)-hydroxide complex. The reaction between cellulose and Schweizer’s reagent is given (Reaction 1). This reagent is classified as a non-derivatizing method of cellulose dissolution because it forms a covalent coordinate bond with copper ions, weakening hydrogen interactions and allowing the cellulose molecule to unravel and disperse (Dias et al., 2020 ). The effects of Schweizer's reagent on cellulose and the mechanism of its dissolution have been extensively described in numerous studies (Dias et al., 2020 ; Hamzavi et al., 2020 ; Przypis et al., 2023 ). Schweizer's reagent is a highly efficient method for removing cellulose from various types of samples. According to research (Yurtsever, 2021 ), the effectiveness of Schweizer's reagent in dissolving cellulose from tea bags is satisfactory, considering that most tea bags consist of cellulose and non-cellulose fibers, with non-cellulose fibers making up 30–35% of the mass. Their study confirms that Schweizer's reagent dissolves only cellulose, leaving non-cellulose material in the mentioned percentage composition after digestion. The study demonstrated that cellulose recovery from toilet paper using Schweizer's reagent was 100%, indicating the reagent's high efficiency in dissolving cellulose (Gupta et al., 2018 ). However, this reagent has not been previously applied to remove cellulose to facilitate microplastic detection in feces samples. 3.3.1. Optimization of the amount of Schweizer’s reagent for efficient cellulose removal from children's feces samples The conditions for using Schweizer’s reagent were optimized to enhance cellulose removal efficiency from children's feces samples. A key aspect of this optimization was determining the optimal ratio between reagent volume and sample mass to ensure effective cellulose digestion and easy filtration. Various combinations were tested, and the results are shown in Table 2 . Table 2 Mass-to-volume ratios of Schweizer’s reagent and sample mass Sample mass (g) Schweizer volume (mL) Mass: Volume ratio Ratio (mL/g) 0.31 20 1:65 64.5 0.60 40 1:67 66.7 0.60 50 1:83 83.3 0.90 50 1:56 55.6 1.00 50 1:50 50 0.65 120 1:185 184.6 0.83 120 1:145 144.6 1.03 120 1:116 116.5 Testing revealed that 120 mL and 50 mL of Schweizer’s reagent per 1 g of sample caused significant filtration difficulties due to increased solution viscosity. While a larger reagent volume dissolves more cellulose, it also increases viscosity, making filtration challenging. These findings indicate that increasing the reagent volume beyond a certain point does not improve the overall process. Among the tested ratios, 40 mL of Schweizer’s reagent per 0.6 g of sample provided the best balance between complete cellulose digestion and effective filtration. This ratio was selected as the optimal condition. While increasing the reagent volume improves cellulose solubility, excessive amounts raise viscosity, hindering filtration. Conversely, using too little reagent may result in incomplete cellulose digestion. Additionally, using 40 mL of Schweizer’s reagent per 0.6 g of sample proved cost-effective, reducing reagent consumption without compromising analysis quality. Thus, while reagent volumes above 100 mL/g enhance cellulose solubility, they also increase viscosity and complicate filtration. On the other hand, volumes below 50 mL/g may lead to incomplete digestion. Both urea, thiourea, KOH reagent, and Schweizer’s reagent effectively remove cellulose. Still, the use of Schweizer’s reagent drastically reduces the amount of cellulose that interferes with microplastic detection via micro FTIR. Schweizer’s reagent proved to be much more effective at removing cellulose than the 8% urea, 8% thiourea, and 8% KOH mixture, as demonstrated by µFTIR filter images after the digestion of children’s feces samples from this study. A comparison of residues after digestion using Schweizer’s reagent and the 8% urea, 8% thiourea, and 8% KOH mixture is presented in Figs. 1A and 1B. Treatment with Schweizer’s reagent results in significantly fewer residual fibers, more uniform particles, and filter-free cellulose traces, which is not the case with the 8% urea, 8% thiourea, and 8% KOH treatment. The digestion residues differ notably, with a higher content of undigested material present after the 8% urea, 8% thiourea, and 8% KOH treatment, highlighting the superiority of Schweizer’s reagent in cellulose removal. Its application in this study effectively reduced the cellulose content, allowing for smoother sample treatment, particularly during filtration using stainless steel filters with 20 µm pores. Schweizer’s reagent removes cellulose more effectively, resulting in significantly fewer residual fibers and more uniform particle distribution. At the same time, the urea-thiourea-KOH mixture leaves a higher amount of undigested material, including visible cellulose residues. The most promising protocol was protocol C, which was chosen for further optimization. Various modifications of Protocol C were tested to ultimately develop Protocol D, which was found to be effective for microplastic isolation. Several different procedures were applied to the samples to establish the final Protocol D. Based on the proven efficiency of Schweizer's reagent in removing cellulose from various sample types, including complete cellulose recovery from toilet paper (Gupta et al., 2018 ) and selective dissolution of cellulose fibers from tea bags (Yurtsever, 2021 ), enzymatic digestion was not necessary. Besides, enzymatic digestion was also excluded because of further limitations: very time-consuming processing; the specificity of the enzymes can hardly be adequate in complex matrices; the necessity of optimization of conditions like pH, temperature, and enzyme concentration (Löder et al., 2017 ; Toto et al., 2023 ; Von Friesen et al., 2019 ); and the high cost of enzymes, which is less economical compared to Schweizer's reagent. Nitric acid digestion was avoided because Schrank et al. ( 2022 ) and Adedapo et al. ( 2024 ) have reported its degradative effects on PA, PET, and PUR polymers, aside from its inducing significant surface modifications in PE and PP. Digestion was not successful without the use of potassium hydroxide, which proved to be an essential reagent for the effective decomposition of organic matter. Our results showed no significant differences when the acidic digestion step is omitted (Fig. 2A and 2B). 3.3 Evaluation of proposed protocol (Protocol D) Acidic digestion with nitric acid was excluded, an essential adjustment to protect the integrity of microplastics. The digestion protocol was further optimized to avoid enzymatic steps and significantly improve the overall digestion time. Enzymatic digestion steps using Viscozyme L and Cellulase were omitted. The final protocol D included pretreatment with Schweizer’s reagent, followed by alkaline and oxidative digestion, thereby eliminating the acid and enzymatic digestion steps. The images show no significant differences when enzymatic and acidic digestion steps are omitted. The comparison of residual material after digestion, shown in Figs. 3A and 3B, clearly demonstrates that excluding acidic and enzymatic steps in digestion does not influence the effectiveness of cellulose removal from the samples. In both cases, the cellulose was effectively removed, which can be explained by the superior efficiency of Schweizer's reagent. This reagent facilitates the complexation of cellulose and its solubilization, making enzymatic treatment unnecessary. Also, the even particle distribution in the filter means that the omission of these steps does not lead to significant changes in the composition or morphology of the residual material after digestion. Chemical mapping of the filter by µFTIR after sample digestion is presented in Figs. 3C and 3D. Figure 3C represents the result of a non-optimized process, whereas 3D represents the result of an optimized process. When digestion is not fully optimized (3C), a more significant amount of residual organic and inorganic material remains on the filter, making the identification and quantification of microplastics more difficult due to spectral interferences. In contrast, the optimized process (3D) significantly reduces the amount of residual material, resulting in a cleaner filter and enabling more precise µFTIR mapping of microplastic particles. Several parameters were examined to validate the digestion method, assess its efficiency, and determine whether microplastic particles are lost during digestion. These parameters include digestion efficiency, recovery rates, and polymer integrity. 3.3.2 Digestion efficiency The efficiency of the proposed digestion protocol for feces samples (protocol D) was evaluated. The results of the digestion efficiency are 99.95% ±and 0.03%. The results indicated a highly efficient digestion process, with minimal residual mass detected on the filters. The slight differences in filter mass post-digestion suggest that nearly all organic material was effectively degraded, leaving behind only trace amounts. Such high efficiency (above 99.9%) demonstrates that protocol D is a highly reliable method for feces digestion, enabling the complete removal of organic matter while preserving the structural integrity of microplastics for further analysis. Compared to literature values, where digestion efficiencies of 98% (Toto et al., 2023 ) and 97% (Yan et al., 2020 ) were reported, the efficiency achieved in this study underscores the superiority of the optimized protocol. This highlights the potential of Schweizer’s reagent as a robust tool for preparing samples for microplastic detection. 3.3.3 Recovery of MPs particles subjected to developed protocol for digestion To evaluate the developed protocol (D) for digestion, the recovery (%) of MP particles after digestion was determined by microFTIR analysis. A polystyrene (PS) standard solution was used, containing approximately 3.6 x 10 4 spherical particles/mL, with an average particle diameter of 100 µm, as confirmed by the manufacturer’s specifications. The standard was diluted to 18 particles per 100 µL, which was directly applied to feces samples. The samples were then digested using protocols B and D, and particle recovery was evaluated through µFTIR characterization. The results of the recovery rate were 128% ± 24%. The recovery test results demonstrated no significant loss of PS standard particles during digestion when protocol D is applied. These values are consistent with those reported in the literature, indicating that the observed recovery range is acceptable. Figure 4A presents an optical image of the filter obtained after digestion of the feces sample with PS spike. PS standard differed from the PS found in the sample and was easily observed (Fig. 4B). A PS standard was also analyzed under an optical microscope (Olympus Microscope BX51M) using UV and visible light to assess potential surface structure and particle dimension changes. The analysis confirmed that there were no significant changes in particle size or surface characteristics (Figures S1 and S2). No structural alterations were observed on the particle surface, which retained its regular spherical shape and exhibited a characteristic reflective pattern. In addition, the recovery test was performed for solid in-house standards PS, PA, PP, HDPE/LDPE, and PET (A list of polymer standards with their average size and density are presented in Supplementary Table S1 ). The recovery values for PS, PA, PP, HDPE, and LDPE were 100% ±10%, indicating complete recovery without variability. In contrast, the recovery value for PET was 89% ± 20%, reflecting more significant variability in the recovery process for this polymer. Therefore, this method proves reliable and suitable for application in the digestion and subsequent analysis of microplastics in children's feces samples. 3.3.4 Polymer integrity To check the influence of the optimized digestion protocol on the integrity of MPs, chemical identification of standard particles (LDPE, HDPE, PP, PVC, PS, PA, PET) was performed before and after the complete digestion procedure by micro-FTIR—besides, FTIR spectra of standard particles after digestion were compared with spectra libraries (Fig. 5). As expected, applying Schweizer’s reagent, alkaline digestion, and oxidative digestion resulted in polymer integrity (in-house standards) ranging from 75–92%, based on the match with spectra from the instrument's database. Similarly, the integrity of the PS Sigma-Aldrich standard ranged from 70–95%, which can be considered well-preserved. These results indicated that the digestion process does not affect the polymer integrity of the standards (PET, PS, PA, PP, HDPE, and LDPE) used for this analysis. Additionally, the maximal weight of children's feces samples that can be effectively digested using this protocol was determined. Seven different weights of feces samples in a range of 0.40- 1.00 g were processed using Protocol D. The maximum feces mass that can be successfully digested under the mentioned conditions of this protocol was 1 g. 3.3.5 Characterization and quantification of microplastic particles in stool samples from Dalmatia The optimized protocol was applied for quantifying and characterizing microplastics in stool samples obtained from the Children's Hospital (n = 14) collected from Dalmatia, a coastal region of Croatia. The sample mass ranged from 0.6 to 0.7 g, corresponding to the optimal range determined during the prior optimization of sample quantity for microplastic analysis. Micro-FTIR analysis enabled the identification, quantification, and characterization of microplastic particles in the examined samples. The analyzed parameters included sample mass, polymer type, spectral match percentage with reference libraries, particle size and size distribution, particle shape, total number of microplastics per sample, and the number of microplastic particles per gram of sample (Table 3 ). Table 3 Characterization of microplastic particles detected in stool samples: sample mass, polymer types, spectral match with reference library, particle size and size range, morphological classification, number of microplastics per sample, and concentration (number of MP particles per gram). PE (polyethylene), LDPE (low-density polyethylene), HDPE (high-density polyethylene), PP (polypropylene), PET (polyethylene terephthalate), PCL (poly(caprolactone)), PS (polystyrene) Sample number Mass of sample [g] Polymer type Match with library (%) Size of MP (µm) Range of size (µm) Shape Number of MPs per sample Number of MP/g 2 0.60 PP 87.15 113.34 x 95.13 100–200 Fragment 4 6.67 LDPE 88.07 109.33 x 98.98 100–200 Spheroid PP 91.07 204.09 x 158.81 200–300 Fragment LDPE 84.32 177.32 x 98.63 100–200 Fragment 9 0.70 PET 76.73 98.24 x 43.53 50–100 Fragment 1 1.43 10 0.69 PE 83.64 101.33 x 89.43 100–200 Fragment 5 7.25 PE 82.19 40.09 x 40.85 0–50 Spheroid PE 80.46 141.44 x 98.22 100–200 Fiber PE 71.97 90.45 x 88.36 50–100 Fragment PE 84.83 139.45 x 127.93 100–200 Fragment 11 0.59 PE 83.72 235.93 x 185.24 200–300 Fragment 2 3.39 PCL 86.5 250.44 x 203.35 200–300 Fragment 12 0.67 PET 73.88 163.83 x 111.28 100–200 Fragment 1 1.49 13 0.85 PS 80.26 49.03 x 45.44 0–50 Spheroid 1 1.18 14 0.75 PCL 78.27 131.04 x 98.35 100–200 Fragment 2 2.67 HDPE 75.96 160.55 x 73.93 100–200 Fiber Microplastics were detected in 50% of the samples, while no microplastics were identified in the remaining 50%. This absence may indicate that microplastics were present below the detection limit of the micro-FTIR spectroscopy method or reflect individual variations in subjects' exposure to microplastics. The number of microplastic particles per sample ranged from 0 to 5, with a corresponding concentration varying between 1.18 and 7.25 particles per gram of sample. MP particles detected in stool samples were classified into four size categories: 0–50, 50–100, 100–200, and 200–300 µm. The majority of particles fell within the 100–200 µm range. The lengths of detected MPs ranged from 40.09 to 250.44 µm, while widths varied between 40.85 and 203.35 µm. Three distinct MP morphologies—fibers, fragments, and spheroids—were identified in the analyzed stool samples (Table 3 ). Fragments were the most prevalent (68.75%), followed by spheroids (18.75%) and fibers (12.50%). The diversity of microplastic shapes is further illustrated in Figure S3, which presents an enlarged view of a silicone filter containing digested stool material. This image was acquired using an optical microscope (Olympus BX51M) under both visible (A) and UV light (B). However, not all particles observed via optical microscopy can be conclusively characterized as microplastics. Therefore, the particles were further analyzed and characterized using a micro-FTIR instrument. Additionally, Figure S3 (C) displays the entire filter, demonstrating an even distribution of particles with a pronounced contrast between the particles and the background. This image was captured using a micro-FTIR microscope. Furthermore, Figure S3 (D) presents a chemical mapping of the particles present on the filter, providing valuable insights into their composition. The micro-FTIR analysis revealed the presence of five different types of MP: polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), poly(caprolactone) (PCL), and polystyrene (PS) (Table 3 ). The most widely distributed and abundant MP type was polyethylene (56.25%), suggesting food and bavarages packaging, including bottled water and plastic bags, and the main source of exposure to MP through ingestion. Conclusion This study presents an innovative approach to efficiently removing cellulose from human feces samples, significantly enhancing digestion processes and the analysis of microplastics. Due to its complex structure and chemical properties, cellulose poses a challenge during digestion and interferes with recording infrared spectra on a micro-Fourier transform spectrometer (µFTIR). In this research, Schweizer's reagent was applied for the first time to remove cellulose from children's feces samples for microplastic detection. Schweizer's reagent successfully complexed cellulose into a soluble form, enabling efficient digestion while preserving the structural integrity of microplastics. Four digestion protocols were evaluated, with the proposed protocol emerging as the most effective for digesting children's feces samples. The digestion efficiency of 99.98% was achieved, demonstrating the protocol's reliability. Recovery testing of polystyrene (PS) microplastic particles showed a recovery value of 128 ± 24%, confirming no significant loss of microplastics during digestion. Furthermore, the polymer integrity of standards, including PET, PS, PA, PP, HDPE, and LDPE, ranged from 75% to 95%. These results confirmed that the digestion conditions did not compromise microplastic polymers’ chemical structure or physical properties. The application of Schweizer's reagent in this context represents a significant advancement, providing a reliable and cost-effective solution for feces digestion. This optimized protocol eliminates harsh acids and enzymatic treatments while ensuring accurate recovery and analysis of microplastics. Microplastic content was determined in 14 feces samples from Dalmatia, of which 7 samples contained microplastics. Among the detected polymers, polyethylene was the most prevalent. The number of microplastic particles per sample ranged from 0 to 5, corresponding to concentrations between 1.18 and 7.25 particles per gram of sample. The most dominant particle shape was fragment (68.75%), while the most common particle size ranged from 40.09 to 250.44 µm in length and from 40.85 to 250.44 µm in width. The main advantage of this method for digesting feces samples lies in its simplified approach. It enables us to digest more significant quantities of samples efficiently with minimal steps. After digestion, the sample contains fewer fibers and cellulose, further simplifying the analysis of microplastics and interpretation of results. This methodology avoids acidic digestion processes, uses fewer chemicals, and provides the possibility of recycling Schweizer's reagent. Declarations Declaration of competing interest The authors declare that there is no conflict of interest. Data availability The data that support the findings of this study are available through the University of Belgrade – Faculty of Chemistry repository of data: https://hdl.handle.net/21.15107/rcub_cherry_6483. Ethics statements This study was approved by the Ethics Committee for the Use of Human Biological Material for Research at the University of Belgrade - Faculty of Chemistry. The ethical approval was granted during an online session on October 10, 2023, for the research titled "Investigation of Microplastic Content in Stool Samples from the Pediatric Population." The study was conducted under the guidance of Dr. Tanja Ćirković Veličković at the University of Belgrade. The approval followed a thorough review of the documentation, including the research request, forms for informed consent, and approval from the Ethics Committee of the Srebrnjak Children's Hospital in Zagreb, Croatia (approval number: 04-930/3-21). The study protocol was in full compliance with ethical standards. Funding This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 965173. Work presented in this manuscript has been partially supported by the Serbian Academy of Sciences and Arts (grant number F-26) and the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract number: 451-03-47/2025-03/200168). AuthorContributions: Miloš Ilić , Methodology, Investigation, Validation, Writing – original draft; Tamara Mutić, Methodology, Investigation, Validation, Writing - original draft; Dragana Stanić-Vučinić , Conceptualization, Visualization, Writing - review and editing; Mirjana Turkalj, Contribution, Writing, Investigation; Ivana Banić , Contribution, Writing, Investigation; Jelena Mutić , Validation, Supervision, Writing- review and editing; Tanja Cirkovic Velickovic , Conceptualization, Funding acquisition, Supervision, Writing - review and editing. Funding This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 965173. The work presented in this manuscript has been partially supported by the Serbian Academy of Sciences and Arts (grant number F-26) and the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract number: 451-03-136/2025-03/200168). 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J Hazard Mater 384:121489. https://doi.org/10.1016/j.jhazmat.2019.121489 Yurtsever M (2021) Are nonwoven fabrics used in foods made of cellulose or plastic? Cellulose/plastic separation by using Schweizer’s reagent and analysis based on a sample of tea bags. Process Saf Environ Prot 151:188–194. https://doi.org/10.1016/j.psep.2021.05.016 Zhang N, Li YB, He HR, Zhang JF, Ma GS (2021) You are what you eat: Microplastics in the feces of young men living in Beijing. Sci Total Environ 767:144345. https://doi.org/10.1016/j.scitotenv.2020.144345 Zhang S, Li F-X, Yu J, Hsieh Y-L (2010) Dissolution behaviour and solubility of cellulose in NaOH complex solution. Carbohydr Polym 81(3):668–674. https://doi.org/10.1016/j.carbpol.2010.03.029 Reaction Reaction 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.tiff floatimage1.png Reaction 1. The reaction between cellulose and Schweizer reagent. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6427909","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":447363024,"identity":"775765d6-5fde-4ea7-98ae-103fb2444556","order_by":0,"name":"Miloš Ilić","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Miloš","middleName":"","lastName":"Ilić","suffix":""},{"id":447363025,"identity":"a6dcbc4f-279e-42ed-b4e1-7c6db6fedfa0","order_by":1,"name":"Tamara Mutić","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Tamara","middleName":"","lastName":"Mutić","suffix":""},{"id":447363026,"identity":"1a0f2751-4334-443c-a843-c89d9c85ca46","order_by":2,"name":"Dragana Stanić-Vučinić","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Dragana","middleName":"","lastName":"Stanić-Vučinić","suffix":""},{"id":447363027,"identity":"2c48e15d-f2cc-47b6-9c98-83fc3f82f7d4","order_by":3,"name":"Mirjana Turkalj","email":"","orcid":"","institution":"Srebrnjak Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mirjana","middleName":"","lastName":"Turkalj","suffix":""},{"id":447363028,"identity":"ba0bff6c-4c3c-4b19-9657-58e94eff0636","order_by":4,"name":"Ivana Banić","email":"","orcid":"","institution":"Srebrnjak Children’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ivana","middleName":"","lastName":"Banić","suffix":""},{"id":447363029,"identity":"c7b517eb-c141-4c44-a5ca-62b909255e18","order_by":5,"name":"Jelena Mutić","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Jelena","middleName":"","lastName":"Mutić","suffix":""},{"id":447363030,"identity":"87c96f72-8e28-4829-ab08-cbb24732fbc3","order_by":6,"name":"Tanja Cirkovic Velickovic","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYFACxgbGBgYGOQYJMJsIDTxQLcakaIEoTGwgWos9++G2jzMqDqf3z24+uoFxRy0Df/sBto8/8NnCk9g8c8OZw7kz7hxLu8F45jiDxJkE5tk8+LRIMDYzPmxLy90gkWN2g7HtGAPDDQZmZrx+gWpJN5DI/wbWIg/UwojXYSAtG9tsEgwkctiAWmoYDIBaGPA67ExiM+OMMzaGM26kmd1IPHOAxxAowoxPC3v78ceMPRUS8vwzkp/d+LijTk7u+OHDeB2GChIYDvMQGTsIUEeS6lEwCkbBKBgZAAA7hku1eMXppAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Belgrade","correspondingAuthor":true,"prefix":"","firstName":"Tanja","middleName":"Cirkovic","lastName":"Velickovic","suffix":""}],"badges":[],"createdAt":"2025-04-11 11:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6427909/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6427909/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82078993,"identity":"05646e72-0aa0-4303-bc1c-ac51a340a285","added_by":"auto","created_at":"2025-05-06 14:10:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":504793,"visible":true,"origin":"","legend":"\u003cp\u003eOptical images of the filters were obtained after the digestion of feces using different protocols: A) after digestion protocol B (with a mixture of 8% urea, 8% thiourea, and 8% of KOH reagent, acid, alkaline, enzymatic, and oxidative digestion) and B) after protocol C (with Schweizer reagent, acid, alkaline, enzymatic, and oxidative digestion). Optical images of silicone filters with a diameter of 1cm and pore size of 1µm were generated by the microFTIR imaging system.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/c393ad123ae017df458ba4f0.png"},{"id":82078994,"identity":"5f4e579f-7e0e-4cea-b651-d55cfb2d4b9d","added_by":"auto","created_at":"2025-05-06 14:10:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":782241,"visible":true,"origin":"","legend":"\u003cp\u003eOptical images of the filters obtained after digestion of feces samples using different protocols: A) after digestion protocol C (with Schweizer reagent, \u003cstrong\u003eacidic,\u003c/strong\u003e alkaline, enzymatic, and oxidative digestion) and B) after modified protocol C (with Schweizer reagent, alkaline, enzymatic and oxidative digestion).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/be79268401b85c0d379ae150.png"},{"id":82079002,"identity":"7a5ee59e-371b-425e-812c-2e48cb40cf92","added_by":"auto","created_at":"2025-05-06 14:10:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1154316,"visible":true,"origin":"","legend":"\u003cp\u003eA) Optical image of the filter obtained after digestion of feces sample by protocol C (with Schweizer reagent, acid, alkaline, enzymatic, and oxidative digestion) and B) Optical image of the filter obtained after digestion of feces sample by protocol D (with Schweizer reagent, alkaline, and oxidative digestion). C) µFTIR chemical mapping of the filter after digestion: (C) non-optimized digestion process with significant residual material. (D) Optimized the digestion process with reduced residual material, allowing for improved microplastic identification.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/bc62a713e10691aecfe9eac5.png"},{"id":82078999,"identity":"0364292d-9f64-4caf-a7ed-8dfd5d92e166","added_by":"auto","created_at":"2025-05-06 14:10:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":602831,"visible":true,"origin":"","legend":"\u003cp\u003eA) Optical image of the filter after digestion of a feces sample spiked with a spherical PS standard. B) Optical image representing the spiked PS standard and real microplastic (PS) detected in the sample.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/e278b85687f1378dd0897bf1.png"},{"id":82768489,"identity":"c5dee751-964e-429b-8d7e-01f2fe059c9d","added_by":"auto","created_at":"2025-05-15 05:33:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4602087,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/f91f0fd8-7c48-4ba2-b440-90d7f8056bce.pdf"},{"id":82079854,"identity":"67f1eecd-4ca8-40fc-9fe6-72d3a7f7403f","added_by":"auto","created_at":"2025-05-06 14:18:20","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":147890,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/8a9632a8d075bba5ebba6d6a.tiff"},{"id":82079855,"identity":"d3fe686e-7432-4c1e-bc79-d257d9eb62c9","added_by":"auto","created_at":"2025-05-06 14:18:20","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24299,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eReaction 1.\u003c/em\u003e The reaction between cellulose and Schweizer reagent.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/df5d5be52b8efbc78b7bb7d5.png"},{"id":82079006,"identity":"bdcc7308-2b6f-4a6a-9313-73eafb686184","added_by":"auto","created_at":"2025-05-06 14:10:20","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":874927,"visible":true,"origin":"","legend":"","description":"","filename":"SUPLEMENTARYmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6427909/v1/763c93b831e83861525ed15e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The application of Schweizer's reagent with FTIR imaging spectroscopic solutions for microplastics advanced analysis of feces samples","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDue to its purpose, wide usability, low cost, and chemical inertness, plastic is widely spread in industry and people's everyday lives (Ho et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Plastic pollution has drastically increased globally in the last two decades, with projections indicating an exponential rise of plastic waste to over 350\u0026nbsp;million tons annually (Ho et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schwabl et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The most significant expansion and exponential increase in human exposure to microplastics was recorded during the COVID-19 pandemic due to increased consumption of single-use plastics, such as face masks, gloves, and disposable coats for virus prevention (Ricciardi et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In most cases, microplastics (MPs) are formed through the degradation and fragmentation of macroplastics, influenced by natural environmental conditions and anthropogenic factors (Jenner et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ricciardi et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Microplastics are not solely produced through fragmentation and degradation (secondary sources), but also leak into the environment by production in various industrial sectors (primary sources) where they have broad applications (Ragusa et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to existing data, microplastics have already entered the food chain. They are found in most marine animals, including the gastrointestinal tracts of fish, shellfish, and crabs, which humans often consume (Ibrahim et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jenner et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Luqman et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mutić et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; P\u0026eacute;rez-Guevara et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Human exposure to microplastic particles most commonly occurs through the consumption of food and drinks contaminated with microplastics and inhaling these particles from the air (Abbasi \u0026amp; Turner, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Approximately 74,000 microplastic particles enter the human body annually through ingestion and inhalation (P\u0026eacute;rez-Guevara et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Microplastics have been detected in saliva, hair, blood, skin surfaces, the human placenta, and lung tissue (Abbasi \u0026amp; Turner, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). One of the earliest signs of human exposure to microplastic particles was their discovery in feces samples, indicating contamination of the gastrointestinal tract (Schwabl et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, examining feces is a crucial step in figuring out whether or not ingested microplastics have been completely or almost entirely ingested and, consequently, whether or not microplastic exposure has taken place. Only a few studies have far analyzed microplastic content in human feces (Abbasi \u0026amp; Turner, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ho et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ibrahim et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Luqman et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Schwabl et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; N. Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe study of microplastics in human feces is still in its early stages, primarily because of the challenges associated with quantifying microplastics and validating reliable methods. Digesting and isolating microplastics from feces represents a highly complex and challenging procedure. Due to this complexity, several methods have been developed for extracting microplastics from feces, including various chemical and enzymatic treatments (Toto et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the validation and harmonization of microplastic quantification methods have not yet been developed.\u003c/p\u003e \u003cp\u003eA methodology for isolating microplastics from human feces samples, involving a series of steps oxidizing agents and acids, was developed. This methodology includes using Fenton's reagent and digestion with 65% nitric acid at 50\u0026deg;C and 70\u0026deg;C (Yan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Toto et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported a method for digesting rat feces samples. This procedure involved acidic digestion using nitric acid and hydrogen peroxide, alkaline digestion using 10% potassium hydroxide, and enzymatic digestion using Viscozyme-L and cellulase. However, a drawback of these procedures lies in the use of nitric acid, which may potentially damage microplastics, as well as in the duration of the process (Toto et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is well known that human feces contain cellulose in a certain amount, as the human diet is largely based on plants that contain plant fibers (Danjo et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e;). Cellulose is a polysaccharide dominant in the plant world, forming the structural foundation of plant cells (Taylor, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The human body cannot break down cellulose because it lacks the enzymes and commensal microbiota necessary for its degradation. As a result, cellulose is excreted in feces in significant amounts (Chesterman et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCellulose represents a significant challenge in microplastic analysis using spectroscopic methods (Olsen et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and it has become crucial to remove cellulose to ensure accurate results efficiently. Traditionally, a urea, thiourea, and KOH mixture has been widely studied as a method for cellulose removal. The effectiveness of this mixture is closely linked to its concentration, with an optimal mixture concentration of 8% typically yielding an efficiency range of 80\u0026ndash;94%, which is considered theoretically satisfactory (Budtova \u0026amp; Navard, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). S. Zhang et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) report that this mixture achieves approximately 91% efficiency in removing cellulose fibers.