A method for rapidly extracting and analyzing microplastics in soil based on density separation method

Abstract

The application relates to the field of environmental monitoring and soil pollution detection, and particularly discloses a method for rapidly extracting and analyzing microplastics in soil based on a density separation method, which aims to solve the problems of low separation efficiency, long operation period, unstable recovery rate, low automation degree and serious organic matter interference in the prior art. The method comprises the steps of sample pretreatment, organic matter removal, density separation, enrichment and purification, density gradient centrifugation and identification and counting, wherein the density separation adopts a multistage density separation system, and saturated sodium chloride solution is used to separate light microplastics, a mixed solution of sodium bromide and calcium chloride is used to separate medium-density microplastics, and a mixed solution of potassium iodide and zinc chloride is used to separate heavy microplastics, and an automatic density separation instrument is used in cooperation with magnetic stirring assistance. By adopting the technical scheme, the light, medium-density and heavy microplastics can be separated step by step, the recovery rate is significantly improved, the operation time is greatly shortened, and various types of microplastics such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate and polyvinyl chloride can be effectively extracted; the automatic density separation instrument is adopted to reduce manual operation links, reduce human errors and improve the repeatability of the method; through hydrogen peroxide pretreatment and density gradient centrifugation double purification, the interference of organic matter and impurities with similar density can be effectively removed; the separation liquid can be recycled and used, the waste liquid can be treated and discharged up to the standard, and the green chemistry concept is met.

Description

Technical Field

[0001] This application belongs to the field of environmental monitoring and soil pollution detection, specifically involving a method for rapid extraction and analysis of microplastics in soil based on density separation. Background Technology

[0002] Microplastics, referring to plastic particles with a diameter of less than 5 millimeters, are recognized by the international community as a global emerging pollutant. Due to their extremely small particle size, large specific surface area, and hydrophobic properties, microplastics readily adsorb persistent organic pollutants from the environment and undergo bioaccumulation and transfer through the food chain, posing a potential long-term threat to ecosystems and human health. Soil, as an important component of the Earth's surface ecosystem, is a significant sink and source of microplastics, and soil microplastic pollution is receiving increasing attention from the academic community and environmental protection departments.

[0003] Density separation is currently the most commonly used technique in the field of soil microplastic extraction. This method is based on the density difference between microplastics and the soil matrix. By preparing a separation solution of a specific density, the microplastics are suspended while the soil particles settle, thereby achieving the separation and extraction of the target material. However, existing technologies suffer from the following drawbacks: traditional density separation solutions, such as saturated sodium chloride solutions, have a density of only about 1.2 g / cm³, which cannot effectively separate high-density microplastics such as polyethylene terephthalate (PET) and polyvinyl chloride (PVC), resulting in a narrow separation range and insufficient method universality; a single separation requires repeated centrifugation and washing operations, with the entire process typically taking more than 24 hours, making it difficult to meet the needs of rapid detection of large batches of samples; microplastics are prone to adsorption, aggregation, or loss during separation, leading to large fluctuations in recovery rates, which generally remain at a low level of 60% to 80%, resulting in poor reproducibility; existing methods are highly dependent on manual operation, with low automation, and significant errors exist between different operators and between different batches operated by the same person, making it difficult to establish standardized detection procedures; impurities such as organic matter and humic acid in the soil easily co-precipitate or adsorb onto the surface of microplastics, severely interfering with subsequent identification and analysis. These technical problems severely restrict the widespread application of soil microplastic detection technology, necessitating the development of a soil microplastic extraction method with high separation efficiency, short operation cycle, strong universality, and high automation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapid extraction and analysis of microplastics in soil based on density separation, which can effectively solve the problems of low separation efficiency, long operation cycle, unstable recovery rate, low degree of automation and serious interference from organic matter in the existing technology.

[0005] To achieve the above objectives, the technical solution adopted by this invention is as follows: a method for rapid extraction and analysis of microplastics in soil based on density separation, comprising the following specific steps: Step 1: Sample pretreatment: Take the soil sample to be tested, air dry it naturally, remove plant roots and gravel and other impurities, grind and sieve to obtain a soil sample within a predetermined particle size range; Step 2: Organic matter removal: Add a predetermined concentration of hydrogen peroxide solution to the soil sample, adjust the pH of the system to a predetermined range, heat in a water bath at a predetermined temperature for a predetermined time to remove organic matter from the soil, and prepare the soil sample and hydrogen peroxide solution according to a predetermined solid-liquid ratio; Step 3: Density separation: Transfer the sample after organic matter removal to a density separation device, add a multi-stage density separation liquid, and perform three-stage density separation sequentially. The first stage uses a saturated sodium chloride solution to separate lightweight microplastics with a density less than the first predetermined density. The second stage uses a mixed solution of sodium bromide and calcium chloride to separate microplastics with densities from the first predetermined density to the second predetermined density. Medium-density microplastics within a preset density range are separated in the third stage using a mixed solution of potassium iodide and zinc chloride to separate heavy microplastics within the third to fourth preset density ranges. Step 4 involves microplastic enrichment and purification: the suspensions obtained from each separation stage are filtered through preset pore size membranes, and the microplastics on the membranes are collected. The membranes are then washed a predetermined number of times with deionized water to remove residual separation liquid. Step 5 involves density gradient centrifugation purification: the collected microplastics are transferred to density gradient centrifuge tubes, and sucrose solutions of preset concentrations are added sequentially to form a gradient. The tubes are centrifuged at preset speeds for a predetermined time to remove impurity particles with densities similar to the microplastics. Step 6 involves microplastic identification and counting: microscopic observation combined with infrared or Raman spectroscopy is used to characterize the microplastics morphologically, perform particle size statistics, and identify polymer types. Step 7 involves quality control: a blank control group and a spiked recovery group are set up, with multiple parallel samples for each batch. The method recovery rate and precision are calculated.

[0006] Preferably, the grinding of the soil sample in step 1 is carried out using a ball mill, with a grinding time of a preset time period and a grinding speed of a preset speed.

[0007] Preferably, in step 2, when removing organic matter, the hydrogen peroxide treatment can be repeated a predetermined number of times until the organic matter is basically removed and the sample color becomes light.

[0008] Preferably, the density separation device in step 3 is an automatic density separator, including a sample inlet, a separation chamber, a stirring system, a temperature control system, a liquid outlet, and an automatic control system; the separation chamber is made of a cylindrical transparent material, and the inner wall is coated with an anti-stick coating to prevent microplastic adsorption.

[0009] Preferably, in step 3, the density separation at each stage is assisted by magnetic stirring, the stirring speed is a preset rotation speed, the stirring time is a preset time period, and the settling time is a preset time period.

[0010] Preferably, in step 3, the suspension of each stage is collected by negative pressure filtration, and the negative pressure value is within a preset negative pressure range.

[0011] Preferably, in step 4, the filter membrane is a pre-burned ashless filter paper or glass fiber filter membrane, the burning temperature is a preset burning temperature, and the burning time is a preset burning time.

[0012] Preferably, after density gradient centrifugation in step 5, a permeation cleaning step is further included: soaking the microplastics in a surfactant solution of a preset concentration for a preset time period to remove trace impurities adsorbed on the surface.

[0013] Preferably, in step 6, the microscopic observation uses a combination of a stereomicroscope and an optical microscope. The stereomicroscope has a first preset magnification for preliminary screening, and the optical microscope has a second preset magnification for morphological observation and particle size measurement.

[0014] Preferably, in step 6, the infrared spectral analysis uses an attenuated total reflection Fourier transform infrared spectrometer with a preset wavenumber range and a preset resolution.

[0015] Preferably, the method further includes microplastic abundance calculation: calculating the microplastic abundance per unit mass of soil based on the sample amount filtered by the filter membrane and the number of microplastics.

[0016] Compared with existing technologies, this invention has the following advantages: A three-stage density separation system enables the stepwise separation of light, medium-density, and heavy microplastics, significantly improving the recovery rate; optimized reagent ratios and processing conditions drastically shorten the overall operation time; the multi-stage density separation liquid covers the density range of common microplastics, effectively extracting various types of microplastics such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, and polyvinyl chloride; the use of an automated density separator reduces manual operation steps, lowers human error, and improves the repeatability and reproducibility of the method; dual purification through hydrogen peroxide pretreatment and density gradient centrifugation effectively removes interference from organic matter and impurities of similar density; the separation liquid can be recycled and reused, and the generated waste liquid can be treated to meet discharge standards, conforming to the concept of green chemistry. Detailed Implementation

[0017] Example 1

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0019] This invention discloses a method for rapid extraction and analysis of microplastics in soil based on density separation, encompassing seven core steps to form a complete technological closed loop. Specifically, the method includes sample pretreatment, organic matter removal, density separation, microplastic enrichment and purification, density gradient centrifugation purification, microplastic identification and counting, and quality control. Each step is described in detail below.

[0020] In step 1, the sample pretreatment stage, the soil sample to be tested is naturally air-dried. The specific operation procedure is as follows: The collected soil sample is spread flat on a dry and clean glass dish or stainless steel tray, placed in a well-ventilated laboratory table or drying oven, and naturally air-dried at room temperature for 48 to 72 hours until the soil sample reaches a constant weight. After natural air drying, plant roots, fallen leaf debris, gravel particles, and visible foreign matter are removed from the soil sample by manual sorting. After sorting, the processed soil sample is transferred to the grinding chamber of a ball mill, and the ball mill is started for grinding. The speed of the ball mill is set to 500 to 800 rpm, and the grinding time is set to 5 to 10 minutes. During the grinding process, the stainless steel grinding balls inside the ball mill collide and rub against the soil sample at high frequency, crushing the soil sample to a predetermined particle size range. After grinding, the soil sample is sieved using a standard sieve, and soil samples with a particle size of less than 2 mm are collected as raw materials for subsequent processing. The upper limit of 2 mm for particle size is based on the standard specifications for soil microplastic analysis, ensuring that the sample particle size meets the processing requirements for density separation.

[0021] In step 2, the organic matter removal stage, a hydrogen peroxide solution with a mass concentration of 10% to 30% is added to the soil sample obtained in step 1. The specific procedure is as follows: Accurately weigh a certain mass of soil sample and add hydrogen peroxide solution according to a solid-liquid ratio of 1:5 to 1:10. The specific solid-liquid ratio is adjusted adaptively according to the organic matter content of the soil sample; a larger solid-liquid ratio is used for soil samples with higher organic matter content, while a lower solid-liquid ratio can be appropriately reduced for soil samples with lower organic matter content. Adjust the pH of the system to the range of 5 to 7 using dilute hydrochloric acid or sodium hydroxide solution. The purpose of adjusting the pH is to create a suitable reaction environment, ensuring the oxidation efficiency of hydrogen peroxide while avoiding excessively vigorous reactions that could cause corrosion of the microplastic surface. Place the pH-adjusted mixture in a constant temperature water bath and heat it at 60 to 80 degrees Celsius for 30 to 60 minutes. During the water bath heating process, hydrogen peroxide decomposes and releases nascent oxygen, which reacts with organic matter and humic acid in the soil, effectively removing organic matter. The effectiveness of organic matter removal can be initially judged by observing the change in sample color. When the sample color changes from dark brown or black to light gray or off-white, it indicates that the organic matter has been largely removed. If the desired effect is not achieved in one treatment, the hydrogen peroxide treatment can be repeated 2 to 3 times. After each treatment, centrifugation, washing, and drying of the sample are necessary until the sample color reaches a light color.

[0022] In step 3, the density separation stage, the soil sample after organic matter removal is transferred to a density separation device for multi-stage density separation. This device is an automatic density separator, comprising an inlet, separation chamber, stirring system, temperature control system, liquid outlet, and automatic control system. The separation chamber is designed as a cylindrical, transparent structure made of borosilicate glass or polycarbonate, facilitating real-time observation of the material's state within the chamber. The inner wall of the separation chamber is coated with an anti-stick coating made of polytetrafluoroethylene or diamond-like carbon film material, effectively preventing the adsorption and adhesion of microplastic particles. The automatic control system is electrically connected to each execution unit, enabling automated operation of the separation process. The separation process proceeds sequentially in a three-stage density separation sequence, with each stage employing magnetic stirring-assisted dispersion technology.

[0023] The first-stage density separation uses a saturated sodium chloride solution as the separation medium, with the solution density controlled within the range of 1.18 to 1.20 g / cm³. After removing organic matter, the soil sample is introduced into the separation chamber, and sufficient saturated sodium chloride solution is added. A magnetic stirring system is then activated for auxiliary dispersion. The magnetic stirring speed is set to 200 to 400 rpm, and the stirring time is 5 to 10 minutes. During stirring, the magnetic rotor in the separation chamber rotates at high speed, creating a uniform turbulent field in the solution, ensuring that the microplastic particles in the soil sample are fully dispersed and in full contact with the separation liquid. After stirring is complete, stirring is stopped, and the system is allowed to stand for 10 to 15 minutes to allow the components with significantly different densities to separate into layers. Under the influence of gravity, lightweight microplastic particles with a density less than 1.3 g / cm³ float to the vicinity of the liquid surface, forming a suspension layer, while particles with a density greater than 1.3 g / cm³ settle to the bottom of the separation chamber. The lightweight microplastics in the suspension layer include low-density polymers such as polyethylene and polypropylene. The suspension is collected by negative pressure filtration, with the vacuum degree controlled within the range of -0.05 to -0.1 MPa. Lightweight microplastics are separated from the separation liquid through a filter membrane.

[0024] The second-stage density separation involves adding a high-density separation solution to the residual sample after the first-stage separation. This high-density separation solution is a mixture of sodium bromide and calcium chloride, with a density controlled within the range of 1.35 to 1.40 g / cm³. The mass ratio of sodium bromide to calcium chloride must be precisely calculated and experimentally verified to ensure that the density of the mixed solution reaches the preset value. After adding the high-density separation solution, a magnetic stirring system is started for dispersion, with stirring parameters consistent with those of the first-stage separation. After settling and stratification, the suspension is collected using negative pressure filtration. This suspension mainly contains medium-density microplastics with a density of 1.3 to 1.6 g / cm³, such as polyethylene terephthalate and nylon, and other medium-density polymers.

[0025] The third-stage density separation is performed on the sample remaining after the second-stage separation. This stage uses a mixed solution of potassium iodide and zinc chloride as the separation medium, with the solution density controlled within the range of 1.55 to 1.70 g / cm³. This high-density separation solution allows denser, heavier microplastic particles, such as high-density polymers like polyvinyl chloride and polyester, to float to the surface. The separation operation maintains the same parameter settings as the first two stages, and the suspension obtained from the third-stage separation is collected by negative pressure filtration.

[0026] In step 4, the microplastic enrichment and purification stage, the suspensions obtained from the three-stage density separation in step 3 are processed separately. First, the suspensions are filtered through a 0.45 μm pore size filter membrane. The filter membrane is a pre-calcined ashless filter paper or glass fiber membrane, with the calcination temperature set to 450-500 degrees Celsius and the calcination time set to 4-6 hours. The purpose of the calcination treatment is to remove organic impurities from the filter membrane, ensuring that the filter membrane itself does not introduce additional contaminants. After filtration, microplastic particles are retained on the filter membrane surface. The filter membrane is transferred to a clean glass petri dish and washed with deionized water 3-5 times, using sufficient deionized water each time to ensure thorough removal of any residual separation liquid from the filter membrane. After washing, the filter membrane is placed in a desiccator to air dry naturally or dried using a low-temperature drying method.

[0027] In step 5, the density gradient centrifugation purification stage, the microplastics collected in step 4 are transferred from the filter membrane to density gradient centrifuge tubes. The specific transfer procedure is as follows: Place the filter membrane in an appropriate amount of deionized water, and use ultrasonic-assisted dispersion or gentle agitation to resuspend the microplastic particles in the deionized water to form a suspension. Transfer the suspension to density gradient centrifuge tubes, ready for density gradient centrifugation. The size of the density gradient centrifuge tubes is selected according to the sample volume; commonly used sizes are 15 mL or 50 mL. Add sucrose solutions with mass concentrations of 5%, 10%, and 20% sequentially to the bottom of the centrifuge tube, forming a density gradient with decreasing concentration from bottom to top. The sucrose solutions must be prepared using analytical grade reagents and deionized water as the solvent to ensure solution purity. The density of the sucrose solution increases with increasing concentration; the density of a 5% sucrose solution is approximately 1.02 g / cm³, a 10% sucrose solution is approximately 1.04 g / cm³, and a 20% sucrose solution is approximately 1.08 g / cm³. The three layers of sucrose solution form a continuous density gradient, enabling effective separation based on the density difference between microplastics and impurity particles.

[0028] Place the prepared density gradient centrifuge tubes in a centrifuge and centrifuge at 4000 to 6000 rpm for 10 to 20 minutes. During centrifugation, under the influence of centrifugal force, microplastic particles migrate to positions with the same density along the sucrose gradient, while impurity particles of similar density are separated to different positions. After centrifugation, carefully remove the centrifuge tubes, avoiding disturbance of the gradient interface. Collect the solution at the target density level, which contains the purified microplastic particles.

[0029] After density gradient centrifugation, a permeation washing step is included to further remove trace impurities from the microplastic surface. The collected microplastics are immersed in a 0.1% to 0.5% sodium dodecyl sulfate solution for 5 to 10 minutes. Sodium dodecyl sulfate, as an anionic surfactant, reduces the interfacial tension on the microplastic surface, thus removing trace impurity particles adsorbed thereon. After immersion, the microplastics are repeatedly washed with deionized water to ensure complete removal of the surfactant.

[0030] In step 6, the microplastic identification and counting stage, the microplastics purified in step 5 are subjected to systematic morphological characterization, particle size statistics, and polymer type identification. Microscopic observation employs a combination of stereomicroscope and optical microscope. The stereomicroscope is set at a magnification of 10 to 45x for preliminary screening and morphological observation of microplastics on the filter membrane. This magnification range allows operators to quickly identify suspected microplastic particles and exclude obvious non-plastic impurities. The stereomicroscope is equipped with a high-resolution digital camera for image acquisition and archiving of target particles.

[0031] The optical microscope was set to a magnification of 100 to 400x for high-magnification observation and precise measurement of suspected microplastics screened by the stereomicroscope. High-magnification observation of the optical microscope reveals the surface morphology characteristics of microplastics, including surface smoothness, cracks, wear marks, and particle shape. Simultaneously, image analysis software accompanying the optical microscope was used to measure the particle size of the microplastics, and the measurement results were automatically recorded and statistical data generated. The particle size distribution of microplastics is an important parameter characterizing their pollution features and is of great significance for assessing ecological risk.

[0032] Infrared spectroscopy analysis was performed using an attenuated total reflectance Fourier transform infrared spectrometer (ATR). The operating principle of this ATR spectrometer is based on the total internal reflection of infrared light on the surface of an ATR crystal. When the sample is in close contact with the ATR crystal surface, the sample molecules absorb infrared light at specific wavenumbers, producing characteristic absorption peaks. The wavenumber range for the infrared spectroscopy analysis was set to 4000 to 650 cm⁻¹, and the resolution was set to 4 cm⁻¹. These parameters cover the characteristic absorption peak regions of common polymer materials. By comparing the infrared spectrum of the sample with the reference spectrum of a standard polymer, accurate identification of the microplastic polymer type can be achieved. Common soil microplastic types include polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyvinyl chloride, and polyamide, each polymer possessing characteristic infrared absorption peaks. For example, polyethylene shows characteristic absorption peaks of methylene groups near 2920 cm⁻¹ and 2850 cm⁻¹; polyethylene terephthalate shows characteristic absorption peaks of carbonyl groups near 1710 cm⁻¹; polyvinyl chloride shows characteristic absorption peaks of carbon-hydrogen bonds near 2900 cm⁻¹, and also shows characteristic absorption peaks of carbon-chlorine bonds in the 600 to 800 cm⁻¹ region.

[0033] For some microplastic samples that are difficult to identify definitively using infrared spectroscopy, Raman spectroscopy can be used for supplementary analysis. Raman spectroscopy obtains molecular vibrational information by analyzing the frequency changes of scattered light from the sample, complementing infrared spectroscopy and enabling effective identification of certain polymers with weak infrared absorption.

[0034] This invention also includes a microplastic abundance calculation step. Based on the sample volume filtered by the filter membrane and the number of microplastics counted, the microplastic abundance per unit mass of soil is calculated according to a preset formula. Let the mass of the soil sample be M (in grams); and the total number of microplastic particles counted on the filter membrane be N. Then, the formula for calculating the microplastic abundance A is: A = NM × 1000 A = MN × 1000

[0035] Where A is measured in units of particles per kg of dry soil. This abundance value reflects the degree of microplastic pollution in soil samples and can be used for comparative analysis between different soil samples as well as for studying the spatiotemporal variation trends of the same soil sample.

[0036] In step 7, the quality control stage, a comprehensive quality control system is established to ensure the accuracy and reliability of the method of this invention. Blank control groups are set up at each stage of sample processing, including reagent blanks, filter membrane blanks, and equipment blanks. The reagent blank is used to detect whether the reagents themselves contain microplastic contamination; the filter membrane blank is used to detect whether the filter membrane meets experimental requirements after ignition treatment; and the equipment blank is used to detect whether glassware, centrifuge tubes, and other equipment used in the experiment introduce exogenous microplastic contamination.

[0037] The spiked recovery group is used to evaluate the recovery rate and accuracy of the method. The specific procedure is as follows: Take a blank soil sample of known mass, add a known quantity and type of microplastic standard to it, and process and analyze it according to the entire process of the method of this invention. The recovery rate of the method is calculated by comparing the actual number of microplastics detected in the spiked recovery group with the number of added microplastics. The recovery rate of the method of this invention can reach 85% to 95%, meeting the technical requirements for environmental sample analysis.

[0038] Three parallel samples were prepared for each batch of samples, and the processing procedures for the parallel samples were exactly the same. Statistical analysis of the parallel samples was performed to calculate the standard deviation and relative standard deviation of the method, which were used to characterize the precision of the method. The relative standard deviation of the method in this invention was controlled within 10%, indicating that the method has excellent repeatability and reproducibility.

[0039] The following is a detailed explanation of the operation process of the present invention using a specific application example.

[0040] A 500g soil sample was collected from the surface layer of an agricultural soil source and air-dried for 72 hours before sorting and ball milling. The ball mill was set to 600 rpm for 8 minutes, and the resulting soil sample had a particle size of less than 2mm after sieving. 50g of the soil sample was accurately weighed and placed in a 1000mL Erlenmeyer flask. 400mL of a 20% hydrogen peroxide solution was added at a solid-liquid ratio of 1:8, and the pH was adjusted to 6 using dilute hydrochloric acid. The Erlenmeyer flask was then heated in a 70°C water bath for 45 minutes to remove organic matter. The sample color changed from dark brown to light gray, indicating that the organic matter was largely removed. If the color did not meet the expected standard, the hydrogen peroxide treatment was repeated once.

[0041] The processed sample was transferred to the separation chamber of the automatic density separator, and saturated sodium chloride solution was added to the preset level. The magnetic stirring system was started, and the speed was set to 300 rpm. After stirring for 8 minutes, the mixture was allowed to stand for 12 minutes. The negative pressure filtration system was started, and the vacuum degree was set to -0.08 MPa. The first-stage suspension was collected. After the first-stage separation was completed, a mixed solution of sodium bromide and calcium chloride was added to the residual sample, and the density was adjusted to 1.38 g / cm³. The second-stage separation was performed using the same stirring and standing parameters, and the second-stage suspension was collected. Finally, a mixed solution of potassium iodide and zinc chloride was added, and the density was adjusted to 1.62 g / cm³. The third-stage separation was performed, and the third-stage suspension was collected.

[0042] The suspensions obtained from the three-stage separation were filtered through pre-ignited ashless filter paper with a pore size of 0.45 μm. The filter membranes were washed four times with deionized water to ensure thorough removal of any residual separation solution. Microplastics on the filter membranes were transferred to density gradient centrifuge tubes, and 5%, 10%, and 20% sucrose solutions were added sequentially to create a density gradient. The tubes were centrifuged at 5000 rpm for 15 minutes. The solutions at the target density gradient were collected, soaked in 0.3% sodium dodecyl sulfate solution for 8 minutes, and washed three times with deionized water.

[0043] The purified microplastics were prepared into slides and initially screened under a stereomicroscope at 20x magnification. Suspected microplastics were then observed under an optical microscope at 200x magnification, and particle size was measured using image analysis software. Subsequently, infrared spectroscopy analysis was performed on an ATR-FTIR spectrometer with a wavenumber range of 4000 to 650 cm⁻¹ and a resolution of 4 cm⁻¹, and polymer types were identified based on characteristic absorption peaks.

[0044] For quality control, two blank controls and three spiked recovery samples were set up for each batch. Known quantities and types of polyethylene, polystyrene, and polyvinyl chloride standard microplastics were added to the spiked recovery samples, and the recovery rates were calculated after processing according to the same procedure. The actual measured recoveries were 92% for polyethylene, 89% for polystyrene, and 86% for polyvinyl chloride, meeting the technical requirements of the analytical method.

[0045] Based on the count of 50 microplastics on the filter membrane, a 50g soil sample was weighed, and the microplastic abundance of the agricultural soil was calculated to be 1000 microplastics / kg dry soil. Infrared spectroscopy analysis showed that the microplastics in the soil sample were mainly polyethylene and polystyrene, accounting for 45% and 30% respectively, with polypropylene accounting for 15% and polyethylene terephthalate accounting for 10%.

[0046] Example 2

[0047] This embodiment provides an alternative technology for the rapid extraction and analysis of microplastics in soil based on density separation, which is suitable for sandy soil samples with low organic matter content.

[0048] The organic matter content of sandy soil samples is usually less than 2%, and the sample color is relatively light, so the soil matrix has relatively little interference with microplastic extraction. Therefore, the processing procedure can be appropriately simplified in the organic matter removal stage. The specific operation is as follows: Take 200g of sandy soil sample, air dry it naturally for 48 hours, and then sort and ball mill it. Set the ball mill speed to 500rpm and the grinding time to 5 minutes. After sieving, obtain soil samples with a particle size of less than 2mm.

[0049] Because sandy soil samples have low organic matter content, hydrogen peroxide treatment can be performed at lower concentrations and for shorter durations to improve efficiency. Accurately weigh 30g of soil sample and place it in a 500mL Erlenmeyer flask. Add 150mL of 10% hydrogen peroxide solution at a solid-liquid ratio of 1:5, and adjust the pH of the system to 5.5 using dilute hydrochloric acid. Heat the Erlenmeyer flask in a 65°C water bath for 30 minutes. Organic matter in sandy soil samples can be effectively removed with lower concentrations of hydrogen peroxide, eliminating the need for repeated treatment.

[0050] The density separation stage maintained the same parameter settings as in Example 1, employing a three-stage density separation system to sequentially separate light, medium, and heavy microplastics. The magnetic stirring speed of the automatic density separator was set to 250 rpm, the stirring time to 6 minutes, and the settling time to 12 minutes. The vacuum degree for negative pressure filtration was set to -0.06 MPa. The sandy soil sample had a coarser particle size, resulting in a relatively faster settling velocity during density separation, which helped to shorten the separation time.

[0051] The microplastic enrichment and purification stage employed a glass fiber membrane for filtration. The membrane was pre-calcined at 480°C for 5 hours to remove organic impurities. The membrane pore size was also 0.45 μm. After filtration, the membrane was washed three times with deionized water.

[0052] The density gradient centrifugation purification stage was set to a centrifugation speed of 4500 rpm and a centrifugation time of 12 minutes. These parameters are suitable for purifying microplastics in sandy soil samples. The permeation washing step used a 0.2% sodium dodecyl sulfate solution for 6 minutes.

[0053] The microplastic identification and counting stage employed a stereomicroscope for batch screening at a magnification of 15x. The stereomicroscope is equipped with an automated scanning platform that allows for systematic scanning of the entire filter membrane, reducing human error. An optical microscope, set to 150x magnification, was used to verify suspected microplastics and measure their particle size.

[0054] Infrared spectroscopy analysis employs transmission mode for sample preparation, where microplastic particles are pressed into thin slices using a diamond cell before detection. This sample preparation method yields higher spectral signal intensity and is suitable for the identification and analysis of microplastic particles with smaller particle sizes.

[0055] For quality control, three parallel samples and two spiked recovery samples were set up for each batch. Polyethylene and polyethylene terephthalate standard microplastics were added to the spiked recovery samples, with recoveries of 94% and 91%, respectively. The matrix interference in sandy soil samples was relatively small, and the method recovery rate was slightly higher than that of Example 1.

[0056] This alternative technology reduces the overall operation cycle to 3 to 4 hours by optimizing the reagent dosage and processing time in the organic matter removal stage, while ensuring the accuracy and reliability of the method.

[0057] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid extraction and analysis of microplastics in soil based on density separation, characterized in that, Includes the following steps: Step 1: Sample pretreatment. The soil sample to be tested is air-dried to remove plant roots, gravel, and other impurities. It is then ground and sieved to obtain a soil sample within a predetermined particle size range. Step 2: Organic matter removal. A predetermined concentration of hydrogen peroxide solution is added to the soil sample. The pH of the system is adjusted to a predetermined range. The sample is then heated in a water bath at a predetermined temperature for a predetermined time to remove organic matter. The soil sample and hydrogen peroxide solution are prepared according to a predetermined solid-liquid ratio. Step 3: Density separation. The organic matter-removed sample is transferred to a density separation device. A multi-stage density separation solution is added, and three stages of density separation are performed sequentially. The first stage uses a saturated sodium chloride solution to separate lightweight microplastics with a density less than the first predetermined density. The second stage uses a mixed solution of sodium bromide and calcium chloride to separate medium-density microplastics with a density between the first and second predetermined densities. The third stage uses a mixed solution of potassium iodide and zinc chloride. Step 4 involves the separation of heavy microplastics with a density ranging from a third to a fourth preset density. Step 5 involves microplastic enrichment and purification, where the suspensions obtained from each separation stage are filtered through membranes with preset pore sizes. The microplastics on the membranes are collected, and the residue is removed by washing with deionized water a predetermined number of times. Step 6 involves density gradient centrifugation purification, where the collected microplastics are transferred to density gradient centrifuge tubes, and sucrose solutions of preset concentrations are added sequentially to form a gradient. The tubes are centrifuged at preset speeds for a predetermined time to remove impurity particles with densities similar to the microplastics. Step 7 involves microplastic identification and counting, using microscopic observation combined with infrared or Raman spectroscopy to characterize the microplastics, perform particle size statistics, and identify polymer types. Step 8 involves quality control, setting up a blank control group and a spiked recovery group. Multiple parallel samples are set up for each batch of samples, and the method recovery rate and precision are calculated.

2. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 1, the soil sample is ground using a ball mill, with a preset grinding time and a preset rotation speed.

3. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 2, when removing organic matter, the hydrogen peroxide treatment can be repeated a predetermined number of times until the organic matter is basically removed and the sample color becomes light.

4. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 3, the density separation device is an automatic density separator, which includes an inlet, a separation chamber, a stirring system, a temperature control system, a liquid outlet, and an automatic control system. The separation chamber is made of a cylindrical transparent material, and the inner wall is coated with an anti-stick coating to prevent microplastic adsorption.

5. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 3, all density separations at each level are achieved using magnetic stirring to assist dispersion. The stirring speed is a preset rotation speed, the stirring time is a preset time period, and the settling time is a preset time period.

6. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 3, the suspension of each stage is collected by negative pressure filtration, and the negative pressure value is within a preset negative pressure range.

7. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 4, the filter membrane is a pre-burned ashless filter paper or glass fiber filter membrane, the burning temperature is a preset burning temperature, and the burning time is a preset burning time.

8. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, After density gradient centrifugation in step 5, a permeation cleaning step is also included, in which the microplastics are soaked in a surfactant solution of a preset concentration for a preset time period to remove trace impurities adsorbed on the surface.

9. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 6, the microscopic observation uses a combination of stereomicroscope and optical microscope. The stereomicroscope is set to a first preset magnification for preliminary screening, and the optical microscope is set to a second preset magnification for morphological observation and particle size measurement.

10. The method for rapid extraction and analysis of microplastics in soil based on density separation according to claim 1, characterized in that, In step 6, the infrared spectral analysis uses an attenuated total reflection Fourier transform infrared spectrometer with a preset wavenumber range and a preset resolution.