- Email: [email protected]
Impact of artificial digestion on the sizes and shapes of microplastic particles
Journal Pre-proof Impact of artificial digestion on the sizes and shapes of microplastic particles Valerie Stock, Christoph Fahrenson, Andreas Thuenemann, Merve Hilal Dönmez, Linn Voss, Linda Böhmert, Albert Braeuning, Alfonso Lampen, Holger Sieg PII:
S0278-6915(19)30800-2
DOI:
https://doi.org/10.1016/j.fct.2019.111010
Reference:
FCT 111010
To appear in:
Food and Chemical Toxicology
Received Date: 21 October 2019 Revised Date:
26 November 2019
Accepted Date: 27 November 2019
Please cite this article as: Stock, V., Fahrenson, C., Thuenemann, A., Dönmez, M.H., Voss, L., Böhmert, L., Braeuning, A., Lampen, A., Sieg, H., Impact of artificial digestion on the sizes and shapes of microplastic particles, Food and Chemical Toxicology (2019), doi: https://doi.org/10.1016/ j.fct.2019.111010. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Together with Holger Sieg, Albert Braeuning, Linda Böhmert, Linn Voss and Alfonso Lampen, Valerie Stock was responsible for the conceptualization of the present study. Andreas Thuenemann developed the methodology for the different digestion steps. Christoph Fahrenson, Merve Hilal Dönmez and Valerie Stock acquired all data shown in the paper. The first draft was created by Valerie Stock and Holger Sieg. This work was supported by the German Federal Institute for Risk Assessment (project 1323-102 and 1329-003).
Impact of artificial digestion on the sizes and shapes of microplastic particles
Valerie Stock1, Christoph Fahrenson2, Andreas Thuenemann3, Merve Hilal Dönmez1, Linn Voss1, Linda Böhmert1,*, Albert Braeuning1, Alfonso Lampen1, Holger Sieg1
1 German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany 2 Technical University Berlin, Electron Microscopy Core Facility, Straße des 17. Juni 135, 10623 Berlin 3 German Federal Institute for Materials Research and Testing, Berlin, Germany
*Corresponding author: Linda Böhmert, German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany. Phone: +49 (30) 18412-3718, E-mail: [email protected]
KEYWORDS: microplastic, oral uptake, particle size, gastrointestinal barrier, artificial digestion
1
ABSTRACT
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Current analyses show a widespread occurrence of microplastic particles in food products and raise
3
the question of potential risks to human health. Plastic particles are widely considered to be inert due
4
to their low chemical reactivity and therefore supposed to pose, if at all only minor hazards.
5
However, variable physicochemical conditions during the passage of the gastrointestinal tract gain
6
strong importance, as they may affect particle characteristics. This study aims to analyze the impact
7
of the gastrointestinal passage on the physicochemical particle characteristics of the five most
8
produced and thus environmentally relevant plastic materials polyethylene, polypropylene, polyvinyl
9
chloride, polyethylene terephthalate and polystyrene. Scanning electron microscopy (SEM) and
10
subsequent image analysis were employed to characterize microplastic particles. Our results
11
demonstrate a high resistance of all plastic particles to the artificial digestive juices. The present
12
results underline that the main stages of the human gastrointestinal tract do not decompose the
13
particles. This allows a direct correlation between the physicochemical particle characteristics before
14
and after digestion. Special attention must be paid to the adsorption of organic compounds like
15
proteins, mucins and lipids on plastic particles since it could lead to misinterpretations of particle
16
sizes and shapes.
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1 INTRODUCTION
The environmental pollution with plastic debris is one of the greatest challenges scientists are facing in recent times. The worldwide production volume of plastics is constantly increasing. Whereas 322 million tons of plastics were produced in 2015, global production grew by 3.85% to 335 million tons in 2016 (PlasticsEurope 2018). With the increase in plastics production, the impact on the environment also increases (Barnes et al. 2009). Intentionally produced microscaled plastic particles, called primary microplastics, or plastic particles originated by UV degradation and other environmental factors (secondary microplastics) can enter human food and beverages through the food chain or by entry into environmental resources like drinking water (Napper et al. 2015; Thompson 2015; van Wezel et al. 2016). To date, there is no uniform microplastic size definition, but a size range between 100 nm and 5 mm is usually assumed. Plastic particles below 100 nm are commonly defined as nanoplastics (EFSA 2016). The bigger size range is rather an environmental issue, but particles below 150 µm in size are considered to be, in principal, systemically bioavailable to humans, and particles below 4 µm can be taken up by intestinal cells (EFSA 2016; Stock et al. 2019). The most commonly produced types of polymers are polypropylene (PP) > polyethylene (PE) > polyvinyl chloride (PVC) and > polyethylene terephthalate (PET) (PlasticsEurope 2018). Recent publications show a contamination of various food products with microplastic particles indicating a widespread exposure (Karami et al. 2017; Liebezeit and Liebezeit 2013; Liebezeit and Liebezeit 2014; Oßmann et al. 2018; Schymanski et al. 2018; Van Cauwenberghe and Janssen 2014; Yang et al. 2015). Microplastics can thereby be enriched in the food chain as well as introduced into foodstuff through the air, which indicates the ubiquitous presence of plastic particles (Allen et al. 2019; Smith et al. 2018). There are initial attempts to standardize analytical methods in order to better estimate human 1
exposure (Hanvey et al. 2017). To date, there are still large knowledge gaps on the hazard of microplastics (EFSA 2016). Plastic materials are usually considered as chemically very unreactive due to their large molecular size. A potential bioreactivity of microplastic particles is therefore more likely to be expected due to the increased surface-to-volume ratio with decreasing particle sizes than due to their chemical properties per se. Moreover, adverse effects from microplastics may result from their ability to function as a carrier for environmental pollutants, from additives like plasticizers and flame retardants or from unreacted residual monomers which may remain in the plastic materials due to incomplete polymerization reactions (Araújo et al. 2002; Bouwmeester et al. 2015; Browne et al. 2007). After oral uptake, particles are subjected to the influence of the various matrices in the digestive tract sections before reaching the intestine. Because the plastic particle size is of great importance for its potential uptake at the intestinal barrier, it is important to understand the influence of the different digestive juices on the particle characteristics (Hussain et al. 2001). Accordingly, it is of significance for a potential uptake whether ingested plastic particles are degraded to smaller fragments during the gastrointestinal passage. Gastric acid in particular might corrode the plastic particles or change their shape, making them potentially more bioreactive. The aim of this work was to investigate the fate of microplastic particles of different plastic materials during the gastrointestinal passage by measuring particle sizes and shapes after the different digestion steps. The present data may serve as a basis for further in vivo experiments in order to assess the potential decomposition of the particles and thus also the resulting effects. This is essential for a reliable future risk assessment of microplastics.