\u003c/p\u003e \u003cp\u003eHowever, a much more efficient method for cellulose removal is the use of Schweizer reagent, which has demonstrated 100% efficiency in removing cellulose, as shown in studies where cellulose was removed from toilet paper (Gupta et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To date, Schweizer reagent has not been applied for cellulose removal from feces samples in order to analyze and isolate microplastics from these samples.\u003c/p\u003e \u003cp\u003eThis study focuses on the development of a novel, fast, and efficient method for extracting microplastics from feces samples and MP counting with their complete chemical characterization by microFTIR spectroscopy (\u0026micro;FTIR). A comprehensive digestion protocol was established to achieve this, combining cellulose removal, alkaline digestion, and oxidative digestion. Initially, we tested four different digestion protocols. The best protocol was further optimized and tested using in-house and commercial microplastic polymer standards.\u003c/p\u003e \u003cp\u003eThe objectives of our study were: (i) to optimize a highly efficient digestion protocol suitable for complex feces samples, introducing Schweizer\u0026rsquo;s reagent for superior cellulose removal, which has not been previously applied in this type of research; (ii) to evaluate a digestion protocol for isolating MPs from feces samples suitable for the counting of MPs and their complete chemical characterization by microFTIR; and (iii) to validate the method efficiency, recovery rate, and the preservation of polymer integrity. At the end, the optimized protocol was applied to 14 feces samples from the Dalmatian coastal region to determine microplastic content. The analyzed microplastic-related parameters included polymer type, particle size and size distribution, particle shape, total number of particles per sample, and particle count per gram of sample.\u003c/p\u003e \u003cp\u003eThe digestion protocol presented here has proven to be suitable for children\u0026rsquo;s feces samples, which is essential as fecal analysis has become an important tool for monitoring the extent of microplastic exposure of live organisms and humans.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1 Sample Collection and Preparation\u003c/h2\u003e\n\u003cp\u003eTo investigate human exposure to microplastics, frozen feces samples from children were obtained from the Srebrnjak Children's Hospital in Croatia. The samples were collected from the population living in Dalmatia, a coastal region of Croatia stretching along the eastern shore of the Adriatic Sea. Each sample was carefully packaged in a 50 mL glass jar with a metal lid and labelled with a unique numeric code on an attached paper strip. Each sample weighed between 4 to 7 grams. These samples were then packed in a cardboard box and stored in a freezer at -20\u0026deg;C until further processing for digestion.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2 Materials and methods\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eChemicals:\u0026nbsp;\u003c/strong\u003eViscozyme from Aspergillus sp, solution, \u0026ge;\u0026thinsp;100 FBGU/g, (Sigma-Aldrich, St. Louis, USA); Cellulase from Trichoderma reesei, aqueous solution, \u0026ge;\u0026thinsp;700 units/g, (Sigma-Aldrich, St. Louis, USA); Glacial acetic acid 100%, p.a. (Zorka pharma, \u0026Scaron;abac, Serbia), Sodium acetate, anhydrous, p.a. (Centrohem, Belgrade, Serbia), KOH (Merck, Darmstadt, Germany); Nitric acid (65%), analytical reagent grade (Merck, Darmstadt, Germany) Hydrogen peroxide 30%, analytical reagent grade (99.9%), (Fisher Scientific, USA); abs EtOH (HPLC grade, Merck, Darmstadt, Germany), Copper(II) sulphate pentahydrate (Merck, Darmstadt, Germany), Ammonia solution, 30%, (Honeywell; North Carolina), NaOH (Merck, Darmstadt, Germany), Urea, reagent grade (99%), (Merck, Darmstadt, Germany), Thiourea, reagent grade (99%), (Merck, Darmstadt, Germany), NaHCO\u003csub\u003e3\u003c/sub\u003e (Merck, Darmstadt, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchweizer reagent\u003c/strong\u003e: Schweizer's reagent is a metal ammine complex with the formula [Cu(NH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e4\u003c/sub\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e2\u003c/sub\u003e](OH)\u003csub\u003e2\u003c/sub\u003e. The reagent was prepared according to \u003cstrong\u003eReaction 1\u003c/strong\u003e by dissolving 6 g of copper(II)-sulphate pentahydrate (CuSO\u003csub\u003e4\u003c/sub\u003e∙5H\u003csub\u003e2\u003c/sub\u003eO) in 75 mL of water under constant stirring until complete dissolution was achieved. A volume of 48 mL of 1 M sodium hydroxide (NaOH) solution was then carefully added dropwise while stirring, forming a light blue gelatinous precipitate. The precipitate was allowed to settle, separated from the solution by filter paper filtration, and washed thoroughly with 20\u0026ndash;30 mL of water to remove residual ions. The washed precipitate was transferred to a clean beaker, and concentrated ammonia solution was added dropwise with continuous stirring until the precipitate was dissolved entirely, forming a clear, deep blue solution. The resulting solution was filtered through a PVDF membrane filter (pore size: 0.22 \u0026micro;m; diameter: 47 mm) to remove any remaining impurities. The prepared Schweizer's reagent was stored in an airtight container and protected from light to ensure stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandards\u003c/strong\u003e: Seven in-house produced MP standards (50\u0026ndash;500 \u0026micro;m) were used: polypropylene (PP), high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyvinylchloride (PVC), polystyrene (PS), polyethylene terephthalate (PET), polycaprolactam (PA-6), were obtained from Ghent University Polymer Department and characterized as described previously (Mutić et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Monodisperse microparticle size PS standard dispersion (Supelco, Product No 59336, approximately 3.6x10\u003csup\u003e4\u003c/sup\u003e particles/ml, of uniform size 100 \u0026micro;m) was purchased from Sigma-Aldrich (Sigma Aldrich, St. Louis, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFilters:\u0026nbsp;\u003c/strong\u003eGF/A glass microfiber filters (Whatman, 1.2 \u0026micro;m, Sigma Aldrich, St. Louis, USA), PTFE membrane filter (0.45 \u0026micro;m, (Hyundai Micro, Seoul, Korea) and stainless-steel filters pore size 20 and 10 \u0026micro;m (Xinmingde Machinery, Henan, China) were used for filtration. Silicone filters for \u0026micro;FTIR, 1 \u0026micro;m pore size (Thermo Fisher Scientific, Waltham, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEquipments:\u0026nbsp;\u003c/strong\u003eFourier transform infrared spectrometer, which couples a microscope (micro-FTIR, iN10 Nicolet, Thermo Fisher Scientific, Waltham, USA), was used for MPs identification. The instrument has an ultra-fast motorized stage and a liquid nitrogen-cooled mercury cadmium telluride detector (MCT detector). Olympus Microscope BX51M Metallurgical (Olympus Corporation, Tokyo, Japan) with Polarization and Differential Interference Contrast (DIC) was used for sample observation and analysis, Laminar flow cabinet (ALPINA, LVAC-K1300).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3 Quality assurance and quality control (QA/QC)\u003c/h2\u003e\n\u003cp\u003eGiven the ubiquitous presence of MP, there is a risk of contamination in the laboratory. Due to this, a cotton lab coat and nitrile gloves were always used during sample processing, digestion, and analysis. Glass containers, metallic spoons, spatula, and tweezers were used in every step and carefully cleaned with Milli-Q water between each sample. The equipment in contact with the samples or the chemical solutions was washed before use and then covered with aluminum foil to avoid microplastic airborne contamination. All the chemicals used in the protocol were pre-filtered with WhatmanTM GF/C (1.2 \u0026micro;m pore size) and kept in glass containers with glass lids. Also, to minimize the risk of contamination, the samples in glass containers were covered with aluminum foil during all the procedures. All the steps in the protocol were performed in a laminar flow cabinet (ALPINA, LVAC-K1300). Positive and blank controls were carried out in triplicate. The feces samples were spiked with a known amount of microplastic, and recovery rates were analyzed. Blank controls, without feces, were carried out in duplicate to evaluate the procedure and background contamination.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4 Development of digestion protocol for analyzing microplastics in feces samples\u003c/h2\u003e\n\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.1 Protocol A: acidic, alkaline, and enzymatic digestion\u003c/h2\u003e\n\u003cp\u003eThe initial protocol, protocol A, combines digestion with acidic, alkaline, and enzymatic digestion (Toto et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The digestion begins with an acidic step using a 30 mL mixture of 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 5% HNO\u003csub\u003e3\u003c/sub\u003e for 24 hours at 40\u0026deg;C. Following this, the samples were filtered using 20 \u0026micro;m stainless steel filters. The next step was alkaline digestion with 30 mL of 10% KOH under the same conditions (24 hours at 40\u0026deg;C). After alkaline digestion, the samples were filtered using 20 \u0026micro;m stainless steel filters. The next step involved enzymatic digestion using a 25 mL mixture of Viscozyme L and Cellulase enzymes in an acetate buffer at pH 5.1 for 24 hours at 40\u0026deg;C. The samples were filtered using 20 \u0026micro;m stainless steel filters, and oxidative digestion was performed using 30 mL of 15% hydrogen peroxide for 16 hours at room temperature. Finally, the samples were transferred to silicone filters for \u0026micro;FTIR analysis and stored in glass Petri dishes until further analysis using \u0026micro;FTIR.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.2 Protocol B: Protocol with pre-step removing cellulose with 8% urea, 8% thiourea and 8% KOH\u003c/h2\u003e\n\u003cp\u003eProtocol B included a pre-treatment step to remove cellulose from samples, as described by (Egea-Corbacho et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). This pre-treatment involved treating the sample with 40 mL of mixture solution containing 8% urea, 8% thiourea, and 8% KOH per 0.1 g of the sample. Feces samples, ranging from 0.3 to 0.6 g, were treated with 120 mL of an 8% mixture solution containing 8% urea, 8% thiourea, and 8% KOH. The mixture was covered with a watch glass and incubated at -20\u0026deg;C for 40 minutes. Following this, the samples were allowed to equilibrate to room temperature. The samples were then filtered through 20 \u0026micro;m stainless steel filters and rinsed with 200 mL of MiliQ water to ensure the quantitative transfer of all material onto the filter. A mixture of 30 mL of 5% HNO\u003csub\u003e3\u003c/sub\u003e and 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added directly in situ on the filter, and digestion was performed for 24 hours at 40\u0026deg;C with constant stirring at 200 rpm. Subsequently, the samples were neutralized to pH 7 using NaHCO\u003csub\u003e3\u003c/sub\u003e, and a 25 mL mixture of Viscozyme L (3.19 units/mL) and Cellulase (32.31 units/mL) enzymes in acetate buffer (pH\u0026thinsp;=\u0026thinsp;5) was introduced. The mixture was incubated for 4 days at 40\u0026deg;C with constant stirring at 200 rpm. The samples were again filtered using 20 \u0026micro;m stainless steel filters and rinsed with 200 mL of MiliQ water. A 30 mL of 10% KOH solution was added directly to the filters, and digestion was conducted for 24 hours at 40\u0026deg;C with constant stirring at 200 rpm. After the alkaline digestion, oxidative digestion was performed using 30 mL of 15% hydrogen peroxide for 16 hours at room temperature. The samples were filtered through 20 \u0026micro;m stainless steel filters, and the filters were sonicated in an ultrasonic bath with 20 mL of 50% ethanol. Finally, the samples were transferred to silicone filters for \u0026micro;FTIR analysis and stored in glass Petri dishes until further analysis using \u0026micro;FTIR.