Rapid separation and extraction method applied to microplastics in plant tissues

Abstract

The invention belongs to the technical field of environmental pollutant analysis and detection, and relates to a rapid separation and extraction method applied to microplastics in plant tissues. The rapid separation and extraction method comprises the following main steps: (1) carrying out pretreatment of removing surface attachments on a collected plant tissue sample; (2) preparing a Fenton digestion solution; (3) carrying out Fenton oxidation digestion and reducing agent reduction on the plant tissue sample; (4) observing and imaging the extracted micro-plastic through a stereoscope; and (5) carrying out microscopic Raman spectrum detection, and establishing a set of standard extraction method for the microplastics in the plant tissues. The method can be used for effectively extracting the microplastics in the plant, is short-term, efficient, economical and practical, is environment-friendly, does not damage the microplastics, and does not influence the subsequent spectrum detection.

Description

Technical Field

[0001] The invention relates to a method for rapid separation and extraction of microplastics in plant tissues, belonging to the technical field of environmental pollutant analysis and detection. Background Art

[0002] Microplastics are plastic particles with a diameter of less than 5mm and are a major carrier of pollution. The problem of soil microplastic pollution is currently a hot topic of research. Existing studies have shown that microplastics are ubiquitous in the soil environment and pose a growing threat to soil ecology. Soil is the foundation for plant growth, and people are paying more and more attention to the harm of microplastic pollution to human health.

[0003] Generally speaking, microplastics refer to plastic particles with a particle size of less than 5 mm, mainly in different forms such as particles, fibers, films, fragments, etc., including primary microplastics and secondary microplastics. Primary microplastics refer to small-sized plastic products produced by industry, such as plastic microbeads contained in cosmetics, as well as plastic particles and resin particles used as industrial raw materials. Secondary microplastics refer to microplastics with smaller particle sizes formed by the physical, chemical and biological weathering processes that cause large plastic waste to split and reduce in volume. Among them, microplastics entering the soil environment will change the physical and chemical properties of the soil (such as soil bulk density, permeability, water holding capacity, and water-stable aggregation, etc.) and biological functions through complex physical and chemical reactions, thereby seriously affecting the growth of plants. Studies have found that microplastics can be absorbed by plants, and can also enter the food chain through the ingestion of soil animals and microorganisms and further transfer and migrate, thereby causing immeasurable impacts on a wider range of life forms. In this process, microplastics are mainly transmitted through the food chain, and plants will ingest microplastic particles in the soil environment through pore adsorption, endocytosis and transpiration. The aggregation, content and distribution of these microplastics are currently hot topics. For the detection of microplastics in plant tissue samples, the interference of organic matter is usually removed by digestion, the microplastic particles in the organic matter are released, and then the microplastic particles are separated from the sample matrix by flotation, filtration, density separation and other methods. Finally, the microplastics are analyzed and detected with the help of special instruments to determine the type and abundance of microplastics. However, the xylem structure, high cellulose content and protein of plant tissues have great limitations on the separation and extraction of microplastics. At present, there is no unified and mature method for the separation, extraction and purification of microplastics in plant tissues, and different methods may lead to different results. This limits the analysis of microplastics to a certain extent. Therefore, it is necessary to study and develop a rapid separation and extraction method for microplastics in plant tissues. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for rapid separation and extraction of microplastics in plant tissues that can quickly separate, enrich and purify microplastics in plants, is economical, practical, environmentally friendly, non-destructive to microplastics, and is highly efficient.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for rapid separation and extraction of microplastics in plant tissues, characterized in that it mainly comprises the following steps:

[0007] Step 1: Pre-treat the collected plant tissue samples to remove surface attachments

[0008] (1) Collect the plant tissue sample to be tested, wash the sample three times with ultrapure water, ensure that it is clean, add anhydrous ethanol to the treated plant tissue sample to cover the sample, place it on a heating furnace, heat it at 59.5°C-60.5°C for 29.5-30.5 minutes, then filter it together with the filtrate onto a filter membrane, use anhydrous ethanol to rinse the sample on the filter membrane into a beaker, repeat three times, then wrap the sample with aluminum foil, place it in an oven, and dry it naturally at 59.5°C-60.5°C for 1.9-2.1 hours to obtain a clean plant tissue sample with surface attachments removed;

[0009] (2) According to the different parts of the plant tissue, the dried plant tissue sample was chopped into particles ≤ 2 mm, then 10 mg of the sample was weighed and placed in a digestion bottle to wait for digestion;

[0010] Step 2: Prepare Fenton digestion solution

[0011] (1) preparing a 2.78% FeSO4 solution by using dilute hydrochloric acid to adjust the pH of 50 mL ultrapure water to 3.01-3.14, then weighing 1.39 g of solid ferrous sulfate using a 1 / 10,000 balance, adding the solution to the solution and stirring to obtain 50 mL of a 2.78% FeSO4 solution;

[0012] (2) Mixing and preparing Fenton digestion solution: add 150 mL of 30% H2O2 solution by mass into a digestion bottle, then use a pipette to add 500 μL of 2.78% FeSO4 solution, the volume ratio of the two being 1:300, and then immediately shake the digestion bottle to evenly mix the FeSO4 and H2O2 to obtain a Fenton digestion solution;

[0013] Step 3: Fenton oxidation digestion and reducing agent reduction of plant tissue samples

[0014] (1) Place the digestion bottle in a shaker and digest for 2 to 2.5 hours at 60°C, 115 rpm and 72W full-spectrum light. After confirming that the plant tissue is completely digested, place the digestion bottle in an ice-water mixture to terminate the digestion reaction;

[0015] (2) Add 15 g of L-ascorbic acid solid to the digestion bottle after the reaction is terminated, and then place it in a shaker with the shaker temperature set to 60°C and the speed set to 115 rpm. Place a thermometer in the digestion bottle to control the reduction reaction temperature at all times. When the temperature is higher than 60°C, place the digestion bottle in an ice-water mixture for 1 to 2 seconds, and then continue to shake for 30 to 40 minutes. After confirming that the digestion solution becomes clear, filter the digestion solution onto a filter membrane with a pore size of 100 nm, rinse the filter repeatedly with ultrapure water for more than 3 times, and filter the rinse solution onto the filter membrane. At this point, the microplastics in the plant tissue sample can be separated and extracted onto the filter membrane;

[0016] Step 4: Use a stereoscope to observe the dried filter membrane under a microscope to obtain the shape, size, surface texture characteristics and position of the microplastics on the filter membrane. Then move the filter membrane under a microscope Raman spectrometer and obtain the Raman spectrum of the corresponding microplastics according to the position determined under the stereoscope. Compare the obtained Raman spectrum with the standard microplastic library for matching degree. Those with a matching degree higher than 80% are judged to be microplastics.

[0017] Preferably, the plant tissue in step 1 includes one or more of flowers, crops, and aquatic plants; the plant tissue samples include one or more of leaves, fruits, roots, and stems of plant tissues.

[0018] Preferably, the concentration of anhydrous ethanol in step 1 is 75%; and the pore size of the filter membrane is 20 μm.

[0019] Preferably, in step 3 (1), the lighting condition is added by installing four 250 cm long full-spectrum light strips with an operating power of 72 W in the cover of the shaker box.

[0020] Preferably, the mass ratio of ice to water in the ice-water mixture in step three (2) is 250 g:300 g to 350 g:300 g.

[0021] In the process of developing the experimental method for efficient separation and extraction of microplastics in soil environment of the present invention, the applicant mainly did the following work:

[0022] 1. Determination of Fenton reagent ratio, addition conditions and subsequent reduction conditions: According to the ratio principle of Fenton reagent, we determined that the optimal reaction ratio of Fenton reagent is 1:300, because the color of the digestion solution is the lightest at 1:300 and remains light in 2 to 3 hours, indicating that the iron ion content in the solution is not much at this time, and the Fenton reaction is in the Fe 2+When the concentration is higher than H2O2, the reaction proceeds continuously and stably, hydroxide is stably produced within 2 to 3 hours, and the overall reaction is in the early and middle stages within 2 to 3 hours and will not enter the late stage. This not only ensures the stable generation of hydroxide, but also ensures that the plant tissue is completely digested. At this ratio, the temperature of the Fenton reagent is controllable; further, we found that in the Fenton reaction, increasing the temperature, oscillating and providing light can significantly increase the reaction rate. In order to ensure that microplastics are not affected by the digestion solution, we finally set the digestion bottle temperature to 60°C, the shaker speed to 115rpm, and the full spectrum light to 100%. Under this condition, based on the previous Fenton reagent ratio, we not only ensure that the digestion will not affect the shape, size, surface texture characteristics and Raman spectroscopy detection recognition rate of microplastics, but also can achieve rapid digestion. 10 mg of plant tissue samples can be completely digested within 85-100 minutes, which is faster and more efficient than any previous method for extracting microplastics in plants, and has no effect on the microplastics themselves; since Fenton reagent will produce more iron compound precipitation after digestion, affecting the subsequent separation and identification of microplastics, the method of adding a reducing agent is adopted to remove the iron compounds in the digestion solution. In order to ensure that the subsequent waste liquid has little pollution to the environment, the method of adding 15g L-ascorbic acid is adopted. The conditions of the reduction reaction and the Fenton oxidation reaction are similar. The temperature of the digestion bottle is controlled at 60°C and the shaking speed is 115rpm. The temperature of the digestion bottle is controlled with an ice-water mixture to achieve complete clarification of the digestion solution in 30 minutes. At this point, the separation and extraction of microplastics in plant tissues can be completed within 2 hours. At the same time, based on the ratio of our Fenton reagent and the subsequent adding ratio of the reducing agent, the hydrogen peroxide and ferrous sulfate we use are much less than the industrial Fenton reagent, and in the subsequent treatment, the reducing agent L-ascorbic acid added by the present invention is also a low-cost, environmentally friendly reducing agent, and the added amount is also small, so the digestion method is economical, fast, efficient and environmentally friendly.

[0023] 2. Verification of the effect of Fenton reagent on microplastic quality, surface morphology, fluorescence intensity and spectral recognition rate: In order to verify whether Fenton reagent has an effect on the surface morphology, weight and spectral data of microplastics, six microplastic standards are needed, high-density polyethylene (HDPE), low-density polyethylene (LDPE), polypropylene (PP) and polyethylene terephthalate (PET), as well as biodegradable plastics polylactic acid (PLA) and polybutylene adipate-butylene terephthalate (PBAT), among which non-degradable polymers correspond to the four microplastics with the highest content in farmland soil, and fully biodegradable polymers are currently two raw materials for fully biodegradable mulch widely used in agricultural planting. These standard microplastic granulation products were purchased from Xifu Zhongxin Packaging Products Co., Ltd. in Shihezi Development Zone, and the particle size was between 2-3mm. Before the test verification, a micro-Raman spectrometer was used for analysis, and the identification matching rate of non-degradable polymers exceeded 95%. Microplastic standards were granular microplastics with a particle size of 0.4-0.5 mm purchased from Guangzhou Zhongxin Plastic Co., Ltd., China. Information on various types of microplastics is shown in Table 1.

[0024] Table 1 Type, shape, size, source and density (g cm) of microplastic standards used in this method -3 )

[0025]

[0026]

[0027] The advantages of the present invention are:

[0028] The present invention provides an efficient separation and extraction experimental method for microplastics in plants that can quickly separate, enrich and purify them, is economical and practical, is environmentally friendly, and is non-destructive to microplastics. The method can completely decompose the organic matter of 10 mg of plant tissue within 85 minutes without damaging the structure of microplastics. The method can be applied to the rapid separation and extraction of microplastics in various plant tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is an operation flow chart of the efficient separation and extraction experimental method in Example 1 of the present invention.