2
2 METHODS 2.1 Chemicals and microplastics
Chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), or Carl Roth (Karlsruhe, Germany) if not otherwise indicated. The 4 µm polystyrene (PS) particles (PFL-4070, Sky Blue fluorescent particles, size 3.6 – 4.5 µm) were purchased from Kisker Biotech GmbH & Co. KG (Steinfurt, Germany), PET (ES 306030) from Goodfellow GmbH (Hamburg, Germany) PE (429015), PP (427861) and PVC (81387) from Sigma-Aldrich (Taufkirchen, Germany). All plastic particles were purchased in pure form from the manufacturers.
2.2 Polypropylene grinding
PP was purchased in granulate form and thus had to be further ground to microplastic particles. This was conducted by Fritsch GmbH using the Pulverisette 14 premium line rotor cutting ball mill (Fritsch GmbH, Idar-Oberstein, Germany). The PP granules were first embrittled in liquid nitrogen. They were then crushed with a maximum rotor speed of >110 m/s and a maximum speed of 22,000 rpm. In this experiment, a rotor with 12 ribs in combination with a sieve ring with 0.5 mm trapezoidal perforation was used for grinding. A quantity of 25 g was ground within 7 minutes.
3
2.3 Artificial in vitro digestion
The artificial in vitro digestion protocol, originally based on the DIN ISO 19738 , has been modified for scientific investigations on nanoparticles and biopolymers (Kästner et al. 2017). The artificial in vitro digestion system essentially considered the 3 digestive compartments mouth, stomach and intestine. The corresponding digestive juices were reproduced according to their respective compositions (table 1).
Table 1: Composition of artificial digestive fluids. Saliva (pH 6.4)
Gastric fluid (pH 2)
Intestinal fluid (pH 7.5)
Substance
c [mg/mL]
Substance
c [mg/mL]
Substance
c [mg/mL]
NaCl
1.667
NaCl
4.143
NaCl
0.3
NaSCN
0.5
KCl
1
CaCl2*2H2O
0.5
Na2SO4
1.833
KH2PO4
0.386
MgCl2*6H2O
0.2
NaHCO3
0.5
Mucin
4.286
NaHCO3
1.0
KCl
1.5
Pepsin
1.429
Ureate
0.3
KH2PO4
2.0
Conc HCl
1 Drop
Trypsin
0.3
CaCl2*2H2O
0.5
10% HCl
2 Drops
Pancreatin
9
Urate
0.33
Bile extract
9
Urea
0.033
NaHCO3 (s)
For titration
4
Alpha-
0.833
Amylase Mucin
2.5
100 mg/mL (PE, PET, PVC), 50 mg/mL (PP) or 10 mg/mL (PS) of the plastic particles were dispersed in 16 mL of synthetic saliva and incubated under agitation at 37 °C in a water bath for 5 min. Prior to the addition of 14 mL of gastric juice, 10 mL of saliva incubation were taken for further analysis. During sampling, the particle suspension was stirred to ensure a constant dispersion of the sample. The gastric juice pH was then set to 2 using hydrochloric acid and the particle mixture was stirred for 2 h at 37°C while checking the pH value every 30 min. Again, a sample of 10 mL was held back for further analyses. Subsequently, 10 mL of artificial intestinal juice were added and set to a pH value of 7.5 using sodium bicarbonate powder. The mixture was then stirred for 2 h and samples of the particle mixtures were collected under agitation for further analyses. After the experiments were started, the digestion enzyme activity was checked by means of control substrates for each digestive juice. Activity of amylases from saliva was verified using amylopectin azure, activity of pepsin from gastric juice by means of an albumin/bromophenol blue complex, lipase activity by using 4-methylumbelliferyl oleate and tryptic activity was confirmed by using azocasein as substrates, respectively. Enzymatic cleavage products were measured photometrically. For positive controls, 50 mg/mL (PE, PET, PVC), 25 mg/mL (PP) or 5 mg/mL (PS) of the plastic particles were incubated in 10 mL of 37% HCl, 68% HNO3 or 100% acetone for 4 hours.
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2.4 Degradation of organic matter
After treatment of the particles with the digestive juices, a part of each sample was taken for treatment with 15% H2O2 for 16 h in order to degrade the organic material from the in vitro digestion (Frias et al. 2018).
2.5 Scanning Electron Microscopy and particle characterization
A drop of each sample was placed on a 5 mm x 7 mm silicon wafer for air-drying. For better absorption, all samples were coated with 5 nm gold. All wafers were examined with a Hitachi S-4000 (Hitachi High-Technologies Corporation, Tokyo, Japan) operated at 20 kV and supplied with a SE detector and a BSE detector. Random images of the polydisperse microplastic particles were obtained by SEM. Images were analyzed with the image processing program ImageJ (Laboratory for Optical and Computational Instrumentation (LOCI) of the University of Wisconsin-Madison, Madison, Wisconsin, USA). At least 100 particles were measured to determine their diameters. Results are given as histograms or mean diameters.
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3 RESULTS 3.1 Undigested particles SEM analysis of undigested microplastic particles showed monodisperse, spherical particles for PS microplastics, whereas the other microplastic materials PE, PP, PET and PVC showed polydisperse porous roundish (PE), shred-shaped (PP) or rather smooth roundish (PVC and PET) particles with diameters between 1 and 200 µm (figure 1). Manual measurement of the particle diameters showed an average particle size of 3.8 µm for PS, 90.1 µm for PE, 67.1 µm for PP, 136.5 µm for PVC and 60 µm for PET.
Figure 1: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics. Size distributions of at least 100 particles, as determined by image analysis are given as histograms with mean diameters.
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3.2 Artificial in vitro digestion
After artificial in vitro digestion, especially PS particles showed changes in their shape and size, developing an irregular surface after saliva treatment which increased over the following digestive steps, accompanied by a growth in particle size. Particle diameters increased up to 20 µm through the different digestion steps (figure 2a). There were no such remarkable changes for the particles of the other plastic materials. Only a few crystalline deposits were visible on the particle surfaces (figure 2be). In order to clarify whether the observed changes in PS particle sizes and shapes were caused by the chemical reaction with the polymer structures by the digestive juices or by the deposition of organic material from the artificial saliva, gastric and intestinal fluid on the particle surfaces, the organic corona had to be removed in a next step (figure 3).
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Figure 2: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics after artificial digestion in artificial saliva (left image), artificial gastric fluid (middle image) and
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artificial intestinal fluid (right image). Diameters of at least 100 particles were measured and average diameters were calculated.
3.3 Degradation of organic matter
Organic material from the digestive juices was degraded with hydrogen peroxide in order to detect their direct influence on the particle characteristics without measuring the organic corona formed by the digestive fluids’ ingredients. Hydrogen peroxide degradation of organic material is used as a relatively fast and mild method for sample preparation of microplastics from environmental samples (Frias et al. 2018). Images of the particles after hydrogen peroxide treatment are shown in figure 3. Here, all particles were again comparable with the undigested particles concerning their size and shape. It therefore is to assume that the altered shape of the PS particles after artificial digestion was not a result of the attack by the digestive juices but rather results from a deposition of organic matter on the particle surface. Due to the smaller size of the PS particles, the deposition optically changes them more than the bigger particles of the other plastic materials used.
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Figure 3: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics after artificial digestion in artificial saliva (left image), artificial gastric fluid (middle image) and
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artificial intestinal fluid (right image) and following protein degradation by 15% H2O2. Diameters of at least 100 particles were measured and average diameters were calculated.
3.4 Digestion with anorganic acids or organic solvent
In order to test the ability of the system to detect changes in particle characteristics and to investigate on the resistance of the different plastic particle materials to more aggressive conditions, the undigested particles were treated with the positive controls 37% HCl, 68% HNO3 and 100% acetone (figure 4). No changes in particle shapes were detected for PE, PP and PET after treatment with each of the positive controls. Also the particle sizes just varied slightly which can be related to the polydispersity of the samples. Pronounced effects on the particle structure and shape (PVC) and also on the size (PS) were only observed for PS and PVC matching their vulnerability to moderately polar solvents such as acetone (Schweitzer 2000).
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Figure 4: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics after
13
treatment with 37% HCl (left image), 68% HNO3 (middle) and 100% acetone (right image). Diameters of at least 100 particles were measured and average diameters were calculated.
4 DISCUSSION
Under in vitro conditions, the cellular uptake behavior of microplastic particles is usually considered exclusively in cell culture medium. In reality, however, plastic particles come into contact with various complex matrices such as saliva, gastric and intestinal juices before reaching the gastrointestinal barrier. Possibly resulting changes in the particle characteristics could include a decomposition of the particles resulting in decreased particle sizes, or resulting in changes in their shapes. As it is the case for metallic micro- and nanoparticles (Sieg et al. 2017), particle sizes and shapes are expected to be of greater importance than chemical identity to assess the bioavailability and direct toxicological impact of microplastic particles. For instance, Zauner et al showed a sizedependent uptake of PS micro- and nanoparticles in different cell lines (Zauner et al. 2001). It is therefore essential for reliable in vitro cellular uptake and translocation studies, as well as for risk assessment, to consider potential deformation or degradation and protein corona formation of the microplastic particles during the digestive process after oral uptake. This issue was investigated in the present study on the basis of the five different particulate plastic materials PS, PE, PP, PET and PVC. The particles were subjected to an artificial in vitro digestion and changes in particle sizes and shapes were investigated by image analysis after each digestion step. Furthermore, the influence of the positive controls hydrochloric acid, nitric acid and acetone was examined. In the first step of the digestion process, the different plastic particles were incubated in artificial saliva. Due to the fact that saliva does not contain proteins or salts that are expected to change plastic particle size or shape, no 14
remarkable effect was expected here. By contrast, gastric juice is a more reactive medium due to its low pH resulting from its hydrochloric acid content of ~10%. Even though previous studies stated that hydrochloric acid at these concentrations does not provoke pronounced effects for plastics, there are also indications that minor changes may occur in the surface texture of PE materials (Ghabeche et al. 2015; Michelot et al. 2017). Especially particulate plastic materials are more prone to chemical attacks due to their larger surface-to-volume-ratios compared to their bulk materials which could promote a faster degradation (Gatoo et al. 2014). With transition from the artificial stomach fluid to the intestinal fluid, the pH increases again to 7.5 and bile extract is added. Here, again, none of the ingredients was expected to degrade the applied plastic materials decisively. An unexpected change in shape and size of 4 µm PS particles was already observed after incubation in artificial saliva. After the use of hydrogen peroxide for degradation of proteins and mucins, it became apparent that these changes were caused by the deposition of organic material from the digestive fluids on the particles. Thus, care must be taken when interpreting the results from artificial digestion with inert particulate material in the low micrometer range. In contrast, this effect was less pronounced for the larger PE, PP, PET and PVC microplastic particles ranging from 60 to 140 µm. In addition, it becomes clear that a high protein deposition takes place. This so-called (protein-) corona is crucial for the biological identity and thus for the uptake rate of the particles (Monopoli et al. 2012). To test the resistance of the materials to higher-concentrated hydrochloric acid than found in gastric juice, the plastic materials were also incubated in 37% hydrochloric acid. Here, no embrittlement was found, as in the study by Ghabeche et al. with incubation of PE in high concentrations of hydrochloric acid (Ghabeche et al. 2015). Therefore two further positive controls, the strong acid nitric acid and the organic solvent acetone, were also tested. Nitric acid also did not decompose any of the materials, while acetone strongly deformed PS particles and fused or swelled them into larger particles. PVC was also prone to acetone resulting in 15
the formation of some smaller PVC particles. The effect of acetone on PS and PVC is well known. Acetone, a semi-polar solvent, attacks and decomposes these semi-polar, non-crystalline plastic materials PS and PVC (Schweitzer 2000). Although this solvent does not play a role in the digestion process, it can be formed in the liver during fat metabolism and might have effects on absorbed and transported plastic particles.
5 CONCLUSION
In this work, we used SEM for investigating effects of gastrointestinal fluids on different plastic particle materials, as state of the art method for characterizing polydisperse large microplastic particles. For the first time, we applied artificial digestion to plastic particles in order to assess whether digestive fluids are capable of decomposing plastic particles into smaller fragments or of modifying their shape and texture. Altogether, it can be concluded that the gastrointestinal passage has no pronounced effects on the particles used here, which provides a more detailed knowledge of the fate of plastic particles at the intestinal barrier and directly links the sizes of orally applied particles with those reaching the intestinal barrier. Also the massive protein adsorption, especially on smaller plastic particles, must be considered for the assessment of plastic particle uptake, toxicity and excretion.
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ACKNOWLEDGMENT The authors thank Lisa Klusmann for technical assistance and Swen Donauer and Wjatscheslaw Oshereljew for professional support. This work was supported by the German Federal Institute for Risk Assessment (project 1323-102 and 1329-003).
DECLARATION OF INTEREST STATEMENT We declare no conflict of interest.
REFERENCES Allen S, Allen D, Phoenix VR, et al. (2019) Atmospheric transport and deposition of microplastics in a remote mountain catchment. Nature Geoscience 12(5):339-344 doi:10.1038/s41561-0190335-5 Araújo PHH, Sayer C, Giudici R, Poço JGR (2002) Techniques for reducing residual monomer content in polymers: A review. Polymer Engineering & Science 42(7):1442-1468 doi:10.1002/pen.11043 Barnes DK, Galgani F, Thompson RC, Barlaz M (2009) Accumulation and fragmentation of plastic debris in global environments. Philosophical transactions of the Royal Society of London Series B, Biological sciences 364(1526):1985-98 doi:10.1098/rstb.2008.0205 Bouwmeester H, Hollman PC, Peters RJ (2015) Potential Health Impact of Environmentally Released Micro- and Nanoplastics in the Human Food Production Chain: Experiences from Nanotoxicology. Environmental science & technology 49(15):8932-47 doi:10.1021/acs.est.5b01090 Browne MA, Galloway T, Thompson R (2007) Microplastic—an emerging contaminant of potential concern? Integrated Environmental Assessment and Management 3(4):559-561 doi:10.1002/ieam.5630030412 EFSA (2016) Presence of microplastics and nanoplastics in food, with particular focus on seafood. EFSA Journal 14(6) doi:10.2903/j.efsa.2016.4501 Frias J, Pagter E, Nash R, et al. (2018) Standardised protocol for monitoring microplastics in sediments, Gatoo MA, Naseem S, Arfat MY, Dar AM, Qasim K, Zubair S (2014) Physicochemical properties of nanomaterials: implication in associated toxic manifestations. BioMed research international 2014:498420 doi:10.1155/2014/498420
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Ghabeche W, Alimi L, Chaoui K (2015) Degradation of Plastic Pipe Surfaces in Contact with an Aggressive Acidic Environment. Energy Procedia 74:351-364 doi:https://doi.org/10.1016/j.egypro.2015.07.625 Hanvey JS, Lewis PJ, Lavers JL, Crosbie ND, Pozo K, Clarke BO (2017) A review of analytical techniques for quantifying microplastics in sediments. Analytical Methods 9(9):1369-1383 doi:10.1039/C6AY02707E Hussain N, Jaitley V, Florence AT (2001) Recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics. Advanced drug delivery reviews 50(1-2):107-42 Karami A, Golieskardi A, Ho YB, Larat V, Salamatinia B (2017) Microplastics in eviscerated flesh and excised organs of dried fish. Scientific Reports 7(1):5473 doi:10.1038/s41598-017-058286 Kästner C, Lichtenstein D, Lampen A, Thünemann AF (2017) Monitoring the fate of small silver nanoparticles during artificial digestion. Colloids and Surfaces A: Physicochemical and Engineering Aspects 526:76-81 doi:https://doi.org/10.1016/j.colsurfa.2016.08.013 Liebezeit G, Liebezeit E (2013) Non-pollen particulates in honey and sugar. Food additives & contaminants Part A, Chemistry, analysis, control, exposure & risk assessment 30(12):213640 doi:10.1080/19440049.2013.843025 Liebezeit G, Liebezeit E (2014) Synthetic particles as contaminants in German beers. Food additives & contaminants Part A, Chemistry, analysis, control, exposure & risk assessment 31(9):15748 doi:10.1080/19440049.2014.945099 Michelot H, Stuart B, Fu S, et al. (2017) The mechanical properties of plastic evidence bags used for collection and storage of drug chemicals relevant to clandestine laboratory investigations. Forensic Sciences Research 2(4):198-202 doi:10.1080/20961790.2017.1335459 Napper IE, Bakir A, Rowland SJ, Thompson RC (2015) Characterisation, quantity and sorptive properties of microplastics extracted from cosmetics. Marine Pollution Bulletin 99(1):178-185 doi:https://doi.org/10.1016/j.marpolbul.2015.07.029 Oßmann BE, Sarau G, Holtmannspötter H, Pischetsrieder