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Is the human body becoming a plastic soup? (Thesis)

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Van’t Hoff Institute for Molecular Sciences

MSc Chemistry

Analytical Sciences

Literature Study

Is the human body becoming a plastic soup?

by

Carlo Roberto de Bruin

12431737

May 2020 12 ECTS

February 2020 - May 2020

Supervisor/1st Examiner: 2nd Examiner:

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Table of contents

A. List of Abbreviations...3

Abstract...4

Introduction...5

1. Human exposure and health effects...7

1.1 Ingestion, inhalation and dermal contact of MNPs...7

1.2 Health effects...9

2. Toxicity testing...12

3. Sample preparation...15

4. Analysis techniques for MNPs...19

4.1 FFF techniques...19

4.2 Chromatography techniques...20

4.3 Light scattering techniques...21

4.4 Microscopy techniques...22

4.5 FTIR spectroscopy...30

4.6 Raman spectroscopy...31

4.7 Thermal analytical techniques...32

4.8 Other potential techniques...33

5. Quantification of MNPs...34

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A.

List of Abbreviations

Abbreviation Definition Abbreviation Definition

AF4 Asymmetrical flow field flow

fractionation PAN Polyacrylonitrile

AFM Atomic force microscopy PBDE Polybrominated diphenyl ethers

AGS Adenocarcinoma gastric cells PCB polychlorinated biphenyls

ALK Alkyd resin PE Polyethylene

ATR Attenuated total reflectance PES Polyethersulfone

BFR Brominated flame retardant PET Polyethylene terephthalate

BPA Bisphenol A PETP Polyethylene terephthalate

CE Capillary electrophoresis POP Persistent organic pollutants

CPE PP Polypropylene

DLS Dynamic light scattering PSD Particle size distribution

EC50 Half-maximal-effective

concentration PSU Polysulfone

EDC Endocrine disruption chemicals PTFE Polytetrafluoroethylene

EHT Electron high tension PVC Polyvinyl chloride

EP Epoxy resin ROS Reactive oxygen species

EVA Ethylene vinyl acetate RY Rayon

FCM Flow cytometry SEC Size exclusion chromatography

FE (SEM-EDS) Field emission SEM-EDS (EDX) Scanning electron

microscopy-energy dispersive x-ray

FFF Field flow fractionation SIM Single ion monitoring

FPA Focal-plane array SPE Solid phase extraction

FTIR Fourier transform infrared TB Toxicity-based

GC Gas chromatography TD Toxicodynamic

HDC Hydrodynamic chromatography TEM Transmission-electron

microscopy

HIS Hyperspectral imaging TED Thermogravimetry-Thermal

desorption

HLB Hydrophilic-lipophilic balance TK Toxicokinetic

HPLC High-performance liquid

chromatography

TOF Time of flight

ICES International Council for the

Exploration of the Sea TPA Terephthalic acid

ICP Inductively coupled plasma

LC Liquid chromatography

LC50 Median lethal concentration

LOD Limit of detection

MALDI Matrix-assisted laser

desroption/ionization

MALS Multiple-angle light scattering

MNPs Micro- and nanoplastics

MS Mass spectrometry

NOEC No observed effect concentration NTA Nanoparticle tracking analysis

Nyl6 Nylon

PA Polyamide

PAA Poly(N-methyl acrylamide)

PAH Polycyclic-aromatic

hydrocarbons

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Abstract

Micro- and nanoplastics (MNPs) degraded from plastic debris in the environment is an emerging topic in the scientific community as they might be hazardous for the environment and human health. More and more MNPs are found in different aquatic organisms (e.g. fish or mussels) and environmental samples such as rivers, soil, sediments and sludge, air samples (e.g. indoor dust). They are being consumed or inhaled by humans and leading to direct exposure. The risk assessment of MNPs in human health is limited due to the lack of knowledge on realistic exposure doses. A standardized quantitation method for such samples can deliver these realistic exposure doses. However, the quantitation is dependent on the development of characterization and identification methods. The current used analytical methodologies are not able to provide a standardized protocol for characterization, identification and quantitation of MNPs, especially for nanoplastics due to their small sizes. The heterogeneity of MNPs and differences in physical weathering in the environment makes comparisons between studies and real samples difficult, due to the lack of standardized methods. This review describes the progress on MNP research from multiple areas that are critical for risk assessment. Firstly, studies on the potential health effects and exposure routes from MNPs and toxic additives are summarized and assessed. Secondly, studies that perform toxicity testing of MNPs are discussed. Thirdly, the sampling and especially sample preparation methods from studies that examine biological and air samples are reviewed. At last, the progress on standardized methods for the separation, characterization, identification and quantitation of MNPs is critically discussed and reviewed. Limitations, advantages and prospects of novel studies are discussed and indicated in all chapters. Currently, studies on microplastics are more accurate and robust than for nanoplastics, due to the lack of methods capable to deal with nanosized plastics. Therefore, studies performed on microplastic that also have potential to be translated to nanoplastics are indicated. New combinations of suitable analytical techniques and more research into nanoplastics is needed for a complete and reliable risk assessment of MNPs on human health.

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Introduction

Plastics are recently produced in extreme quantities over the entire globe, and their production has increased for the past decades. In 1950 the production consisted of 1.5 million tons, this increased to 359 million tons in 2018. The is expected to have tripled by the year of 2050, which will cause a significant increase in plastic pollution (1). Our obsession with plastic can be attributed to its extremely low cost, versatility, inertness, and durability. Due to these properties, plastic has almost limitless applications. In 2016, approximately 40% of the plastic produced in Europe was directed towards single use applications (2). Currently, plastics are used in all kinds of products such as: packaging, clothing, electronics, industrial materials or office supplies and despite of plastic recycling it causes a lot of plastic litter and debris. Not only are there many different polymers used in consumer grade products, most of these products contain co-polymers and additives, such as stabilizers and plasticizers, to tailor the functionality of the plastic to its intended use. Therefore, plastic must be sorted after collection, a time consuming and potentially expensive process. Some additives, such as flame retardants, are toxic and plastics which contain them are certainly not suited to be recycled into things such as children’s toys or food packaging. It is for this reason that virgin plastic is often preferred to recycled plastic by manufactures (3). Of all the plastic that has ever been produced, roughly 12% gets incinerated for energy recovery while 60% is simply disposed of and allowed to accumulate in landfills and the natural environment (4).

Plastic debris does not decompose naturally and fractionates into increasingly much small particles, micro- and nanoplastics (MNPs). MNPs are more reactive and potentially more harmful to humans and other life forms (5). Gigault et al. demonstrated in vitro that UV-light exposure can result in the progressive degradation of marine polyethylene (PE) and polypropylene (PP) microplastic fragments, leading to the formation of nanoplastics (6). The size range of MNPs (Fig.1) is discussed by multiple authors. In another study Gigault et al. proposed the following definition: “nanoplastics are particles within a size ranging from 1 to 1000 nm resulting from the degradation of industrial plastic objects and can exhibit colloidal behavior(7).