[0030] Figure 2 It is a schematic diagram of the technical operation process of the efficient separation and extraction experimental method in Example 1 of the present invention.

[0031] Figure 3 This is a display of the extraction of PE environmental samples from cotton stalks in Example 1 of the present invention. As shown in the figure, the extracted PE environmental samples can be clearly seen.

[0032] Figure 4This is the identification demonstration of the PE environmental sample of the cotton stalk in Example 1 of the present invention. The Raman spectrum matching degree of the PE environmental sample shown in the figure is 83%. DETAILED DESCRIPTION

[0033] The present invention is described in detail below with reference to specific embodiments, but this is not intended to limit the technical content of the present invention.

[0034] The main instruments and reagents used in the examples are as follows:

[0035] (1) Reagents:

[0036] Ferrous sulfate (FeSO4·5H2O): purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.;

[0037] Hydrogen peroxide (H2O2): 30% by mass, analytical grade, purchased from Chengdu Kelong Chemicals Co., Ltd.;

[0038] L-Ascorbic acid (C6H8O6): purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0039] (2) Experimental instruments:

[0040] GZX-9140MBE drying oven, manufacturer: Shanghai Boxun Company, China;

[0041] JL-UPR-III-10T Jinlan series ultrapure water purifier, manufacturer: Sichuan Jinlan Technology Co., Ltd., China

[0042] SHA-BA constant temperature water bath oscillator, manufacturer: Changzhou Guowang Instrument Manufacturing Co., Ltd., China;

[0043] GM-1.0A vacuum filtration device, manufacturer: Tianjin Jinteng Company, China;

[0044] MeiLen MTB 200 electronic balance, manufacturer: Shenzhen MeiMo Electronics Co., Ltd., China;

[0045] V 2.0 Zeiss stereo microscope, manufacturer: Zeiss, Germany;

[0046] inVia confocal Raman spectrometer, manufacturer: Renishaw, UK.

[0047] Microplastic identification and analysis method: Use a stereo microscope to observe the purified microplastic samples prepared in the example, and record the shape and color characteristics; use a micro-Raman spectrometer to perform spectral identification on suspected microplastic particles.

[0048] Embodiment 1:

[0049] An efficient separation and extraction experimental method for microplastics in soil environment, step 1: pretreatment of the collected cotton tissue samples to remove surface attachments

[0050] (1) Collect the cotton tissue sample to be tested, wash the sample with ultrapure water three times, ensure that it is clean, add anhydrous ethanol to the treated plant tissue sample to cover the sample, place it on a heating furnace, heat it at 60°C for 30 minutes, and then filter it together with the filtrate onto a filter membrane, use anhydrous ethanol to rinse the sample on the filter membrane into a beaker, repeat three times, and then wrap the sample with aluminum foil, place it in an oven, and dry it naturally at 60°C for 2 hours to obtain a clean plant tissue sample with surface attachments removed;

[0051] The concentration of anhydrous ethanol is 75%;

[0052] The pore size of the filter membrane is 20 μm;

[0053] (2) Chop the dried cotton tissue sample into pieces with a particle size of ≤2 mm, then weigh 10 mg of the sample, place it in a digestion bottle, and wait for digestion;

[0054] Step 2: Prepare Fenton digestion solution

[0055] (1) preparing a 2.78% FeSO4 solution by using dilute hydrochloric acid to adjust the pH of 50 mL of ultrapure water to 3.01 to 3.14, then weighing 1.39 g of solid ferrous sulfate (FeSO4·5H2O) using a 1 / 10,000 balance, adding the solution to the solution and stirring to obtain 50 mL of a 2.78% FeSO4 solution;