M, Christiansen SH, Dicke W (2018) Small-sized microplastics and pigmented particles in bottled mineral water. Water Research 141:307-316 doi:https://doi.org/10.1016/j.watres.2018.05.027 PlasticsEurope (2018) Plastics-the Facts 2017: An analysis of European plastics production,demand and waste data. Brussels, Belgium, p 16 Schweitzer P (2000) Mechanical and Corrosion-Resistant Properties of Plastics and Elastomers. In: Schweitzer PA (ed). CRC Press, Fallston, Maryland, p 29-168 Schymanski D, Goldbeck C, Humpf H-U, Fürst P (2018) Analysis of microplastics in water by micro-Raman spectroscopy: Release of plastic particles from different packaging into mineral water. Water Research 129:154-162 doi:https://doi.org/10.1016/j.watres.2017.11.011 Sieg H, Kästner C, Krause B, et al. (2017) Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials. Langmuir 33(40):10726-10735 doi:10.1021/acs.langmuir.7b02729 Smith M, Love DC, Rochman CM, Neff RA (2018) Microplastics in Seafood and the Implications for Human Health. Current environmental health reports 5(3):375-386 doi:10.1007/s40572018-0206-z Stock V, Bohmert L, Lisicki E, et al. (2019) Uptake and effects of orally ingested polystyrene microplastic particles in vitro and in vivo. Archives of toxicology 93(7):1817-1833 doi:10.1007/s00204-019-02478-7
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Thompson RC (2015) Microplastics in the Marine Environment: Sources, Consequences and Solutions. In: Bergmann M, Gutow L, Klages M (eds) Marine Anthropogenic Litter. Springer International Publishing, Cham, p 185-200 Van Cauwenberghe L, Janssen CR (2014) Microplastics in bivalves cultured for human consumption. Environmental Pollution 193:65-70 doi:https://doi.org/10.1016/j.envpol.2014.06.010 van Wezel A, Caris I, Kools SA (2016) Release of primary microplastics from consumer products to wastewater in the Netherlands. Environmental toxicology and chemistry 35(7):1627-31 doi:10.1002/etc.3316 Yang D, Shi H, Li L, Li J, Jabeen K, Kolandhasamy P (2015) Microplastic Pollution in Table Salts from China. Environmental science & technology 49(22):13622-7 doi:10.1021/acs.est.5b03163 Zauner W, Farrow NA, Haines AM (2001) In vitro uptake of polystyrene microspheres: effect of particle size, cell line and cell density. Journal of controlled release : official journal of the Controlled Release Society 71(1):39-51 doi:10.1016/s0168-3659(00)00358-8
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Highlights: •
Plastic particles of different materials were subjected to artificial digestion
•
Digestive fluids didn’t decompose the particles or modified their shapes and sizes
•
A distinct protein corona is formed on plastic particles after artificial digestion
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0278-6915(19)30800-2
DOI:
https://doi.org/10.1016/j.fct.2019.111010
Reference:
FCT 111010
To appear in:
Food and Chemical Toxicology
Received Date: 21 October 2019 Revised Date:
26 November 2019
Accepted Date: 27 November 2019
Please cite this article as: Stock, V., Fahrenson, C., Thuenemann, A., Dönmez, M.H., Voss, L., Böhmert, L., Braeuning, A., Lampen, A., Sieg, H., Impact of artificial digestion on the sizes and shapes of microplastic particles, Food and Chemical Toxicology (2019), doi: https://doi.org/10.1016/ j.fct.2019.111010. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Together with Holger Sieg, Albert Braeuning, Linda Böhmert, Linn Voss and Alfonso Lampen, Valerie Stock was responsible for the conceptualization of the present study. Andreas Thuenemann developed the methodology for the different digestion steps. Christoph Fahrenson, Merve Hilal Dönmez and Valerie Stock acquired all data shown in the paper. The first draft was created by Valerie Stock and Holger Sieg. This work was supported by the German Federal Institute for Risk Assessment (project 1323-102 and 1329-003).
Impact of artificial digestion on the sizes and shapes of microplastic particles
Valerie Stock1, Christoph Fahrenson2, Andreas Thuenemann3, Merve Hilal Dönmez1, Linn Voss1, Linda Böhmert1,*, Albert Braeuning1, Alfonso Lampen1, Holger Sieg1
1 German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany 2 Technical University Berlin, Electron Microscopy Core Facility, Straße des 17. Juni 135, 10623 Berlin 3 German Federal Institute for Materials Research and Testing, Berlin, Germany
*Corresponding author: Linda Böhmert, German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany. Phone: +49 (30) 18412-3718, E-mail: [email protected]
KEYWORDS: microplastic, oral uptake, particle size, gastrointestinal barrier, artificial digestion
1
ABSTRACT
2
Current analyses show a widespread occurrence of microplastic particles in food products and raise
3
the question of potential risks to human health. Plastic particles are widely considered to be inert due
4
to their low chemical reactivity and therefore supposed to pose, if at all only minor hazards.
5
However, variable physicochemical conditions during the passage of the gastrointestinal tract gain
6
strong importance, as they may affect particle characteristics. This study aims to analyze the impact
7
of the gastrointestinal passage on the physicochemical particle characteristics of the five most
8
produced and thus environmentally relevant plastic materials polyethylene, polypropylene, polyvinyl
9
chloride, polyethylene terephthalate and polystyrene. Scanning electron microscopy (SEM) and
10
subsequent image analysis were employed to characterize microplastic particles. Our results
11
demonstrate a high resistance of all plastic particles to the artificial digestive juices. The present
12
results underline that the main stages of the human gastrointestinal tract do not decompose the
13
particles. This allows a direct correlation between the physicochemical particle characteristics before
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and after digestion. Special attention must be paid to the adsorption of organic compounds like
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proteins, mucins and lipids on plastic particles since it could lead to misinterpretations of particle
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sizes and shapes.
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1 INTRODUCTION
The environmental pollution with plastic debris is one of the greatest challenges scientists are facing in recent times. The worldwide production volume of plastics is constantly increasing. Whereas 322 million tons of plastics were produced in 2015, global production grew by 3.85% to 335 million tons in 2016 (PlasticsEurope 2018). With the increase in plastics production, the impact on the environment also increases (Barnes et al. 2009). Intentionally produced microscaled plastic particles, called primary microplastics, or plastic particles originated by UV degradation and other environmental factors (secondary microplastics) can enter human food and beverages through the food chain or by entry into environmental resources like drinking water (Napper et al. 2015; Thompson 2015; van Wezel et al. 2016). To date, there is no uniform microplastic size definition, but a size range between 100 nm and 5 mm is usually assumed. Plastic particles below 100 nm are commonly defined as nanoplastics (EFSA 2016). The bigger size range is rather an environmental issue, but particles below 150 µm in size are considered to be, in principal, systemically bioavailable to humans, and particles below 4 µm can be taken up by intestinal cells (EFSA 2016; Stock et al. 2019). The most commonly produced types of polymers are polypropylene (PP) > polyethylene (PE) > polyvinyl chloride (PVC) and > polyethylene terephthalate (PET) (PlasticsEurope 2018). Recent publications show a contamination of various food products with microplastic particles indicating a widespread exposure (Karami et al. 2017; Liebezeit and Liebezeit 2013; Liebezeit and Liebezeit 2014; Oßmann et al. 2018; Schymanski et al. 2018; Van Cauwenberghe and Janssen 2014; Yang et al. 2015). Microplastics can thereby be enriched in the food chain as well as introduced into foodstuff through the air, which indicates the ubiquitous presence of plastic particles (Allen et al. 2019; Smith et al. 2018). There are initial attempts to standardize analytical methods in order to better estimate human 1
exposure (Hanvey et al. 2017). To date, there are still large knowledge gaps on the hazard of microplastics (EFSA 2016). Plastic materials are usually considered as chemically very unreactive due to their large molecular size. A potential bioreactivity of microplastic particles is therefore more likely to be expected due to the increased surface-to-volume ratio with decreasing particle sizes than due to their chemical properties per se. Moreover, adverse effects from microplastics may result from their ability to function as a carrier for environmental pollutants, from additives like plasticizers and flame retardants or from unreacted residual monomers which may remain in the plastic materials due to incomplete polymerization reactions (Araújo et al. 2002; Bouwmeester et al. 2015; Browne et al. 2007). After oral uptake, particles are subjected to the influence of the various matrices in the digestive tract sections before reaching the intestine. Because the plastic particle size is of great importance for its potential uptake at the intestinal barrier, it is important to understand the influence of the different digestive juices on the particle characteristics (Hussain et al. 2001). Accordingly, it is of significance for a potential uptake whether ingested plastic particles are degraded to smaller fragments during the gastrointestinal passage. Gastric acid in particular might corrode the plastic particles or change their shape, making them potentially more bioreactive. The aim of this work was to investigate the fate of microplastic particles of different plastic materials during the gastrointestinal passage by measuring particle sizes and shapes after the different digestion steps. The present data may serve as a basis for further in vivo experiments in order to assess the potential decomposition of the particles and thus also the resulting effects. This is essential for a reliable future risk assessment of microplastics.
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2 METHODS 2.1 Chemicals and microplastics
Chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), or Carl Roth (Karlsruhe, Germany) if not otherwise indicated. The 4 µm polystyrene (PS) particles (PFL-4070, Sky Blue fluorescent particles, size 3.6 – 4.5 µm) were purchased from Kisker Biotech GmbH & Co. KG (Steinfurt, Germany), PET (ES 306030) from Goodfellow GmbH (Hamburg, Germany) PE (429015), PP (427861) and PVC (81387) from Sigma-Aldrich (Taufkirchen, Germany). All plastic particles were purchased in pure form from the manufacturers.
2.2 Polypropylene grinding
PP was purchased in granulate form and thus had to be further ground to microplastic particles. This was conducted by Fritsch GmbH using the Pulverisette 14 premium line rotor cutting ball mill (Fritsch GmbH, Idar-Oberstein, Germany). The PP granules were first embrittled in liquid nitrogen. They were then crushed with a maximum rotor speed of >110 m/s and a maximum speed of 22,000 rpm. In this experiment, a rotor with 12 ribs in combination with a sieve ring with 0.5 mm trapezoidal perforation was used for grinding. A quantity of 25 g was ground within 7 minutes.
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2.3 Artificial in vitro digestion
The artificial in vitro digestion protocol, originally based on the DIN ISO 19738 , has been modified for scientific investigations on nanoparticles and biopolymers (Kästner et al. 2017). The artificial in vitro digestion system essentially considered the 3 digestive compartments mouth, stomach and intestine. The corresponding digestive juices were reproduced according to their respective compositions (table 1).
