An efficient method for extracting microplastics from feces of different species

Journal Pre-proof An efficient method for extracting microplastics from feces of different species Zehua Yan, Huajin Zhao, Yanping Zhao, Qiande Zhu, Ruxia Qiao, Hongqiang Ren, Yan Zhang

PII:

S0304-3894(19)31443-8

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121489

Reference:

HAZMAT 121489

To appear in:

Journal of Hazardous Materials

Received Date:

31 July 2019

Revised Date:

16 October 2019

Accepted Date:

16 October 2019

Please cite this article as: Yan Z, Zhao H, Zhao Y, Zhu Q, Qiao R, Ren H, Zhang Y, An efficient method for extracting microplastics from feces of different species, Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121489

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Title page Title An efficient method for extracting microplastics from feces of different species

Authors: Zehua Yan1, Huajin Zhao1, Yanping Zhao2, Qiande Zhu3, Ruxia Qiao1, Hongqiang

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Ren1, Yan Zhang1*

Affiliations:

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1. State Key Laboratory of Pollution Control and Resource Reuse, School of the

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Environment, Nanjing University, Nanjing, Jiangsu 210023, China 2. School of Environment, Nanjing Normal University, Nanjing 210023, China

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3. State Key Laboratory of Hydrology-Water Resources and Hydraulic Engineering,

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Nanjing Hydraulic Research Institute, Nanjing, Jiangsu 210029, China

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Corresponding author:

Yan Zhang: [email protected]

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ORCID ID: 0000-0002-4762-6639

Graph abstract

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An efficient method for extracting MPs from feces of different species is developed. 97% of MPs are recovered from feces and no effect on features of the MPs are observed. Different types of MPs are identified in the feces of IBD patients.

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  

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Highlights

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ABSTRACT

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Concerns have been raised regarding the ingestion of microplastics (MPs) by numerous organisms including humans. However, no efficient and standardized

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methods are available for extracting MPs from feces. In this study, we introduce a novel approach with high digestion efficiency that involves using Fenton’s reagent

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and nitric acid to remove feces solids. Firstly, Fenton’s reagent was used to degrade

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small solids and decompose large solids into small pieces. Secondly, nitric acid was used to digest the remaining solids and filters. Furthermore, absolute ethyl alcohol was used to remove the mineral residues wrapped on the plastic surfaces and disperse MPs. By using this method, 97.78 % MPs can be recovered from human and chicken feces, and no significant changes were observed in the physical and Raman spectral 2

properties of different polymer types of MPs. This method has also been verified by extracting MPs from field feces. Overall, the proposed method can efficiently digest feces solids and extract MPs with higher recovery rate, less intermediate steps and less damage, which can serve as an economical and feasible method for the detection of MPs in the feces of different species.

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Key words: microplastics; extraction methods; feces; Fenton’s reagent; nitric acid

1. Introduction

Microplastics (MPs, < 5 mm) are mainly generated through two sources: (1)

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primary MPs are produced in the micron size and widely used in personal care

products such as cosmetics, facial cleansers, detergents, sunscreens, and drug vectors;

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(2) secondary MPs originate from the breakdown of larger plastics through physical

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wave-action, photodegradation, and biodegradation in the environment [3, 4]. MPs have been widely detected in different environments and MPs pollution has become a

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major focus of research worldwide [1, 2]. In addition, MPs have been detected in various organisms such as birds, fish, and mussels [5-7]. Direct exposure to plastic

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products such as plastic bottles and food packaging bags which may bring MPs into

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the bottled water and food and increase the likelihood of MPs being accidentally ingested by humans [8, 9]. Although many studies speculate that humans are most likely to ingest MPs

through food and drinking water [10, 11], these conclusions are all extrapolation and assumptions based on limited literature [12, 13]. Precise information is rare about the 3

number and characteristics of MPs in human body. However, due to ethical restrictions and technical barriers, it is inconvenient to directly quantify microplastics in the guts of human and large mammals. Fortunately, feces serve as ideal samples for investigating interactions between MPs and gut and intestinal flora [14] and may provide direct evidence of human ingestion of MPs. While a few studies have attempted to identify MPs in animal feces [5, 15, 16], efficient and optimized

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extraction methods remain limited.

MP compositions can be identified by Raman and Fourier transform infrared

(FTIR) spectroscopy [17, 18]. However, it is difficult to directly distinguish MPs from

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organic and inorganic mixtures, which stand as the main technical obstacles to

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identifying MPs from environmental or biological samples. Different ways to separate MPs from organic matter have been introduced, such as physical removal and

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chemical digestion [19, 20]. Physical removal, including filtration and ultrasonic extraction, involves several intermediate steps, during which a considerable number

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of MPs may be lost [21]. Moreover, MPs are wrapped with complex solids in feces,

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which cannot be completely eliminated by physical removal. Chemical digestion is an alternative method, and it involves using hydrogen peroxide (H2O2) [5, 15, 22], nitric

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acid (HNO3) [23], potassium hydroxide (KOH) [24], sodium hydroxide (NaOH) [25] or enzymes [19] to digest organic matter. Nevertheless, these reagents are usually used alone to digest organic matter through time-consuming procedures. At the meanwhile, high digestion temperature and strong chemical reactions will damage MPs [19]. Therefore, efficient and relatively gentle methods are urged for the 4

extraction of MPs from feces. According to previous studies, density separation is always applied to separate MPs after digestion [22, 26]. However, it is not suitable for separating MPs from fecal digestion solution. According to our preliminary experiments, mineral colored digestion residues still wrapped on the plastic surfaces after density separation (using 1.8 g cm-3 NaI) so that it was difficult to identify MPs by visual observation and

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Raman spectra. Some studies have used pyrolysis-gas chromatography mass spectrometry (Py-GC-MS) [19] and thermal extraction desorption gas

chromatography mass spectrometry (TED-GC-MS)[27] to identify MPs. However,

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these methods can only detect a few types of plastic and provide no information about

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the number, size or shape of MPs. Hyperspectral imaging has been used to directly

and complex composition.

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identify MPs in the fish gut [6], but it is not suited to examining feces due to its niff

This study aimed to develop an efficient method for digesting feces and

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extracting MPs involving the following: (1) using chemical digestion methods to

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destroy solids in feces efficiently while minimizing damage to MPs; (2) using absolute ethyl alcohol to dissolve residual organic matter and disperse MPs; (3)

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simplifying intermediate steps to reduce the possible loss of MPs. Digestion efficiency, recovery rates, digestion effects, and validation with field feces were also evaluated.

2. Materials and methods 2.1. Feces sample collection 5

In this study, the feces of human, chicken, and zebrafish were collected. For human feces, the excreted feces were directly loaded into prewashed 2 L brown glass bottles and sealed with wooden lids. For chicken feces, prewashed 2 L brown glass containers were placed under the cages. After cacation, the glass containers were tightly sealed with wooden lids. For zebrafish feces, prewashed 1 L brown glass container with filter cotton was placed in the circulating water tank of the aquarium,

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tightly sealed the upper opening with wooden lids. The feces collected on the filter

cotton were transferred to prewashed 1 L brown glass containers by metal spoon for subsequent tests.

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2.2. Sample digestion

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The glass containers containing feces samples were connected to a vacuum freeze dryer (FreeZone 2.5, LABCONCO, USA) to remove moisture. Lyophilized

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feces samples were then placed in 1 L glass beakers covered with tin-foil to prevent contamination with a magnetic stirrer. Fenton’s reagent was added to each beaker

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according to the dry weight (d.w.) of samples shown in Table 1.

