Literature Study
Master Chemistry: Analytical Sciences Master Thesis
Microplastics in Humans and Animals
How to identify and detect microplastic in biological samples?
Kyara van Aken
12 EC
Student ID UvA: 12841277
Student ID VU: 2699123
February-March 2021
Supervisor/ 1st Examiner: 2nd Examiner:
Dr. Freek Ariese Dr. Heather A. Leslie
2
Abstract
Micro- and nanoplastics (MNPs) find their fate in the environment. MNPs are degraded from plastic debris or produced as small particles in a product. These particles might be dangerous for the environment and the human health. MNPs have been found in drinking water, food, and the air, this results in direct exposure routes of MNPs to humans and animals. The knowledge about the risks of MNPs is limited. For risk assessment, particles should be quantified and identified, however no standardized protocol is yet utilized in the scientific world. The identification, characterization, and quantification of these particles is difficult, due to the many plastic types, the broad size range, and the matrix properties.
This review describes the methods that are currently being utilized in the research of MNPs in biological samples. The most important human exposure routes of MNPs are ingestion, inhalation, and dermal contact. Furthermore, MNPs are distributed within the body and can be found in every important organ. For the analysis of these organs, sample preparation is necessary. Chemical digestion is a common technique to digest the organic matter. KOH or enzymatic digestion are the most suitable for the digestion of the organic matter and do not alter the plastic particles. Also, other preparation methods are described. The method of choice depends on the research question and often preparation techniques are combined. Moreover, several techniques are reviewed for the separation, identification, and quantitation of MNPs. Combinations of techniques are the most suitable for a
standardized protocol. For example, a TGA-FTIR-GC/MS technique can identify and quantify the microplastics within a sample. Nonetheless, this method is relatively new and should be further investigated for MNPs. The last chapter of this review describes why it is necessary to have a standardized protocol, examples of toxicity testing are critically reviewed. Lack of control experiments and the use of unrealistic high dosages in a short time for in vitro and in vivo tests show the importance for a standardized method. Furthermore, the high dosages shows why the increase of plastic debris should stop. Also, PS microbeads are commonly used in toxicity testing, while fibers are the most abundant form in the environment and other plastic types probably have a different toxicity.
3
Abbreviations
A4F Asymmetric flow field flow fractionation AFM Atomic force microscopy
BPA Bisphenol A
CARS Coherent anti-stokes Raman scattering CE capillary electrophoresis
DEHP di(2-ethylhexyl) phthalate DLS Dynamic light scattering
DSC Differential Scanning Calorimetry EDS Energy-dispersive X-ray spectroscopy ePS extruded polystyrene
FCM Flow cytometry FFF Field Flow Fractioning FTIR Fourier transform infrared GC Gas chromatography
GPC Gel permeation chromatography HDC Hydrodynamic chromatography
ICP-MS Inductively coupled plasma - mass spectrometry IR Infra-red
LC Liquid chromatography LDPE Low density polyethylene LOD Limit of detection
LOQ Limit of quantification
MADLS Multi angle dynamic light scattering MALS Multi angle light scattering
MNPs Micro and nano plastics MP Microplastic
MS Mass spectrometry
MS/MS Tandem mass spectrometry NP Nano plastic
4 PA Poly amide PA-12 Nylon 12 PA-6 Nylon 6 PAN Polyacrylonitrile PBT Polybutylene PC Polycarbonate PE Polyethylene
PET Polyethylene terephthalate PLA Polylactic acid
PMMA Poly methyl methacrylate
POM Polyoxymethylene
PP Poly propylene
PS Polystyrene
PTFE Polytetrafluoroethylene
PUR Polyurethane PVC Poly vinyl chloride Py Pyrolysis
SAPEA Science Advice for Policy by European Academies SEC Size exclusion chromatography
SEM Scanning electron microscopy SRS Stimulated Raman spectroscopy TDS Thermal desorption spectroscopy TED Thermal extraction and desorption TEM Transmission electron microscopy TGA Thermogravimetric analysis TRPS Tunable Resistive Pulse Sensing UV Ultraviolet
5
Index
Abstract ... 2
Abbreviations ... 3
1 Introduction ... 6
2 Exposure and distribution ... 8
2.1 Ingestion, inhalation, and dermal contact ... 8
2.2 Distribution ... 10 3 Sample preparation ... 12 4 Analysis of microplastics ... 16 4.1 Separation ... 17 4.2 Identification ... 19 4.3 Quantitation ... 25
5 Toxicity and risks ... 27
6 Conclusion and further prospects ... 30
7 Literature... 31
6
1 Introduction
The annual plastic production increases every year. The global plastic production in 1950 was 1.5 million metric tons, whereas in 2019 368 million metric tons of plastic was manufactured.1 Humans utilize plastic products in their daily life, plastics are often chosen
because they are cheap, durable, and can be used for numerous applications. Currently, plastics are used in packaging, clothes, electronics, toys, and many other products. Plastic pellets are the building blocks for nearly all plastic products. These preproduction pellets may escape into the environment during every stage of its lifecycle, like production, transportation, and final product manufacturing. In addition, some plastics from cosmetics or other products cannot be removed by wastewater treatment plants and may end up in drinking water or the ocean.2 Many different kinds of polymers are utilized to manufacture
plastic products. Moreover, these products often contain additives such as stabilizers, plasticizers, antioxidants, or UV absorbers. These additives are added to improve the
product usage or appearance. Certainly not all additives should be used in food packaging or toys, because some might be toxic.3 Nevertheless, every year half of the produced plastic is
discarded after one time usage and subsequently become waste. Figure 1 shows the direct plastic debris in the ocean. Plastics may be recycled; however, this is difficult as many kinds of plastic are collected together. This plastic mix cannot be reused for the same quality of products, luckily science is working on techniques to separate different kinds of plastics.4 Not
all plastics are collected and some particles will find their fate in the environment. After some time, the larger plastic particles will be degraded due to sun radiation or ocean waves and become micro or nano plastics (MNPs). Particles with this origin are called secondary microplastics. Primary microplastics are specifically made in the nano or microplastic size, they are used in toothpaste, scrubs, cosmetics, and clothing. Microplastics are particles smaller than 5 mm and they originate from primary or secondary sources. The size range of nanoparticles is not well defined, although the size range between 1-100 nm is often utilized.5 Nonetheless, 1000 nm is also often used as an upper limit for nanoplastics.6
Figure 1 Several products with different sizes of ocean debris of plastic. However, plastic particles can also degrade to form smaller particles. Furthermore, the sizes about what are nano, micro, mesoplastics, etc. may not correspond to every article.7
7
In 1997 captain Charles Moore discovered large quantities of plastic in the North Pacific Ocean. High plastic concentrations have been found in many more places in the ocean, in the south pacific a plastic area larger than Germany and France combined has been
discoverd.8 Furthermore, MNPs have been found in areas where little or no human activity
is, like deep in the ocean, on the top of the mount Everest, the north and south pole and many other places.9–11 This shows that MNPs are able to travel long distances, due to their
characteristic properties such as persistence, buoyancy, small size, and low density.10 There
is a growing concern about MNPs in the environment, because of the potential physical and chemical harm to organisms.12 Furthermore, MNPs have been found within the body of
humans and animals. Even, some researchers show that the MNPs move within the food chain.12,13 Some studies have found adverse effects of MNPs on the health. Information
about the effects on humans of MNPs is limited, due to the fact that humans are not
intended for toxicity testing. Nonetheless, information about the toxicity can be determined with models, animal studies, and some human samples. Various animals may contain a few comparable human metabolic pathways, which can be used to estimate the toxicity.14
However, many things are still unclear about the toxicity, intake, distribution, and excretion of MNPs in humans and animals. Moreover, there are still no standardized
protocols for the methods or the recording of results. This thesis will focus on the analytical techniques that can be used for the identification and quantification of MNPs in biological samples. Several techniques have different applications, the advantages and disadvantages of every method will be described. In addition, the minimum particle size that can be measured with the technique will be reported. Chapter two will describe the exposure of MNPs, this will mainly focus on the ingestion, inhalation, and dermal contact. In addition, this chapter will give extra information about the distribution of the MNPs and will clarify where these particles have been found within the body. Chapter three will discuss the sample preparation of biological samples to detect the MNPs. The analysis of the particles will be described in chapter four and five. However, chapter five will focus more on the toxicity testing whereas chapter four focuses on the techniques that can be used to separate, identify, and quantify MNPs.
8
2 Exposure and distribution
This chapter will discuss the exposure routes of microplastic into humans and animals. Furthermore, this chapter describes the distribution of microplastics within the body. The three main routes of microplastics to enter the tissues are: ingestion, inhalation, and dermal contact.
2.1 Ingestion, inhalation, and dermal contact
The main route for microplastics to enter the body is considered ingestion, which means that it is present in food and drinks.2,15 Microplastics have been found in several food
products like salt,16 mussels,17 fish,18,19 sugar, 20 and many other products. However, dust on
food might have a larger impact of the plastic ingestion than the food itself.21 A study of Cox
et al. (2019) estimated that the annual plastic consumption is 39,000 to 52,000 particles per person with an American diet. 90,000 particles MPs could be added when people only drink from bottled water, when compared to only 4000 particles MPs from only tap water.22 The
dust from 39 Chinese cities were analyzed for plastics, PET MPs were detected in all the samples and PC MPs were detected in 70% of the samples.23 These discoveries make plastic
ingestion very likely and shows a route for microplastics to enter the gastrointestinal tract. Moreover, MPs has been found in human stool.24 This confirms the hypothesis that people
involuntarily ingest microplastics and that it enters the gastrointestinal tract. Zebrafish were used to analyze the intestinal toxicity of microplastic fibers. This research concluded that zebrafish could absorb MPs, especially in an early life stage. MPs were detected in the zebrafish gut, which causes intestinal damage and toxicities. More serious effects were observed with longer MP fibers, such as a decreased food intake.25 Zebrafish are often used
to study the possibly human toxic effects. They are used because of their optical transparency, genetic manipulability, and translational potential. Furthermore, the development of their immune system is well known.14
Another important exposure route for microplastics is inhalation. Microplastics are released into the air via synthetic clothes, textiles, shedding or abrasion of materials, plastic combustion, and landfill.2,15 Prata. (2018) found that the most abundant MPs in the
atmosphere are PP, PE, PS, and PET.26 The particles can be easily transported by the wind
and distributed around the globe. Besides, these particles have a small size and can easily be inhaled. The inhalation has been estimated to be 26-130 airborne MPs per person per day.2
However, estimations about the inhaled particles may differ. This is due to the different sample method and the lack of standardized measuring methods. Furthermore, this also depends on the cleaning schedule, activities, furniture material, weather conditions and the season. In general, the respiratory system can filter the microplastics. Particles smaller than 2.5 µm will remain primarily in the lungs and are able to pass the respiratory barrier.27
According to Chen et al. (2020) airborne microplastics are mainly from synthetic textiles and the dominant shape in the atmosphere are fibers. Fibers with a length larger than 250 µm have been observed in human lungs and may cause chronic and acute inflammation.28
Macrophages are part of the immune system and attack unknown particles. When
nanoparticles enter the respiratory system, the MPs are attacked by the macrophages and translocated to the circulation and lymphatic systems.15 MP fibers are able to pierce the
macrophage, which may cause inflammatory reactions. The length of fibers in the air have been measured predominant between 200 and 600 µm,29 this means that the fibers found in
9
lungs may be inhaled from the air. A research demonstrated that 50 nm PS NPs might have a cytotoxic and genotoxic effect on the respiratory system and macrophages.30 A route for
inhaled microplastics to the digestive tract is via the mucus, larger particles stick to the mucus and may enter the digestive tract.
Lastly, a probably less significant exposure route is dermal contact. Nevertheless, this exposure route should not be disregarded without any evidence of the toxicity. Dermal exposure happens when the skin absorbs the microplastics from personal care products or dust. Microplastic are too big to transverse the dermal barrier. However, NPs that are smaller than 100 nm can be absorbed by the skin. Often this route is associated with monomers and additives of plastics.2,15 At this moment there are no studies that quantify
the plastic intake of dermal contact.31 Microplastics are often used for emulsifying agent or
cheap filler in personal care product. Microbeads are the small plastic balls, with a diameter less than 1 micrometer. Nevertheless, the exposure via dermal contact will be decreased in many developed countries as microbeads in personal care and many other products are banned.32 Moreover, NPs are used in medication. The particles are utilized to transport the
drug within the body to achieve the desired effect. These small particles are able to cross cell membranes and probably enhance chemicals’ bioavailability.5,33 Another concern is about
the additive DEPH present in the blood bags for blood transfusion. This additive has been found in the blood in the bags and may be a potential risk.34 Figure 2 displays a summary of
the exposure routes and potential health risks for humans.2
Figure 2 Summary of the routes of exposure and potential health risks for humans. Potential risks that are not discussed in this chapter will be more explained in chapter 5. 2
10
2.2 Distribution
After the uptake of microplastics via the respiratory or gastrointestinal tract, particles may translocate throughout the body. Microplastics have been found in biological samples, however little is known about the distribution of MPs within the body. The distribution of microplastics was determined in scallops. Nine different tissues for MPs between 5 µm and 1 mm were analyzed, figure 3 shows the anatomy of a scallop and the measured tissues. The MP concentration were the highest in the anus, intestine and kidney, respectively. In the gill, muscle, kidney, gonad, hepatopancreas, hemolymph and mantle MPs were all found in similar, lower concentrations.35 This indicates that higher concentrations can be found in the
gastrointestinal tract for scallops. Moreover, the microplastics accumulation of several tissues of freshwater fish red tilapia has been determined. PS microplastics did accumulate the most in the following order: gut > gills > liver ≈ brain. The microplastics disturbed the metabolisms in the liver and inhibited the neurotransmitter (AChE) activity in the brain. This suggests neurotoxicity of MPs to freshwater fish.36 In another research fluorescent PS
microplastics were fed to mussel, subsequently crabs eat these mussels. The microplastics were found in the stomach, gills, testes, and brain of the crab after one hour and after 21 days. The number of MPs decreased over time only in the stomach and gills. When the mussel consumption increased the number of MPs also increased. However the number of microplastics did not increase with a higher dose in the brain, gills, and testes, perhaps the excretion rate is fast which may result in a limited uptake rate.37 Other researchers also
found MNPs in the brain of crabs and seabass, they will get in the bloodstream after uptake trough the gills, gut, and possibly the lungs. MPs can be absorbed and enter the circulatory system through the epithelia and M cells in the intestine.38–40 The main adsorptive part of
the gastrointestinal tract is the small intestine.41 For example, in the gastrointestinal tract of
the common dolphinfish was the most abundant microplastic PE, followed by PS and PP.42
11
Microplastics are also found in mammals. Doyle-McCullough et al. (2007) researched the uptake of labelled fluorescent PS microparticles with a diameter of 2 µm in vivo. Moreover, labelled MPs can easily be distinguished from accidental microplastics in
laboratory experiments. The microparticles uptake in the small intestinal ranges from 0.12 to 0.32% of the administered dose in rats, mice, and guinea pigs. The uptake was independent of the species; however, the uptake differs with age. Young adult males have a much higher intake when compared to younger and older age groups.43 The analysis of the digestive tract
is likely dependent on the origin and volume of the gut as the microplastics are likely not evenly distributed. Microplastics are also distributed in the human body; with animal testing the distribution and risks of plastics can be estimated. An example of animal testing is that mice were fed for 28 days with fluorescent PS MPs, this causes accumulation in the testis which decreases the fertility.44 Furthermore, pigmented MPs have been found in the human
placenta, in the maternal, foetal, and amniochorial membranes. This research states that it is unknown how the MPs reach the bloodstream and if the particles are from the
gastrointestinal or respiratory system.45 Summarized, this chapter shows that MPs can be
found in every major organ in the body and also can be spread within the body with for example the circulation system.
12
3 Sample preparation
Micro and nano plastics can be found in air, food, and cosmetics. As a result of the exposure routes, it has also been found in the human body. This chapter will discuss methods that are used for the isolation and extraction of biological samples. Sample preparation is necessary as microplastics consists of organic material. This preparation of MPs is challenging due to the large variety of plastics, the small particle size in diverse sample concentrations, and the pollution in the air.18 The technique of choice depends on
the type of matrix, the particle size, and the research question. In general, smaller particles are more difficult to separate from the matrix.46 In addition, polymers can be categorized in
the groups hydrolysable or non-hydrolysable, this is depending on the presence or absence of an ester or amide group. Non-hydrolysable polymers, such as PE, PP, and PVC, are more difficult to degrade. Hydrolysable polymers, such as PET, PA, and PUR, will degrade more easily as there are biodegradation pathways present in the molecule.47 This should be
considered when a method is developed as degradation is unfavorable. The most commonly used extraction methods can be divided into three categories, namely: chemical, physical, and manual.48 Figure 4 shows the research trends of microplastic between 2012 and 2018.
The majority of research has been conducted on aquatic animals. Furthermore, often multiple separation methods are utilized for the sample preparation of organisms.
Microplastics have been found in animals under natural and laboratory conditions. Biological samples should be obtained differently, depending on the purpose of the
research. Commercially available products such as mussels, fish, and other food products are easy to obtain. Furthermore, the plastic intake of laboratory animals can be controlled with the food they eat and desired tissues can be analyzed after a specific amount of days.43Wild
animals should be captured before analysis, most analyzed wild animals are aquatic species. When the living animals are captured they should be dried and frozen at -20oC.49 This is
recommend as defecation occurs between 30 minutes to 150 hours after collection.50
Defecation is often not desirable, for example when the whole MP intake of fish is measured or information about the environment is desired. However, the gastrointestinal tract of edible fish is cleaned before human consumption. This means that defecation is favorable when the human intake should be determined.
Moreover, samples should be handled carefully because of the high risk of plastic contamination. Firstly, use of plastic lab equipment should be avoided for minimalization of the pollution.51 Animals may be captured with polymer ropes, nets or traps. In this case
animals should be exposed for a minimal period and a reference sample of the gear should be retained. Additionally, airborne contamination should be avoided with thorough cleaning, covering samples and equipment, and wearing polymer free clothes.50 Furthermore, in the
laboratory glassware should be rinsed before use, cotton lab coats should be worn, and blanks without tissue and airborne MP particles in Petri dish should be analyzed. The Petri dish should be exposed to airborne microplastic for some time and analyzed with for
example a stereomicroscope. The fibers and particles should be recorded and photographed. In the end, all the fibers and particles found in the blank and the Petri dish should be
subtracted from the levels found in the samples.42 A good way to decrease the airborne MPs
pollution is to handle the samples in a clean room, which is designed to minimize airborne contamination during sample preparation and analysis.52
13
The extraction of microplastics should be based on a difference between the matrix and the analyte. Often plastics are less dense and more hydrophobic than the biological matrix. Currently, no effective hydrophobicity-based extraction for MPs in biota has been described. However, in other fields very small particles are successfully measured with hydrophobic interaction chromatograpy.48 Acid, base or oxidative digestion with nitric acid (HNO
3),
potassium hydroxide (KOH), or hydrogen peroxide (H2O2) can well digest the biological
samples and leave behind the plastic particles. Also stronger acids and bases can be used for the digestion of biological samples.50,53 Advantages of an acid digestion is that it speeds up
the digestion, nonetheless it can also cause damage to the plastic or dissolve pH-sensitive polymers. However, H2O2 should have no effect of the structure of the polymers.54
Dissection is often used for microplastic analysis of the digestive tract and is the
predominant method for the analysis of larger aquatic animals or the whole body of smaller organisms. The dissection of the gastrointestinal tract with subsequently a quantification of the plastic particles is commonly utilized. This method is relatively cheap and can accurately detect particles larger than 500 µm. However, smaller particles are able to translocate within the organism between the tissues.48,50 Fixation and cryosection methods are often used to
analyze the translocation of the MNPs.48
Figure 4 Overview of the studied organisms and separation methods for MNP analysis in biological samples of 45 studies between 2012 and 2018.48 This figure shows that most studies are conducted for aquatic life and mostly chemical
separations methods are utilized. A combination of separation methods is also possible.
According to Figure 4, chemical digestion is the most (62%) utilized technique for the sample preparation of microplastics in biological samples. Dehaut et al. (2016) researched several solvents for the digestion of the biological sample. It is important that the solvent does not degrade the plastic particles, digest the biological tissues efficiently, and is preferable inexpensive for frequently use of the method. Acid digestion methods with HCl and HNO3 were not suitable, because PP, PE, PA-12, and PS were degraded during this
procedure. Nitric acid is not recommended to utilize for digestion, because it slightly colors the plastic yellow and degrades polyamide. Besides, 10 M NaOH also degrades PC and PET. Enzymatic digestion was not tested in this research, because of digestion efficacy for issues
14
and difficulties with the implementation. More suitable methods were alkaline digestion with 0.24 M NaOH and 10% KOH with an incubation and filtration. Nevertheless, the NaOH degraded the cellulose acetate polymer and did not digest the tissues well. According to the research the method with 10% KOH showed the most promising results for the extraction of microplastics for fish, mussels and crab tissues.55 Another research compares H
2O2 and KOH
digestion in mussel and fish samples. The interlaboratory recovery rate is for KOH 99.67% and for H2O2 88.75%. When the recovery rate, time consuming, technical difficulties, and
costs are considered, digestion with 10% KOH is the most optimal method.56 Nonetheless,
Schirinzi et al. (2020) investigated microplastic digestion in dolphinfish. This research compared the digestion with KOH/HNO3 and H2O2/HNO3. The KOH mixture partially
degraded PC and PET microplastics and the recovery rate of particles smaller than 300 µm was 18%, probably working with a lower concentration and temperature may improve the recovery. However, higher efficiency was observed with KOH/HNO3,also working with a
higher temperature (60oC) reduced the incubation time.42 According to Löder et al. (2015)
samples are well purified with the use of different technical enzymes (before FT-IR
analysis).51 Enzymatic digestion is not hazardous and does not dissolve or degrade the plastic
particles, as the enzymes are especially developed to hydrolyze proteins and break down tissues. Advantages of enzymatic digestion are: easy and fast methodology, low chemical hazard, high recovery rates, short digestion time, no physical alternation of most polymers, and usable in combination with FTIR or Raman.57 However, enzymes are more expensive
when compared to chemical digestion techniques, especially when utilized with larger samples.49 Other disadvantages are that it often needs to be heated or boiled, the method is
more complex, often pH depended, more time-consuming, and the enzymes are not common in the lab.57 On the other hand, von Friesen et al. developed a promising tissue
digestion method with pancreatic enzymes and a pH buffer (Tris). This method has a high throughput, requires minimal handling, has low costs, and no additional risks for the performer or the environment when compared to potassium hydroxide digestion. Furthermore, this method digested the tissues better when compared to 10% KOH.58
Karlsson et al. (2017) developed another enzymatic method (proteinase K) in combination with CaCl2 (for activation of the enzyme) and an oxidative treatment with H2O2, this method
reported a recovery of 97% and barely altered the polymer weight (<1%). The enzymatic protocol was compared to a microwave digestion, however this method appeared to be less suitable as sample preparation method for biota (recovery of 34% and average change in polymer weight of 18%).59
Furthermore, biological samples contain a lot of biomass, this makes it more difficult to apply physical techniques such as filtration. Filtration could easier be used with low solid content.48 When filtration is utilized, the balance must be found between the ability to
capture small particles and filter clogging which decreases the sample throughput. Minimization of filter clogging can be achieved with sequential filtration when using
increasingly smaller pore sizes. Figure 5b shows the set-up of this method. Nevertheless, this method is mostly suitable for nanoparticles as the larger particles will stay behind the
coarser filters. After a digestion, filtration of biological samples can be utilized to filter
undigested tissue, inorganic residue, and the microplastics. Research suggested a 0.2 and 0.7 µm glass fibre filters, 5 µm cellulose nitrate filters, 5 µm cellulose acetate membranes, or a 50 or 250 µm mesh.50 Larger filters are more rapid, but will have a greater loss of smaller
particles. The disadvantage of glass fiber filters is that they can shed and contaminate the sample.
15
Although, density separation is commonly utilized in water and sediment studies, only a few times it is used in biota studies. An example of the set-up of this protocol can be found in Figure 5a. Some studies used NaCl to separate the less dense particles and a centrifuge has also been used for the separation of MPs. However, these separation methods can also be combined. Percoll (suspended colloidal silica nanoparticles) is often used for density gradient centrifugation. Density separation is mostly useful before a digestion. Saturated salts can separate less dense particles well from inorganic matter.50 NaCl is recommended by
the European Marine Strategy Framework Directive, because it is cheap and safe to use. However, the use of NaCl could lead to an underestimation of more dense particles. NaI and ZnCl2 might be a good alternative, due to their ability to separate more dense particles.48
However, density separation with ZnCl2 in combination with the oil extraction protocol is not
advisable for biota, this salt corroded the surface of PET particles and changes the spectra of PP and LDPE.54 Liu et al. (2020) combined chemical digestion (10% KOH), density separation
(NaI), and filtration (25 mm cellulose nitrate filter) for quantification of microplastics in mussels. The combination of the three techniques made it is easier to distinguish the MPs, due to larger differences in the results.60
Figure 5 Overview of examples of density-based and sized-based separation methods. (a) is a density separation and (b) is a sequential separation (dead-end filtration), noted should be 0.1 µm is filtered twice.48
16
4 Analysis of microplastics
This chapter will review separation, characterization, identification, and quantitation techniques for microplastics in biological samples. Figure 6 describes the most used techniques for the separation, identification, and characterization of MNPs. Besides, the figure also shows the particle size of the samples that can be analyzed. There are methods that provide semi-quantitative data. For instance the number of plastic particles are counted with a microscope.12, 20,61 However, it is possible to quantitate micro- and nanoplastics with
for example fluorescence measurements.43 Table 1 in the appendix shows an overview of all
the techniques that are described in this thesis.
Figure 6 Techniques for the characterization, identification, and separation of micro- and nanoplastics. Sorted for suitable particle sizes that can be measured. CE, SEC, and FFF are techniques to separate particles based on size. Raman and FTIR microscopy can identify the particles but also determine the morphology. TEM, SEM, optical microscopy, DLS, and MALS are techniques to characterize the particles on size and morphology. CE may be useful for analysis of nanoparticles, however it has not been very well explored for the analysis of MNPs. 46
17
4.1 Separation
Figure 6 shows several separation techniques, the separation of microplastics is based on the size and morphology of the particles. Flow systems are attractive as the size
separation of the MNPs can be achieved relatively fast. Furthermore, these techniques can easily be coupled to on-line detectors. This provides real-time information about the type, amount, and sizes of the polymers.46 Nonetheless, most of these techniques are utilized for
much higher concentrations than found in the environment.48
Field flow fractionation (FFF) is an emerging technique for the separation of nano particles. This technique differs from chromatography because there is no stationary phase.62 The separation takes place in a trapezoidal channel with a carrier liquid. The
particles are separated based on the difference in diffusion coefficient, depending on a perpendicular external field. Utilized external fields are thermal, electric, magnetic, centrifugal and crossflow. Thermal FFF is suitable to separate MPs based on molecular weight and molecular composition. Sedimentation FFF separates particles with electrolytes based on the difference in density. The most common technique is asymmetrical flow-FFF (A4F), which separates particles based on their size.63 Several detectors may be used such as
UV-vis, fluorescence, MALS, DLS, and ICP-MS. These techniques can provide information on the particle size distribution or the number of particles. Figure 7 shows a schematic overview of FFF. This figure shows the parabolic flow, due to this and the external field particles are separated on size and therefore elute at different times. This technique is quite powerful as it is able to measure a broad range from 1 nm up to 100 µm.46,63 Furthermore, it is a
non-destructive and relatively fast method. Nevertheless, drawbacks of this method are the high sample dilution occurring during migration in the channel to the detector. This limits the sensitivity of the detector. Another limitation is the molar mass range, because the lower limit is set by the membrane pore size. Mostly a minimal size of 10 kDa is used for the lowest molar mass of analytes.62 Monikh et al. (2019) reported a successful fractionation with
AF4-MALS of PS nanoplastics spiked to eggshells. The nanoparticles had a size of 60, 200, and 600 nm. In addition, the obtained fractions from the FFF are well suited to utilize for GC-MS identification.64 Another study spiked fish with 100 nm PS and PE nanoparticles. This method
compared an enzymatic digestion with proteinase K and an acid digestion with nitric acid. The enzymatic approach was more suitable, because it degrades the fish matrix but does not affect the plastic particles. This study demonstrated that it was possible to separate the PS nanoparticles from the matrix with A4F and determine their size with MALS. However, it was not possible to detect the PE nanoparticles, due to an elevated light scattering background. This shows that this method is not suitable for all types of nano plastics and it may require adjustments for the analysis of other particles.65
18
Figure 7 Schematic overview of asymmetric-flow field-flow fractionation. This picture shows the parabolic flow of FFF and that the smaller particles elute earlier than the larger particles.48
Chromatography is a complementary technique to FFF, this technique utilizes a stationary phase to separate the analytes and can be used for many applications. The separation of the analytes is commonly based on the difference in polarity of the
compounds. However, the analysis of MNPs with this analytical technique is challenging due to the large size of the polymers, the low polarity of the polymers which makes it hard to dissolve, and the interaction between the analyte and the stationary phase. This makes reversed phase chromatography often not suitable for the analysis of MNPs. Moreover, chromatography is not able to provide direct information on the number of particles. However, it may give valuable information regarding the concentration of microplastics in a sample.66 LC MS can be used for many applications, nonetheless not often for MNP analysis.
Nevertheless, Zhang et al. (2020) developed a method to quantify PC and PET MPs in dust samples. Only particles smaller than 150 µm were collected, subsequently the polymers were depolymerized. For the analysis, a common C18 column was utilized, electrospray was the ionization source, and the detector was a triple quadrupole MS. Significant positive correlations were found for the concentration of PET and PC monomers. MPs were found in dust samples all over the world.67,68 Chromatographic techniques that separates particles on
their size are size exclusion and hydrodynamic chromatography. The separation of SEC is based on the hydrodynamic volume of particles, the smaller particles diffuse further into the porous bead structure of the stationary phase and elute later than the bigger particles.46
Another name for SEC is gel-permeation chromatography. It has been used for separation of nanoparticles at a size between 2-200 nm. HDC separates analytes based on their size in the solution. Separation takes place in an open tube or a packed column with inert nonporous beads. Particles are separated on size due to the parabolic flow profile. Particles move the fastest in the middle and the slowest near the walls of the tube. As a result smaller analytes are able to get closer to the walls when compared to the larger particles, due to this larger analytes will elute earlier.69 A study compared A4F to HDC for the separation of gold
nanoparticles. The resolution of A4F was better when compared to HDC, nevertheless HDC recoveries were significantly better than the recovery of A4F.70,71 However, neither SEC nor
19
4.2 Identification
Figure 6 displays several techniques for the characterization and identification of microplastics. Currently these techniques are utilized for the chemical identification, size, and morphology characterization. The most utilized method is visual microscopy, often in combination with FTIR or Raman spectroscopy. Microscopy reveals the morphology and density, nonetheless chemical characteristic should be determined with more advanced analytical techniques.50
Microscopic identification methods for microplastics consists mostly of two steps. First physical characterization with a microscope followed by chemical characterization with for example spectroscopy. Microscopy techniques are used to determine the morphology and size of MPs. Often plastics are classified by their size, shape, and color. Size is often based on the longest dimension. The MP shape is mostly described in five categories: fragments, fibers, beads, foams, and pellets. Stereomicroscopy is mainly utilized for the identification of plastic-like particles, it provides surface texture and structural information for particles larger than ±500 µm. Stereomicroscopy is reported as an easy and cheap method for single analysis of MPs. Besides, software can be utilized for the image analysis for quantification of the particles.72,73 However, transparent particles smaller than 100 µm are difficult to
analyse.74 The visual counting and analysis with a microscope may result in an
overestimation or underestimation of the plastic particles present in a sample. Colored particles are better visible than transparent particles and the MNPs are not equally
distributed in the biological samples. This means that some images contain MNPs and some not, especially for low concentration samples, this phenomenon is called sparse. Sparse can result in a large statistical variability.
In addition, human errors should be minimalized, with for example standardized protocols.49,75 On the other hand, scanning electron microscopy (SEM) is able to give
extremely clear images of the plastic-like particles. A high intensity electron beam scans the sample and gives a detailed image (less than 0.5 nm) of the surface. The electrons react with the sample to provide an image. SEM can be combined with energy-dispersive X-ray
spectroscopy (EDS) and is able to provide the elemental composition of the particle. The advantage is that inorganic particles can be distinguished from the organic polymers. Nevertheless, SEM-EDS is expensive, the sample size that can be analyzed per run is small, and the sample preparation is time-consuming.50, 74,76,77 Figure 8 shows an example of a
particle from a Myctophid stomach misidentified with microscopy but correctly identified with SEM-EDS.78 Transmission electron microscopy (TEM) utilizes a higher electron
acceleration voltage on a very thin analyte which provides more information on the interior. However, the visualization of MNPs with TEM is challenging, due to the amorphous structure of polymers, which requires heavy-metal stains.46
20
Currently dark-field hyperspectral microscopy is presented for the analysis of MNPs. This technique measures in the VIS-NIR range (400-1000 nm). Label free PS particles with a minimum size of 100 nm have successfully been detected in vivo. This technique allows quantitative identification of ingested MPs by nematodes.79 Recently a paper was published
for the identification and characterization of MNPs with fluorescence lifetime imaging microscopy (FLIM). This is a microscopic technique to detect the fluorescence lifetime of various plastics at an excitation wavelength of 440 nm and relies on the autofluorescence of the plastics. This method can characterize plastics. However, incubation at elevated
temperature can cause a variation in fluorescence lifetime. This raises the question if this behavior is related and in which extent to the use of additives or structural
recrystallization.80
The most used identification technique is FTIR. This technique provides information on the chemical bonds of the particles. The signal is dependent on the change in electric dipole moment during vibration, which makes it sensitive to polar functional groups. Carbon-polymers can easily be identified with a unique spectrum. The measured spectrum is compared to a reference spectrum for the identification of the particle. FTIR is suitable for differentiation of particles and for the evaluation of aging particles with the observation of surface oxidation. Transmission FTIR measurements require transparent samples and filters and are limited by the thickness of the sample. Small microparticles down to 5 µm can be
Figure 8 a) shows an optical microscopy image fragment from Myctophid stomach that is potentially misidentified as plastic b) SEM/EDS does not reveal a significant carbon peak, although a large peak for Ca is observed which suggests that the particle is a shell fragment. c) High magnification SEM image shows the porous structure of the shell structure 78
21
measured with micro-FTIR (FTIR equipped with a microscope).75,76 Furthermore, attenuated
total reflectance FTIR (ATR-FTIR) and focal plane array FTIR (FPA-FTIR) are utilized for the identification of microplastics. ATR-FTIR is suitable for the identification of irregular particles larger than 500 µm.76,81 This technique is fast and the reflectance mode can be measured.
The disadvantage of measuring the reflectance is that irregular particles can give unrecognizable spectra caused by a refractive error. However, ATR in combination with microscopy is able to measure directly on the sample. This means that it can operate without manual handling and might be effective for particles smaller than 500 µm.51
FPA-FTIR utilizes filter paper and screening with infrared light and pixels (like a digital camera) and makes it possible to get many spectra with one measurement.51,76 FTIR is limited by the
diffraction limited spot size (e.g. 5 µm at 1000 cm-1), samples should be dried prior to
analysis as water is IR active, and the samples should be purified for evident spectra.
Another imaging technique is Raman spectroscopy, FTIR and Raman are complementary techniques. Raman is observed when molecular vibrations in Raman-active material cause scattering of light. A vibration is Raman active when there is a change is polarizability. For simple molecules this means that an IR active vibration is often Raman inactive, however this rule does not apply for larger molecules due to the increase of vibrations within the
molecule. The measured Raman spectra should also be compared to reference spectra for identification. When Raman is combined with a microscope (micro-Raman), it is able to identify particles smaller than 1 µm.46,82 Furthermore, a study showed a method to identify
particles down to 100 nm with Raman imaging. Raman imaging works with a confocal Raman microscope and a scanning laser beam. This method was also tested for dust particles; however, some limitations became clearer. The particle size that can be measured is limited by the collected Raman signal, the stage-stepping resolution, the pixel size, and laser spot size. The imaging resolution should be increased, however this is challenging.83 A drawback
of Raman is that fluorescent samples cannot easily be measured. Shorter wavelengths contain more energy which means more Raman signal, unfortunately also the fluorescent signal increases. Another problem of FTIR and Raman is that a limited area can be analyzed per run, the spot size of the analysis decreases with shorter wavelengths. Kumar et al. (2021) combined a FPA based micro-FTIR and micro-Raman to analyze MPs in mussels with a size between 3 and 5000 µm. The MP samples were size fractionated with a stainless-steel filter, samples smaller than 50 µm were analyzed with micro-Raman spectroscopy and particles larger than 50 µm with FPA-based micro-FTIR.17 The broader range that can be measured by
combining these two methods gives a better estimation of the abundance of MPs in samples. It is important to be able to measure a broad range due to the growing plastic pollution in the environment. Large plastic particles will degrade over time and become smaller. More advanced Raman techniques are also used for the detection of MNPs, these techniques are capable of real-time monitoring of microplastics. The difference between these techniques and conventional Raman spectroscopy is that coherent anti-stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) use multiple photon energies to address the molecular vibrations. CARS and SRS techniques scan the sample faster than conventional Raman spectroscopy, however obtained spectra are less complete. CARS is a microscopy label-free technique. This technique is capable of detecting polymer particles within a biological tissue with subcellular precision.82 Nonetheless, CARS may be hindered by
pigments in natural specimens.53 A CARS study detected nanoplastics of the acrylic
co-polymer with the size of 80 nm.84 SRS also shows a potential for the fast, label free analysis
22
harbor sediment sample and PET in nail polish. The size of the particles were a few tens of microns. Drawbacks of this method were that the method was optimized for only five calibrated polymers, this limits the spectral information. Furthermore, the sharp depth and focus can hinder the detection or cause deformation of the images. The spatial resolution was limited to 12 µm, due to the opening image processing function which removes small pixel clusters below that size.85 These advanced techniques should be further explored for
their usefulness for the identification of MNPs.
Furthermore, several light scattering techniques could be utilized for the
characterization of microplastics. MNPs can be characterized with dynamic light scattering or multi-angle light scattering. These are high-throughput techniques for the analysis in
aqueous media. MALS determines parameters such as gyration and radius by the average intensities of scattered incident light at various angles, whereas DLS measures the Brownian motion via a time-dependent fluctuation in scattering intensity. However, the Brownian motion is dependent on viscosity of the analyte and the scattering is dependent on the refractive index. Both are quite difficult to measure.86 DLS is the most widely used technique
in the nanometric range to determine the particle size distribution. DLS is commonly used for particles with a size between 800 nm and 5 µm. Moreover, when multiple angles are used instead of one angle (MADLS) smaller particles down to 1 nm can be measured. MALS is able to measure particles in a range between 100 nm and 3000 µm. A disadvantage of these measurement techniques is the low size resolution, due to that particles are measured together instead of one by one. This makes it hard to distinguish multiple polymers in the same sample.87 Adding a fractionation step before the analysis helps to significantly increase
the resolution power. A4F may be used for the fractionation before light scattering
techniques. Nevertheless, this technique is not useful to detect particles in the micrometer range. Tunable resistive pulse sensing (TRPS) is single particle technique and can measure the particle size distribution and concentration of NPs. This technique contains one pore where the nano or microparticles can pass, when a particle passes the pore a signal is measured. TRPS is able to detect particles with a size from 40 nm to 20 µm with a high resolution. However, measurement parameters should be changed to cover the whole range.88
Additionally, thermal techniques can be useful for the identification of MNPs. These techniques are capable of identifying and quantifying microplastics based on the
degradation products of the MNPs. Different techniques can be used to gain information about the plastics. Differential scanning calorimetry is useful to determine thermal
properties of a polymer, this technique measures the change of the analyte when heated. A comparable technique is TGA, it measures the weight loss of an analyte with a small balance as function of the increased temperature. These techniques determine the stability of the analytes at high temperatures. When DSC is coupled to TGA it is possible to identify more polymers. However, it is hard to identify these techniques when they have overlapping phase transition signals. In addition, thermal-analytical techniques has been used for the separation of the decomposition of MNPs.55,89 Pyrolysis gas chromatography is another
thermal technique. Herein a sample is introduced into a heated chamber and broken down into smaller stable fragments. This chamber contains an inert gas or a vacuum.
Subsequently, the fragments are separated by chromatography with typically a MS detector. Mass spectra of the fragments can be analyzed to identify the polymers, as every polymer has specific fragments. Nevertheless, this is a destructive technique, the samples need to be placed manually, and the data analysis is time-consuming. This makes it difficult to analyze
23
large numbers of microplastics. Furthermore, the within-lab reproducibility is challenging with py-GC/MS and depended on the sample preparation, pyrolysis type, and pyrolysate transfer.48 TGA can be coupled to FTIR or GC/MS to determine the thermal properties and
gaseous decomposition products. FTIR can monitor in real time qualitative analysis and GC/MS for both qualitative and quantitative analysis. Liu et al (2020) researched
microplastics in mussels with TGA-FTIR-GC/MS. The combination of these techniques is able to provides a strong discriminatory power. This method is able to determine the type of polymer and the polymeric mass in the sample.60 However, these techniques do not
determine the count of particles nor the size of the particles. Nonetheless, with an estimated average size the particle number can be calculated. Another thermal technique is thermal extraction and desorption (TED), it utilizes lower temperatures and can identify smaller molecules. When this method is combined with pyrolysis or thermogravimetry techniques it is able to provide information about smaller volatile compounds or additives in the plastics.66
Goedecke et al. (2020) evaluated several thermo analytical techniques for the detection of microplastics in environmental samples. TED-GC/MS is the most suitable technique for samples with unknown matrix and unknown variable kinds and volumes of plastics. TGA-FTIR is a robust method and useful for samples with known kinds and volumes of polymers. However, TED-GC/MS is currently not able to detect PVC MPs, TGA-MS may be the future solution to this problem.90 Thermo analysis is an often utilized technique and will be more
highlighted for the quantitation in paragraph 4.3.
Analytical techniques that are barely described for the analysis of microplastics are flow cytometry (FCM) and atomic force microscopy (AFM). The technique FCM is able to measure and detect the physical and chemical characteristics of a liquid stream of cells or particles. This technique can be utilized to detect extracellular vesicles and populations of plastic particles in seawater. However, this technique is not suitable for the mass based concentration measurement.88 NP can be analyzed with nano FCM, unfortunately this
method is not suitable in the micron range. 88 This method was successfully utilized in a
study where microalgae were exposed to fluorescent labelled PS microplastics. This shows that FCM can be utilized in toxicity studies for MNPs.91,92 The other technique AFM is a
scanning probe technique which analyzes the surface of the analyte on atomic scale with a tiny tip. The tip-analyte interaction is important for the measurement, this can cause a cantilever deflection and measured with a photodiode detector. This technique can be used to establish the characterization for example the degradation and aging of MNPs. When compared to SEM, AFM is able to characterize surface morphology, stiffness, adhesiveness, hydrophobicity, conductivity, magnetization, and work function.77 Advantages of AFM are
that the sample can be preserved, the analysis can be conducted in the open air, and it is a relatively simple sample preparation when compared to other techniques. AFM has been applied for studies of the characterization of MNPs. Nevertheless, the scanning time of a sample is relatively slow, the probe has limited motion flexibility, and the tip-sample interactions may introduce artifacts. However, there are some emerging techniques with faster scanning rates even faster than SEM.77 AFM in combination with Raman or IR can
enable characterization in the nanoscale. For example, in a study AFM is combined with IR to determine the thermal and mechanical properties of aged microplastics. Figure 9 shows an example of the results for ageing MPs with AFM-IR. They conclude that the surface of aged microplastics changed from smooth to rough and the glass transition point decreased.93
24
Figure 9 From top to bottom: Topographical image, AFM-IR image at C=O peak 1706 cm-1, and frequency image. A) unaged
and B) aged microplastics; C) three dimensional images of unaged (top) and aged (bottom) MPs. The AFM-IR strength of aged (B) MPs was much higher than the unaged (A). The more purple the higher and the browner the lower the surface of
25
4.3 Quantitation
The quantitation of microplastics is difficult, due to the lack of standardized protocols. Semi-quantitative data can be obtained with a microscope. The particles are counted by people, with a high risk of false negatives or positives. Software is highly recommended to reduce the errors. However, particles smaller than 500 µm should not be counted with this technique. Techniques such as microscopy determine the number of particles in a sample. This provides in depth information such as size, surface, count, and shape. Rivers et al. (2019) reviewed many articles and concluded that surface area was the most accurate parameter to describe MNPs. He suggested that the results should described per km2 or m3,
so it is easier to compare results.94
Chromatography is mostly used for the quantitation of analytes. Paragraph 4.1 showed a quantitation of microplastics with LC/MS. The PET and PC from dust were depolymerized with an alkali-assisted thermal hydrolysis in environmental samples. The measured
concentration in the digestive of mussels was lower than expected, in contrast to the quite high concentration in the clam. The recoveries of the method were between 87% and 97%, which shows that this method is quite reliable. However, the monomers of these polymers may be added to a polymer as additive. This may cause overestimation of the concentration in the sample.68 Müller et al. (2020) described another method to measure PET in
environmental samples. The samples were extracted with an alkaline extraction
(depolymerization) and measured with an LC-UV (wavelength at 240 nm) and compared to a TED-GC/MS method. The calculated LOD and LOQ of the LC-UV method were 1.55 and 6.05 mg kg-1, respectively. Both methods gave comparable results and are faster than the more in
depth analysis which give information about the count, size, and shape.95 Figure 10 provides
a schematic view of the LC quantification of MNPs.
Figure 10 Summarized explanation of the research of the determination of PC and PET with depolymerization extraction and liquid chromatography mass spectrometery.68
Thermo analytical techniques can identify and quantify MNPs. Dümichen et al (2015) presented a method with thermal extraction with TGA, which was connected to solid-phase adsorber and subsequently analyzed with TDS-GC-MS. This method was able to quantify PE MPs in environmental matrices in one step. Furthermore, it provided the analysis of complex matrices due to the TGA in comparison with Py-GC/MS.96 Liu et al (2020) analyzed an
26
mussels. The recoveries for spiked mussels were higher than 97% and PE has been found as most abundant MP in the mussels. This study showed that this method is capable to analyze PE, PP, PVC, and PS.60 These techniques are able to quantify MNPs with their mass
concentration. Becker et al. (2020) provided the first interlaboratory comparison of several thermo analytical methods. Acceptable recovery and reproducibility were measured among the participants performing Py-GC/MS, TGA-FTIR, and TED-GC/MS.97
Fluorescence is another technique that can quantify the particles due to the staining. Often this technique is used for laboratory animals to determine the translocation of the particles within the body. Animals are typically fed with fluorescent polystyrene
microbeads.82,98 Nevertheless, a study tested whether the commercial fluorescent-labelled
nano PS are able to leach their fluorophores and auto fluorophore. This study found that the particles can leach their fluorophores and the fluorophore can accumulate within the tissues of a zebrafish larvae. Furthermore, green autofluorescence was observed for larvae who were not exposed to plastic particles.99 This research shows the importance of control
groups and that studies without it are unreliable. Another research concluded that Nile Red was the best stain for microplastics with excitation wavelengths at 254 and 470 nm.
Preferably, staining protocols stain the polymers and fibers without staining the natural organic matter in a sample. When the organic matter is stained an additional removal may be required. Muscles of shrimps and fish presented fluorescence when stained with Nile Red. However, the researchers declare that microplastics are not likely to be abundant in muscle tissue since they are readily consumed by organisms. This means that the staining dye only cause fluorescence in muscle tissues under 254 and 365 nm light and not in other organic matter.100 Another review states that Nile Red is inconsistent and not plastic-specific.
Although, it could be effective for clean samples.48 With the help of a software program and
the fluorescent intensity the amount, size, and shape of particles could be determined. Moreover, the combination with spectroscopy techniques such as FTIR could be powerful tool for the quantitation and identification.100
Another emerging technique for the quantification of MNPs is AF4. This technique can separate MNPs, deliver the particle size distribution, and the fraction can be collected. This technique is powerful in combination with other techniques. Paragraph 4.1 already
described A4F-MALS methods. The LOD of this method is 52 µg particles / gram fish for PS particles of 100 nm. It is expected that the LOD will increase with smaller particles.65 When
coupled to MALS it also gives directly the particle size. A4F may also be coupled to
fluorescence microscopy. This separation of the particles with A4F may be a solution for the auto-fluorescence from cells and tissues. Also, the MPs could be coupled to elemental labelling.65 However, this method is not suitable to measure particles in the micrometer
range.88
This chapter has described many analysis techniques that can be utilized for the separation, identification, characterization, and quantitation of MNPs. All the described identification and characterization techniques are complementary to each other. Moreover, this chapter describes combinations of techniques. The combination of the techniques is able to give a more complete image of the distribution and toxicity. The choice of analytical techniques depends on the research question. Known (stained) microplastics in laboratory animals are more easily to detect than the unknown microplastics in wild animals. The particles are more difficult to measure in wild animals, because they are exposed to a larger variety of plastics that differ in type, shape, size, and are often exposed to lower
27
5 Toxicity and risks
Multiple researchers showed the presence of MNPs in humans and animals. Chapter 2.2 described the exposure and distribution of MNPs. These particles may have effect on the health of their host. The WHO and SAPEA state that there is lack of information about the toxicity of MNPs to human. However, both conclude that there is no risk of MNPs to humans and animals.101 This chapter will review the knowledge on potential human health effects
and will describe several techniques that are utilized to determine the toxicity. Microplastic consumption may lead to oxidative stress, abrasion, satiation, ulcers, decreased growth rate, and reproducibility.5 Therefore, it is essential to research the effects
of the MNPs. NPs can pass cell membranes; this means that potentially toxic compounds may be found in the cells. Besides, various researchers showed the toxicity of MNPs in animals.43,99 Forte et al. (2016) investigated in vitro the interaction of PS nanoparticles within
the biological system. Particles with a size of 44 nm and 100 nm in the gastric
adenocarcinoma cells were evaluated. The smaller particles accumulate more rapidly and efficiently in the cytoplasm when compared to the larger particles.102 Abarghouei et al
(2021) investigated the effect of PS microbeads in goldfish at a size of 0.25 or 8 µm at 300 mg/L for 168 hours. The MPs have the ability to be absorbed by the gill, liver, and intestines of goldfish. The highest concentrations were found in the intestines and livers for the smaller particles, probably due to the fact that the gills are earlier exposed to the particles and smaller particles can more easily pass through the organs and enter the bloodstream.103
These researches show that smaller particles are potentially more dangerous and concluded that size matters. Smaller particles are likely to be more dangerous and probably accumulate within the body.
Toxicity testing determines the degree to which a substance negatively impacts the normal biological function of an organism. These tests should reveal the species, organ, and dose specific toxic effects of the analyte. The toxicity of a compound can be tested with the accidental exposures to the substances, in vitro testing with cells or cell lines, or in vivo exposure on test animals.104 Accidental exposure routes can be determined when the MP
concentration is determined. Chapter 2 described that people consume 39,000 to 52,000 particles, drink 4,000 to 90,000 particles from water, and inhale 9,490 to 47,450 particles per year.2,22 However, this will not give enough information about the toxicity of the MNPs. In
vivo experiments utilize laboratory animals, they live in a controlled environment. Figure 11 shows an in vivo experiment with PS microplastic in marine zooplankton, these animals are at the bottom of the food chain.105 Another example of an in vivo test researched
fluorescent microfibers of various lengths in water flea, shrimp, and zebrafish were
investigated. Mostly microbeads are utilized for in vivo experiments, however fibers are the most abundant form of MPs in the environment.106 Li et al (2020) described a in vivo
research with beads, in this study mice were fed with PE MPs with 6, 60, or 600 µg every day for 5 weeks. The numbers of gut microbial species, bacterial abundance, and flora diversity were increased for the mice who ingest the highest concentration microplastics.
Furthermore, PE MP can induce intestinal dysbacteriosis and inflammation as this is seen for the mice who were fed the highest concentration.107 Additionally, the long term effects of PS
beads were tested in nematodes. Nematodes with different life-history responded
differently to the presence of the beads. Nevertheless, their responses were not correlated to their gut concentrations. The presence of MPs significantly accelerated the population
28
growth of the Acrobeloides nanus and had subtle impacts on long term multigenerational tests.108 Moreover, Hou et al. (2021) researched the effect of microplastics on the ovary in
rats. The rats were exposed to 0.5 µm PS MPs with a hematoxylin-eosin staining in the concentrations of 0.015, 0.15 or 1.5 mg/kg/day. This research concluded that PS MPs have an adverse effect on the ovary and may be a potential health factor for female infertility. These negative effects may be caused by oxidative stress.109 Many animal studies show that
the exposure to MNPs leads to impairment in oxidative and inflammatory intestinal balance and disturbs the gut microbiota.110
Figure 11 The zooplankton had eaten PS microplastics and these MPs are also found in the feces. 105
However, there are a lot of concerns about using laboratory animals, because animals often need to be killed or hurt for the research, for example when the inside of the gut is analyzed. A more ethical approach is in vitro testing, this implies testing with cells or cell lines that are like human cells. A study of Walczak et al. (2015) exposed Caco-2 and HT29-MTX cells to pristine and ingested 50 nm PS NPs with different charges. The translocation of the particles significantly increased for in vitro digested NPs. Some nanoparticles decreased the amount of protein in the corona.111 Stock et al. (2019) analyzed the uptake and effects of
microplastic in vitro using human intestinal epithelial cell line Caco-2 and in rodents in vivo. Different sizes of spherical fluorescent PS of 1, 4 and 10 µm were used to study the uptake and transport. The in vivo study was utilized to analyze the transport at the intestinal
epithelium and oxidative stress response. The mice were fed 3 times a week for 28 days with 4.55*107 particles of the different sizes. The effect of the particles on the macrophage
polarization were in vitro tested with a human cell line THP-1, to detect possible impact on the intestinal immune cells. This study demonstrated that a minor fraction of the particles has been absorbed by the cellular uptake and no lesions or inflammatory responses were detected. Furthermore, the particles did not interfere with the differentiation and activation of the human macrophage model. Altogether, this research suggested that the oral exposure to PS MPs under these conditions does not pose relevant or acute health risks to humans.41
Besides, this research shows the relevance of combining in vitro and in vivo measurements and that they are complementary to each other. Nevertheless, in vivo and in vitro studies often utilize high concentrations which are not representable for the daily intake. More studies with realistic doses should be conducted.112 Furthermore, a critical review of