Table 1: Composition of artificial digestive fluids. Saliva (pH 6.4)
Gastric fluid (pH 2)
Intestinal fluid (pH 7.5)
Substance
c [mg/mL]
Substance
c [mg/mL]
Substance
c [mg/mL]
NaCl
1.667
NaCl
4.143
NaCl
0.3
NaSCN
0.5
KCl
1
CaCl2*2H2O
0.5
Na2SO4
1.833
KH2PO4
0.386
MgCl2*6H2O
0.2
NaHCO3
0.5
Mucin
4.286
NaHCO3
1.0
KCl
1.5
Pepsin
1.429
Ureate
0.3
KH2PO4
2.0
Conc HCl
1 Drop
Trypsin
0.3
CaCl2*2H2O
0.5
10% HCl
2 Drops
Pancreatin
9
Urate
0.33
Bile extract
9
Urea
0.033
NaHCO3 (s)
For titration
4
Alpha-
0.833
Amylase Mucin
2.5
100 mg/mL (PE, PET, PVC), 50 mg/mL (PP) or 10 mg/mL (PS) of the plastic particles were dispersed in 16 mL of synthetic saliva and incubated under agitation at 37 °C in a water bath for 5 min. Prior to the addition of 14 mL of gastric juice, 10 mL of saliva incubation were taken for further analysis. During sampling, the particle suspension was stirred to ensure a constant dispersion of the sample. The gastric juice pH was then set to 2 using hydrochloric acid and the particle mixture was stirred for 2 h at 37°C while checking the pH value every 30 min. Again, a sample of 10 mL was held back for further analyses. Subsequently, 10 mL of artificial intestinal juice were added and set to a pH value of 7.5 using sodium bicarbonate powder. The mixture was then stirred for 2 h and samples of the particle mixtures were collected under agitation for further analyses. After the experiments were started, the digestion enzyme activity was checked by means of control substrates for each digestive juice. Activity of amylases from saliva was verified using amylopectin azure, activity of pepsin from gastric juice by means of an albumin/bromophenol blue complex, lipase activity by using 4-methylumbelliferyl oleate and tryptic activity was confirmed by using azocasein as substrates, respectively. Enzymatic cleavage products were measured photometrically. For positive controls, 50 mg/mL (PE, PET, PVC), 25 mg/mL (PP) or 5 mg/mL (PS) of the plastic particles were incubated in 10 mL of 37% HCl, 68% HNO3 or 100% acetone for 4 hours.
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2.4 Degradation of organic matter
After treatment of the particles with the digestive juices, a part of each sample was taken for treatment with 15% H2O2 for 16 h in order to degrade the organic material from the in vitro digestion (Frias et al. 2018).
2.5 Scanning Electron Microscopy and particle characterization
A drop of each sample was placed on a 5 mm x 7 mm silicon wafer for air-drying. For better absorption, all samples were coated with 5 nm gold. All wafers were examined with a Hitachi S-4000 (Hitachi High-Technologies Corporation, Tokyo, Japan) operated at 20 kV and supplied with a SE detector and a BSE detector. Random images of the polydisperse microplastic particles were obtained by SEM. Images were analyzed with the image processing program ImageJ (Laboratory for Optical and Computational Instrumentation (LOCI) of the University of Wisconsin-Madison, Madison, Wisconsin, USA). At least 100 particles were measured to determine their diameters. Results are given as histograms or mean diameters.
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3 RESULTS 3.1 Undigested particles SEM analysis of undigested microplastic particles showed monodisperse, spherical particles for PS microplastics, whereas the other microplastic materials PE, PP, PET and PVC showed polydisperse porous roundish (PE), shred-shaped (PP) or rather smooth roundish (PVC and PET) particles with diameters between 1 and 200 µm (figure 1). Manual measurement of the particle diameters showed an average particle size of 3.8 µm for PS, 90.1 µm for PE, 67.1 µm for PP, 136.5 µm for PVC and 60 µm for PET.
Figure 1: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics. Size distributions of at least 100 particles, as determined by image analysis are given as histograms with mean diameters.
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3.2 Artificial in vitro digestion
After artificial in vitro digestion, especially PS particles showed changes in their shape and size, developing an irregular surface after saliva treatment which increased over the following digestive steps, accompanied by a growth in particle size. Particle diameters increased up to 20 µm through the different digestion steps (figure 2a). There were no such remarkable changes for the particles of the other plastic materials. Only a few crystalline deposits were visible on the particle surfaces (figure 2be). In order to clarify whether the observed changes in PS particle sizes and shapes were caused by the chemical reaction with the polymer structures by the digestive juices or by the deposition of organic material from the artificial saliva, gastric and intestinal fluid on the particle surfaces, the organic corona had to be removed in a next step (figure 3).
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Figure 2: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics after artificial digestion in artificial saliva (left image), artificial gastric fluid (middle image) and
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artificial intestinal fluid (right image). Diameters of at least 100 particles were measured and average diameters were calculated.
3.3 Degradation of organic matter
Organic material from the digestive juices was degraded with hydrogen peroxide in order to detect their direct influence on the particle characteristics without measuring the organic corona formed by the digestive fluids’ ingredients. Hydrogen peroxide degradation of organic material is used as a relatively fast and mild method for sample preparation of microplastics from environmental samples (Frias et al. 2018). Images of the particles after hydrogen peroxide treatment are shown in figure 3. Here, all particles were again comparable with the undigested particles concerning their size and shape. It therefore is to assume that the altered shape of the PS particles after artificial digestion was not a result of the attack by the digestive juices but rather results from a deposition of organic matter on the particle surface. Due to the smaller size of the PS particles, the deposition optically changes them more than the bigger particles of the other plastic materials used.
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Figure 3: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics after artificial digestion in artificial saliva (left image), artificial gastric fluid (middle image) and
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artificial intestinal fluid (right image) and following protein degradation by 15% H2O2. Diameters of at least 100 particles were measured and average diameters were calculated.