[0056] (2) Mixing and preparing Fenton digestion solution: Add 150 mL of 30% H2O2 solution to the digestion bottle, and then use a pipette to add 500 μL of 2.78% FeSO4 solution, the volume ratio of the two is 1:300, and then immediately shake the digestion bottle to evenly mix the FeSO4 and H2O2 to obtain the Fenton digestion solution;

[0057] Step 3: Fenton oxidation digestion and reducing agent reduction of plant tissue samples

[0058] (1) Place the digestion bottle in a shaker and digest for 85-100 minutes at 60°C, 115 rpm and 72W full-spectrum light. After confirming that the cotton tissue is completely digested, place the digestion bottle in an ice-water mixture to terminate the digestion reaction;

[0059] (2) Add 15 g of L-ascorbic acid solid to the digestion bottle after the reaction is terminated, and then place it in a shaker and shake at 60°C and 115 rpm. Place a thermometer in the digestion bottle to control the reduction reaction temperature at all times. When the temperature is higher than 60°C, place the digestion bottle in an ice-water mixture for 1 to 2 seconds, and then continue to shake for 30 minutes. After confirming that the digestion solution becomes clear, filter the digestion solution onto a filter membrane with a pore size of 100 nm, rinse the filter repeatedly with ultrapure water for more than 3 times, and filter the rinse solution onto the filter membrane. At this point, the microplastics in the cotton tissue sample can be separated and extracted onto the filter membrane;

[0060] Step 4: Use a stereoscope to observe the dried filter membrane under a microscope to obtain the shape, size, surface texture characteristics and location of the microplastics on the filter membrane, such as Figure 3 As shown in the figure, the circle is the microplastic we extracted from the plants; then the filter membrane is moved under the micro-Raman spectrometer, and the Raman spectrum of the corresponding microplastic is obtained according to the position determined under the stereoscope. The matching software WiRE is used to compare the obtained Raman spectrum with the standard microplastic library. The microplastics with a matching degree higher than 80% are judged as microplastics. Figure 4 We got it Figure 3 After comparing the Raman spectrum of the sample in the circle with the standard spectrum library, the recognition rate of PE is 83%, indicating that we have successfully extracted the microplastics in the plant tissue.

[0061] Embodiment 2:

[0062] The difference from Example 1 is that the effect of the Fenton reagent provided by the present invention on microplastics is further verified.

[0063] Microplastic particles (standards of HDPE, LDPE, PET, PP, PBAT, and PLA) were prepared and added to a pre-cleaned glass conical flask. To investigate the effect of Fenton's reagent on microplastics, 150 mL of 30% hydrogen peroxide solution and 500 μL of 0.1 mol / L ferrous sulfate solution were added under two experimental conditions: (1) ambient / room temperature (temperature maintained between 20°C and 25°C); (2) at 40°C with a rotation speed of 115 rpm; and (3) at 60°C with a rotation speed of 115 rpm. Each experimental condition was repeated three times, and three particles of each polymer type were tested in each replicate, for a total of 108 particles. After exposure, the particles were thoroughly rinsed with purified water. Subsequently, the particles were air-dried in a petri dish for 48 h and subjected to physical and chemical characterization, i.e., particle mass, size, surface morphology, and infrared spectroscopy. Each particle was photographed using a macroscopic zoom stereo microscope at a magnification of 8.6 times to visually observe changes in particle surface morphology. The mass of each particle before and after treatment was recorded using a balance with a measurement accuracy of 0.1 mg. Finally, a micro-Raman spectrometer was used to analyze the spectral changes of the particles before and after the addition of Fenton's reagent.

[0064] The above image data, weight data, area data and spectral data were subjected to significant difference analysis. The analysis results showed that the size, shape, surface texture characteristics, weight and spectral recognition rate of these particles did not change significantly before and after digestion, indicating that this digestion method is a non-destructive extraction method for microplastics in plants.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapid separation and extraction of microplastics in plant tissues, characterized in that: The main steps are as follows: Step 1: Pre-treat the collected plant tissue samples to remove surface attachments: (1) Collect the plant tissue sample to be tested, wash the sample three times with ultrapure water, ensure that it is clean, add anhydrous ethanol to the treated plant tissue sample to cover the sample, place it on a heating furnace, heat it at 59.5°C-60.5°C for 29.5-30.5 minutes, then filter it together with the filtrate onto a filter membrane, use anhydrous ethanol to rinse the sample on the filter membrane into a beaker, repeat three times, then wrap the sample with aluminum foil, place it in an oven, and dry it naturally at 59.5°C-60.5°C for 1.9-2.1 hours to obtain a clean plant tissue sample with surface attachments removed; (2) According to the different parts of the plant tissue, the dried plant tissue sample was chopped into particles ≤ 2 mm, then 10 mg of the sample was weighed and placed in a digestion bottle to wait for digestion; Step 2: Prepare Fenton digestion solution: (1) preparing a 2.78% FeSO4 solution: adjusting the pH of 50 mL of ultrapure water to 3.01-3.14 using dilute hydrochloric acid, then adding 1.39 g of solid ferrous sulfate weighed using a 1 / 10,000 balance, and stirring to obtain 50 mL of 2.78% FeSO4 solution; (2) Mixing and preparing Fenton digestion solution: add 150 mL of 30% H2O2 solution by mass into a digestion bottle, then use a pipette to add 500 μL of 2.78% FeSO4 solution, the volume ratio of the two being 1:300, and then immediately shake the digestion bottle to evenly mix the FeSO4 and H2O2 to obtain a Fenton digestion solution; Step 3: Fenton oxidation digestion and reducing agent reduction of plant tissue samples: (1) Place the digestion bottle in a shaker and digest for 85-100 minutes at 60°C, 115 rpm and 72W full-spectrum light. After confirming that the plant tissue is completely digested, place the digestion bottle in an ice-water mixture to terminate the digestion reaction; (2) Add 15 g of L-ascorbic acid solid to the digestion bottle after the reaction is terminated, and then place it in a shaker with the shaker temperature set to 60°C and the speed set to 115 rpm. Place a thermometer in the digestion bottle to control the reduction reaction temperature at all times. When the temperature is higher than 60°C, place the digestion bottle in an ice-water mixture for 1 to 2 seconds, and then continue to shake for 30 to 40 minutes. After confirming that the digestion solution becomes clear, filter the digestion solution onto a filter membrane with a pore size of 100 nm, rinse the filter repeatedly with ultrapure water for more than 3 times, and filter the rinse solution onto the filter membrane. At this point, the microplastics in the plant tissue sample can be separated and extracted onto the filter membrane; Step 4: Use a stereoscope to observe the dried filter membrane under a microscope to obtain the shape, size, surface texture characteristics and position of the microplastics on the filter membrane. Then move the filter membrane under a microscope Raman spectrometer and obtain the Raman spectrum of the corresponding microplastics according to the position determined under the stereoscope. Compare the obtained Raman spectrum with the standard microplastic library for matching degree. Those with a matching degree higher than 80% are judged to be microplastics.

2. The method for rapid separation and extraction of microplastics in plant tissues according to claim 1, characterized in that: The plant tissues in step 1 include one or more of flowers, crops, and aquatic plants; the plant tissue samples include one or more of leaves, fruits, roots, and stems of plant tissues.

3. The method for rapid separation and extraction of microplastics in plant tissues according to claim 1 or 2, characterized in that: The concentration of anhydrous ethanol in step 1 is 75%; the pore size of the filter membrane is 20 μm.

4. The method for rapid separation and extraction of microplastics in plant tissues according to claim 3, characterized in that Step 3 (1) The lighting condition is added by installing four 250 cm long full-spectrum light strips with an effective power of 72W in the shaker box cover.

5. The method for rapid separation and extraction of microplastics in plant tissues according to claim 4, characterized in that: The mass ratio of ice to water in the ice-water mixture in step 3 (2) is 250 g:300 g to 350 g:300 g.

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