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Nanoplastics are polydisperse in physical properties and heterogeneous in composition. This is, because their occurrence and production are highly dependent on the degradation of microplastics. Nanoplastics present colloidal behavior which can induce aggregation in a medium, the aggregation is depending on the physical and chemical conditions of the dispersion medium such as the ionic strength, pH, temperature and UV light etc. Due to their smaller radius, the ratio of surface area to bulk material is significantly larger for nanoplastics. Mattsson et al. provided a helpful way of understanding this relationship stating: “if a normal plastic shopping bag is totally transformed into particles with a diameter of 40 nm, they will theoretically yield a surface area of 2600 m2” (9). This enables the same mass of plastic to have a much larger negative impact through the adsorption and transportation of increased amounts of persistent organic pollutants (POPs).

MNPs represent a major concern for society and among scientists, due to the potential negative effects on ecosystems and organisms. MNPs have the tendency to accumulate in different matrices like soil (10), freshwater (11), sediment (12), fish tissue (13) and air (14). Due to different studies performed on organisms potential human health issues are hypothesized such as: oxidative stress, neurotoxicity and chronic inflammation (15). Humans can be exposed to MNPs via multiple routes such as, inhalation, ingestion and dermal contact, which might lead to the above-mentioned health effects. Figure 2 provides an overview of potential pathways and particle toxicity for microplastics in the human body. Information on the effects of MNPs in humans is limited due to ethical constrains, as human samples are not intended for use in toxicity testing. Nevertheless, several animals contain (few) human metabolic pathways which can be used for estimating the potential toxicity of MNPs and the effects of exposure using models. Currently, zebrafish are often used in toxicity studies, as it is unique among vertebrate model organism systems. Zebrafish have high genetic homology to humans, with approximately 70% of human genes and about 82% of potential human disease-related genes (16), which makes them strongly related in terms of health effects. However, knowledge on the concentrations of MNPs present in different environmental compartments and the concentration levels in organisms after exposure is still limited, mainly due to the lack of standardized methodologies, making accurate risk assessment of human health complicated.

Despite the increase in research focusing on MNPs as environmental pollutants, there is still a lack of standardization among the studies reported. This leads to difficulties in comparing results and potentially even questionable data. Therefore, standardized analytical methodologies to characterize, identify and quantify MNPs in environmental samples are needed. Hereby, realistic exposure doses of MNPs should be tested, which will enable to fully assess the risk of MNPs on human health in an accurate manner. In addition, there is more progress on methods for analyzing microplastics than analyzing nanoplastics. The characterization of MNPs provides detailed information that could help the process of improving identification and quantification techniques for MNPs. For characterization, light scattering (e.g. DLS) and imaging by microscopy (e.g. SEM-EDS) seem to have great potential to provide a particle size distribution (PSD) and visual details from the geometry and surface characteristics. However, the distinction between different kinds of plastics in different samples have to be kept in mind when answering the analytical question. The sampling and sample treatment need precautions due to contamination issues (e.g. lab equipment) of plastics. Sample treatment techniques like chemical digestion, enzymatic digestion and density separation in combination with filtering and incubation are widely used (17). It is of great importance to isolate the MNPs properly to prevent aggregation, contamination and matrix effects. Due to the small size of MNPs this is a highly complicated task, especially for nanoplastics. In addition, separation methods like gas and liquid chromatography (GC and LC) or field flow fractionation (FFF) are occasionally used for identification and size-based separation, respectively. For detailed characterization, identification and quantification,

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spectroscopic analysis, FFF, thermo analytical methods and also LC- and GC-MS methods have been reported and are discussed in chapter 6. All the mentioned studies have their advantages and disadvantages. How we can benefit from the qualities of each technique depends on the analytical question to be answered.

The samples that can have a direct influence on human health are food products (e.g. fish and other eatable products), tap water, air (e.g. indoor dust) samples and even packaging of food and beverages. These samples are in accordance with the exposure routes ingestion, inhalation and dermal contact. Therefore, the aim of this review is to summarize the potential human health effects from MNP exposure and in vivo/in vitro models used for toxicity testing. Furthermore, various techniques from sampling, sample treatment, characterization, identification and quantification of MNPs in biological and air samples are discussed. All the current methodologies that have been reported in MNP studies are critically discussed and reviewed. The known and unknown potential possibilities, limitations and challenges for the future are indicated and discussed.

1.

Human exposure and health effects

Humans are exposed to MNPs every day, due to the extensive plastic litter that is released in the environment. Most of the exposure is through the ingestion of food containing MNPs or through the inhalation of MNPs in the air (indoor and outdoor dust). Exposure through dermal contact is also plausible, due to plastics contained in care products, textiles or in dust. The exposure to MNPs can lead to negative health effects. In addition, additives or compounds that may leach, desorb or adsorb from or to MNPs can also lead and/or contribute to negative health effects. These topics are discussed in this section.

1.1

Ingestion, inhalation and dermal contact of MNPs

Ingestion and inhalation are the most significant exposure routes of MNPs into humans, contributing the most to negative health effects. Cox et al. estimated the intake of 39.000-52.000 microplastic particles for each person/year, based on food consumption (18). In addition, Catarino et al. estimated the fiber exposure of 13.731–68.415 particles/year/capita during a meal through dust fallout in a household (19). While the plastic production keeps increasing, the same applies for the intake of microplastics and as a result the intake of nanoplastics will also increase due to the degradation of microplastics. Intake of MNPs through food or through the mucociliary clearance after inhalation may cause negative effects (e.g. inflammatory response) on the gastrointestinal system. Microplastics have been found in multiple food samples, for example table salt (20), sugar (21), beer, tap water, sea salt (22) and nanoplastics were recently found in fish samples from a local supermarket in Beijing (23). However, microplastics accumulate in the gills, liver and guts of fish, which are not usually consumed by humans. On the other hand, mussels are consumed as a whole and MNPs accumulate in mussels as well (24). This raises the concern of bioaccumulation of MNPs in the food chain, despite of the unknown risks for human health. Contaminated food is one of the greatest concerns in public media, in addition, other studies determined that exposure routes from packaging could lead to contamination of food as well. For example, Schymansky et al. (25) provided a research on the release of plastic particles from different packaging into mineral water, which found more micro-sized plastic particles

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in returnable plastic bottles compared to single-use plastic bottles. The average microplastic content was 118 ± 88 particles/L in returnable, but only 14 ± 14 particles/L in single-use plastic bottles. In addition to the potential adverse effects of ingested MNPs, the large surface area of MNPs can easily adsorb other harmful pollutants with the potential to be transported into organs once ingested (26), these pollutants are discussed in section 3.2.

Air is contaminated with MNPs by multiple sources like fibers from clothes, abrasion of materials (e.g. plastic sheets and tires). The particles are easily transported by the wind and are very persistent. Humans are exposed to MNPs every day through breathing. Multiple studies estimated the inhalation of microplastic particles per day. For instance, Prata studied the consequences of the inhalation of airborne microplastics and estimated that 26-130 particles could be inhaled per day for each individual (27). This estimation was based on measured particles in the studies from Dris et al. (14)(28) and the human tidal volume 6 L per min estimated by Guyton and Hall (29). While Vianello et al. estimated a value of 272 inhaled particles per day for each individual (30). In this study a thermal manikin was used to reproduce the respiratory system which gives a different estimation of inhaled particles. This kind of sampling is likely to be more capable of estimating inhaled particles compared to vacuum cleaners and pumps, as it simulates human intake. However, the number of inhaled particles will be different for each sample location, because MNPs are not equally distributed around the globe. This was shown in the study from Zhang et al. where HPLC-MS/MS was used to quantify microplastics (PET and PC) in indoor dust samples from 12 countries (31). In addition, sampling procedures (e.g. thermal manikin or vacuum cleaner) and types of samples (e.g. indoor or outdoor dust) will influence the number of inhaled particles as well. Comparison between studies that estimate inhaled particles can only be possible with standardized sampling procedures and the same sample types.

Microplastic exposure through dermal contact is less plausible due to their size, therefore, it is not likely that they are able to cross the dermal barrier. In contrast, nanoplastics are much smaller in size and could potentially transverse the dermal barrier, although this was not proven yet (32). However, due to the lack of knowledge on the properties and toxicity of these particles, this possibility should not be underestimated. Because, cosmetics containing nanoplastics, dust particles in the air or polluted water may be potential exposure routes for nanoplastics across the dermal barrier. Nanoplastics are smaller in size and have a higher surface to volume ratio than microplastics independent of the exposure route. Therefore, nanoplastics are potentially more harmful as it is easier for them to cross membranes and other barriers in the human body. In addition, it has been shown that nanoplastics are capable to penetrate cell membranes (33), which can cause changes of behavior from fish shown by another study of Mattson et al. (34). The combination of higher surface area and increased biological mobility gives them the potential to be much more dangerous than microplastics.

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Figure 2: Overview of the potential pathways and particle toxicity for microplastics in the human body (15)

1.2

Health effects of MNPs

MNPs may cause detrimental health effects on humans and organisms throughout the ecosystem, furthermore the additives or adsorbed compounds may also enhance the toxicity of MNPs, however it is dependent on the amount of exposure and susceptibility of the immune system. Contradictory, is the fact that the use of carbon nanotubes and fullerenes is increasing in the biomedical field (35)(36), due to their unique properties The use of nanoparticles is a promising tool for drug delivery and food additives due to their ability to cross membranes and translocation in the human body. Forte et al. produced a study with polystyrene (PS) nanoparticles in adenocarcinoma gastric cells (AGS), where smaller sized particles (44nm) accumulated faster and more efficiently in the cytoplasm of AGS compared to larger size particles (100nm) (37). Which means that the size determines the concentration in certain body tissues, therefore, the smallest nanoplastics might cause the most damage to human health. Prata et al. (10) provided a review on the possible health effects of microplastics on humans. Microplastics can cause many effects such as oxidative stress, cytotoxicity, endocrine disruption, immune disruption and neurotoxicity. These effects may be more severe for nanoplastics, due to faster accumulation in the cytoplasm and more efficient translocation, however, more research is needed on the effects of nanoplastics. Nowadays, animal studies are used to provide knowledge on the health effects and toxicity of MNPs, these studies will be discussed in chapter 4. As a critical point, most toxicology studies use a high dose of MNPs for determining toxicity, while the daily intake is estimated orders of magnitude below this dose (38). Therefore, the estimated health effects for environmental exposure are currently not realistic, however, these studies still give an overview of possible health effects. In addition, the plastic production is still increasing and the amount of MNPs in the environment will increase over time. Hence, it would be likely for daily doses of MNPs to increase as well. In another study from Shen et al. it was reported that not only the particle size has an influence on the toxic effects, but also the charge of the particles. Positively charged nanoplastics showed more significant effects on the normal physiological activity of cells than negatively charged nanoplastics. In addition, the release of additives and contaminants like POPs adsorbed on MNPs poses more significant threats to organisms than MNPs themselves (39).

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Additives may leach from the plastic into the surrounding environment, which also could be the human body. Leaching will primarily occur at the surface of the plastic particles and the body fluids or tissue, therefore, leaching of additives from plastic particles might induce and contribute to potential health effects (40)(15). For example, plastic additives like phthalates, brominated flame retardants (BFRs) and bisphenol A (BPA) are most abundant and can be a concern to human health (41). Phthalates are esters and mainly used as plasticizers to increase the flexibility, longevity, durability and transparency of the plastic. Additionally, they are used as solvents and fixatives in fragrances, but they are also used as additives in cosmetics or personal care products. Hence, it is possible to enter the human body through inhalation or dermal contact. Phthalates are among the endocrine disrupting chemicals (EDCs) and can cause health effects in fetuses, infants, children and adults when they are present in the human body (42). BFRs are added to plastics to reduce the flammability in products, these compounds have an inhibitory effect on combustion chemistry. Therefore, BFRs are added to clothes, electronics and furniture to protect humans and the environment in daily life. However, polybrominated diphenyl ethers (PBDEs) are a major class of BFRs that can have adverse effects on human health because of their bioaccumulative and persistent behavior. Due to this, these compounds can cause endocrine disruption and neurobehavioral effects (43). BPA is used as a monomer in the production of polycarbonate plastics and epoxy resins lining. It is also used in many consumer goods like water bottles, food containers and sport equipment, therefore, ingestion is the main exposure route. BPA is also classified as endocrine disruptor and can cause damage to reproductive system, immune system and neuroendocrine system, etc. In addition, BPA can also induce carcinogenesis and mutagenesis in animal models, which has been showed in different studies (44). Chen et al. investigated the uptake of BPA in the presence and absence of nanoplastics in adult zebrafish, and the results have shown that the presence of nanoplastics caused a significant increase in the uptake of BPA, which caused neurotoxicity (45). The mentioned studies above confirm the need of further research on the physical and chemical weathering that can cause breakdown of MNPs and on the interactions of digestive fluid and lipids with plastic matrices within organism bodies. This is needed for more accuracy in the determination of leaching chemicals and the assessment for their risk. Besides the potential hazards of additives in plastics, other compounds that may adsorb to or desorb from plastics may also cause potential health effects.

The hydrophobicity of plastic in combination with the high surface area as a micro- or nanoparticle, makes them great as adsorbents for compounds including POPs such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) or heavy metals (46). These kind of compounds, similarly to MNPs, can also induce carcinogenesis and mutagenesis (47), and therefore, the combination of these compounds may enhance the toxicity of the whole complex. On the other hand, the presence of additives in plastics may also reduce the adsorption of PAHs to MNPs by competing for the adsorption sites. In this context, Lijing Lui et al. compared the adsorption of PAHs on micro-and nanoplastics by an experiment with PS, micro-and concluded a higher adsorption to nanoplastics due to the higher surface to volume ratio (48). This finding is relevant because the abundance/amount of nanoplastics will increase over time due to the continuous degradation of (micro)plastics, leading to nanoplastics which could potentially be more harmful to humans. The influence of nutrition on the development of diseases or protection of those that are associated with exposure to POPs has also been studied. Recently, Żwierełło et al. published an extensive review on the influence of polyphenols (49). The results of in vitro and in vivo animal studies show a significant impact, a healthy diet rich in polyphenols might play a protective role against the toxic effects of POPs. Many antioxidants are polyphenols, which are found in spices, dried herbs, seeds, nuts, vegetables, fruits and many other food sources. The protective effect of polyphenols arises from their impact on the level and activity of the components of the antioxidant system, enzymes involved in the elimination of xenobiotics, and

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therefore, on the level of reactive oxygen species (ROS). For example, quercetin is a polyphenolic compound that can effectively reduce the negative health effects of PCBs. Selvakumar et al. studied the influence of quercetin on PCBs extensively, where rats were exposed to 50mg/kg quercetin together with a mixture of 2mg/kg PCBs (50). It showed that levels of endogenous hydrogen peroxide and peroxide in homogenates obtained from the brain, cerebellum and hippocampus were reduced compared to the control groups. In addition, reduced oxidative stress and DNA damage were observed with an increased activity of antioxidant enzymes which support the excretion of POPs. However, more human clinical trials are necessary to determine the real potential of polyphenols in the protection and prevention of POP-related diseases, because tests are only performed on rodents. Logically, testing on humans demands extreme caution and needs to be performed in a controlled way with suitable technologies. In addition, polyphenols might undergo metabolic changes during digestion and their metabolites do not always retain the same properties, which could make daily intake doses unrealistic. Therefore, this aspect needs more research on the mechanisms of polyphenols in humans and specific doses of daily intake should be mentioned and investigated. Although, these tested toxic compounds are additives from plastics, a research on the influence of polyphenols on MNPs is also a future research that will be interesting.

Other potential hazards can occur due to the adsorption of heavy metals to MNPs. Liao and Yang conducted a study on microplastics that serve as vector for Chromium (Cr) in an in-vitro human digestive model (51). Cr can be released in the gastric and intestines phase, but in accordance with the calculated daily intake of Cox et al., the released amounts would not pose any hazards for human health (18). However, for nanoplastics this could be different due to a probable higher adsorption of any metal. Furthermore, the adsorption ability for different pollutants is also dependent of diverse factors such as salinity, temperature, pH, dissolved organic matter and the physical-chemical properties and aging of MNPs (52). In the environment, there is variance within these factors, therefore, more research is needed on different environmental samples in conjunction with a standardized sample treatment and characterization method. This may provide the solution for the accurate determination of mixture toxicity from MNPs with the mentioned pollutants. Recently the potential of MNPs as organism vector has also been studied by Shen et al. (53) and partly by Deschênes and Ells (54).

The accumulation of nanoplastics in biofilms is a great concern, because biofilms are involved in many biological and organic processes from fundamental mechanisms in plant production, water remediation units and food production. In addition, to establish a complete risk assessment for ecological health this needs to be considered as well.

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2.

Toxicity testing

In the 16th century a scientist named Paracelsus made the statement: “the dose makes the poison”. This principle defines that all things are poisonous eventually, only the dose determines their toxicity, a famous statement among toxicologists. This also applies for MNPs, and in toxicology, several predictive models have been invented to estimate certain endpoints that are relevant for organisms such as, no observed effect concentration (NOEC) or median lethal concentration (LC50), etc. Many studies rely on animal testing, in vivo and in vitro models are used to test the toxicity of compounds and estimate human health effects. However, using animals for this purpose is complex due to qualitative and quantitative differences. For example, the mechanism of excretion (qualitative) and ratio of excreted concentration (quantitative) can differ for each compound between humans and animals (55). Therefore, dose estimation requires careful consideration about the difference in pharmacokinetics and pharmacodynamics among species. Nair and Jacob (56) provided a simple practice guide to perform dose conversion between animals and human, where allometric scaling was used to convert doses between different species. In this approach the exchange of drug dose is based on normalization of dose to body surface area. Due to the unique characteristics (biochemical process, anatomical, physiological) among species, the possible differences in pharmacokinetics or physiological time is accounted by allometric scaling. To explore qualitative and quantitative differences and interactions of toxic compounds within organisms, toxicity-based-toxicokinetic/toxicodynamic (TBTK/TD) modelling can be used. TBTK/TD modelling is a powerful mechanistic approach clarifying fate and behaviours of specific toxicants, facilitating to translate exposure to time course of toxic effects on related biomarkers, for example the inhibition of cytochrome P450. Where TK refers to the change in concentration over time and TD defines the effects of the toxicants. Currently, TBTK/TD modelling is the most promising tool for human health risk assessment. In a study from Yang et al., a TBTK/TD model was used to quantify organ-bioaccumulation and biomarker responses from PS microplastic particles in mice, that generally serve as mammalian terrestrial model organism (57). It was observed that PS microplastics (5-20µm) caused neurotoxicity effects, inflammation and altered metabolomics, although the severity is likely to depend on the size of the microplastics. This was implied, because the threshold concentration of the 5 µm PS was higher (8 ± 5 μg g−1) than the threshold concentration of 20 µm PS (0.71 ± 0.14 μg g−1) among the

most sensitive biomarker. This implication was also observed in studies from Schirinzi and from Gopinath which are described below.

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Figure 3: Schematic overview of TBTK/TD modeling of PS microplastic in mice (57)

In Figure 3, a schematic overview of the TBTK/TD model, which consists of different steps, is shown. After modelling and parameterization (EC50, etc.), a three-parameter Weibull model was used to estimate a threshold value for each biomarker that was tested. The extrapolation from mice to human was conducted by an algorithm, which was based on the well-constructed guidelines of interspecies dose conversion by the US Food and Drug Administration, where a safety factor of 10 was used to allow variabilities from the algorithm. This model offers a framework for microplastic exposure in mammals and offers an algorithm for the extrapolation from animals to humans for health risk assessment perspective, which also have the potential to be used for nanoplastics. Amereh et al. (58) produced an in vivo study where rats were exposed to PS nanoparticles, which is one first studies of nanoplastic exposure on mammals. With the use of semen biomarkers and blood samples, evident alterations in reproductive hormones and gene expression patterns were indicated. Which means that nanoplastics can cause endocrine disruption. As a critical point, the rats were kept in plastic cages which could lead to contamination and false positives. The mouse and especially zebrafish are commonly used for in vivo studies to model human disease. A mouse has similarities in physiology and anatomy which are matched by substantial genetic homology. Despite of anatomical similarities, zebrafish are also genetically related to humans and can be used to investigate human infection by

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injecting a corresponding site that best suits the research question (59). Zebrafish have high genetic homology to humans, with approximately 70% of human genes and about 82% of potential human disease-related genes. Due to this, zebrafish can reveal fundamental concepts of microbial pathogenesis and host defense, which may help to develop innovative therapies to combat human infection. Therefore, zebrafish are not intended to replace animal models like mice, but are complementary with each other. For example, Sökmen et al. investigated the exposure of PS nanoplastics (20nm) to zebrafish embryos, where it was proved that these particles can reach the brain and bioaccumulate there, which lead to oxidative DNA damage in the brain. But more studies are needed to clarify the bioaccumulation of nanoplastics into mammals, however these studies demonstrate the potential danger of nanoplastics. Future toxicity studies should include different types of MNPs of various shapes and sizes instead of using exclusively commercially available PS nanospheres. This is crucial for the risk assessment of MNPs.

Animal testing is not promoted due to ethical issues, and therefore, in vitro studies that can provide complementary valuable information are used. Mammalian cell lines have proven to be excellent models for the determination of cytotoxicity of potential harmful compounds to human health. In a study from Schirinzi et al. human cerebral and epithelial cells were exposed to different concentrations of PS (10 µm) and PE (3-16 µm) spherical microplastics (60). Based on this study, they did not pose any significant cytotoxicity in the two cell lines; only oxidative stress was observed at high concentration levels ranging from 1 to 10 mg/L. PS exposure resulted in higher ROS generation in both cell cultures which was probably related to the average smaller size of the PS particles. In another study, Gopinath et al. (61) exposed human blood cells to different forms (virgin, isolated and coronated) of PS nanoplastics (100 nm) in ranging concentrations from 10 to 100 μg/mL Conformational changes in blood protein, cytotoxicity, genotoxicity and hemolysis were observed after different exposure durations (4h or 24h). In addition, there was a significant decrease in cell viability and also damage to the DNA structure. One disadvantage of in vitro studies, is the lack of insights in the bioaccumulation process of MNPs, because this process may influence the cytotoxicity. For example, the question still remains if it possible for microplastics to degrade in the body of a human or animal into nanoplastics along its excretion process. Most of the toxicity studies described above make use of fluorescence detection and fluorescently labelled particles for the identification and quantification of MNPs. These methods are able to visualize the translocation of the particles and to determine concentrations. The use of fluorescence detection and fluorescence labelling methods are critically discussed in chapter 6 and 7.

Until now, the used doses of exposure in most studies (in vitro and in vivo) seem unrealistic for environmental exposure of MNPs. In addition, variation in results may be explained by differences in chemical nature of MNPs such as; size, shape, surface chemistry, other physicochemical properties and different exposure routes. Gray and Weinstein investigated the influence of different sizes and shapes of microplastics (PS, PE, PP), it turned out that the mortality of shrimps was highest when exposed to fiber shaped PP microplastics instead of spheres and fragments. Due to this, all studies can only provide indicative results related to human health (62). Therefore, adequate sample treatment, characterization, identification and especially quantitation methods are highly needed. This will provide a better understanding of the physicochemical properties from different kind of plastics and realistic doses of exposure. Which will realize more accuracy in toxicity testing and will improve the risk assessment of MNPs on human health, especially with the use of toxicity models. In the upcoming chapters the progress of analytical methodologies is reviewed and discussed.

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3.

Sample preparation

MNPs were found in food, air and organisms in multiple studies, these samples are strongly related to human health via ingestion or inhalation, therefore the focus in this review is on biological and air samples. Multiple studies reported the use of different sampling procedures and treatments on both kind of samples. The sampling procedure and treatment are of great importance to reliably identify and quantify MNPs. Multiple approaches involving digestion of matrix by acid, alkaline and enzymatic treatment. These sampling procedures and treatments are discussed below.

Plastics are everywhere nowadays and therefore MNPs can be found in a broad spectrum of samples. For instance, biological samples used for studies such as fish or mussels from a market or other eatable food products, can easily be obtained. Specific cell lines or different body parts can be isolated and treated for analysis to the wishes of the researcher involved. In addition, air samples can be collected through a stand-alone pump (63), vacuum cleaner (31) or with innovative technologies such as a breathing thermal manikin (30), after and during these sampling methods, filters were used for collection of MNPs. However, all samples should be homogenized and contamination must be avoided. Contamination is the main issue in any sampling procedure around MNP studies, because plastic equipment is used everywhere and, therefore, there is a strong risk of contamination. It is of great importance to identify potential sources that can contaminate the samples and prevention measures should be taken. For example, tools and setups should not contain any plastics but non-polymer materials to avoid systematic contamination, always work under a laminar flow hood and after sampling the samples need to be shielded against airborne contamination. Airborne contamination like synthetic fibers from atmospheric fall out, clothing or gear is probably the most difficult to avoid, it also seems unlikely to completely avoid plastics in all used factors. Therefore, a thorough blank value and recovery evaluation of the method should be performed at all times. Air samples need to be treated extra carefully due to consisting of fibers, the used filtration system needs to be thoroughly washed between samples and used filters need to be exposed to very high temperatures in order to remove fibers and other contaminants, because airborne contamination is always present (64). Living organism samples such as fish need some treatment after collection as well, the samples have to be frozen at -21℃ according to ICES (65), or could also be preserved in fixatives like formaldehyde or ethanol (66). This is because a living organism will start decomposing after 30 minutes (67), hereafter the sample is not representative anymore. Another difficult aspect for both biological and air samples is the broad spectrum of samples, and every sample is different due to their environment, sex, age, wet weight, length, shape, size or amount etc. All these attributes should be noted if possible, to study their influence and to be able to compare with other studies around the whole world. Standardized sampling procedures will make comparisons easier for all kinds of samples involving MNPs. Hermsen et al. (66) provided a standardized protocol for the detection of ingested microplastics in biota. It comprises some requirements for each step in a method, from sampling to detection. It is recommended to follow these requirements before setting up a method, especially for contamination control and the use of positive (spiked samples) and negative controls (blanks). For the detection of nanoplastics this protocol may needs to be adjusted.

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Figure 4: Overviews of the kinds of animals and of multiple sample preparation techniques that are used for microplastic research (68)

After the sampling procedure, the samples can be treated via multiple methods such as density separation, chemical digestion or enzymatic digestion. Depending on the type of sample, different methods or approaches need to be applied due to their difference in composition. For example, air samples are mostly treated through density separation with ZnCl or NaI. Allen et al. (see Table 1) (69) tested a sample treatment on five atmospheric deposition samples from a mountain. The used sample treatment was based on other studies. They used 10mL of 30% H2O2 for the removal of organic matter and the procedure was repeated after 7 days, this time using 5mL of 30 % H2O2. After 14 days the samples were filtered and density separation with ZnCl2 was performed. Two sets of blanks were used to test the sampling procedure and sample treatment on the presence of possible microplastic contamination. Loss of sample was controlled by taking photographs before and after the treatment with an Olympus SC30 camera which was attached to the stereomicroscope. However, no results on the recovery were given and this protocol is lengthy, requiring 14 days, which might not be suitable for standardization. In addition, Dris et al. also used density separation with ZnCl2 in combination with filters without the removal of organic matter, although other type of samples (indoor and outdoor air) were used (see Table 1) (63). However, this study did also not provide results about the recovery of the particles after sample treatment which is a crucial detail to determine the quality of the sample treatment method. In this context, Prata et al. (64) used a method that first removed the organic matter with 15% H2O2 (see Table 1). After 8 days of organic matter removal, samples were filtered in a washed glass fiber filter and afterwards transferred in a NaI solution for density separation. This method delivered 94.4% recovery of PS spiked in common textile fibers and was used on real indoor and outdoor samples. In summary, the mentioned studies all state to be suitable for microplastic fibers in air samples. The method from Prata et al. is more suitable due to the high recovery which is desired for standardization. However, this method is time consuming, which is not ideal for standardization. Nevertheless, as long as blanks are used to test the sampling procedure and the sample treatment for each set of samples to control contamination, all these methods are suitable. In addition, spiked samples with standards should be used to test the recovery of the sample treatment and results should be noted. Very recently, Sobhani et al. also detected nanoplastics in paint-polishing dust samples, where it was sufficient to add the samples in a H2SO4:H2O2 (2:1,v/v) solution which was kept

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overnight (70).This method looks promising for the characterization and identification of MNPs and it is further discussed in chapter 6.

Biological samples are treated differently than airborne samples, due to different composition and physical and chemical properties. The organic matrix comprises tissues or an organism, therefore, other protocols have been applied. Dehaut et al. reviewed and tested protocols for the sample treatment of tissues from fish, crabs and mussels. In this study, six protocols were tested on degradation of the plastic polymers and digestion of tissue. Three protocols were not suitable, these protocols included multiple acid digestion treatments with HCl, HNO3 and HNO3:HClO4. The acid treatments degraded the plastic polymers involving PE, PP, PA-12 and PS. In the second experiment the other three protocols were tested on a wider range of plastic polymers. Protocol 5 that used 10M of NaOH was not suitable as it degraded PC and PET. The protocols that were suitable used alkaline digestion with 0.24M NaOH and 10% KOH with incubation and filtration. However, 0.24M NaOH degraded the cellulose acetate polymer and did not provide good digestion of the tissues as clogging of the filter occurred. The treatment with 10% KOH was presented most suitable for the extraction of MNPs from fish, mussel or crab tissue. In addition, a recovery of 100% was obtained for cod fillets, saithe whole alimentary tracts and mussels. Although, degradation of cellulose acetate also occurred at this protocol. However, it was less intensively than the treatment with 0.24M NaOH. Barboza et al. successfully used the digestion protocol with 10% KOH from Dehaut et al. for microplastics in the gastrointestinal tract, gills and dorsal muscle of three fish species. The most common polymers found were PE, polyester and rayon. The incubation was performed at 60°C for 24 hours, but this was not efficient for gill samples. Therefore, gill samples were incubated at 40°C for 72 hours which was successfully used in the study of Karami et al. (see Table 2) (71). Therefore, it seems that treatments have to be altered for specific parts of tissue which is not desired for a standardized protocol. Rist et al. (72) tested multiple treatments on exposed Daphnia Pulex to MNPs; alkaline digestion with NaOH, 30% H2O2 treatment, acid digestion (nitric acid, HNO3), 25% tetramethyl ammonium hydroxide (TMAH) and an enzymatic digestion with Proteinase K. Strong agglomeration of particles and loss of particle fluorescence were the consequences of the treatments with NaOH, H2O2 and HNO3. The use of TMAH resulted in an incomplete dissolution of the tissues and Proteinase K only gave minor agglomeration of the particles, however, the particle fluorescence signal was completely maintained. The used protocol for enzymatic treatment was less time consuming relative to alkaline and acid digestion (73). Agglomeration was measured by the z-average size and polydispersity index, both values need to be low as possible to obtain a homogenous sample with small nanoplastics. Alkaline digestion with KOH was not tested in this protocol while it often reported in the literature. However, alkaline digestion with NaOH resulted in significant loss of fluorescence and more agglomeration. Therefore, it appears that enzymatic digestion is more suitable for the analysis of MNPs with fluorescence detection. However, when using thermal fragmentation and spectroscopy techniques, digestion with KOH appears to be suitable as treatment protocol which was shown in the study of Dehaut et al. However, enzymatic treatment was not tested in this protocol as it was assumed to be difficult to implement and present digestion efficacy issues.

After all, it is clear that the used sample treatment should not alter the MNPs, and therefore, the chosen sample protocol is of great importance for the analysis. For example, with use of optical microscopy and dynamic light scattering it has been shown that aggressive methods such as acid, alkaline or H2O2 treatment can cause aggregation of the particles. The aggregation could be caused by the significant change of the ionic strength. Furthermore, these treatments could also have negative effects on the fluorescence signal of labeled MNPs (e.g. in toxicology experiments). Enzymatic digestion is milder than acid, alkaline and H2O2 treatment, therefore, it is likely to be more suitable as treatment protocol before fluorescence or light scattering analysis, it has been demonstrated to cause

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no or less aggregation of the particles. However, validation of sampling and sample treatment protocols is necessary. The used sample treatment protocol depends on the type of sample and the available analytical techniques. Several preconcentration steps of the sample are always necessary due to the very low amounts of MNPs in real samples. Several methods can be used for this purpose such as membrane filtration, ultrafiltration, ultracentrifugation, or evaporation of solvent.

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4.

Analysis techniques for MNPs

In this chapter the methods for the separation, characterization and identification of MNPs are reviewed and discussed. Figure 5 shows an overview of the techniques reported in the literature. In the subchapters below, the following techniques will be discussed: FFF, chromatography, light scattering, microscopy, spectroscopy, thermal fragmentation and other potential techniques. In addition, Table 1 & 2 provide an overview of the recent studies from analyzing MNPs in air and biological samples.

4.1 FFF techniques

Field-flow fractionation (FFF) is one of the emerging techniques for the size characterization of nanoplastics. FFF does not belong to chromatography techniques as it does not use any stationary phase and has multiple variants that can be used such as, thermal, electric, magnetic and cross-flow FFF. The separation take place in an empty channel and it is based on differences in the diffusion coefficients of the analytes, being caused by an external field, applied perpendicularly to the axial parabolic flow (75). Due to the parabolic flow profile and the external field, particles are localized in different regions of the channel. Therefore, the particles will move towards the detector at different speeds and exiting the channel at different times. The power of FFF is the broad range of particles that can be measured and because this technique is non-destructive and relatively fast. In addition, agglomeration behavior of MNPs can be studied using FFF(76). The most common variant is asymmetrical flow-FFF (AF4), which can be coupled to multiple detectors like UV-vis, refractive index, fluorescence, multi-angle light scattering (MALS) and dynamic light scattering (DLS) (77). These detection techniques can provide information on number of particles and particle size distributions for the characterization of MNPs. However, the development and optimization of an AF4 -based method requires a good understanding of the technique (78). Monikh et al. reported the use of AF4-MALS to successfully fractionate and characterize PS nanoparticles (60, 200, 300, 600nm) spiked (100mg/L) in eggshells (see Table 2) (79). This method shows the potential of AF4 for the characterization of nanoplastics. Moreover, the authors state that the fractions can be further analyzed by GC-MS for polymer identification and perhaps also for quantification purposes. However, egg shells are not related to human health as they are normally not consumed by humans. Correia and Loeschner also used AF4-MALS for the analysis of fish tissue samples which were spiked with 100 nm PS particles (final concentration of 5.2 µg/mL) (80). Figure 6 shows the overlayed fractograms obtained by analyzing dye red aqueous fluorescent spherical polystyrene nanoparticles (FIPSNP) in ultrapure water and fish. There were also non-spiked fish samples added to the sample sequence. The fractogram shows the capability of AF4 to separate the PS nanoparticles from the matrix and the peak shows minor deviation with the PS nanoparticles in ultrapure water. In this study Proteinase K was used for the digestion of matrix, which showed to prevent aggregation, whereas acid treatment with nitric acid induced aggregation of the PS nanoplastics in the studied samples. Therefore, enzymatic digestion was again more suitable than acid treatment. In addition, DLS was used to study the possible effects of aggregation, although an increased spiked particle mass (23 mg/g fish) was used to assure the detectability of the particles. This demonstrates the importance of an adequate sample treatment procedure for reliable and accurate characterization, identification and quantification. The authors reported that after optimizing the carrier liquid composition for the AF4 experiments, it was also possible to analyze PE nanoparticles. However, it was not possible to detect the PE nanoparticles when spiked (10 µg/µL) to fish samples .The authors attribute this to an elevated light scattering background signal from the organic fish residues in the AF4 running conditions. This means that every

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developed method for a certain type of nanoplastic and may not be applicable to other types of nanoplastics, which makes it complicated to standardize these protocols for multiple kinds of plastics, unless suitable studies are performed a wide variety of MNPs. Hence, AF4 demonstrates to be suitable for analyzing nanoplastics. However, this technique it is less likely to be useful to separate microplastics as the elution mode will be changed from normal to steric. This phenomenon can occur with particles around and above 1µm, in which large particles undergo stronger forces from the laminar flow (81) and therefore, larger particles will elute faster than smaller particles. To prevent this when analyzing nanoplastics, a filtration step at the inversion point is needed to exclude the larger particles which can be studied by complementary techniques.

Figure 6: AF4-LS fractograms of the pristine FlPSNPs in ultrapure water, fish spiked with FlPSNPs and non-spiked fish (80).

4.2 Chromatography techniques

A complementary technique to FFF is chromatography, which makes use of a stationary phase to separate the analytes. However, this kind of separation pose more challenges for the analysis of MNPs, as interactions with the stationary phase may occur. Nanoplastics are expected to have rougher surfaces compared to microplastics, due to their origin from fragmentation, which may increase the interactions with the stationary phase. Overall, the small size of nanoplastics can also cause problems, as the pore size of the stationary phase may not suffice, and because of this, analytes may have less interaction with the stationary phase. Recently, reversed phase high performance liquid chromatography (HPLC) coupled to mass spectrometry has been used for the separation and quantitation of microplastics (PC and PET) in air (indoor dust) and biological (mussels and clams) samples (82). Other chromatographic techniques such as size exclusion chromatography (SEC)(83) and hydrodynamic chromatography (HDC)(84) have been reported for the separation of engineered nanoparticles. Hence, they may be also suitable for the separation of nanoplastics. Pirok et al. combined HDC and SEC in a comprehensive 2D-LC system, where the combined two-dimensional

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distribution of particle size and molecular size of PS and polyacrylate particles was obtained successfully (85). This combination of techniques is promising; however, the optimization of two-dimensional separations is very time consuming and much more complicated compared to a one-dimensional system.

4.3 Light scattering techniques

MNPs in the environment can be very different in physical and chemical properties, for example in hydrodynamic radius, zeta potential, geometry and surface characteristics. These parameters have an influence on the identification and quantitation of MNPs, therefore, detailed characterization is necessary. The analytical question determines the technique that needs to be used. Light scattering is used in many studies, for example DLS can deliver a PSD in the range of 1nm to 3µm. DLS measures the intensity of the scattering from the light-particle interaction as a function of time (86). A laser beam passes through a suspension, where particles cause fluctuations due to their Brownian motion. Through autocorrelation, the fluctuations can be connected to the different hydrodynamic size of the particles, which delivers the PSD. In addition, DLS is also capable of the determination of the zeta potential of the particles through the detection of frequency shifts in the scattered light (87). The zeta potential is a value that provides information on the stability of a suspension. MNPs may undergo weathering in the environment which can cause changes in surface charge. Therefore, the zeta potential might describe the ageing of MNPs. For example, González-Fernández et al. used DLS to measure the zeta potential and size of MNPs in Pacific oyster (88). Although DLS is a relatively easy to use technique it is limited to a very low particle concentration due to multiple scattering effects, where photons are scattered multiple times before reaching the detector. Despite the broad range of particle sizes that can be measured, a mixture of particle sizes may cause problems, and the measured radii can be skewed towards higher sizes (81). This is because larger particles will scatter with more intensity than smaller particles, therefore, the signals of large particles will cover the signals of the small particles. Because of this, small particles will be overlooked and therefore, DLS can only measure average size. Another problem might be caused by contamination of dust fibers or formed aggregates from the sample matrix. This could be a difficult issue for biological and air samples, and therefore, strict measures should be taken for sample preparation to use this detection technique properly, as described in chapter 5. Another approach that uses (static) light scattering is MALS, this technique measures the scattered light from the sample by different angles and can determine the molecular weight and the average size of molecules in a solution. MALS is often coupled online with AF4. For instance, Gigault et al. reported the use of AF4-MALS for the characterization of nanoplastics (89). In this study different AF4 conditions were tested for the analysis of PS particles of sizes between 1-800nm. The developed method was able to characterize submicron populations in fish samples. However, when the same AF4 method was applied in a smaller range, only for particles between 200 and 800nm, the selectivity decreased significantly compared to the results obtained for particles 1-800nm. Therefore, the elution profile was tuned in four different subfraction (variating smaller size ranges) methods to enable the separation of both <200nm and 200-800nm PS. In these subfraction methods, it was found that a constant cross-flow rate enhanced the selectivity. The developed method and additional four subfraction methods combined, may be used to study all the submicron populations in fish samples. This study demonstrates the advantages of AF4 coupled to MALS, and the developed methods were also used in a more recent follow up study where the degradation of microplastics to nanoplastics was studied (90). This study is from El Hadri and is described in section 4.5 FTIR spectroscopy further below.

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Nanoparticle tracking analysis (NTA) is a light scattering technique complementary to DLS. Both techniques calculate the hydrodynamic size of particles based on the measured Brownian motion. NTA uses a microscope and a high-sensitivity videocamera which makes it possible to visualize (video image) and record every particle. Therefore, it can determine the hydrodynamic size of each individual particle instead of average size data as generated by DLS (91). On the other hand, very polydisperse particles (10nm-1mm) or a very narrow size range (1-10nm) cannot be measured with suitable accuracy, which makes NTA more limited on size range. Hou et al. compared both techniques, where it turned out that DLS was more suitable to study large particles (>1000nm), while NTA was more accurate in detecting small particles (>1000nm)(92). Therefore, it may be hypothesized that DLS is more suitable for microplastics and NTA for nanoplastics, although the mentioned study did not focus on plastics, but on cerium oxide nanoparticles. Lambert and Wagner successfully characterized PS, PLA, PP, PE and PET in the range of 30-2000nm in two studies with NTA (93)(94) and it was showed that NTA is capable to quantitate MNPs with common used models due to its software. These models and their applications are further described in Bayat et al. 2015 (95), Weipeng et al. 2015 (96) and Yang et al. 2012 (97). The main disadvantage of NTA is that it does not provide images of individual particles, and because of this, the structure of the particles cannot be evaluated. Luckily, other techniques like optical microscopy, scanning electron microscopy (SEM) or transmission electron microscopy (TEM) are fit for this purpose and make the characterization of MNPs more complete.

4.4 Microscopy techniques

Microscopy techniques provide information on the morphology of a sample, this includes parameters like geometry and surface characteristics. Optical microscopy is a technique used for single particle analysis of microplastic particles, where the stereomicroscope is often reported in literature. The stereomicroscope is used for visual identification which is a fast, simple and cheap technique. However, it was reported that this technique is limited due to the difficulty of distinguishing microplastics from other small organic/inorganic debris particles which may lead to false positives and false negatives (98). In addition, a stereomicroscope is not capable of visualizing nanoplastics due to restricted diffraction limits. Combining microscopy with other identification techniques can confirm the presence of microplastics, where a stereomicroscope is used for visual sorting and identification is performed by FTIR or Raman spectroscopy. In combination with such techniques, optical microscopy can be a valuable tool, although it is more time consuming. Liu et al. conducted a study on suspended atmospheric microplastics, where a stereomicroscope was used for visualizing of the particles and µ-FTIR was used for the identification of microplastics (see Table 1) (99). Multiple types and sizes of microplastics were successfully identified and measured through an Image J software program after importing high-resolution photographs. In toxicology experiments fluorescence microscopy is often used for the tracking and translocation of MNPs. Forte et al. performed an in vitro study where human gastric cells were exposed to unmodified PS nanoparticles (37). In this study, fluorescence microscopy was used to track and localize dyed PS nanoparticles the observed intensities were compared with the maximal intensity to calculate exact concentrations. Rist et al. performed an in vivo study where mussels were exposed to PS beads of 2µm and 100nm and fluorescence was used for the same principle (see Table 2) (24). The quantification of MNPs with use of fluorescence is discussed in chapter 7.

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Besides optical microscopy, electron microscopy (EM) is a very powerful technique for detailed information of MNPs. Electron microscopy uses an electron beam that scans the sample, where the electrons cause many interactions with the sample which can be detected and give very specific information about the sample. It can observe very small differences between the wavelengths of high energy electrons which illustrates its resolution, which makes it possible to image nanosized particles (100). EM can be divided into the techniques of TEM and SEM. TEM uses a higher electron acceleration voltage and a very thin sample, providing more information on the interior of the sample. While SEM uses a lower voltage, which causes interactions in the surface of the sample. Additionally, SEM can give information on morphology, ageing and origin of the sample. Both techniques have high resolution and are mostly coupled to energy dispersive X-ray spectrometer (EDS) allowing visualization of the sample whilst simultaneously gaining qualitative information on the elemental composition. SEM-EDS is powerful combination for the characterization of MNPs, however there are some limitations as well. The technique is expensive and very time consuming with many sample preparation steps, hence limiting the number of samples that may be analyzed in a given timeframe. In addition, SEM cannot provide colored images which means that the colors of particles cannot be used as identifiers. EDS can detect trace amounts of specific elements (including Na, Al, Ca etc.), therefore, it may determine the presence of additives by the chemical signature of these elements. The major limitation of EDS spectra is its inability to differentiate between elemental signatures originating from the polymer and elemental signatures originating from additives (101). Gniadek and Dąbrowska acknowledge these limitations, but also indicate the use of conventional FE-SEM to bypass some limitations. FE-SEM with low electron high tension (EHT) provides high resolution images of microplastic samples without prior sample preparation. In this way sample preparation and investigation can also be done fast, easy and at low cost. However, small samples are needed to diminish charging effects and EDS normally uses high voltages which cannot be applied in this situation because it could cause a sample charging effect as well. Therefore, Gniadek and D ąbrowska used a moderate voltage instead, which can only be used when light elements are taken under consideration (102). Due to this, adjusted conventional FE-SEM also has limitations, but this study shows the potential of SEM-EDS for the characterization of MNPs. However, to adjust the technique properly, knowledge and experience with the technique are needed.

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Figure 7: An example of the analysis of microplastics by SEM-EDS, images of microplastics with the attached spectrum (103)

Dehghani et al., conducted a study where dust samples were collected from the streets in Tehran, Iran and analyzed with a fluorescence microscope and SEM-EDS(103). Samples were treated with H2O2 to remove organic matter and then the samples were separated on density with ZnCl2. After the sample treatment, the samples were analyzed with a fluorescence microscope, SEM-EDS and afterwards the confirmed microplastics were counted with a binocular microscope. Fluorescence microscopy did not provide satisfactory results due to interferences (fluorescence agents from paper) and the sample preparation may have caused loss of sample. On the other hand, SEM-EDS did provide useful results on surface characteristics and elemental composition (see Figure 7). Smooth surfaces were observed for microplastics and glass spheres, which were distinguished with the binocular microscope. The EDS spectrum demonstrated trace amounts of Na, Mg, Ca, Al and Si. These elements give an indication which microplastics are present, because these antioxidants are present in additives of certain plastics, but is not a guarantee. These elements are often used in PP, PS and PE to slow down the oxidation cycle. However, the use of SEM-EDS may not suffice, because some plastics or additives in plastics might have small differences in elemental composition which cannot be distinguished by SEM-EDS alone. Therefore, for accurate characterization and identification of microplastics it would be preferable to combine microscopy and FTIR or Raman. This was also recommended by Song et al., who compared FTIR with microscopy as the two methodologies are complementary (98). However, for nanoplastics (and also microplastics) SEM-EDS may be combined with other spectroscopic techniques like FTIR and Raman. This may be a very powerful combination for complete characterization and identification, despite of increasing analysis time and costs. In addition, the use of TEM-EDS may be more explored for the characterization of MNPs, as it is used rarely.

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Table 1: Overview of studies for the characterization and identification of MNPs in air samples

Type of polymer and size range

Samples Sample treatment Analysis techniques Detected levels Ref.

PS

330-383 µm Indoor and outdoor air

Removing organic matter with H2O2. Density separation with

NaI.

Visual identification, Use of Nile red,

µ-Raman spectroscopy. - (64)

PET, PVC and Polyamides, 50-250µm

Indoor dust All samples were filtered. Densityseparation with ZnCl

2.. µ-FTIR coupled with ATR

Counting through software histolab coupled to stereomicroscope: Indoor air: 600 fibers, Outdoor air: 40

fibers, Dust fall: 350 fibers (63)

PET, PE, PES, PAN, PAA, RY, EVA, EP, ALK 23-9955 µm

Suspended atmospheric microplastics

Filtered during sampling with KB-120F type intelligent middle flow total suspended particulate sampler (Jinshida, Qingdao)

Stereomicroscopy and µ-FTIR

The sizes of microplastics were measured using the Image J software program (version 1.51j8) after importing high resolution photographs. SAMP abundances ranged from 0 to 4.18 n/m3, with a mean concentration of

1.42 ± 1.42 n/m3 (mean ± standard deviation)

(99)

PE, Polyester, Nyl6 11 µm

Collecting air samples with a breathing thermal manikin that

simulates human

metabolic rate and breathing.

Enriched membrane flushed with ethanol, deposited on a ZnSe window and dried

FPA-micro-FTIR imaging Concentrations between 1.7 and 16.2 particles m−3 (30)

PP, LD-PE, UPVC, PET, PA, PS

400 µm

Spiked water and air

samples - Near IR and Refractive index - (104)

PET, PC

Building blocks: TPA and BPA.

<150 µm

Indoor dust samples Alkalidepolymerization with KOH and 1-assisted heating pentanol

HPLC-MSMS

In all dust samples:

PET concentrations of 38–120,000 µg/g PC concentrations of <0.11–1700 µg/g (31) PS, PP, PE, PVC and PET 50-600 µm Atmospheric deposition samples

Filtered, removing organic matter with H2O2 and density separation with ZnCl.

Visual microscopy inspection and µ-Raman spectroscopy

Visual counting and using Image J software

Daily fall out ±69 microplastic particles (69)

HPLC-MSMS

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Building blocks: TPA and BPA

0.075−0.15 mm

Indoor dust depolymerization of PC and PET into

bisphenol A and p-phthalic acid (BPA and PTA), SPE Poly-SeryHLB 6 cc/200 mg PS 600, 300 100 nm Paint-polishing dust samples

Removal of organic matter with a

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