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.

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© 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: Linda.Boehmert@bfr.bund.de

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

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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18

19

20

21

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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.

5
 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.

6
 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.

7
 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 2b-

e). 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).

8
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

9
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.

10
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

11
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).

12
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 size-

dependent 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.

16
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: