Review of Microplastic Distribution, Toxicity, Analysis Methods, and Removal Technologies

: Microplastic contamination has become a problem, as plastic production has increased worldwide. Microplastics are plastics with particles of less than 5 mm and are absorbed through soil, water, atmosphere, and living organisms and ﬁnally affect human health. However, information on the distribution, toxicity, analytical methods, and removal techniques for microplastics is insufﬁcient. For clear microplastic analytical methods and removal technologies, this article includes the following: (2) health effects and toxicity of microplastics were researched. (3) The sampling, pretreatment, and analytical methods of microplastics were all reviewed through various related articles. (4) The various removal techniques of microplastics were categorized by wastewater treatment process, physical treatment, chemical treatment, and biological treatment. This paper will be of great help to microplastic analysis and removal techniques.


Introduction
In 2018, global production of plastic products reached almost 360 million metric tons/ year [1]. Out of the 2.5 billion metric tons of solid waste generated by 192 countries in 2010, about 275 Mt consisted of plastics [2]. Once a large plastic item is thrown away in nature, it degrades into smaller plastics and it becomes microplastics (MPs). Microplastics are small plastic particles smaller than 5 mm, and plastic particles smaller than 1 µm are defined as nanoplastics [3][4][5]. Microplastic particles exist in water, are consumed by living things, and can affect human health. Currently, microplastics are detected in various areas, such as streams, rivers, seas, drinking water, and even food in the world [5]. Microplastics can be divided into two parts: primary microplastics and secondary microplastics [6]. Primary microplastic is defined as plastic made of 5 mm or less according to a specific purpose, and it is often included in toothpaste, face wash, cosmetics, industrial abrasives [7,8], and 3D-printer particles [9]. Secondary microplastics were large at the time of production and manufacturing, but the large plastics were crushed or degraded through physical, chemical, and biological weathering, due to the environment, and became microplastics, such as straw foam and mulching vinyl [8]. As such, pollution from microplastics are becoming a serious problem around the world, and many studies on microplastics such as distribution, toxicity, analysis, and removal are being conducted by many researchers. Among the 192 countries of the world, 44 countries have carried out research regarding microplastics; the studies looking at the impacts on organisms have mostly targeted fish (38%), whereas few studies on other highly affected organisms, such as turtles (1%), have been conducted [10].

Microplastic Distribution in Marine
Research on microplastics started with the ocean contamination in early 2014. Bakir et al. (2014) concluded that circulated contamination zones are easily formed in semi-enclosed seas, such as the Mediterranean coast [31]. Since 2014, many studies on microplastics in the ocean have confirmed the number of microplastic particles in surface water. Research on microplastic pollution in the ocean has mainly focused on the number of microplastic particles in the seawater layer, especially surface water. Iosobe et al. (2015) investigated the concentration of microplastics (<5 mm in size) and mesoplastics (>5 mm in size) in the East Asian sea, around Japan, in 2014, and the total concentration was 1,720,000 MPs/km 2 , which is higher than 63,320 MPs/km 2 in the world oceans and 63,320 MPs/km 2 in the North Pacific Ocean [32]. Ressell and Webster (2021) collected surface-sea-level samples from the Scottish seas from 2014 to 2020, using a neuston net, and the concentrations of microplastics ranged from 0 to 91,128 MPs/km 2 , and 50 percent of the fragmented plastics were identified as microplastics. Due to the different geographic and temporal ranges of the data, trends in microplastics could not be identified [33]. Pan et al. (2018) collected microplastics from 18 sites in the Western North Pacific Ocean from August to September 2017 and found an average of 1 × 10 4 MPs/km 2 . The MP type was identified as polyethylene (57.8%) > polypropylene (36.0%) > nylon (3.4%) [34]. Among these studies, there are not enough cases in which the total amount of microplastics on the surface of seawater is comparatively analyzed by region. In addition, among the particle sizes in reported microplastic studies, few cases of 0.3 mm or less size have been reported. The reason is that most of the sampling methods for the seawater layer used a mesh size of 333 µm [34,35]. Lindeque et al. (2020) hypothesized that most of the grids used for sampling in the marine sector are 333 µm in size, which may lead to an underestimation of microplastics. They checked the concentration of microplastics in each sample after collecting samples, using 100, 333, and 500 µm nets at the same sampling site. As a result, it was confirmed that, under the statistical significance level, the sample using the grid mesh of 100 µm contained 2.5 and 10 times more microplastics, respectively, than the samples using the grid mesh of 333 and 500 µm [35]. These study results confirmed that there is a limit to the scope of microplastic collection depending on the size of the mesh used for sampling in the microplastic pollution research.
Research on plastic trapping in marine coastal vegetation ecosystems is still in its early stage.  investigated the behavior of microplastics in intertidal and subtidal zones planted with marine plants, such as seagrasses. This was known as the first study on Z. marina to identify microplastics [36]. Piarulli et al. (2020) confirmed the presence of microplastics in salt marshes and 96% of the 330 samples analyzed did not contain microplastics. Suspension and facultative deposit-feeding bivalves (0.5-3%) contains a little compared with omnivores (95%). Although the incidence of MPs was low, the distribution of MP size, shape, and polymer was varied [25]. More studies investigated the presence of microplastics in vegetation, seagrasses and algae in the canopy [37,38]. Cozzolino et al. (2020) divided two intertidal zones and two subtidal zones to determine the degree of microplastic contamination in vegetation developed in Ria Formosa lagoon in Portugal. The microplastic abundance in the sediment was assessed. According to the results of the investigation, the entrapment effect of microplastics in the sediment and crown layer was higher in the subtidal zone than in the intertidal vegetation zone, and the presence or absence of vegetation did not affect the microplastic concentration [39]. As the current research focuses on only one type of vegetation zone, there is a limit to ascertaining the degree of contamination of microplastics according to the vegetation characteristics.
As research on microplastics in the marine sector expands and research cases are accumulated, additional research on the occurrence, distribution, and characteristics of microplastics considering climate and spatial differences in the marine environment is needed.

Microplastic Distribution in Land
The research on microplastic contamination on land has been relatively insufficient compared to the ocean research. Research studies on freshwater and wastewater treatment plants, using water as a medium among terrestrial environments, are evaluated to be relatively numerous compared to soil and air. Tibbetts et al. (2018) conducted a microplastic survey from a heavily urbanized catchment, the River Tame, and four of its tributaries, which flow through the city of Birmingham, UK [40]. All sediment sampled was found to contain microplastics with an average abundance of 165 MPs/kg [40].  investigated the distribution of microplastics in surface water, fish, and sediment near a sewage treatment plant (STP) in the Tanchon stream, one of the main tributaries flowing into the Han River, Korea, and they found that microplastics concentrations in water varied spatially and temporarily, ranging between 5.3 and 87.3 MPs/m 3 (31.4 ± 28.5 MPs/m 3 ). In fish, the concentration in upstream and downstream sites was 7.3 ± 7.3 MPs/ kg and 12.4 ± 17.9 MPs/kg. In sediment, microplastic concentration in upstream and downstream sites was 493.1 ± 136.0 MPs/kg and 380.0 ± 144.2 MPs/kg [41]. Kosuth et al. (2018) tested the presence of anthropogenic particles in 159 samples of globally sourced tap water, 12 brands of Laurentian Great Lakes beer, and 12 brands of commercial sea salt. A total of 81% were found to contain anthropogenic particles in the tap water. They indicated that the average person ingests over 5800 particles of synthetic debris from these three sources annually, with the largest contribution coming from tap water (88%) [42]. Mason et al. (2018) investigated eleven globally sourced brands of bottled water, purchased in 19 locations in nine different countries, and found that 93% of them contained microplastic synthetic polymer particles among the 259 total bottles [43].
Recent studies have highlighted the discussion that freshwater serves as a major route and source of microplastics between land and ocean [44][45][46]. Freshwater serves as a major route and source of microplastics transport between land and ocean. Ockelford et al. (2020) pointed out that up to 80% of plastic in the oceans comes from river networks, especially during periods of flooding [44]. Galloway et al. (2017) found that most of microplastics are accumulated in the ocean, and that land is one of the primary sources of microplastics in the ocean [45]. Therefore, microplastic pollution by freshwater is highly dependent on human activity, and factors such as population density and industrialization in the river area can be a major factor affecting marine microplastic pollution [46]. Newly noteworthy research resulted in terrestrial freshwater research that rainfall, such as river overflow or heavy rains due to flooding, affected the movement of microplastics on land [47]. Lebreton et al. (2017) observed that between 1.15 million and 2.41 million tons of plastic waste currently enters the ocean every year from rivers, with over 74% of emissions occurring between May and October [29]. Hitchcock (2020) emphasized the effect of storms on microplastic pollution in aquatic ecosystems. He measured the abundance of microplastics during the storm and found that the abundance of microplastics was more than 40 times higher during the storm than before the storm. The maximum microplastic abundance in this study was about 17,833 particles/m 3 during the peak of the storm. During the two days of heavy rain, the concentration of microplastics increases from 400 particles/m 3 before the storm to a maximum of 17,383 particles/m 3 . The study results may be influenced by microplastics present in the atmosphere, or it may be influenced by the concentration of microplastics in the ocean at the point of occurrence of heavy rain clouds [48]. However, it is difficult to estimate the specific route and source based only on the research. Accordingly, freshwater is more accessible to humans than the ocean, so if it is adjacent to an industrialized or urbanized area, a higher degree of microplastic pollution than the ocean might appear.
The migration of microplastics through the soil has been attributed to the recycling of sewage sludge [49], the use of plastic mulching vinyl [50], the use of organic fertilizers [51], and land application from wastewater treatment plant discharges [41], and other irrigation methods using contaminated river water [52,53] have contributed to the transfer and accumulation of microplastics in agricultural and natural soils. Studies on land contamination of microplastics include a study on open land in an industrial area [54], a study on the concentration of microplastics in soil samples in an open land of flood area in Switzerland [55], and a study on mixed agricultural land [56]. Microplastics, which are confirmed at a high rate, are found more in sludge than in treated wastewater, so reuse of sludge can serve as a source of microplastics in soil. Corradini et al. (2019) confirmed the presence of microplastics in land-use areas which are closely related to human activities. In relation to agricultural soil, research continues to be published that soil is a major storage area for microplastics due to the use of fertilizers recycled from wastewater treatment plant sludge [56]. Regarding the distribution of microplastics in soil, Dioses-Salinas et al. (2020) explained similarities in the morphological type and size of the microplastics in terms of the product purpose. They considered that the characteristics of the source of microplastics, the topography of the surrounding area, hydrological properties, the presence of currents, and the extraction and identification methods used in the survey analysis could affect the difference in microplastic concentrations in individual surveys [52]. Sediment is a particle accumulated in the environment by a medium, and it is difficult to explain it independently as soil [57]. In this regard, sediment should be considered as the main environment in the soil sector, considering the characteristics of the medium of solid particles [57]. Harris (2020) also argues that plastic particles are a solid and transportable material, so it is desirable to look at them as one of the sediments physically [57]. To summarize the above content, microplastic research is being actively conducted in the soil field, especially on agricultural land. Since sludge deposited with microplastics in wastewater treatment facilities can diffuse into the soil, it is necessary to check the use of sludge recycling fertilizers used in agricultural land.

Microplastic Distribution in Air
As freshwater is considered to be a major part of the migration of microplastics to the ocean, studies on the degree of microplastic pollution in the air field are insufficient compared to the water and sediment fields. However, light microplastics can easily be transported through the air to other environments on land and in the ocean. Yukioka et al. (2020) investigated the abundance of microplastics in road dust in three cities to identify the occurrence and characteristics of microplastics in surface road dust in Kusatsu City, Japan; Danang City, Vietnam; and Kathmandu City, Nepal [58]. The abundance of microplastics among road dust in the three cities surveyed was 0.10 to 39.6 MPs/m 2 , and the color, shape, and polymer type of microplastics were all different by region. The proportion of rubberlike microplastics was higher in the dust samples taken from the road than in the samples extracted from the dust present in the general environment of the surveyed areas [58]. It can be inferred that vehicle tires, hoses, shoes, and electrical insulation may be potential microplastic sources of road dust [59]. These findings are also consistent with the findings of Vogelsang et al. (2018), who estimated that the main source of microplastics from road dust would include the surface wear of automobile tires and paint for pavement road markings [60]. However, detailed information to explain the occurrence and distribution of microplastics in road dust in this study was lacking, and it was difficult to specify the source of microplastics in road dust. In order to explain the occurrence of microplastics distributed in the air and to estimate the distribution and pollution level of microplastics by region or environmental characteristics, more research investigations should be conducted. In the study of , the condition of microplastics among indoor and outdoor dust in 39 cities in China was investigated and the daily human exposure due to indoor dust intake was estimated. They determined that the indoor dust concentration was 1550 to 120,000 mg/kg, the outdoor dust concentration was 212 to 9020 mg/kg, and the daily human exposure by indoor dust intake was 6480 ng/kg-bw/day, estimated to be 17,300 ng/kg-bw/day of PET MPs in children [61]. To summarize, it is only possible to confirm that air is one of the pathways contributing to the movement and diffusion of microplastics. Accordingly, more research should be conducted in order to identify the clear source of origin, migration route, spatial distribution characteristics, and toxicity to the human body.
Microplastic contamination is being studied in all areas of the environment as it includes most parts, such as water, soil, and air. Eriksen

Microplastic Toxicity
Microplastics are themselves a problem, but organic contaminants adsorbed on microplastics can also be a problem [23]. The recent research on microplastic toxicity has begun considering the mixed toxicity with other substances. Zhou et al. (2020) studied that microplastics and pharmaceutical and personal care products (PPCPs) could be toxic to organisms when they were exposed in a mixed form [63]. Microplastics could play a role of carrier to mediate PPCPs. Weber et al. (2021), mixed toxicity experiments of microplastics and copper were performed on Lymnaea stagnalis (water snails) and showed no change in toxicity [64]. Gao et al. (2019) checked the capability and characteristics of heavy metals on microplastics in marine environment and found that polyvinyl chloride, polypropylene, polyethylene, polyamides and polyformaldehyde could adsorb lead, copper and cadmium and the heavy metals showed higher absorbance on PVC and PP particles compared with PA, PE and POM [65]. Xu et al. (2021) performed a mixed toxicity test with zebrafish (Danio rerio) and microplastics, although they did not find any lethal effect, but it was reported that the mixed exposure was correlated with the concentration of microplastics in survival, body length, and heart rate [66]. This may be supported by a study by Lu et al. (2018), who reported that microplastics increase cadmium accumulation in the liver, intestine, and gills of zebrafish and cause oxidative damage and inflammation [67]. Yang et al. (2020) showed that Chlorella pyrenoidosa exhibits antagonism when nonylphenol and microplastics are mixed [68]. Although the results of mixed toxicity are diverse, it is worth that the smallersize plastics could aggravate with prolonged exposure time on algal growth. Stock et al.
(2019) performed in vivo experiments in mice and in vitro experiments, using human cells. An oxidative stress response was observed in male mice, suggesting an effect on intestinal immune cells. However, no histological lesions were observed. Experiments using cells confirmed that fine particles can be absorbed into cells. However, the study concluded that oral exposure to microplastics did not cause serious health effects in mammals [69].  also investigated the effect of microplastics, using human Caco-2 cells, and reported that nanoscale microplastics could increase cytotoxicity [70]. Pop et al. (2021) concluded that bisphenol A (BPA) had a direct or indirect effect on chlorophyll in water microorganisms, and it was confirmed that it was a problem for cell-membrane division, integrity, and survival [14]. Bhatnagar and Anastopoulos (2016) reported that BPA was used as an endocrine disruptor as a major raw material in cosmetics and was found in surface water, groundwater, wastewater, and landfill runoff [15]. Wang and Qian (2021) confirmed that phthalates negatively affect the endocrine system and various organ functions, especially the reproductive organs, such as pregnancy, child growth, and child development [71]. Since there are few experimental studies on microplastic metabolism in the human body, it is judged that caution is needed in the interpretation of the results. In addition, studies related to the toxic effects of microplastics, using organisms and cell lines, are being actively conducted, and many research papers on toxicity have been published recently.
Recently, several studies have been conducted to evaluate the risk of microplastics in ecosystem. Adam et al. (2019) calculated the predicted-no-effect concentration (PNEC) as the fifth percentile of the probabilistic species sensitivity distribution, based on 53 values from 14 freshwater species and found that ecological risks cannot be entirely excluded in Asia, where 0.4% of the RCR values were above 1 [72]. Besseling et al. (2019), preliminary sensitivities for each species of marine water, estuary water, and freshwater were derived and risk assessment was performed. In this study, 168 toxicity data from a total of 66 studies were reviewed, and the HC 5 (Hazardous Concentration for 5% of the species) value was determined to be 2.0 ng/L. Based on the species sensitivity distribution, it was found that there is a potential for harm to sensitive species in some hotspot areas [73]. Xu et al. (2018) performed an initial exposure assessment to surface water in the estuary of the Changjiang River in China and the nearby East China Sea. In this study, microplastic sampling was performed at 29 points, and the shape, size, color, and type of microplastics were measured [74]. In addition, they established the risk level criteria for microplastic pollution, using PLI (Pollution Load Index) shown in Table 2. Although risk assessments for microplastics are continuously being conducted on humans and ecology, it is considered that more research is needed for a standard of concentration, and accuracy in microplastic risk.

Microplastic Analysis
Scientific methods to quantify and differentiate microplastics are needed to more systematically understand the impact of microplastics on the environment [75]. The measurement and analysis technology for microplastics has not yet been established, and the currently used analysis technologies have limitations in terms of detection limit and speed [76]. Accordingly, many researchers are actively working on developing more accurate microplastic analysis [75]. The analysis process of water microplastics can be roughly categorized into sampling, pretreatment, and analysis. Figure 4 is the procedure for sampling and analyzing microplastics.

Sampling
There are various types of microplastic sampling methods, and they are applied differently, depending on water (freshwater and seawater), sediment, soil, cosmetics, and living organisms. However, this paper deals with the most widely used water and sediment sampling.

Water Sampling
Sampling of microplastics in water is highly dependent on the density, shape, properties, water flow, and depth of the microplastics [77]. In particular, the floating state of plastics depends on the salinity density according to the location and depth of sampling, so it is important to understand the density according to the salinity before collection. For microplastic sampling in water, net sampling and bulk sampling are widely used. Nets used for the sampling are manta trawl, neuston net, bongo net, and microplastic traps [77,78]. In bulk sampling, a specific volume is sampled using a water collection container [78]. Figure 5 shows the types of nets for a net sampling. A net sampling method provides the pros of a large area sampling and large volume sampling by a boat [79,80]. At this time, a flow meter should be attached to the net so that the amount of water can be clearly expressed in m 3 . However, cons are the size of collected microplastics that limited depending on the size of the mesh [81]. The mesh of manta trawl is generally 330 µm mesh, so it is impossible to collect fine plastics smaller than this [78]. Using a nylon net (100 µm), more microplastics can be collected, but if the size of the mesh is less than 100 µm, it can be quickly clogged by plankton, so it is important to collect it at a low speed to avoid from fast clogging [80]. Vermaire et al. (2017) confirmed the difference between 0.1 MPs/L by nylon net and 0.00135 MPs/L by manta net, in which nylon nets (100 µm) collected microplastics almost 100 times higher than manta nets (330 µm) [82]. Nevertheless, manta net, neuton net, and bongo net mesh sizes (330~350 µm) are widely used, because they can collect a large volume of water. If a plankton net with a small mesh is used, a higher concentration of microplastic can be collected than other nets, but sampling is possible only within 1 min, ensuring a low flow rate [83]. A bulk sampling or grab sampling is collected as much as a fixed volume, and the sampling range is narrower than the net sampling. As an alternative technology, SubCtech developed a microplastic sampler based on a pump on a ship. It provides sampling at high vessel speed and instantaneous size separation, using low power water pump [84]. These represents that the measured amount of microplastics could vary depending on the sampling methods [77]. To summarize the above, net sampling is mainly used to collect the amount of microplastics in a large area, and a net of 100-350 µm is used depending on the size of the microplastics. When collecting microplastics in a limited space or in a narrow area, grab sampling or bulk sampling is recommended, and a large number of samples is recommended.

Sediment Sampling
The distribution of microplastics in the sediment is uneven and can vary greatly depending on the collection point, number of collections, and depth of extraction [85]. For example, when sediment is collected from a tidal line with high microplastic accumulation, a high concentration of microplastic can be observed [86]. When the depth of sampling is low, the concentration of microplastics can be high, and when the depth of the sample is 15 cm or more (subsoil), it can show low concentration [87]. Accordingly, the standard method used for soil collection is recommended for the sampling method. Soil sampling is limited to the topsoil layer (0~15 cm), and in general, 5 to 10 samples are collected in a zigzag pattern, or 1 representative point of collection and 4 points are selected, and a total of five samples are mixed and tested [88]. Sediment sampling equipment is a general shovel, grab sampler, sediment core sampler, and auger sampler, as shown in Figure 6. In addition, the weight g or volume L of the sample has a great influence on the representativeness of the sample, so 300-500 g is recommended, as with the soil sampling method [89]. The samples collected in this way are sealed in glass bottles of 300 g or more, tested in a refrigerated state at 0 to 4 degrees, and transported to the laboratory [89]. The sample transported to the laboratory is dried at 40 • C for 72 h to correct the moisture content and the moisture content is checked. For soil samples, Tomas et al. suggested sieving to 1, 2, and 5 mm in compliance with standard mesh sizes of commercially available test sieves [90]. Standard sieve sizes vary from 37.5 to 0.075 mm, so 0.5 or 0.15 mm sieve is recommended to collect smaller microplastics.

Pretreatment
The collected samples contain microplastic particles and various other particulate materials, such as sand, clay, and microorganisms. These samples with other pollutants rather than plastics require pretreatment. The method of pretreatment is largely divided into two parts as density separation and oxidation. The density separation use NaCl or other chemicals to separate microplastics from other materials. The oxidation uses H 2 O 2 or other chemicals to remove natural organic matter [91]. The density separation and oxidation can be performed first or later depending on the type of samples and degree of contamination. For high contaminated microplastic samples, a pretreatment process is required to prevent interference with the measurement of inorganic particles before instrument analysis [92]. Microplastics and inorganic particles are very small in size and are difficult to remove with the naked eye, so separation is mainly used using a difference in density [92]. Many chemicals, such as NaCl, NaBr, NaI, CaCl 2 , ZnCl 2 , and Na 6 [H 2 W 12 O 40 ], have been used for the density difference separation of microplastics. The solution concentration of the density difference for the separation of various plastics is NaCl solution with a density of 1.2 kg/L, sodium polytungstate with a density of 1.4-1.8 kg/L, CaCl 2 with a density of 1.3-1.5 kg/L, NaI with a density of 1.6-1.8 kg/L, ZnCl 2 with a density of 1.5-1.7 kg/L, ZnBr 2 with a density of 1.7 kg/L [92][93][94]. Separation by using the difference in density has a problem, in that the recovery rate is as low as 40% when the particle size of the microplastic is less than 1 mm [95]. Recently, with the development of a method to increase the microplastic recovery rate, even small plastic particles can be recovered at the level of 96-99% [92,96]. Density difference separation takes a long time, such as treatment time (2-5 h) and settling time (24-72 h), so some studies are being conducted to save time, such as centrifugation and air bubbling [94,97]. However, a standardized recovery method for microplastic is not established, and so it is necessary to develop a standardized method for microplastic density separation.
In order to remove natural organic matter (NOM) on the surface of microplastics after separating plastics from inorganic particles, 30% hydrogen peroxide, Fenton oxidation, acid decomposition, base decomposition, and enzyme decomposition are used alone or in combination [91,95]. Hydrogen peroxide is excellent in removing organic matter, but it is not suitable for the pretreatment of plastics made of polyethylene and polypropylene; mixed hydrogen peroxide, sulfuric acid, or hydrochloric acid are often used [90,92,95]. The H 2 O 2 oxidation results corroborated the findings from previous studies, in that there was polymer degradation despite the effective removal of organic matter from the samples (sludge, 80-86%; soil, 96-108%) [91]. When using acids, low pH can lead to microplastic damage [95]. NaOH alkaline digestion resulted in significant degradation of PET and PC particles but removed 61-67% of the organic matter from the sludge samples and 64-68% from the soil samples. KOH alkaline digestion had minimal impact on the plastic particles; however, it only removed 57% of the organic matter from the sludge samples and 35% from the soil samples [95]. Another method for decomposing organic matter is using enzymes. Cloe M. et al. (2014) used enzymatic digestion techniques, which removed >97% of plankton in seawater, without any damage to the microplastic [98]. However, the method using the enzymatic digestion techniques requires a pretreatment time of about 6 days, and oxidation of NOM removal of microplastics may take 24 h-30 days, depending on the type of sample. Accordingly, it is necessary to standardize a method that can minimize plastic damage and reduce time during pretreatment. Figure 7 shows a simple pretreatment step before analysis.

Analysis Method
Among the microplastics analysis, the simplest method is to determine the number of microplastics by staining them with 10 ug/mL Nile Red reagent in 10% dimethyl sulfoxide, with a 10 min incubation time and using a counting program [88]. However, it is not easy to consistently dye microplastics of various properties, it is difficult to identify the types of microplastics, and the pretreated samples may contain substances other than microplastics, so the accuracy is low [99]. Kang et al. (2020) compared the Nile Red method to FTIR, with an accuracy of 78% [99]. Shim et al. (2016) found that the recovery rate of polyethylene (100-300 µm) spiked to pretreated natural sand was 98% in the NR stating method, which was not significantly different with Fourier-transform infrared spectroscopy (FTIR) identification [100]. The NR staining method was suitable for discriminating fragmented polypropylene particles from large numbers of sand particles in laboratory experiment [100]. Another easy method is to find the number of moles of solute in a sample by using Beer's law based on the UV-Vis measurement result [101].
This method is capable of quantitative analysis of microplastics to some extent, but qualitative analysis is not possible. In addition, if the microplastic sinks to the floor or jumps on the surface, it can cause various errors. Accordingly, the types of methods that are currently widely used can be broadly divided into two types, namely non-destructive methods and destructive methods [85]. Non-destructive analysis includes microscopy, microscopy with spectroscopy, microscopy with FTIR, and microscopy with Raman spectroscopy [102][103][104][105]. Destructive analysis includes liquid chromatography with mass spectrometry (LC/MS/UV) [106,107], gas chromatography connected with mass spectrometry (GC/MS), subsuming pyrolysis/gas chromatography/mass spectrometry (Pyr/GC/MS), and thermal desorption gas chromatography (TD/GC/MS) [108][109][110]. Among them, the most commonly used methods are a microscopy with FTIR, Raman, and Pyr/GC/MS. The analysis of microplastics is not clearly defined, so many methods are still being used and studied. Tables 3 and 4 review the currently used analysis methods. Recently, for rapid analysis of microplastic, a charge-coupled device (CCD) camera, photodiode [111], dynamic light scattering (DLS) [112], nanoparticle tracking analysis (NTA) [113], fluorescence spectroscopy [114], and atomic force microscopy (AFM) [115]     4.3.1. FTIR Spectroscopy FTIR spectroscopy is a non-destructive testing method that analyzes a polar functional group such as a carbonyl group by using the degree of scattering of a substance, using infrared rays of a specific wavelength [127,128]. Previously, it was difficult to analyze microplastics under 500 µm and contaminated plastics with FTIR spectroscopy, but recently, microplastics as small as 10 to 5 µm can be analyzed by using a new micro(µ)-FTIR microscopy [104]. FTIR spectroscopy can quickly measure all the frequencies of the infrared source at the same time through pretreated filter paper [117]. However, this method still takes a long time to count small size plastics with irregular shapes and is difficult to apply to samples less than 20 µm [116,117]. Although the condition setting of the FTIR spectroscopy differs depending on the analytical device and microplastic analysis items, the general settings are shown in Table 5. Conform both by mass and by particle count. Use standards to find recovery rate [116,[129][130][131]

Raman Spectroscopy Method
Raman spectroscopy is a method to determine the presence and type of plastic by scattering light generated when the molecules of microplastic particles are excited in vibrational, rotational, and other states [132]. Micro-Raman spectroscopy can analyze microplastics with a large area and high resolution [133], and it offers high selectivity and reproducibility and requires low sample amounts with a minimal sample preparation and shot data collection time [132,133]. This method is effective, but when the sample is contaminated by organic matter, it acts as an interfering factor for fluorescent materials, so it must be completely removed during pretreatment [96]. Raman spectroscopy is often used with FTIR spectroscopy, because it is difficult to measure a sample of 1 to 50 µm in FTIR. Raman spectroscopy is often used to identify below 10 µm and is capable of resolving particles down to 1 µm or less [132]. Primpke et al. (2020) measured seawater by using FTIR and Raman spectroscopy, and Raman confirmed the number of microplastics by about 23% higher than that of FTIR, and, in particular, it showed a high microplastic discovery rate at ≤500 µm [109]. General Raman spectroscopy settings are offered in Table 6. Conform both by mass and by particle count. Use standards to find recovery rate [102,103,105,121,132] Currently, a Laser Direct Infrared (LDIR) chemical imaging system [134] has been developed that improves the analysis time and resolution, which are the disadvantages of FTIR and Raman spectroscopy, and improves the analysis speed.

GC/MS Method
A GC/MS method is a destructive method to determine sample of microplastics by heat-treating the sample and analyzing the gas [104]. There are two types of GC/MS analysis of microplastics: Pyr/GC/MS and TD/GC/MS. In both cases, the amount of microplastics is analyzed by the number of ions released through pyrolysis or thermal desorption [110]. Advantages of GC/MS over FTIR and Raman spectroscopy can analyze both qualitative and quantitative analysis of small microplastics size (<10 µg) with a small sample volume of microplastics, because of its high sensitivity [135]. However, GC/MS is destructive, resulting in the total loss of the particle and subsequently eliminating further particle analysis [125]. Plus, GC/MS allows the analysis of a whole MP particle, in contrast with Raman and FTIR (in reflection mode), which only analyze the surface of the MP particle, being sensitive to interference caused by additives such as pigments [124,136]. Table 7 shows general setting conditions for microplastic analysis using TD and Pyr/GC/MS

Wastewater Treatment Plants (WWTP)
Industrial wastewater, domestic wastewater, agricultural wastewater, and livestock wastewater contain many microplastics, and wastewater treatment plants cannot remove all microplastics. These wastewaters include micro pellets from cosmetic preparations, especially facial scrubs and textile fibers, the latter due to the shedding of particles during the textile washing process [123]. The wastewater is moved to a municipal wastewater treatment plant, where large plastics are removed; however, microplastic or nanoplastic particles still remain after several water treatment processes, and some of microplastics are still discharged to the final effluent [140]. Accordingly, the efficiency of removing microplastics in wastewater treatment plants has emerged as an important issue, and many studies are being conducted.
Typical WWTP processes are divided into preliminary treatment, primary treatment, secondary treatment, and tertiary treatment, and the process chart is as shown in Figure  8. Preliminary treatment consists of a screen and a sedimentation tank, mainly removing large and large plastics. Primary treatment mainly consists of aeration and sedimentation, and it removes light plastics or heavy plastics by skimming and sedimentation. Secondary treatment is mainly biological treatment to remove organic material. Biological treatment consists of an anaerobic tank, anoxic tank, an aerobic tank, and a settling tank, and most of the microplastics >500 µm are removed [137,141,142]. Tertiary treatment is mainly used as an option in WWTPs and consists of a process of removing phosphorus and nitrogen by using chemicals. At this time, it is possible to increase the removal rate of microplastics by using several coagulants [76,137]. Table 8 confirmed the MPs' removal rate according to the process of WWTPs.  The size and concentration of microplastics in the wastewater entering the wastewater treatment plant require more research depending on the location of the wastewater treatment plant, the type of wastewater, and the purpose of treatment. The concentration of microplastics in the effluent from the wastewater treatment plant was 1 MPs/L or less in many studies when the primary, secondary, and tertiary treatments were completed. In addition, it is difficult to ascertain a clear average removal rate of the reviewed studies because the microplastic treatment rate varies at each treatment stage, but the concentration of the inlet is different. Most of the high concentration of microplastics is removed from the primary treatment and contained in the sludge, so the use of recycled fertilizers by the sludge can cause secondary pollution of microplastics.

Physical Removal Technology
Physical methods for removing microplastics include flotation, sedimentation, and filtration, but filtration is a representative method. Filtration includes various methods, such as screening, diskfilter, sandfilter, and membrane filtration (microfiltration, MF; ultrafiltration, UF; nanofiltration, NF; dynamic membrane, DF; and reverse osmosis, RO). Screening is used in both general WWTP and drinking-water treatment plants (DWTP). The screening method generally removes large plastic particles by filtering and sedimentation. The microplastic removal rate by the screening method was confirmed to be from about 40% to about 80% [134,141,142]. Diskfilter is often used in WWTP. Simon et al. (2019) showed that the diskfilter removed up to 89.7% of microparticles less than >10 µm [149]. A sandfilter is used in both WWTP and DWTP. Wolff et al. (2021) showed microplastic removal rates of 99.2% ± 0.29% and 99.4% ± 0.15% by a rapid sandfiltration [150]. Most of the microplastic removal using the membrane filtration shows an efficiency of more than 90%, and it appears to be particularly effective in removing microplastics larger than 10 µm [88,99,[101][102][103]119]. However, when microplastics are effectively removed by using the membrane method, membrane contamination can be accelerated by the deposition of microplastics, which can accelerate contamination of other organic matters in the membrane [130]. Accordingly, when using a membrane, a pretreatment process must be installed in order to prevent excessive membrane contamination of organic matters and microplastics. Table 9 provides a positive physical removal technology for microplastic removal

Chemical Removal Technology
The method of using chemicals for microplastics has been studied in various ways, but in general, the most representatively used method is coagulation/precipitation in water treatment. In particular, there are many differences in the amount of microplastics removed, depending on the type of coagulant, the amount of coagulant, and coagulation retention time. Accordingly, many studies are being conducted to find the optimal coagulant type and conditions, and more clear studies for microplastic removal are needed in the future. In Figure 9, Lapointe et al. (2020) compared the removal rates of polyester (PEST), weathered PE, and pristine PE through a Jar test, using aluminum-based coagulants and polyacrylamide (PAA). When aluminum-based coagulants 2.73 mg Al/L and PAM 0.3 mg PAM/L coagulants were administered to 500 MPs/L water, the optimal removal rates were similar. The removal efficiency of microspheres was PE = 82% of 140 µm, PS = about 80% of 140 µm, PE = about 88% of 15 µm, and PEST fiber = 99% [155]. Table 10 presents a microplastic removal by chemical removal technology.  The review results were found to be able to efficiently remove microplastics by combining PAM with Al-based and iron-based coagulants up to 99% dependent on the microplastic size, number, and water conditions, and it was confirmed that electrocoagulation was also effective in removing microplastics.

Biological Removal Technology
Among the microplastic removal methods, biologic methods include activated sludge treatment, aerobic and anaerobic digestion, lagoons, and septic tanks. In activated sludge systems, bacteria are known to trap microplastics <0.5 mm [158]. However, the activated sludge system only plays an imposing role of trapping microplastics in water, and it is difficult to degrade plastics with a short residence time (7-14 h) in WWTPs [158]. Liu et al.
(2019) virgin microplastics do not significantly affect the activities of ammonia oxidizing bacteria, nitrite oxidizing bacteria, and phosphorus accumulating organisms [159]. Cunha et al. (2020) used 10 mg/L of fresh Cyanothece sp. and found microplastic removal rate of up to 47% [160]. Canniff and Hoang (2018) confirmed the growth rate by exposure to PE beads, using Daphnia magna, and confirmed that the PE intake rate increased as the particle concentration and exposure time increased. In particular, it was confirmed that R. subcapitata exposed to PE beads grew more than those without exposure [161]. As such, the removal of microplastics by using a biologic method is generally low in efficiency, and secondary contamination of microplastics in sludge or sedimentation can be increased. The effect of microplastics on the performance of the bioreactor system should not be overemphasized [162]. Therefore, it can be concluded that high microplastic removal efficiency by biological methods is not very positive.

Conclusions
As worldwide plastic production has been increasing, plastic contamination has caused global deterioration of the environment, due to the long plastic-decomposition time [3,4,160]. As a result, microplastic contamination is becoming a global issue, but monitoring, toxicity, analysis, and removal technology are still insufficient for researchers. Therefore, this paper reviewed the distribution, toxicity, analysis method, and removal technologies of microplastics and summarizes the results as follows. - Microplastics with the high contamination were reported as PET, PU, PS, PVC, PP, PE, and PA. -Contamination paths of microplastics include agricultural wastewater, industrial wastewater, litter, sewage treatment plant, household personal products, road runoff, fishing waste, and atmosphere decomposition, which finally flow into the sea to pollute sea creatures and are absorbed by humans. - The pollution of microplastics around the world is sharply increasing, and it is appearing in drinking water, sewage water, rivers, seas, soil, and everywhere. Finally, microplastics will cause a huge problem in the near future. -Although the toxicity of microplastics has not been studied much, plastics, such as PS, PVC, PP, etc., could still cause problems in human health, and several researchers are conducting research on the risk of microplastics. -Analysis of microplastics were divided into sampling, pretreatment, and analysis parts. Water sampling and sediment sampling were discussed in the sampling part.
In the pretreatment part, how to deal with the density difference separation and how to remove other contaminants rather than microplastics were discussed in detail. In the analysis method part, most of used microplastic analytical methods and methods for a possible application for a microplastic analysis were summarized in detail. The various techniques for the removal method of microplastics were summarized in WWTP, physical, chemical, and biological technologies. Each technique for microplastic removal rate was summarized in several tables. In particular, the microplastic removal rate in WWTP was found to be more than 70% after secondary treatment, although there was a difference depending on the research papers.
In conclusion, microplastic contamination is scattered around the world and directly affects human life. Accordingly, research on microplastic contamination, analysis, monitoring, and removal technology is insufficient, so deeper interest from researchers and national policies are urgently needed for overcoming microplastic contamination.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in Review of Microplastic Distribution, Toxicity, Analysis Methods, and Removal Technologies.

Conflicts of Interest:
The authors declare no conflict of interest.