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Fenton’s reagent was composed of 30% hydrogen peroxide (H2O2) and an iron catalyst solution prepared with 20 g iron (II) sulfate heptahydrate in 1 L ultrapure

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water (Milli-Q with a 0.22 μm Millipak final filter). The iron catalyst solution and H2O2 was added into glass beakers in turn according to the volume ratio of 1:2.5. For each addition, the volume of H2O2 did not exceed 50 mL. The digestion lasted less than 5 h below 40℃. After that, the solution was vacuum filtered using mixed cellulose ester membrane filters (47 mm diameter, 1 μm pore size) which are 6

composed of cellulose nitrate and cellulose acetate (CN-CA) [28]. The beakers and filtration devices were rinsed and filtered for three times. The used CN-CA filters were placed in 1 L glass beakers and 65% nitric acid (HNO3) was added and incubated in 50°C water bath for 30 min. The temperature was then increased to 70°C and kept for 10 min to digest more resistant solids in the feces. The CN-CA filters can be fully digested by HNO3. For filtration, the solution was diluted at a 1:2 ratio (v:v)

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with ultrapure water and filtered through polytetrafluoroethylene (PTFE) membrane filters (47 mm diameter, 1 μm pore size). The beakers and filtration devices were

L glass beakers for next extraction analysis.

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2.3. Sample extraction

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rinsed and filtered again for three times. The used PTFE filters were placed into the 1

After digestion, 100 mL of absolute ethyl alcohol was added to the 1 L glass

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beakers containing used PTFE filters mentioned above, followed by ultrasonic treatment (100 KHz) for 10-15 min. Then, the PTFE filters were removed (rinsed with

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absolute ethyl alcohol for three times) and the rest solution was vacuum filtered

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through new PTFE filters again. The extracted particles on the PTFE filters were kept for characterization analysis.

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2.4. Efficiency of digesting feces To evaluate the reduction efficiency of feces solids through our methods, human,

chicken and zebrafish feces were used. Human feces were collected from healthy volunteers. Chicken feces were collected from a local farm. Zebrafish feces were collected from our laboratory. The influence of feces weight on the digestion was 7

tested. Five grams of human and chicken feces and 2 g of human, chicken and zebrafish feces were used. Five grams of zebrafish feces was not detected because insufficient zebrafish feces can be collected and the moisture of the zebrafish feces was very high. For 5 g of feces, 500 mL of 30% H2O2 with iron catalyst and 150 mL of 65% HNO3 were used. For 2 g of feces, 200 mL of 30% H2O2 with iron catalyst and 100 mL of HNO3 were used. The feces were digested as described above. Three

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replicates were performed. After digestion, the filters were weighed and the digestion efficiencies were calculated. 2.5. Recovery rates detection

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Different polymer types and sizes of MPs were respectively mixed with 5 g

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(d.w.) human feces and 5 g (d.w.) chicken feces. Zebrafish feces were not tested in this experiment because 5 g (d.w.) feces is too much for zebrafish. Three types of MPs

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were used (Figure S1): polystyrene (PS, 250 μm), polyethylene (PE, 150 μm), and polyvinyl chloride (PVC, 75 μm). Three replicates were conducted and 30 particles

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(10 particles for each type of MPs) were used for each replicate. The particles were

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digested and extracted as described above. After treatment, the number of MPs was counted with a microscope and the recovery rates of MPs were calculated. Raman

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spectra of 3 particles of each polymer type were detected before and after the treatment. The detection method is described in 2.6. 2.6. Effects of digestion on different types of plastic The effects of the digestion process were tested on five widely used polymers, including PE, PS, PVC, polypropylene (PP), and polyethylene terephthalate (PET) 8

[20, 29]. The test particles were purchased from a plastic raw material factory (Guangzhou, China). These particles have a particle size of 2-4 mm, which is convenient to quantify the number of particles and identify the degradative changes of the particle surface. For each plastic type, 3 replicates were carried out using 3 particles for each replicate (total of 45 particles tested). The samples were digested as described in section 2.2. The same types of undigested plastic particles were used as

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control. After digestion, the particles were removed from the beakers, rinsed thoroughly with ultrapure water, and left to air dry in Petri dishes.

To examine possible changes in physical characteristics, the particles were

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measured using a Nikon SMZ 1000 microscope set to ×10 and ×80 with DT 2000

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software (vers.2.0). Images were photographed to assess the changes in color and surface area of MPs before and after treatment. The changes in particle masses were

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also recorded surface area degradation. To examine the changes in spectroscopic properties, the particles were analyzed using Raman spectra (Renishaw, inVia-Reflex)

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before and after digestion. The space resolution was measured as less than 0.5 μm

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within a wavenumber range of 200 nm to 1050 nm. The spectra were normalized before comparation.

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2.7. Extraction of MPs from field samples Field feces samples, including human and chicken feces were collected to verify

the effectiveness of our method. Ten human feces samples (1-5 g d.w. for each

sample) were collected from Inflammatory Bowel Disease (IBD) patients admitted to a gastroenterology clinic of the Second Affiliated Hospital of Nanjing Medical 9

University (Nanjing, China). All operations performed meet the hospital’s ethical review requirements. Ten chicken feces samples (5 g d.w. for each sample) were collected from a local farm (Nanjing, China). Digestion and extraction were performed as described above. At the meanwhile, five blank control (only ultrapure water without feces) were performed to evaluate the possible contamination during the procedure [25]. All the suspected MPs on the filters were examined using

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microscope and Raman spectra. The samples’ spectra were compared with library data in onboard software of inVia-Reflex. 2.8. Quality control

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Laboratory clothes made from plastic-free materials and cotton gloves were worn

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during the experiment. All labware was washed three times with ultrapure water (Milli-Q with a 0.22 μm Millipak final filter) before the experiment and was inspected

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under a microscope (Nikon SMZ 1000) to make sure no MPs contamination. CN-CA and PTFE filters were tested by Raman spectra before the experiment to ensure no

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MPs contamination.

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2.9. Statistical analysis

Results are presented as mean ± SD. An ANOVA test was conducted to compare

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the digestion efficiency and recovery rate of MPs in different feces. T-test was performed to compare the differences in masses and sizes of MPs in the control and treatment groups. P < 0.05 was considered statistically significant. All analyses were performed using SPSS Statistics (vers.19). 3. Results 10

3.1. Digestion efficiency of feces Human feces before and after digestion are shown in Figure 1A and Figure 1B. The digestion efficiencies for the feces of different species are shown in Figure 1C. In general, the digestion efficiencies all exceed 97% for the tested feces samples. There was no significant difference in the digestion efficiency of feces among different species. In addition, there was no significant difference in the digestion efficiency of

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feces with different weights. 3.2. Recovery rates of MPs

The recovery rates of MPs were detected by mixing three types of MPs and

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feces. After the treatment, MPs could be clearly observed on the PTFE filters (Figure

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2A-C). The recovery of MPs for human and chicken feces is shown in Figure 2D. The recovery of PE-, PS-, and PVC-MPs in human feces was 100%, 100%, and 93.33%,

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respectively. The recovery of PE-, PS-, and PVC-MPs in chicken feces was 100%, 96.67%, and 96.67%, respectively. The overall recovery rate of all types of MPs was

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97.78% for both human feces and chicken feces. For the same species, no significant

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difference was found among different plastic types. For the same plastic types, no significant difference was found between different species. According to the Raman

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spectra, no recognizable differences for the three types of particles were observed before and after the extraction process (Figure 3).

3.3. Effects of digestion on MPs. All types of plastic appeared largely resistant to the digestion process (Table 2). After digestion, the weight of particles changed from -0.40 ± 0.00% to 1.85 ± 0.00%. 11

The equivalent circle diameter changed from -0.74 ± 0.01% to 2.28 ± 0.02% and the minimum diameter changed from -0.69 ± 0.01% to 2.38 ± 0.01%. No significant changes (p > 0.05, t-test) in the masses or sizes of the MPs were observed before and after the digestion process, indicating that digestion caused little physical damage to the MPs. In addition, no degradation was observed on the surfaces of MPs (Figure 4) and

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no spectral changes were observed for the PE-, PP- and PET-MPs (Figure 5A-C).

However, slight changes in color (clear to yellow) were observed for the PVC and PS particles (Figure 4D, E). The Raman spectra of PS-MPs showed no changes after

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digestion (Figure 5D) which was consistent with previous studies [25]. Although the

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Raman spectra of PVC-MPs underwent slight changes due to the digestion, the PVCMPs could still be clearly identified (Figure 5E).

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3.4. MPs extraction from field feces

To verify the introduced method, MPs were extracted from field feces collected

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from local hospital and farm. After treatment, all feces were fully digested as

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described above. No plastic contaminants were observed in the blank samples (n = 5). All MPs on the filters were observed under a microscope and identified by Raman

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spectra. Occurrence of MPs for chicken and IBD patients was 50% (5 out of 10) and

40% (4 out of 10), respectively (Figure 6). MPs found in the chicken feces were confirmed to be nylon fibers and polyethylene terephthalate (PET) particles (Figure S3A and S3B). MPs found in the human feces were identified as polybutylene terephthalate (PBT) and poly (vinyl alcohol-co-vinyl butyral) (PVB) particles (Figure 12

S3C and S3D). In addition, the particle size limit of in this method was approximately 1 μm depended on the pose size of filters and space resolution of Raman spectra. 4. Discussion 4.1. Feces digestion efficiency Digestion is the first step to separate MPs from environmental and biological samples. Various chemicals can be used for the digestion. For instance, 10%

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potassium hydroxide (KOH) has been used to digest biological tissues [24, 30]. However, it is not suitable to digest feces (Figure S2A) because of the different

compositions. Biological tissues are dominated by proteins [22] while feces are

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composed of 75% water and 25% solids [14]. The solids in feces can be further

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divided into proteins, nitrogenous matter and undigested matter such as polysaccharides and fibers. KOH is more suitable for digesting proteins that are easy

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to hydrolyze in alkaline conditions [31]. While the polysaccharides, fibers and inorganic solids in feces are difficult to completely digested by KOH. In addition,

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alkaline hydrolysis cannot eliminate the niff of feces in a short time.

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HNO3 digestion has been proven to be an efficient means to remove organic matter [19, 32], but it requires higher temperatures and longer times and may destroy

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MPs [23, 33, 34]. Even if the reaction temperature and time were adjusted to 50℃ for 30 min or 70℃ for 10 min, HNO3 digestion cannot fully remove the feces solids (Figure S2B). Fenton reaction can generate hydroxyl radicals and is widely applied to degrade contaminants in water [35]. Fenton’s reagent has been used to extract MPs via digestion of sludge, soil, and organism samples [22, 34], however, it cannot fully 13

digest feces solids even use higher temperatures and larger volumes (Figure S2C). The last feces solids including inorganic solids (phosphate, intestinal secretions, and dried ingredient of digestive juice) [36] and oxidation products of larger molecules (small chlorinated alkanes, n-paraffins, and short-chain carboxylic acids) [37] are resistant to oxidation by Fenton reaction. These results suggested that it is difficult to completely digest feces wrapped on the microplastic surface by using single agent.

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To completely digest feces and effectively extract MPs, different digestion

reagents need to be used together. The digestion process in this study involved two

phases: Fenton’s reagent digestion and HNO3 digestion. Due to the rapid reaction rate,

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Fenton’s reagent can quickly reduce the niff of feces in the first phase [38].

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Meanwhile, macromolecular solids can be decomposed into small pieces by Fenton’s reagent [39], which is conductive to the second digestion phase. To reduce the effect

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of digestion on the properties of MPs, Fenton reaction was conducted at below 40 ℃ in our experiments. Increasing temperature can reduce time for digestion, but it is

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harmful to the original characteristics of MPs. High temperature also requires a longer

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cooling time and produces more foam. To prevent the foam overflowing during the Fenton reaction, less than 5 g (d.w.) feces is appropriate in the first phase. In addition,

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the digested samples were filtered using CN-CA filters which can be completely dissolved by HNO3 in the second digestion phase to avoid possible loss of MPs. In the second digestion phase, HNO3 was used to digest the remaining feces

solids and CN-CA filters. HNO3 and H2O2 have been used together to digest biota samples [40], but the mixed solution is easy to spill over [23]. Moreover, H2O2 has a 14

lower reaction rate compared with Fenton’s reagent and is difficult to eliminate the niff of feces. HNO3 and NaOH can also digest fish gut and muscle samples and take a shorter time (< 6 h) than our method [25]. However, our method is adapted to digest feces of more complex composition. Similar with the first digestion phase, temperature is also a key factor for the second digestion phase. According to previous studies [41], we tried 60℃ as the digestion temperature, but the residual organic

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matter could not be completely removed in a short time. So, a higher temperature is required. But the temperature should not exceed 80℃, otherwise the surface

properties of MPs will be severely modified [25]. Finally, 70℃ was selected as the

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maximum temperature for digesting. As a result, the digestion efficiencies of feces of

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chicken, human and zebrafish all exceed 95% and limited effects were induced on the characteristics of MPs. These results suggested that our digestion method is efficient

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and practical. Note that this study only tried fecal samples of limited species. To adapt to more species samples, the final digestion temperature can be further optimized in

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the range of 60-70℃.

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4.2. MPs extraction efficiency

In our preliminary experiment, sodium iodide solution (NaI, 1.8 g cm-3) was used

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for the density separation of MPs from residual feces solids following previous studies [26], but the separation was not optimal and the cost was relative high (Figure S2D). The 96% (v/v) ethyl alcohol has been used as a density separation reagent to extract high density MPs from biological materials [42]. Ethyl alcohol has also been widely used as a solvent for drugs and organic matters [43, 44]. Thus, in this study, 15

absolute ethyl alcohol was used to dissolve the remaining organic matter wrapped onto the surfaces of MPs and to separate MPs from the mineral residues after digestion. Overall, the recovery of all types of MPs exceeded 97% and no significant changes in the properties of MPs were observed after the extraction process, which indicated that our method is effective to extract MPs with no damage. 4.3. Effects of digestion on MPs.

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To evaluate the effects of digestion on MPs, five types of MPs were directly

treated by digestion reagents without feces. The digestion process has no effect on

PS-, PE-, PP- and PET-MPs, only PVC-MPs were affected based on Raman spectra.

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Previous studies have demonstrated that PVC is more easily to be degraded by HNO3

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[19], which was used in our method. While, the same digestion process caused no obvious spectra changes for the MPs extracted from the feces. On one hand, the

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reaction with feces wrapped on the surface of MPs consumed the reagents and limited their effects on MPs. On the other hand, the presence of feces may have also blocked

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the reagents from full contact with the MPs [23].

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4.4. Extraction MPs from field feces. Chicken and human feces from local farm and hospital were used to verify the

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digestion and extraction methods. Nylon fibers and PET particles were found in the chicken feces. The spectra of nylon fibers presented low similarity rates to the standard substance, which may attributed to the ageing of plastics [33]. The digestion and extraction process may also lead to changes in the spectra of nylon fibers, which was not evaluated in this study. The spectra of PET particles present very similar rates 16

to those of the standard substance indicating little effect was caused on these particles. For human feces, PVB and PBT plastics were found. These plastics are widely used in our daily life [46-48]. These MPs in the feces may come from MPs contaminated air, drinking water or food [49]. In addition, PBT plastics are commonly used in medicine [50] indicating that medical devices could be a source of MPs for patients. Some particles in the human feces (Figure S4) could not be identified by

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Raman spectra and their chemical composition could be further confirmed by the

combination of multiple techniques such as FTIR, Py-GC-MS, and TED-GC-MS in future studies.

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5. Conclusions

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In summary, digestion with H2O2 and HNO3 serves as an efficient and feasible way to fully digest feces with limited effects on the physical and optical features of

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most MPs. MPs can also be successfully extracted from feces by using absolute ethyl alcohol. Moreover, using this method, different polymer types and

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shapes of MPs have been successfully detected in chicken and IBD patient feces. The

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results illustrate that our method is effectively adapted for analyzing the levels and properties of MPs remaining in feces. In the future, FTIR characterization could be

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included to assess the modification of the sampled plastics.

Competing interests The authors declare no competing interests.

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Declaration of Interest Statement The authors declare no competing interests. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21777068, 41605129). We also want to thank Professor Faming Zhang of The Second Affiliated Hospital of Nanjing Medical University for providing human feces

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samples for this study.

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Figure caption Figure 1. Human feces before (A) and after (B) digestion and digestion efficiency of human, chicken and zebrafish feces (C). Figure 2. Recovery rates for three types of MPs (PS, PE, and PVC). (A) The PS-MPs on the filters after extraction. (B) The PE- and PS-MPs on the filters after extraction. (C) The PE and PVC-MPs on the filters after extraction. (D) Recovery for each type

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of MPs in human and chicken feces after extraction (No error bars existed for PE and PS in human feces and PE in chicken feces which mean that the recovery was 100% for all triplicates tested). Red arrows indicate MPs.

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Figure 3. Raman spectra of the three types of MPs (PE, PS, and PVC) used in the

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recovery rate detection. Black lines represent Raman spectra of MPs before digestion and red lines represent Raman spectra of MPs after digestion.

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Figure 4. Micrograph images (×10 and ×80) of different types of MPs before and after the digestion. (A) PE-MPs, (B) PET-MPs, (C) PP-MPs, (D) PS-MPs, (E) PVC-

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MPs. The particles were photographed in microscope (×10) and the surface details of

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particles were photographed in microscope (×80) and are shown in the upper right corner of the panel.

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Figure 5. Raman spectra of five types of MPs before and after the digestion. (A) PEMPs, (B) PP-MPs, (C) PET-MPs, (D) PS-MPs, (E) PVC-MPs. Black lines represent Raman spectra of MPs before digestion and red lines represent Raman spectra of MPs after digestion.

Figure 6. MPs detected in field feces. (A) Nylon fibers extracted from field chicken 25

feces. (B) PET particles extracted from field chicken feces. (C) PBT particles extracted from field human feces. (D) PVB particles extracted from field human

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feces. Red arrows indicate MPs.

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Table 1. Reagents and filters used for different sample weights. Fenton’s reagent

(g, d.w.)

a

﹤1

100

40

50

2

1-2

200

80

80

4

2-3

300

120

100

6

3-4

400

160

120

8

4-5

500

200

150

H2O2 (mL)

b

HNO3 (mL)

Number of CNCA filters

Fe2+ (mL)

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a

c

Sample weight

10

30% hydrogen peroxide. b Iron catalyst solution, 20 g of iron (II) sulfate heptahydrate in 1L of

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filtered ultrapure water. C 65% nitric acid.

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f Mass (mg)

Equivalent circle diameter (μm)

Minimum diameter (μm)

pr

Polymer type

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Table 2. Characteristic changes of different MPs caused by digestion a.

Color change

After digestion

Before digestion

After digestion

Before digestion

After digestion

Before digestion

After digestion

PE

0.03±0.00%

-0.01±0.00%

-0.74±0.01%

-0.64±0.00%

-0.69±0.01%

-0.49±0.02%

no

no

PVC

1.85±0.00%

-0.03±0.00%

-0.69±0.00%

-1.84±0.01%

-0.56±0.00%

-0.73±0.01%

yellowing

no

PP

0.03±0.00%

0.03±0.00%

-0.71±0.01%

0.24±0.01%

-1.11±0.01%

0.07±0.01%

no

no

PET

0.58±0.01%

0.08±0.00%

2.28±0.02%

-0.43±0.00%

2.38±0.01%

-0.79±0.00%

no

no

PS

-0.40±0.00%

0.03±0.00%

0.58±0.02%

0.49±0.02%

0.41±0.02%

yellowing

no

Pr

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Results are presented as the mean ± SD of the three replicates per treatment (three particles per replicate).

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Before digestion

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