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.3 Protocol C: Protocol with pre-step removing cellulose with Schweizer reagent\u003c/h2\u003e\n\u003cp\u003eThis protocol includes a preliminary step for cellulose removal using Schweizer\u0026rsquo;s reagent, followed by four main digestion steps: acidic digestion, enzymatic digestion, alkaline digestion, and oxidative digestion.\u003c/p\u003e\n\u003cp\u003eFeces samples were treated with 40 mL of Schweizer\u0026rsquo;s reagent for 40 minutes at room temperature under constant stirring at 200 rpm. After the treatment, the samples were filtered through 20 \u0026micro;m stainless steel filters to remove undigested residues and rinsed with 200 mL of Milli-Q water to ensure the quantitative transfer of all material onto the filter.\u003c/p\u003e\n\u003cp\u003eSubsequently, 30 mL of a 5% HNO\u003csub\u003e3\u003c/sub\u003e and 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e mixture was added in situ directly onto the filter, and digestion was performed for 24 hours at 40\u0026deg;C with constant stirring at 200 rpm. The samples were then neutralized to pH 7 using NaHCO\u003csub\u003e3\u003c/sub\u003e, after which 25 mL of a Viscozyme L (3.19 units/mL) and Cellulase (32.31 units/mL) enzyme mixture in acetate buffer (pH 5) was introduced. The enzymatic digestion step was carried out for 4 days at 40\u0026deg;C with constant stirring at 200 rpm. After enzymatic digestion, the samples were filtered again using 20 \u0026micro;m stainless steel filters and rinsed with 200 mL of Milli-Q water. Then, 30 mL of a 10% KOH solution was added directly to the filters, and alkaline digestion was conducted for 24 hours at 40\u0026deg;C with constant stirring at 200 rpm. This was followed by oxidative digestion using 30 mL of 15% hydrogen peroxide, which was performed for 16 hours at room temperature. Finally, the samples were filtered through 20 \u0026micro;m stainless steel filters, and the filters were subjected to ultrasonic treatment in 20 mL of 50% ethanol. The processed samples were then transferred to silicone filters for \u0026micro;FTIR analysis and stored in glass Petri dishes until further analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.4 Protocol D: Final optimized protocol for cellulose removal and microplastic detection in human feces\u003c/h2\u003e\n\u003cp\u003eThe final protocol D is based on three digestion steps: applying Schweizer's reagent and two main steps: alkaline digestion (with 10% KOH) and oxidative digestion (with 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003eThe feces samples were treated with 40 mL of Schweizer\u0026rsquo;s reagent for 40 minutes at room temperature under constant stirring at 200 rpm. After treatment, the samples were filtered through 20 \u0026micro;m stainless steel filters, rinsed with 200 mL of Milli-Q water, and subjected to alkaline digestion using 30 mL of 10% KOH for 24 hours at 40\u0026deg;C directly on the filter with constant stirring at 200 rpm. Subsequently, the samples were filtered again through 20 \u0026micro;m stainless steel filters, rinsed with 200 mL of Milli-Q water, and both filters were transferred to an Erlenmeyer flask for oxidative digestion. This step was carried out at room temperature using 30 mL of 15% hydrogen peroxide for 16 hours. After oxidative digestion, the samples were filtered again through 20 \u0026micro;m stainless steel filters. The filters were then sonicated in an ultrasonic bath with 20 mL of 50% ethanol to ensure optimal recovery. Finally, the processed samples were transferred to silicone filters, stored in glass Petri dishes, and prepared for \u0026micro;FTIR analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.5 Evaluation of a protocol for isolation MPs from feces samples for microFTIR analysis\u003c/h2\u003e\n\u003cp\u003eThe protocol regarding digestion efficiency for MP analysis, recovery rates, and polymer integrity was evaluated by recording microFTIR spectra.\u003c/p\u003e\n\u003cp\u003eThe digestion efficiency was expressed in percentages (%), such as the ratio between matrix residue after digestion and sample weight before digestion. The weight of the matrix residue was determined by the weight difference of the dried filter (silicone filter used for \u0026micro;FTIR 1.0 \u0026micro;m). The dry weight of each filter, obtained by placing filters at 60 \u003csup\u003e0\u003c/sup\u003eC for 24 h, was measured before and after digestion to assess the proportion of remaining organic matter on each filter after digestion. For each sample, digestion efficiency (E) was calculated according to the formula: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{E}=\\left(\\frac{\\varvec{W}-\\varvec{D}}{\\varvec{W}}\\right)\\:\\varvec{x}100\\varvec{\\%}\\)\u003c/span\u003e\u003c/span\u003e where: W= a mass of the feces sample, F\u0026thinsp;=\u0026thinsp;a mass of the filter before digestion, R\u0026thinsp;=\u0026thinsp;a mass of the filter after digestion, D=R\u0026minus;F\u0026ndash; the difference in filter mass.\u003c/p\u003e\n\u003cp\u003eTo perform the recovery test, solid in-house standards were prepared by suspending microplastic particles in Milli-Q water. A few drops of Milli-Q water were added to a clean microscope slide containing the solid plastic standards, creating a suspension of particles. Individual microplastic particles were visually detected, carefully selected, and transferred into the sample using an inoculating loop. This method ensured precise sample spiking with a known number of microplastic particles, enabling accurate recovery efficiency evaluation. The recovery rate (%) was calculated as a ratio of the count of particles lying on filters after digestion and the number of particles subjected to digestion for each polymer type. In addition, a PS standard solution was used, containing approximately 3.6 x 10\u003csup\u003e4\u003c/sup\u003e spherical particles/mL, with an average particle diameter of 100 \u0026micro;m, as confirmed by the manufacturer\u0026rsquo;s specifications. The standard was diluted to 18 particles per 100 \u0026micro;L. The feces samples were spiked with 100 \u0026micro;L of this standard, and recovery rates were analyzed (positive control).\u003c/p\u003e\n\u003cp\u003eBlank controls, without a feces sample, were carried out to evaluate the procedure and background contamination.\u003c/p\u003e\n\u003cp\u003eTo check the influence of the optimized digestion protocol on the integrity of MPs, chemical identification of standard particles (LDPE, HDPE, PP, PVC, PS, PA, PET) was performed before and after the complete digestion procedure by micro-FTIR. FTIR spectra of standard particles after digestion were compared with spectra libraries.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.6. Microplastic identification\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative analysis and polymer identification with microFTIR spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative analysis and polymer identification were conducted using the ThermoFisher Nicolet iN10 FTIR microscope (ThermoFisher, USA). Fast mapping of the silicone filter was performed in reflection mode, covering an area of 1 cm \u0026times; 1 cm and utilizing a cooled MCT detector. The resulting chemical maps were analyzed for each filter, with a focus on peak height intensity. Spectral acquisition was conducted at a resolution of 8 cm⁻\u0026sup1;, with 16 scans collected per measurement point.\u003c/p\u003e\n\u003cp\u003eAll particles were manually examined, irrespective of their composition. A particle was classified as plastic if its spectral match with the reference library exceeded 70%. The spectral database comprises over 185,000 spectra from more than a hundred different libraries, including commercially available and in-house collected datasets.\u003c/p\u003e\n\u003cp\u003eParticle dimensions were determined using microscopy imaging and a digital ruler tool to measure the longest dimension (maximum Feret diameter). The aperture was set to 50 \u0026micro;m, with a detection limit of 10 \u0026micro;m.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Initial tests\u003c/h2\u003e\n \u003cp\u003eIn order to evaluate and optimize sample preparation methods for the isolation of MP from the feces samples, a literature study on extraction protocols was carried out. Different approaches were systematically evaluated and optimized to address the specific challenges associated with feces digestion. A comparison of tested methods regarding different steps is presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTested protocols\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStep\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtocol A\u003c/p\u003e\n \u003cp\u003e(Yan 2020)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtocol B\u003c/p\u003e\n \u003cp\u003e(Toto 2023)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtocol C\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtocol D (new protocol)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePre-treatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120 mL of a mixture containing 8% urea, 8% thiourea, and 8% KOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40 mL of Schweizer reagent; 40 minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40 mL of Schweizer reagent; 40 minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcidic digestion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 5% HNO\u003csub\u003e3\u003c/sub\u003e and 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours at 40\u0026deg;C, stirred at 200 rpm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 5% HNO\u003csub\u003e3\u003c/sub\u003e and 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours at 40\u0026deg;C, stirred at 200 rpm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 5% HNO\u003csub\u003e3\u003c/sub\u003e and 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours at 40\u0026deg;C, stirred at 200 rpm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlkaline digestion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 10% KOH; 24 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 10% KOH; 24 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 10% KOH; 24 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 10% KOH; 24 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnzymatic digestion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetic buffer (pH\u0026thinsp;=\u0026thinsp;5) with 25 mL Viscozyme and cellulase. Incubation at 40\u0026deg;C for 4 days, stirred at 200 rpm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetic buffer (pH\u0026thinsp;=\u0026thinsp;5) with 25 mL Viscozyme and cellulase. Incubation at 40\u0026deg;C for 4 days, stirred at 200 rpm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetic buffer (pH\u0026thinsp;=\u0026thinsp;5) with 25 mL Viscozyme and cellulase. Incubation at 40\u0026deg;C for 4 days, stirred at 200 rpm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eOxidative digestion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; 16 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e;16 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e;16 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 mL of 15% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e;16 hours\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal time\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 days\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe initial protocol, protocol A, was adapted from the method described by Toto et al. (\u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) for rat feces digestion and modified. The reason for starting with this protocol was that it involves steps that have shown promising results when applied to human feces samples, making it a suitable candidate for adaptation in our study.