3.4 Digestion with anorganic acids or organic solvent
In order to test the ability of the system to detect changes in particle characteristics and to investigate on the resistance of the different plastic particle materials to more aggressive conditions, the undigested particles were treated with the positive controls 37% HCl, 68% HNO3 and 100% acetone (figure 4). No changes in particle shapes were detected for PE, PP and PET after treatment with each of the positive controls. Also the particle sizes just varied slightly which can be related to the polydispersity of the samples. Pronounced effects on the particle structure and shape (PVC) and also on the size (PS) were only observed for PS and PVC matching their vulnerability to moderately polar solvents such as acetone (Schweitzer 2000).
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Figure 4: Scanning electron microscopic images of (A) polystyrene, (B) polyethylene, (C) polypropylene, (D) polyvinyl chloride and (E) polyethylene terephthalate microplastics after
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treatment with 37% HCl (left image), 68% HNO3 (middle) and 100% acetone (right image). Diameters of at least 100 particles were measured and average diameters were calculated.
4 DISCUSSION
Under in vitro conditions, the cellular uptake behavior of microplastic particles is usually considered exclusively in cell culture medium. In reality, however, plastic particles come into contact with various complex matrices such as saliva, gastric and intestinal juices before reaching the gastrointestinal barrier. Possibly resulting changes in the particle characteristics could include a decomposition of the particles resulting in decreased particle sizes, or resulting in changes in their shapes. As it is the case for metallic micro- and nanoparticles (Sieg et al. 2017), particle sizes and shapes are expected to be of greater importance than chemical identity to assess the bioavailability and direct toxicological impact of microplastic particles. For instance, Zauner et al showed a sizedependent uptake of PS micro- and nanoparticles in different cell lines (Zauner et al. 2001). It is therefore essential for reliable in vitro cellular uptake and translocation studies, as well as for risk assessment, to consider potential deformation or degradation and protein corona formation of the microplastic particles during the digestive process after oral uptake. This issue was investigated in the present study on the basis of the five different particulate plastic materials PS, PE, PP, PET and PVC. The particles were subjected to an artificial in vitro digestion and changes in particle sizes and shapes were investigated by image analysis after each digestion step. Furthermore, the influence of the positive controls hydrochloric acid, nitric acid and acetone was examined. In the first step of the digestion process, the different plastic particles were incubated in artificial saliva. Due to the fact that saliva does not contain proteins or salts that are expected to change plastic particle size or shape, no 14
remarkable effect was expected here. By contrast, gastric juice is a more reactive medium due to its low pH resulting from its hydrochloric acid content of ~10%. Even though previous studies stated that hydrochloric acid at these concentrations does not provoke pronounced effects for plastics, there are also indications that minor changes may occur in the surface texture of PE materials (Ghabeche et al. 2015; Michelot et al. 2017). Especially particulate plastic materials are more prone to chemical attacks due to their larger surface-to-volume-ratios compared to their bulk materials which could promote a faster degradation (Gatoo et al. 2014). With transition from the artificial stomach fluid to the intestinal fluid, the pH increases again to 7.5 and bile extract is added. Here, again, none of the ingredients was expected to degrade the applied plastic materials decisively. An unexpected change in shape and size of 4 µm PS particles was already observed after incubation in artificial saliva. After the use of hydrogen peroxide for degradation of proteins and mucins, it became apparent that these changes were caused by the deposition of organic material from the digestive fluids on the particles. Thus, care must be taken when interpreting the results from artificial digestion with inert particulate material in the low micrometer range. In contrast, this effect was less pronounced for the larger PE, PP, PET and PVC microplastic particles ranging from 60 to 140 µm. In addition, it becomes clear that a high protein deposition takes place. This so-called (protein-) corona is crucial for the biological identity and thus for the uptake rate of the particles (Monopoli et al. 2012). To test the resistance of the materials to higher-concentrated hydrochloric acid than found in gastric juice, the plastic materials were also incubated in 37% hydrochloric acid. Here, no embrittlement was found, as in the study by Ghabeche et al. with incubation of PE in high concentrations of hydrochloric acid (Ghabeche et al. 2015). Therefore two further positive controls, the strong acid nitric acid and the organic solvent acetone, were also tested. Nitric acid also did not decompose any of the materials, while acetone strongly deformed PS particles and fused or swelled them into larger particles. PVC was also prone to acetone resulting in 15
the formation of some smaller PVC particles. The effect of acetone on PS and PVC is well known. Acetone, a semi-polar solvent, attacks and decomposes these semi-polar, non-crystalline plastic materials PS and PVC (Schweitzer 2000). Although this solvent does not play a role in the digestion process, it can be formed in the liver during fat metabolism and might have effects on absorbed and transported plastic particles.
5 CONCLUSION
In this work, we used SEM for investigating effects of gastrointestinal fluids on different plastic particle materials, as state of the art method for characterizing polydisperse large microplastic particles. For the first time, we applied artificial digestion to plastic particles in order to assess whether digestive fluids are capable of decomposing plastic particles into smaller fragments or of modifying their shape and texture. Altogether, it can be concluded that the gastrointestinal passage has no pronounced effects on the particles used here, which provides a more detailed knowledge of the fate of plastic particles at the intestinal barrier and directly links the sizes of orally applied particles with those reaching the intestinal barrier. Also the massive protein adsorption, especially on smaller plastic particles, must be considered for the assessment of plastic particle uptake, toxicity and excretion.
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ACKNOWLEDGMENT The authors thank Lisa Klusmann for technical assistance and Swen Donauer and Wjatscheslaw Oshereljew for professional support. This work was supported by the German Federal Institute for Risk Assessment (project 1323-102 and 1329-003).
DECLARATION OF INTEREST STATEMENT We declare no conflict of interest.
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Highlights: •
Plastic particles of different materials were subjected to artificial digestion
•
Digestive fluids didn’t decompose the particles or modified their shapes and sizes
•
A distinct protein corona is formed on plastic particles after artificial digestion
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: