Method for analyzing microplastics and method for separating and recovering microplastics for analysis

Abstract

This method for analyzing microplastics having a size of 500 μm or less in a sample comprises: a microplastic recovery step for separating and recovering microplastics by filtering the sample; and an analysis step for analyzing the microplastics recovered in the microplastic recovery step. In the method, an instrument used for the recovery in the microplastic recovery step is washed with an organic solvent having a specific gravity smaller than that of water, and the microplastics remaining on the surface of the instrument that is in contact with the sample are captured and recovered.

Description

Method for analyzing microplastics and method for separating and recovering microplastics for analysis

[0001] The present invention relates to a method for analyzing microplastics of a size of 500 μm or less in a sample and a method for separating and recovering microplastics for analysis.

[0002] In recent years, the presence of microplastics in the marine environment has become a matter of great concern. Microplastics originate from plastic waste such as plastic shopping bags, convenience store lunch boxes, and plastic bottle caps. They are discarded or left untreated after overflowing from trash cans, and then enter rivers and sewers, or are blown by the wind, eventually ending up in the ocean.

[0003] Because microplastics have adverse effects on the environment and human health, the number of microplastic particles in the ocean has been investigated according to guidelines and carefully verified through comparative experiments between laboratories (see Non-Patent Documents 1 to 3). In addition, more than 8,000 trawl surveys have been conducted to date to quantify the amount of microplastics in the world's upper oceans (see Non-Patent Document 4).

[0004] Isobe , A. , Buenaventura , NT , Chastain , S. , Chavanich , S. , Cozar , A. , DeLorenzo , M. , Hagmann , P. , Hinata , H. , Kozlovskii , N. , Lusher , AL , Marti , E. , Michida , Y. , Mu , J. , Ohno , M. , Potter , G. , Ross , PS , Sagawa , N. Shim , WJ , Song , YK , Takada , H , Tokai , T , Torii , T , Uchida , K , Vassillenko , K , Viyakarn , V , Zhang W , (2019). An interlaboratory comparison exercise for the determination of microplastics in standard sample bottles, Tues. Pollut. Bull., 146, 831-837.Cadiou, J.-F., Gerigny, O., Koren, S., Zeri, C., Kaberi, H., Alomar, C., Panti, C., Fossi, MC, Adamopoulou, A., Digka, N., Deudero, S., Concato, M., Carbonell, A., Baini, M., Galli, M., Galgani, F., (2020). Lessons learned from an intercalibration exercise on the quantification and characterization of microplastic particles in sediment and water samples, Mar. Pollut. Bull., 154, 111097.Cowger, W., Booth, AM, Hamilton, BM, Thaysen, C, Primpke, S, Munno, K, Lusher, AL, Dehaut, A, Vaz, VP, Liboiron, M., Devriese, LI, Hermabessiere, L., Rochman, C., Athey, SN, Lynch, JM, Frond, HD, Gray A., Jones, OAH, Brander, S., Steele, C., Moore, S., Sanchez, A., and Nel, H., (2020). Reporting Guidelines to Increase the Reproducibility and Comparability of Research on Microplastics. Appl. Spectrosc., 74, 1066-1077.Isobe, A., Azuma, T., Cordova, MR, Cozar, A., Galgani, F., Hagita, R., Kanhai, LD, Imai, K., Iwasaki, S., Kako, S., Kozlovskii, N., Lusher, AM, Mason, SA, Michida, Y., Mituhasi, T., Morii, Y., Mukai, T., Popova, A., Shimizu, K., Tokai, T., Uchida, K., Yagi, M., Zhang, W., (2021). A multilevel dataset of microplastic abundance in the world's upper ocean and the Laurentian Great Lakes. Micropl. & Nanopl. 1:16.

[0005] However, there is currently no established method for measuring microplastics that are smaller than a few hundred micrometers, and the development of such a method has been desired.

[0006] The object of the present invention is to provide a method capable of accurately analyzing microplastics of a size of 500 μm or less in a sample.

[0007] In the course of investigations to solve the above problems, the present inventors first discovered that the analysis of microplastics with a size of 500 μm or less among microplastics has the following problems.

[0008] The number of microplastic particles is thought to be underestimated because microplastics are captured and lost in sampling and experimental equipment. Specifically, some microplastics remain on filters, the inner walls of containers, tubes, etc. during the processing process and are lost, so it is likely that the estimated amount is lower than the actual amount.

[0009] As a result of various investigations into the above-mentioned problems, the inventors discovered that when recovering microplastics, by washing the equipment used with an organic solvent having a lower specific gravity than water and supplementing and recovering the microplastics remaining on the sample contact surface of the equipment, it is possible to more reliably separate and recover microplastics in the sample and analyze them with high accuracy, which led to the completion of the present invention.

[0010] That is, the present invention is as follows: [1] A method for analyzing microplastics of a size of 500 μm or less in a sample, comprising: a microplastics recovery step of filtering the sample to separate and recover microplastics; and an analysis step of analyzing the microplastics recovered in the microplastics recovery step, wherein the method is characterized in that an instrument used for recovery in the microplastics recovery step is washed with an organic solvent having a specific gravity smaller than that of water, and microplastics remaining on the sample-contacting surface of the instrument are captured and recovered.

[0011] [2] The method for analyzing microplastics described in [1] above, wherein the microplastics to be analyzed are plastics other than the material of the instrument.

[0012] [3] The method for analyzing microplastics according to [1] or [2] above, characterized in that the microplastics to be analyzed include at least polyethylene, polypropylene, polystyrene, polyester polyol, ethylene vinyl acetate, ethylene propylene rubber, acrylonitrile butadiene styrene, and polyethylene terephthalate.

[0013] [4] The method for analyzing microplastics according to any one of [1] to [3] above, wherein the organic solvent having a specific gravity smaller than that of water is ethanol.

[0014] [5] The method for analyzing microplastics according to any one of [1] to [4] above, wherein the microplastics to be analyzed have a size of 300 μm or less.

[0015] [6] The method for analyzing microplastics according to any one of [1] to [5] above, wherein the filtration in the microplastics recovery step is gravity filtration. [7] The method for analyzing microplastics according to [6] above, wherein the gravity filtration is siphon filtration.

[0016] [8] A method for separating and recovering microplastics for analysis from a sample in order to analyze microplastics of 500 μm or less contained in the sample, comprising washing an instrument used to separate and recover microplastics from the sample with an organic solvent having a specific gravity lower than that of water, and capturing and recovering microplastics remaining on the sample contact surface of the instrument. [9] The method for separating and recovering microplastics for analysis described in [8] above, wherein the organic solvent having a specific gravity lower than that of water is ethanol.

[0017]

[10] A method for analyzing microplastics present in water that are 500 μm or smaller in size, comprising: a sampling step of collecting a sample containing microplastics from water; a microplastic recovery step of filtering the sample collected in the sampling step to separate and recover microplastics; and an analysis step of analyzing the microplastics recovered in the microplastic recovery step, wherein the method comprises washing an instrument used for recovery in the microplastic recovery step with an organic solvent having a specific gravity less than that of water, and capturing and recovering microplastics remaining on the sample contact surface of the instrument.

[11] A method for separating and recovering microplastics from a sample collected from water in order to analyze microplastics 500 μm or smaller contained in the sample, wherein the method comprises washing an instrument used for separating and recovering microplastics from the sample with an organic solvent having a specific gravity less than that of water, and capturing and recovering microplastics remaining on the sample contact surface of the instrument.

[0018] According to the microplastic analysis method and the method for separating and recovering microplastics for analysis of the present invention, microplastics of a size of 500 μm or less can be more reliably separated and recovered from samples and analyzed with high accuracy.

[0019] FIG. 1 is a flow diagram of one embodiment of the microplastic analysis method of the present invention.

[0020] The method for analyzing microplastics according to the present invention is a method for analyzing microplastics of a size of 500 μm or less in a sample, and comprises a microplastic recovery step of filtering the sample to separate and recover microplastics, and an analysis step of analyzing the microplastics recovered in the microplastic recovery step. The method is characterized in that the equipment used for recovery in the microplastic recovery step is washed with an organic solvent having a lower specific gravity than water, and microplastics remaining on the sample contact surface of the equipment are supplemented and recovered.

[0021] In addition, the microplastic analysis method of the present invention may include processes other than the microplastic recovery process and analysis process, such as a preparation process or a sample collection process carried out before the microplastic recovery process.

[0022] In the preparation process, for example, from the perspective of preventing contamination (hereinafter simply referred to as contamination), measures are taken to prevent the contamination of samples if there is a risk of microplastics or other substances being mixed in from the outside. That is, the number of microplastic particles may be overestimated due to plastic particle contamination in the analytical environment, so measures are taken to prevent such particle contamination.

[0023] Furthermore, the sample collection process is a process of collecting samples from water such as the sea when analyzing microplastics in seawater, river water, lake water, etc.

[0024] In addition, the method for separating and recovering microplastics for analysis according to the present invention is a method for separating and recovering microplastics from a sample in order to analyze microplastics of 500 μm or less contained in the sample, and is characterized in that the tool used to separate and recover the microplastics from the sample is washed with an organic solvent having a lower specific gravity than water, and the microplastics remaining on the sample contact surface of the tool are captured and recovered.

[0025] The microplastic analysis method and the method for separating and recovering microplastics for analysis of the present invention are novel techniques that use an organic solvent with a specific gravity lower than that of water to wash the equipment used to separate and recover microplastics to be analyzed from a sample containing microplastics. The use of such an organic solvent allows for more reliable recovery of microplastics. In other words, washing with an organic solvent with a specific gravity lower than that of water makes it easier for fine microplastics with low specific gravity to disperse in the organic solvent, allowing the microplastics to be effectively washed away from the walls of the equipment.

[0026] The sample to be treated in the microplastic analysis method and the method for separating and recovering microplastics for analysis of the present invention is not particularly limited as long as it contains microplastics, and examples include water collected from the sea, rivers, lakes, drinking water, etc., as well as blood and soil. By analyzing water collected from the sea, rivers, lakes, etc., the state of environmental pollution can be understood.

[0027] The size (particle diameter) of the microplastics contained in the sample is 500 μm or less, but a smaller size of 300 μm or less is preferable because the effects of the present invention are more pronounced. On the other hand, the lower limit of the size is not particularly limited, but is, for example, about 10 μm.

[0028] These microplastics are, for example, tiny plastics that float in water or on the water surface, and specific examples of the materials used include polyethylene (PE), polypropylene (PP), polystyrene (PS), polyester polyol (PEP), ethylene vinyl acetate (EVA), ethylene propylene rubber (EPDM), acrylonitrile butadiene styrene (ABS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), and nylon (polyamide: PA).

[0029] The microplastics to be analyzed are preferably made of a material other than the material of the equipment used for collecting microplastics. For example, polyvinyl chloride is often used in equipment, so it is preferable to exclude it from the analysis target in order to perform a more accurate analysis. If equipment made of polyvinyl chloride is not used, it is preferable to analyze polyvinyl chloride. In other words, it is preferable that the equipment used for collecting microplastics is made of a material other than the microplastics to be analyzed.

[0030] Examples of organic solvents having a specific gravity lower than that of water include alcohols such as methanol, ethanol, isopropanol, butanol, pentanol, and hexanol, as well as acetone and hexane. Among these, ethanol is preferred because it is easy to handle and inexpensive. Two or more organic solvents having a specific gravity lower than that of water may be used in combination. The organic solvent having a specific gravity lower than that of water may contain water, but the water content is preferably 30% by mass or less, more preferably 10% by mass or less, and particularly preferably substantially free of water.

[0031] Each step of the present invention will be described below with reference to the drawings. As shown in Figure 1, the microplastic analysis method of the present invention includes, for example, a preparation step (S1), a sample collection step (S2), a microplastic recovery step (S3), an organic matter decomposition step (S4), and an analysis step (S5). In this embodiment, a case will be described in which water collected from the sea is used as a sample containing microplastics, but the same applies when the sample is water collected from a river, lake, etc.

[0032] Each step will be specifically described below.

[0033] [Preparation Process] The preparation process is a process carried out to prevent contamination at various stages, such as during sample collection and microplastic recovery and analysis, in order to prevent the contamination of samples with microplastics and other substances from the external environment. For example, to avoid dust in the air, a clean booth or clean room may be installed inside the ship or analysis room. Furthermore, since the bottles containing the reagents may be made of the plastics being analyzed, it is preferable to filter the reagents beforehand and transfer them to glass bottles to prevent contamination by plastics and other substances from the reagents used in the analysis. It is preferable to use ultrapure water (Milli-Q water) for the water used in the analysis.

[0034] [Sample Collection Step] The sample collection step is a step of collecting water from the sea. For example, when sampling on a ship, a Niskin bottle (Niskin sampler) or the like is used to collect a sample from the surface layer of seawater.

[0035] [Microplastics Recovery Process] The microplastics recovery process is a process in which a sample is filtered to separate and recover microplastics. Specifically, the collected sample is first transferred from a Niskin bottle (Niskin collector) to a container. At this time, the sample is transferred from the Niskin bottle to a polycarbonate container via a silicone tube (a silicone rubber tube) to prevent the sample from coming into contact with air. Here, the tools used in this process, such as the tool used to transfer the sample from the Niskin bottle, are preferably made of a material other than the microplastics contained in the sample that are the subject of analysis.

[0036] The sample transferred to the container is then filtered. Gravity filtration, which can be performed at a slow filtration rate to prevent damage to fragile microplastics, is preferred. Microplastics degraded in nature are fragile, and if some of the microplastics are broken down by physical or chemical stimuli during sample processing, the number of microplastic particles may be overestimated. This must be prevented. Siphon filtration, which utilizes the siphon principle, is particularly preferred as a gravity filtration method. For example, the sample is initially sucked up from the top of the container using a silicone tube with a vacuum pump, and then filtered at a slow rate using the siphon principle. It is recommended to use a stainless steel filter for filtration. The mesh (openings) of this filter are appropriately selected depending on the size of the microplastics to be collected. This allows microplastics to be collected on the filter.

[0037] After the above filtration, the tools used for collection in this process are washed with an organic solvent with a specific gravity lower than that of water to capture and collect any microplastics remaining on the sample-contacting surfaces of the tools. Specifically, after washing the tools, the organic solvent containing the microplastics used for washing is filtered by gravity filtration using the filter used for this collection to capture and collect any microplastics remaining on the tools. This allows for more reliable collection of microplastics remaining on the tools.

[0038] As described above, by using an organic solvent with a lower specific gravity than water as a cleaning solution, microplastics with a lower specific gravity are more easily mixed in the cleaning solution without floating, making them easier to remove from the sample contact surface of the instrument, and it is thought that microplastics can be recovered more effectively. As will be described later, when washing with water, the recovery rate of microplastics was about 35%, while when washing with ethanol, it was about 90%, showing a dramatic improvement in recovery rate. Note that an organic solvent with a lower specific gravity than water and a lower specific gravity than all of the microplastics to be analyzed is particularly preferable as a cleaning solution.

[0039] The above-mentioned instruments include those that come into direct contact with the sample from collection to filtration, specifically the above-mentioned silicone tubes, containers, filters, filter holders, etc. It is preferable to wash all of these instruments with an organic solvent, but it is also possible to wash only some of the instruments with a large amount of microplastics attached, for example. Note that the above-mentioned filters are preferably washed with heat using an organic solvent (particularly ethanol) to remove lipids mixed in with the microplastics.

[0040] [Organic matter decomposition step] The organic matter decomposition step is a step of decomposing and removing organic matter other than microplastics attached to the filter. Note that, although it is preferable to perform this treatment, it may be omitted.

[0041] For example, the filter containing microplastics collected in the microplastic recovery process is subjected to low-temperature two-stage digestion. This reduces physical and chemical damage to the microplastics. Conventional methods can be used for two-stage digestion, such as "Alfonso, MB, Takashima, K., Yamaguchi, S., Tanaka, M., Isobe, A., 2021. Microplastics on plankton samples: multiple digestion techniques assessment based on weight, size, and FTIR spectroscopy analyses, Mar. Pollut. Bull., 173, 113027."

[0042] After the decomposition process, the filter is rinsed sequentially with ultrapure water and an organic solvent with a specific gravity less than that of water, and the organic solvent containing the microplastics is collected in a container. Note that since there is a possibility that the microplastics may remain attached to the filter, the filter is further subjected to, for example, an ultrasonic cleaner to separate the microplastics from the filter, and the organic solvent containing the microplastics is collected in a container.

[0043] [Analysis Step] The analysis step is a step of analyzing the microplastics treated in the organic matter decomposition step (or the microplastic recovery step if the organic matter decomposition step is omitted). For example, a micro-FTIR (infrared spectrophotometer) is preferably used to identify the type of polymer in the microplastics. First, the organic solvent containing the microplastics obtained in the organic matter decomposition step and recovered in a container is poured onto an analytical filter, and the microplastics are accumulated on the analytical filter. To improve analytical accuracy, it is preferable to concentrate the microplastics within a small area in the center of the filter. It is also preferable to wash the equipment used in this step, such as the container, with an organic solvent with a specific gravity lower than water and then pour it onto the analytical filter to more reliably recover the microplastics.

[0044] Furthermore, since it is necessary to eliminate wrinkles and flexures in the filter and the analytical errors that result from these, it is preferable to dry the filter by, for example, placing the filter on a metal ring-shaped jig and fixing the periphery with a ring-shaped rubber (rubber band).

[0045] The filter is placed on the sample support of the micro-FTIR, and the polymer type, particle number, size, etc. of the microplastics are examined. While there are no particular limitations on the type of microplastics to be analyzed, it is preferable to limit the analysis to those that are not used as materials for the instruments in the method of the present invention and that are likely to be present in relatively large quantities in the ocean, etc., as this shortens the processing time. Specific examples include one or more of polyethylene, polypropylene, polystyrene, polyester polyol, ethylene vinyl acetate, ethylene propylene rubber, acrylonitrile butadiene styrene, and polyethylene terephthalate.

[0046] When using micro-FTIR for analysis, it is preferable not to perform density separation, which is usually performed when analyzing particles larger than 500 μm to separate plastics from pebbles, etc. This is because excessive deposition or coloring of the NaI used in density separation on the filter can lead to a decrease in the spectral signal. However, density separation may be performed when using other analytical methods.

[0047] Furthermore, after the organic matter decomposition process, or even after density separation, the sample may be divided for subsampling. For samples with high concentrations of microplastics, the sample can be divided to facilitate analysis in the subsequent analysis process.

[0048] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. In order to minimize contamination of the sample (microplastics), damage to the microplastics, and loss of the microplastics, analysis of microplastics in the sample was carried out using the following procedures.

[0049] 1.1 Preparation process (prevention of contamination) To obtain accurate results of microplastic abundance, it is very important to avoid contamination with other microplastics and non-plastic dust and fibers in the analysis room, etc. These small substances cause noise in the spectral signals used to identify plastic particle polymers and also significantly increase the processing time for particle counting by FTIR.

[0050] Therefore, when collecting seawater samples, a clean booth was installed on board to prevent the inclusion of suspected microplastics in the air. Similarly, on land, filtration, two-stage digestion, and other processes were performed in a clean bench. An electrostatic eliminator was installed in the clean bench to prevent external dust from adhering to the experimental equipment. Three HEPA filters were also installed to continuously remove airborne dust. Furthermore, both on board and on land, experimental equipment made from non-plastic materials, such as silicone resin, fluororesin, glass, and metal, was used.

[0051] Ultrapure water (Milli-Q water) produced by an ultrapure water production system was used for all experiments, including cleaning all equipment and preparing reagents. Polyethersulfone, which is rarely found in nature, was selected as the final filter material for the ultrapure water production system, and the polystyrene water intake cover was removed because it is the polymer of the microplastics being analyzed.

[0052] Digestion (10% KOH, 30% H ２ O ２The reagents used for the 0.05M Fe(II) solution and 99.5% ethanol were also potential sources of contamination due to the plastic caps of the reagent bottles and plastics released during the manufacturing process. Therefore, all reagents were filtered through a 1 μm polytetrafluoroethylene (PTFE) membrane filter and transferred to a rinsed glass reagent bottle covered with a PTFE cap. Specifically, the filtration process was repeated until FTIR microscopy analysis showed that the reagents contained no microplastics. Finally, the microplastic-free reagents were stored in glass bottles with PTFE caps. Hereinafter, unless otherwise noted, "ethanol" refers to the 99.5% ethanol solution filtered using the above method.

[0053] 1.2 Sampling Process (Microplastic Sampling on Board) Surface seawater was collected from the ship using eight L-Niskin bottles made of polyvinyl chloride (PVC) under conditions that minimized contamination. Seawater was transferred directly from the Niskin bottles to polycarbonate containers via silicone tubing, preventing exposure to air. The containers containing the seawater samples were immediately covered with rubber stoppers to avoid contamination.

[0054] 1.3 Microplastic Recovery Process (Filtration of Microplastics from Seawater Samples) Filtration of microplastics from seawater samples was carried out on a clean bench (analysis room) using a filtration device with a stainless steel filter (pore size 10 μm, diameter 47 mm). Seawater was transferred from a polycarbonate container to the filtration device via a silicone tube (inner diameter 7.94 mm). Specifically, a vacuum pump was used only initially, and thereafter, the seawater was transferred to the filtration device and filtered using the siphon principle to maintain a filtration rate slow enough to prevent the microplastics from breaking down.

[0055] After filtering the seawater, the silicone tube and the polycarbonate container were washed with ethanol to remove any microplastics that had adhered to the surface. The ethanol used for washing was then filtered through the filter.

[0056] To remove lipids, the stainless steel filters were immersed in ethanol for 1 minute and heated to 60°C using a hot plate stirrer. The stainless steel filters were then washed with water by suction filtration. All filters were stored in containers made of perfluoroalkoxyalkane (hereinafter referred to as PFA).

[0057] 1.4 Organic matter decomposition process (two-stage digestion) In this protocol, a two-stage digestion was performed at low temperature to reduce physical and chemical damage to microplastics. The stainless steel filters in the PFA containers were immersed in 30 mL of 10% KOH solution in a tall beaker at 40 °C for 72 hours, and then immersed in 60 mL of 30% KOH. ２ O ２ The filter was then oxidized and digested with 20 mL of 0.05 M Fe(II) at 40°C or below. The stainless steel filter was then rinsed with ultrapure water and ethanol and collected in a tall beaker. Specifically, the washed stainless steel filter was placed in a PFA container containing 30 mL of ethanol solution. The capped PFA container was then placed in an ultrasonic cleaner for 1 minute to separate any microplastics that may still be attached to the filter. The ethanol solution containing the microplastics was then transferred to a tall beaker and stored for further processing.

[0058] 1.5 Analysis Process (Identification of Microplastics by Micro-FTIR) The type of plastic polymer was analyzed using a micro-FTIR (Nicolet iN10 MX, Thermo Fisher Scientific). Generally, a micro-FTIR scans a small area (8 mm x 8 mm in "focal plane array (FPA)" mode). Here, a PTFE filter was placed on a metal ring-shaped jig, fixed around its periphery with a ring-shaped rubber, and dried in a petri dish on a clean bench. Finally, the PTFE filter was placed on the micro-FTIR sample support, and the type of microplastic polymer was identified.

[0059] In order to shorten the processing time, the types of polymers to be identified using micro-FTIR were determined in advance to be those that may exist in the ocean. The plastic polymers targeted in this study were those with a lower density than seawater (up to 1.025 g / cm), such as polyethylene, polypropylene, polyester polyol, ethylene vinyl acetate, and ethylene propylene rubber. ３ ) and the density was 0.96 to 1.05 g / cm ３ Polystyrene was also included in the study because of the possibility of detecting foamed polystyrene fragments (lighter than seawater). Furthermore, polyethylene terephthalate is less likely to travel long distances in the ocean because its density is heavier than seawater, but polyester SMP fibers may reach the upper ocean layers through atmospheric deposition.

[0060] The polymer type, particle number, and size of microplastics on a PTFE filter were investigated using micro-FTIR scan images (FPA mode, transmission mode, 715-4000 / cm range, 8 / cm resolution, acquired with 16 cumulative scans) of an 8 mm x 8 mm filter area, and the polymer spectra were compared with those of the library. The selection criteria were a hit quality index of 60% or higher (correlation coefficient 0.6; statistically significant value) for the infrared absorption spectra of plastic particles, and detection of all expected spectral peaks for each polymer type.

[0061] On the other hand, microplastics with very high hit quality indices (e.g., 90% or higher), such as those that are undegraded or degraded to the same extent as those in libraries of UV- or heat-damaged polymers, are unlikely to exist in nature and may be derived from contamination. However, it is difficult to determine a unique best hit quality index appropriate for marine microplastics. Therefore, overestimation due to contaminated microplastics during observation was reduced by subtracting the number of particles detected in the contamination test. Size was defined as the ferret diameter of each particle displayed on the monitor and measured using the image processing software provided with the micro-FTIR. The size of microplastics measured by micro-FTIR was confirmed by measuring the particles with a stereo microscope (Olympus, Japan, SZX7) and confirmed to have a reliable LOD of 10 μm or greater.

[0062] Using the method described above, measurements were taken of each type of microplastic polymer at two locations near Japan. Table 1 shows the particle count and concentration (number of particles per liter of seawater), while Table 2 shows the particle count and concentration (number of particles per liter of seawater) for each size range. The concentration is shown in parentheses next to the particle count.

[0063]

[0064]

[0065] As described above, it was found that microplastics with a size of 500 μm or less can be separated, collected, and analyzed.

[0066] The following describes the tests and results of confirming the effectiveness of the method of the present invention: 1. Confirmation of the effectiveness of the method of washing with an organic solvent having a specific gravity smaller than that of water In this test, the test was carried out three times using spherical red polystyrene beads (Thermo Fisher, RD100T) with a diameter of 100 μm.

[0067] First, 100 red beads were mixed with 5 liters of ultrapure water, followed by filtration (step 1.3 above), two-stage digestion (step 1.4 above), and final filtration through a PTFE filter for spectroscopic analysis (step 1.5 above). The number of beads on the filter was then counted using a stereomicroscope, and the recovery rate (R100) was calculated. Furthermore, the ethanol was replaced with ultrapure water to rinse the equipment, and this particle recovery rate was compared.

[0068] As a result, when ethanol was used for cleaning, the recovery rate was about 88%, but when ultrapure water was used instead of ethanol, the recovery rate dropped sharply to 33.6%. This is thought to be because microplastics, which have a higher specific gravity than ethanol, were more likely to move from the sample contact surface of the instrument when they came into contact with ethanol, allowing them to be recovered. Note that a similar trend was observed for organic solvents other than ethanol that have a lower specific gravity than water.

[0069] 2. Confirmation of the effect of siphon filtration in the present invention The destruction rate of polystyrene beads by siphon filtration was confirmed to be less than 2%. Compared to the average destruction rate of about 9% in the case of conventional filtration using a mechanical pump, it can be seen that the destruction of microplastics is sufficiently suppressed by applying siphon filtration.

[0070] The present invention is industrially useful because it enables accurate analysis of microplastics with a size of 500 μm or less in a sample.

Claims

1. A method for analyzing microplastics with a size of 500 μm or less in a sample, comprising: a microplastic recovery step of filtering the sample to separate and recover the microplastics; and an analysis step of analyzing the microplastics recovered in the microplastic recovery step. The method is characterized in that the instrument used for recovery in the microplastic recovery step is washed with an organic solvent having a specific gravity smaller than that of water, and the microplastics remaining on the sample contact surface of the instrument are supplemented and recovered.

2. The method for analyzing microplastics according to claim 1, wherein the microplastics to be analyzed are plastics other than the material of the instrument.

3. The method for analyzing microplastics according to claim 1 or 2, wherein the microplastics to be analyzed include at least polyethylene, polypropylene, polystyrene, polyester polyol, ethylene vinyl acetate, ethylene propylene rubber, acrylonitrile - butadiene - styrene, and polyethylene terephthalate.

4. The method for analyzing microplastics according to claim 1 or 2, wherein the organic solvent having a specific gravity smaller than that of water is ethanol.

5. The method for analyzing microplastics according to claim 1 or 2, wherein the microplastics to be analyzed have a size of 300 μm or less.

6. The method for analyzing microplastics according to claim 1 or 2, wherein the filtration in the microplastic recovery step is gravity filtration.

7. The method for analyzing microplastics according to claim 6, wherein the gravity filtration is siphon filtration.

8. A method for separating and recovering microplastics from a sample for analyzing microplastics with a size of 500 μm or less contained in the sample. The method is characterized in that the instrument used for separating and recovering the microplastics from the sample is washed with an organic solvent having a specific gravity smaller than that of water, and the microplastics remaining on the sample contact surface of the instrument are supplemented and recovered.

9. The method for separating and recovering microplastics for analysis according to claim 8, wherein the organic solvent having a specific gravity smaller than that of water is ethanol.

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