\u003c/p\u003e\n \u003cp\u003eProtocol A was applied, which included acid, alkaline, enzymatic, and oxidative digestion. In most studies, acidic conditions and potent oxidizing agents were employed to digest feces samples and detect microplastics. However, numerous studies have examined the impact of strong acids and oxidizing agents on the integrity of microplastics (Adedapo et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ortiz et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schrank et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sipps et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). For instance, 65% nitric acid has been shown to be particularly harmful to polymers such as polyamide (PA), polyethylene terephthalate (PET), and polyurethane (PUR), leading to their complete dissolution at elevated temperatures (Schrank et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, significant surface changes have been observed in polyethylene (PE) and polypropylene (PP) after exposure to nitric acid, which reacts with functional groups on the surface of microplastics, resulting in molecular-level alterations) (Schrank et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Adedapo et al. (\u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) emphasized that polypropylene (PP) is highly susceptible to degradation under the influence of nitric acid, with substantial degradation occurring at both room and elevated temperatures.\u003c/p\u003e\n \u003cp\u003eHowever, filtration was impossible immediately after the first step of acid digestion with hydrogen peroxide and nitric acid due to the high cellulose content, which clogged the 20 \u0026micro;m stainless steel filter. Therefore, this protocol was not further optimized.\u003c/p\u003e\n \u003cp\u003eOptimization efforts were directed toward removing cellulose from the samples before applying the combined digestion protocol.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Optimized Pre-Treatment as an Additional Step for Superior Cellulose Removal\u003c/h2\u003e\n \u003cp\u003eEfficient digestion is critical for ensuring accurate downstream analyses, such as polymer identification through \u0026micro;FTIR spectroscopy, and underscores the importance of selecting appropriate reagents and protocols in microplastic research. To achieve high digestion efficiency, pretreatment protocols were essential in overcoming challenges related to cellulose removal.\u003c/p\u003e\n \u003cp\u003eTo address this issue, an additional step was introduced to reduce the cellulose content in the samples. These pre-treatments aim to remove a significant amount of cellulose, which causes issues during digestion, further treatment, and the filtration of digest through a 20 \u0026micro;m stainless steel filter. Therefore, comparing these two methods and concluding which pretreatment method is more compelling is crucial.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eProtocol B\u003c/strong\u003e included a pretreatment step for cellulose removal with a mixture of 8% urea, 8% thiourea, and 8% KOH, followed by acid, alkaline, enzymatic, and oxidative digestion. \u003cstrong\u003eProtocol C\u003c/strong\u003e included Schweizer\u0026rsquo;s reagent as the pretreatment step, followed by acid, alkaline, enzymatic, and oxidative digestion.\u003c/p\u003e\n \u003cp\u003eIn most studies examining the efficiency of the 8% urea, 8% thiourea, and 8% KOH mixture, the efficiency itself depends on the concentration of the mix. An optimal concentration of 8% usually provides an efficiency range of 80\u0026ndash;94%, which is theoretically satisfactory (Budtova \u0026amp; Navard, \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). According to Zhang et al., (\u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), the efficiency of cellulose fiber removal with this mixture is approximately 91%.\u003c/p\u003e\n \u003cp\u003eSchweizer\u0026apos;s reagent consists of copper(II)-hydroxide dissolved in ammonia, forming the tetraamminediaquacopper(II)-hydroxide complex. The reaction between cellulose and Schweizer\u0026rsquo;s reagent is given (Reaction 1). This reagent is classified as a non-derivatizing method of cellulose dissolution because it forms a covalent coordinate bond with copper ions, weakening hydrogen interactions and allowing the cellulose molecule to unravel and disperse (Dias et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The effects of Schweizer\u0026apos;s reagent on cellulose and the mechanism of its dissolution have been extensively described in numerous studies (Dias et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hamzavi et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Przypis et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSchweizer\u0026apos;s reagent is a highly efficient method for removing cellulose from various types of samples. According to research (Yurtsever, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), the effectiveness of Schweizer\u0026apos;s reagent in dissolving cellulose from tea bags is satisfactory, considering that most tea bags consist of cellulose and non-cellulose fibers, with non-cellulose fibers making up 30\u0026ndash;35% of the mass. Their study confirms that Schweizer\u0026apos;s reagent dissolves only cellulose, leaving non-cellulose material in the mentioned percentage composition after digestion. The study demonstrated that cellulose recovery from toilet paper using Schweizer\u0026apos;s reagent was 100%, indicating the reagent\u0026apos;s high efficiency in dissolving cellulose (Gupta et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eHowever, this reagent has not been previously applied to remove cellulose to facilitate microplastic detection in feces samples.\u003c/p\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1. Optimization of the amount of Schweizer\u0026rsquo;s reagent for efficient cellulose removal from children\u0026apos;s feces samples\u003c/h2\u003e\n \u003cp\u003eThe conditions for using Schweizer\u0026rsquo;s reagent were optimized to enhance cellulose removal efficiency from children\u0026apos;s feces samples. A key aspect of this optimization was determining the optimal ratio between reagent volume and sample mass to ensure effective cellulose digestion and easy filtration. Various combinations were tested, and the results are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMass-to-volume ratios of Schweizer\u0026rsquo;s reagent and sample mass\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample mass (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSchweizer volume (mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMass: Volume ratio\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRatio (mL/g)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e184.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e144.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eTesting revealed that 120 mL and 50 mL of Schweizer\u0026rsquo;s reagent per 1 g of sample caused significant filtration difficulties due to increased solution viscosity. While a larger reagent volume dissolves more cellulose, it also increases viscosity, making filtration challenging. These findings indicate that increasing the reagent volume beyond a certain point does not improve the overall process.\u003c/p\u003e\n \u003cp\u003eAmong the tested ratios, 40 mL of Schweizer\u0026rsquo;s reagent per 0.6 g of sample provided the best balance between complete cellulose digestion and effective filtration. This ratio was selected as the optimal condition. While increasing the reagent volume improves cellulose solubility, excessive amounts raise viscosity, hindering filtration. Conversely, using too little reagent may result in incomplete cellulose digestion.\u003c/p\u003e\n \u003cp\u003eAdditionally, using 40 mL of Schweizer\u0026rsquo;s reagent per 0.6 g of sample proved cost-effective, reducing reagent consumption without compromising analysis quality. Thus, while reagent volumes above 100 mL/g enhance cellulose solubility, they also increase viscosity and complicate filtration. On the other hand, volumes below 50 mL/g may lead to incomplete digestion.\u003c/p\u003e\n \u003cp\u003eBoth urea, thiourea, KOH reagent, and Schweizer\u0026rsquo;s reagent effectively remove cellulose. Still, the use of Schweizer\u0026rsquo;s reagent drastically reduces the amount of cellulose that interferes with microplastic detection via micro FTIR.\u003c/p\u003e\n \u003cp\u003eSchweizer\u0026rsquo;s reagent proved to be much more effective at removing cellulose than the 8% urea, 8% thiourea, and 8% KOH mixture, as demonstrated by \u0026micro;FTIR filter images after the digestion of children\u0026rsquo;s feces samples from this study. A comparison of residues after digestion using Schweizer\u0026rsquo;s reagent and the 8% urea, 8% thiourea, and 8% KOH mixture is presented in Figs. 1A and 1B. Treatment with Schweizer\u0026rsquo;s reagent results in significantly fewer residual fibers, more uniform particles, and filter-free cellulose traces, which is not the case with the 8% urea, 8% thiourea, and 8% KOH treatment. The digestion residues differ notably, with a higher content of undigested material present after the 8% urea, 8% thiourea, and 8% KOH treatment, highlighting the superiority of Schweizer\u0026rsquo;s reagent in cellulose removal. Its application in this study effectively reduced the cellulose content, allowing for smoother sample treatment, particularly during filtration using stainless steel filters with 20 \u0026micro;m pores.\u003c/p\u003e\n \u003cp\u003eSchweizer\u0026rsquo;s reagent removes cellulose more effectively, resulting in significantly fewer residual fibers and more uniform particle distribution. At the same time, the urea-thiourea-KOH mixture leaves a higher amount of undigested material, including visible cellulose residues.\u003c/p\u003e\n \u003cp\u003eThe most promising protocol was protocol C, which was chosen for further optimization.\u003c/p\u003e\n \u003cp\u003eVarious modifications of Protocol C were tested to ultimately develop Protocol D, which was found to be effective for microplastic isolation. Several different procedures were applied to the samples to establish the final Protocol D.\u003c/p\u003e\n \u003cp\u003eBased on the proven efficiency of Schweizer\u0026apos;s reagent in removing cellulose from various sample types, including complete cellulose recovery from toilet paper (Gupta et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) and selective dissolution of cellulose fibers from tea bags (Yurtsever, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), enzymatic digestion was not necessary. Besides, enzymatic digestion was also excluded because of further limitations: very time-consuming processing; the specificity of the enzymes can hardly be adequate in complex matrices; the necessity of optimization of conditions like pH, temperature, and enzyme concentration (L\u0026ouml;der et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Toto et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Von Friesen et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e); and the high cost of enzymes, which is less economical compared to Schweizer\u0026apos;s reagent.\u003c/p\u003e\n \u003cp\u003eNitric acid digestion was avoided because Schrank et al. (\u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Adedapo et al. (\u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) have reported its degradative effects on PA, PET, and PUR polymers, aside from its inducing significant surface modifications in PE and PP. Digestion was not successful without the use of potassium hydroxide, which proved to be an essential reagent for the effective decomposition of organic matter.\u003c/p\u003e\n \u003cp\u003eOur results showed no significant differences when the acidic digestion step is omitted (Fig. 2A and 2B).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Evaluation of proposed protocol (Protocol D)\u003c/h2\u003e\n \u003cp\u003eAcidic digestion with nitric acid was excluded, an essential adjustment to protect the integrity of microplastics. The digestion protocol was further optimized to avoid enzymatic steps and significantly improve the overall digestion time. Enzymatic digestion steps using Viscozyme L and Cellulase were omitted. The final protocol D included pretreatment with Schweizer\u0026rsquo;s reagent, followed by alkaline and oxidative digestion, thereby eliminating the acid and enzymatic digestion steps.\u003c/p\u003e\n \u003cp\u003eThe images show no significant differences when enzymatic and acidic digestion steps are omitted. The comparison of residual material after digestion, shown in Figs.\u0026nbsp;3A and 3B, clearly demonstrates that excluding acidic and enzymatic steps in digestion does not influence the effectiveness of cellulose removal from the samples. In both cases, the cellulose was effectively removed, which can be explained by the superior efficiency of Schweizer\u0026apos;s reagent. This reagent facilitates the complexation of cellulose and its solubilization, making enzymatic treatment unnecessary. Also, the even particle distribution in the filter means that the omission of these steps does not lead to significant changes in the composition or morphology of the residual material after digestion.\u003c/p\u003e\n \u003cp\u003eChemical mapping of the filter by \u0026micro;FTIR after sample digestion is presented in Figs. 3C and 3D. Figure 3C represents the result of a non-optimized process, whereas 3D represents the result of an optimized process. When digestion is not fully optimized (3C), a more significant amount of residual organic and inorganic material remains on the filter, making the identification and quantification of microplastics more difficult due to spectral interferences. In contrast, the optimized process (3D) significantly reduces the amount of residual material, resulting in a cleaner filter and enabling more precise \u0026micro;FTIR mapping of microplastic particles.\u003c/p\u003e\n \u003cp\u003eSeveral parameters were examined to validate the digestion method, assess its efficiency, and determine whether microplastic particles are lost during digestion. These parameters include digestion efficiency, recovery rates, and polymer integrity.\u003c/p\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2 Digestion efficiency\u003c/h2\u003e\n \u003cp\u003eThe efficiency of the proposed digestion protocol for feces samples (protocol D) was evaluated. The results of the digestion efficiency are 99.95% \u0026plusmn;and 0.03%. The results indicated a highly efficient digestion process, with minimal residual mass detected on the filters. The slight differences in filter mass post-digestion suggest that nearly all organic material was effectively degraded, leaving behind only trace amounts. Such high efficiency (above 99.9%) demonstrates that protocol D is a highly reliable method for feces digestion, enabling the complete removal of organic matter while preserving the structural integrity of microplastics for further analysis. Compared to literature values, where digestion efficiencies of 98% (Toto et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) and 97% (Yan et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) were reported, the efficiency achieved in this study underscores the superiority of the optimized protocol. This highlights the potential of Schweizer\u0026rsquo;s reagent as a robust tool for preparing samples for microplastic detection.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.3 Recovery of MPs particles subjected to developed protocol for digestion\u003c/h2\u003e\n \u003cp\u003eTo evaluate the developed protocol (D) for digestion, the recovery (%) of MP particles after digestion was determined by microFTIR analysis.\u003c/p\u003e\n \u003cp\u003eA polystyrene (PS) standard solution was used, containing approximately 3.6 x 10\u003csup\u003e4\u003c/sup\u003e spherical particles/mL, with an average particle diameter of 100 \u0026micro;m, as confirmed by the manufacturer\u0026rsquo;s specifications. The standard was diluted to 18 particles per 100 \u0026micro;L, which was directly applied to feces samples. The samples were then digested using protocols B and D, and particle recovery was evaluated through \u0026micro;FTIR characterization. The results of the recovery rate were 128% \u0026plusmn; 24%. The recovery test results demonstrated no significant loss of PS standard particles during digestion when protocol D is applied. These values are consistent with those reported in the literature, indicating that the observed recovery range is acceptable.\u003c/p\u003e\n \u003cp\u003eFigure 4A presents an optical image of the filter obtained after digestion of the feces sample with PS spike. PS standard differed from the PS found in the sample and was easily observed (Fig.\u0026nbsp;4B).\u003c/p\u003e\n \u003cp\u003eA PS standard was also analyzed under an optical microscope (Olympus Microscope BX51M) using UV and visible light to assess potential surface structure and particle dimension changes. The analysis confirmed that there were no significant changes in particle size or surface characteristics (Figures \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). No structural alterations were observed on the particle surface, which retained its regular spherical shape and exhibited a characteristic reflective pattern.\u003c/p\u003e\n \u003cp\u003eIn addition, the recovery test was performed for solid in-house standards PS, PA, PP, HDPE/LDPE, and PET (A list of polymer standards with their average size and density are presented in Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). The recovery values for PS, PA, PP, HDPE, and LDPE were 100% \u0026plusmn;10%, indicating complete recovery without variability. In contrast, the recovery value for PET was 89% \u0026plusmn; 20%, reflecting more significant variability in the recovery process for this polymer.\u003c/p\u003e\n \u003cp\u003eTherefore, this method proves reliable and suitable for application in the digestion and subsequent analysis of microplastics in children\u0026apos;s feces samples.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.4 Polymer integrity\u003c/h2\u003e\n \u003cp\u003eTo check the influence of the optimized digestion protocol on the integrity of MPs, chemical identification of standard particles (LDPE, HDPE, PP, PVC, PS, PA, PET) was performed before and after the complete digestion procedure by micro-FTIR\u0026mdash;besides, FTIR spectra of standard particles after digestion were compared with spectra libraries (Fig. 5). As expected, applying Schweizer\u0026rsquo;s reagent, alkaline digestion, and oxidative digestion resulted in polymer integrity (in-house standards) ranging from 75\u0026ndash;92%, based on the match with spectra from the instrument\u0026apos;s database. Similarly, the integrity of the PS Sigma-Aldrich standard ranged from 70\u0026ndash;95%, which can be considered well-preserved. These results indicated that the digestion process does not affect the polymer integrity of the standards (PET, PS, PA, PP, HDPE, and LDPE) used for this analysis.\u003c/p\u003e\n \u003cp\u003eAdditionally, the maximal weight of children\u0026apos;s feces samples that can be effectively digested using this protocol was determined. Seven different weights of feces samples in a range of 0.40- 1.00 g were processed using Protocol D. The maximum feces mass that can be successfully digested under the mentioned conditions of this protocol was 1 g.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.5 Characterization and quantification of microplastic particles in stool samples from Dalmatia\u003c/h2\u003e\n \u003cp\u003eThe optimized protocol was applied for quantifying and characterizing microplastics in stool samples obtained from the Children\u0026apos;s Hospital (n\u0026thinsp;=\u0026thinsp;14) collected from Dalmatia, a coastal region of Croatia. The sample mass ranged from 0.6 to 0.7 g, corresponding to the optimal range determined during the prior optimization of sample quantity for microplastic analysis.\u003c/p\u003e\n \u003cp\u003eMicro-FTIR analysis enabled the identification, quantification, and characterization of microplastic particles in the examined samples. The analyzed parameters included sample mass, polymer type, spectral match percentage with reference libraries, particle size and size distribution, particle shape, total number of microplastics per sample, and the number of microplastic particles per gram of sample (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacterization of microplastic particles detected in stool samples: sample mass, polymer types, spectral match with reference library, particle size and size range, morphological classification, number of microplastics per sample, and concentration (number of MP particles per gram). PE (polyethylene), LDPE (low-density polyethylene), HDPE (high-density polyethylene), PP (polypropylene), PET (polyethylene terephthalate), PCL (poly(caprolactone)), PS (polystyrene)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMass of sample\u003c/p\u003e\n \u003cp\u003e[g]\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolymer type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMatch with library (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSize of MP (\u0026micro;m)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRange of size (\u0026micro;m)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eShape\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of MPs per sample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of MP/g\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" align=\"left\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.34 x 95.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" align=\"left\"\u003e\n \u003cp\u003e6.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDPE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.33 x 98.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpheroid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e204.09 x 158.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u0026ndash;300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDPE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e177.32 x 98.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.24 x 43.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u0026ndash;100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" align=\"left\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.33 x 89.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" align=\"left\"\u003e\n \u003cp\u003e7.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.09 x 40.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026ndash;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpheroid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e141.44 x 98.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFiber\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.45 x 88.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u0026ndash;100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.45 x 127.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e235.93 x 185.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u0026ndash;300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e3.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250.44 x 203.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u0026ndash;300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e163.83 x 111.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.03 x 45.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026ndash;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpheroid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.04 x 98.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFragment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHDPE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e160.55 x 73.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFiber\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eMicroplastics were detected in 50% of the samples, while no microplastics were identified in the remaining 50%. This absence may indicate that microplastics were present below the detection limit of the micro-FTIR spectroscopy method or reflect individual variations in subjects\u0026apos; exposure to microplastics. The number of microplastic particles per sample ranged from 0 to 5, with a corresponding concentration varying between 1.18 and 7.25 particles per gram of sample.\u003c/p\u003e\n \u003cp\u003eMP particles detected in stool samples were classified into four size categories: 0\u0026ndash;50, 50\u0026ndash;100, 100\u0026ndash;200, and 200\u0026ndash;300 \u0026micro;m. The majority of particles fell within the 100\u0026ndash;200 \u0026micro;m range. The lengths of detected MPs ranged from 40.09 to 250.44 \u0026micro;m, while widths varied between 40.85 and 203.35 \u0026micro;m.\u003c/p\u003e\n \u003cp\u003eThree distinct MP morphologies\u0026mdash;fibers, fragments, and spheroids\u0026mdash;were identified in the analyzed stool samples (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Fragments were the most prevalent (68.75%), followed by spheroids (18.75%) and fibers (12.50%). The diversity of microplastic shapes is further illustrated in Figure S3, which presents an enlarged view of a silicone filter containing digested stool material. This image was acquired using an optical microscope (Olympus BX51M) under both visible (A) and UV light (B). However, not all particles observed via optical microscopy can be conclusively characterized as microplastics. Therefore, the particles were further analyzed and characterized using a micro-FTIR instrument. Additionally, Figure S3 (C) displays the entire filter, demonstrating an even distribution of particles with a pronounced contrast between the particles and the background. This image was captured using a micro-FTIR microscope. Furthermore, Figure S3 (D) presents a chemical mapping of the particles present on the filter, providing valuable insights into their composition.\u003c/p\u003e\n \u003cp\u003eThe micro-FTIR analysis revealed the presence of five different types of MP: polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), poly(caprolactone) (PCL), and polystyrene (PS) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The most widely distributed and abundant MP type was polyethylene (56.25%), suggesting food and bavarages packaging, including bottled water and plastic bags, and the main source of exposure to MP through ingestion.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents an innovative approach to efficiently removing cellulose from human feces samples, significantly enhancing digestion processes and the analysis of microplastics. Due to its complex structure and chemical properties, cellulose poses a challenge during digestion and interferes with recording infrared spectra on a micro-Fourier transform spectrometer (µFTIR). In this research, Schweizer's reagent was applied for the first time to remove cellulose from children's feces samples for microplastic detection. Schweizer's reagent successfully complexed cellulose into a soluble form, enabling efficient digestion while preserving the structural integrity of microplastics.\u003c/p\u003e\n\u003cp\u003eFour digestion protocols were evaluated, with the proposed protocol emerging as the most effective for digesting children's feces samples. The digestion efficiency of 99.98%\u0026nbsp;was achieved, demonstrating the protocol's reliability. Recovery testing of polystyrene (PS) microplastic particles showed a recovery value of 128 ± 24%, confirming no significant loss of microplastics during digestion. Furthermore, the polymer integrity of standards, including PET, PS, PA, PP, HDPE, and LDPE, ranged from 75% to 95%. These results confirmed that the digestion conditions did not compromise microplastic polymers’ chemical structure or physical properties.\u003c/p\u003e\n\u003cp\u003eThe application of Schweizer's reagent in this context represents a significant advancement, providing a reliable and cost-effective solution for feces digestion. This optimized protocol eliminates harsh acids and enzymatic treatments while ensuring accurate recovery and analysis of microplastics.\u003c/p\u003e\n\u003cp\u003eMicroplastic content was determined in 14 feces samples from Dalmatia, of which 7 samples contained microplastics. Among the detected polymers, polyethylene was the most prevalent. The number of microplastic particles per sample ranged from 0 to 5, corresponding to concentrations between 1.18 and 7.25 particles per gram of sample. The most dominant particle shape was fragment (68.75%), while the most common particle size ranged from 40.09 to 250.44 µm in length and from 40.85 to 250.44 µm in width.\u003c/p\u003e\n\u003cp\u003eThe main advantage of this method for digesting feces samples lies in its simplified approach. It enables us to digest more significant quantities of samples efficiently with minimal steps. After digestion, the sample contains fewer fibers and cellulose, further simplifying the analysis of microplastics and interpretation of results. This methodology avoids acidic digestion processes, uses fewer chemicals, and provides the possibility of recycling Schweizer's reagent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available through the University of Belgrade \u0026ndash; Faculty of Chemistry repository of data: https://hdl.handle.net/21.15107/rcub_cherry_6483.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee for the Use of Human Biological Material for Research at the University of Belgrade - Faculty of Chemistry. The ethical approval was granted during an online session on October 10, 2023, for the research titled \u0026quot;Investigation of Microplastic Content in Stool Samples from the Pediatric Population.\u0026quot; The study was conducted under the guidance of Dr. Tanja Ćirković Veličković at the University of Belgrade. The approval followed a thorough review of the documentation, including the research request, forms for informed consent, and approval from the Ethics Committee of the Srebrnjak Children\u0026apos;s Hospital in Zagreb, Croatia (approval number: 04-930/3-21). The study protocol was in full compliance with ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project has received funding from the European Union\u0026rsquo;s Horizon 2020 research and innovation programme under grant agreement No 965173. Work presented in this manuscript has been partially supported by the Serbian Academy of Sciences and Arts (grant number F-26) and the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract number: 451-03-47/2025-03/200168).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorContributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMilo\u0026scaron; Ilić\u003c/strong\u003e, Methodology, Investigation, Validation, Writing \u0026ndash; original draft; \u003cstrong\u003eTamara Mutić,\u0026nbsp;\u003c/strong\u003eMethodology, Investigation, Validation, Writing - original draft;\u003cstrong\u003e\u0026nbsp;Dragana Stanić-Vučinić\u003c/strong\u003e, Conceptualization, Visualization, Writing - review and editing; \u003cstrong\u003eMirjana Turkalj,\u0026nbsp;\u003c/strong\u003eContribution, Writing, Investigation; \u003cstrong\u003eIvana Banić\u003c/strong\u003e, Contribution, Writing, Investigation; \u003cstrong\u003eJelena Mutić\u003c/strong\u003e,\u0026nbsp;Validation,\u0026nbsp;Supervision, Writing- review and editing; \u003cstrong\u003eTanja Cirkovic Velickovic\u003c/strong\u003e, Conceptualization, Funding acquisition, Supervision, Writing - review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project has received funding from the European Union\u0026rsquo;s Horizon 2020 Research and Innovation Programme under grant agreement No 965173. The work presented in this manuscript has been partially supported by the Serbian Academy of Sciences and Arts (grant number F-26) and the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract number: 451-03-136/2025-03/200168).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbasi S, Turner A (2021) Human exposure to microplastics: A study in Iran. 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Carbohydr Polym 81(3):668\u0026ndash;674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2010.03.029\u003c/span\u003e\u003cspan address=\"10.1016/j.carbpol.2010.03.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Reaction","content":"\u003cp\u003eReaction 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6427909/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6427909/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe content, characteristics, and distribution of microplastics (MP) in feces samples are crucial for the investigation of human exposure and health risks. Feces, being rich in cellulose, is a particularly complex matrix for MP analysis. Numerous studies of microplastics in different matrices show how to remove organic matter from samples, but there are very few studies on the removal of cellulose.\u003c/p\u003e \u003cp\u003eIn this study, an efficient protocol for the digestion of children\u0026rsquo;s feces was developed and optimized as a combination of the innovative cellulose removal treatment of samples with previously known alkaline/oxidation treatments. To remove the cellulose, 40 mL of Schweizer's reagent was added for every 600 mg of dry sample for 40 mins. After that, the samples were passed through a 20 \u0026micro;m mesh sieve and washed with ultra-pure water. Samples were then subjected to alkaline digestion using 10% KOH for 24 hours at 40\u0026deg;C, followed by oxidative digestion using 30 mL of 15% hydrogen peroxide for 16 hours. MP content was determined in 14 feces samples from the Croatian region of Dalmatia, of which 7 samples contained MP. The number of MP ranged from 0 to 5, corresponding to concentrations between 1.18 and 7.25 particles per gram of sample. Among the detected polymers, polyethylene was the most prevalent (56% of particles) and the most dominant particle shape was fragment (68.75%). In comparison to alternative methods used for MP analysis in human feces, tour method efficiently remove cellulose and allow digestion of the matrix in a cost-effective and time efficient manner, allowing subsequent analysis by microFTIR.\u003c/p\u003e","manuscriptTitle":"The application of Schweizer's reagent with FTIR imaging spectroscopic solutions for microplastics advanced analysis of feces samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 14:10:15","doi":"10.21203/rs.3.rs-6427909/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc797735-7717-40c7-95d5-e1d861f868c8","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-15T05:24:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 14:10:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6427909","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6427909","identity":"rs-6427909","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}