Visual quantitative method for microplastic standard recovery

Abstract

The invention relates to the technical field of scientific evaluation of a micro-plastic standardized extraction method, in particular to a visual quantitative method for micro-plastic standard recovery. Image J image processing software is adopted to count results obtained after two times of photographing, experimental errors caused by human factor interference can be avoided, and scientificity and objectivity are achieved; the black-matrix white grid filter membrane is adopted for filtering, so that photos shot for multiple times can form a complete filter membrane photo, and the problems of missed recording and repeated counting of micro-plastics caused by experimental operation can be effectively avoided; in addition, the counting mode based on the photos can also avoid the problem of low data quality caused by subjective consciousness of the experimenter.

Description

Technical Field

[0001] The present invention relates to the technical field of scientific evaluation of microplastic standard extraction methods, and particularly to a visualization and quantification method for microplastic spike recovery. Background Art

[0002] The scientific evaluation of the standard extraction method of microplastics in environmental media has always been a lacking part in the pretreatment field, restricting the accuracy of its environmental abundance evaluation and also hindering the research on the impact mechanism of microplastics on the ecosystem. It is still impossible to prove whether the previous microplastic pretreatment methods can completely or effectively separate and extract microplastics in environmental media. Although there have been a large number of reports on the qualitative and quantitative analysis of microplastics in environmental media, the extraction efficiency of microplastics by this pretreatment method and whether there is loss of microplastics during extraction and transfer are still unknown.

[0003] In this regard, existing research has evaluated the scientificity of pretreatment methods by adding and then recovering microplastics to environmental media, but there are certain problems in the types, addition, recovery, and counting methods of spiked microplastics. Usually, the too large particle size or lack of characteristics of spiked microplastics will lead to high recovery difficulty or being easily interfered by microplastics in environmental samples. At the same time, the microplastic counting results are greatly affected by the subjectivity of experimenters. All the above problems will ultimately result in the lack of persuasiveness of microplastic recovery rate data.

[0004] In view of the defects in the prior art, there is an urgent need for a scientific, objective and easy-to-implement evaluation method to measure the scientificity of microplastic pretreatment methods, so as to provide an important basis for determining future environmental microplastic extraction methods. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a visualization and quantification method for microplastic spike recovery. The quantitative analysis method of the present invention is scientific, objective and simple to implement.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides a visualization and quantification method for microplastic spike recovery, including the following steps: After taking the first photo of the microplastic standard particle solution, perform the first counting to obtain the first counting result; After mixing the above microplastic standard particle solution and environmental sample, perform pretreatment and digestion treatment in sequence to obtain the digested environmental sample; Mix the digested environmental sample and saturated salt solution and perform solid-liquid separation to obtain the supernatant; Filter the supernatant with a black background and white grid filter membrane to obtain a microplastic spike recovery sample on the surface of the black background and white grid filter membrane; After taking a second photo of the spiked recovery sample of microplastics on the surface of the black background and white grid filter membrane, a second count is performed to obtain the second counting result; Calculate the recovery rate: Recovery rate = Second counting result / First counting result; Both the first count and the second count are performed using the Image J image processing software in the computer.

[0007] Preferably, the concentration of the microplastic standard particle solution is 0.5 - 2 ppm.

[0008] Preferably, the microplastic standard particles in the microplastic standard particle solution are microplastic standard particles with a specific shape and / or color; The particle size of the microplastic standard particles is 1 - 1000 μm.

[0009] Preferably, the amount of the microplastic standard particles and the environmental sample in the microplastic standard particle solution is (50 - 800) : 1 g.

[0010] Preferably, the solid-liquid separation is carried out in a density separation beaker; A spout is provided 3 cm above the top of the side wall of the density separation beaker.

[0011] Preferably, the saturated salt solution includes one or more of saturated zinc chloride solution, saturated zinc bromide solution, saturated sodium chloride solution, saturated sodium iodide solution, and saturated calcium chloride solution; The dosage ratio of the digested environmental sample to the saturated salt solution is 1 g : (10 - 200) mL.

[0012] Preferably, the saturated zinc chloride solution is poured into the digested environmental sample through a glass rod for drainage.

[0013] Preferably, the method of solid-liquid separation is static settlement, and the static settlement time is 5 - 8 h.

[0014] Preferably, the filtration method is suction filtration; After the suction filtration is completed, it also includes suction filtration washing with a surfactant solution.

[0015] Preferably, the first photo and the second photo are independently taken using a microscope or a macro lens; The microscope is a 10 - 180 times zoom microscope connected to the computer through a USB interface; The macro lens is a 10 - 40 times mobile phone micro-focusing lens.

[0016] The present invention provides a visualization and quantification method for microplastic spiking recovery, comprising the following steps: After the first photographing of the microplastic standard particle solution, the first counting is performed to obtain the first counting result; after mixing the above microplastic standard particle solution and the environmental sample, pretreatment and digestion treatment are successively carried out to obtain the environmental sample after digestion treatment; after mixing the environmental sample after digestion treatment and the saturated salt solution, solid-liquid separation is carried out to obtain the supernatant; the supernatant is filtered by a black background and white grid filter membrane, and a microplastic spiking recovery sample is obtained on the surface of the black background and white grid filter membrane; after the second photographing of the microplastic spiking recovery sample on the surface of the black background and white grid filter membrane, the second counting is performed to obtain the second counting result; calculate the recovery rate: recovery rate = second counting result / first counting result; both the first counting and the second counting are performed by the Image J image processing software in the computer. The present invention uses the Image J image processing software to count the results after two photographings, which can avoid experimental errors caused by human factors and has scientificity and objectivity; using a black background and white grid filter membrane for filtration can form a complete filter membrane photo from multiple photographed photos, effectively avoiding the problems of missed counting and repeated counting of microplastics caused by experimental operations, and the counting method based on photos can also avoid the problem of low data quality caused by the subjective awareness of the experimenter. Description of the Drawings

[0017] Figure 1 is a schematic flow chart of the quantitative analysis method of the present invention; Figure 2 is a schematic structural diagram and a physical diagram of the density separation beaker adopted by the present invention; Figure 3 is a physical diagram of the microscope or macro lens adopted by the present invention; Figure 4 is a physical diagram of the low-magnification microscope photograph before and after spiking the PE standard (75 - 95 μm and 125 - 150 μm) in Example 1 of the present invention; Figure 5 is a physical diagram of the low-magnification microscope photograph before and after spiking the standard products PA (coarse 50 μm, length 1 - 5 mm) and PBT (coarse 65 μm, length 1 mm) in Example 2 of the present invention; Figure 6 is a schematic flow chart of the operation steps of the spiking recovery of the present invention. Detailed Embodiments

[0018] As Figure 1 shown in the schematic flow chart and as Figure 6For the operation step schematic flow chart, the present invention provides a visualization and quantification method for microplastic spiking recovery, including the following steps: After taking the first photo of the microplastic standard particle solution, perform the first counting to obtain the first counting result; After mixing the above microplastic standard particle solution and the environmental sample, perform pretreatment and digestion treatment in sequence to obtain the digested environmental sample; Mix the digested environmental sample and the saturated salt solution and then perform solid-liquid separation to obtain the supernatant; Filter the supernatant with a black background and white grid filter membrane to obtain a microplastic spiking recovery sample on the surface of the black background and white grid filter membrane; After taking the second photo of the microplastic spiking recovery sample on the surface of the black background and white grid filter membrane, perform the second counting to obtain the second counting result; Calculate the recovery rate: Recovery rate = Second counting result / First counting result; Both the first counting and the second counting are performed by using the Image J image processing software in the computer.

[0019] In the present invention, unless otherwise specified, all raw materials are commercially available products well-known to those skilled in the art.

[0020] In the present invention, after taking the first photo of the microplastic standard particle solution, perform the first counting to obtain the first counting result.

[0021] In the present invention, the concentration of the microplastic standard particle solution is preferably 0.5 - 2 ppm, more preferably 1 ppm. In the present invention, the microplastic standard particle solution preferably includes a solution prepared by mixing plastic standard particles and ethanol. In the present invention, the particle size of the microplastic standard particles is preferably 1 - 1000 μm, more preferably 50 - 150 μm, and most preferably 75 - 95 μm or 125 - 150 μm. The present invention has no special limitation on the shape or color of the microplastic standard particles. In the present invention, the shape of the microplastic standard can be fiber, flake, microbead or block.

[0022] In the present invention, the process of the first photo taking is preferably to transfer the microplastic standard particle solution to a clean petri dish with a pipette gun, and use a microscope or a macro lens (the physical diagram is as Figure 3Take a photo as shown). In the present invention, the microscope is preferably a 10-180 times zoom microscope connected to a computer through a USB interface; the macro lens is preferably a 10-40 times mobile phone micro-focus lens. In the present invention, taking a photo with the above microscope or macro lens can capture the photographed content in the form of 1-10 groups of photos, so as to avoid the time cost required for counting due to the small field of view of a high-power microscope commonly used in laboratories, which requires taking 100-1000 groups of photos.

[0023] In the present invention, the process of the first counting is preferably to import the photos obtained after the first photo taking into the Image image processing software, and then click Plugins, Analyze, Cell Counter, Cell Counter, and Initialize in sequence, and then click the mouse to count.

[0024] After obtaining the result of the first counting, in the present invention, after mixing the above microplastic standard particle solution and the environmental sample, pretreatment and digestion treatment are carried out in sequence to obtain the digested environmental sample.

[0025] In the present invention, the environmental sample is preferably soil, sludge, river bottom sediment or biological tissue; the present invention has no special limitation on the source of the environmental sample, and any environmental sample containing microplastics from conventional sources well-known in the art can be used.

[0026] In the present invention, the dosage of the microplastic standard particles in the microplastic standard particle solution and the environmental sample is preferably (50-800):1 g, more preferably (200-300):1 g, and most preferably 200:1 g.

[0027] In the present invention, the mixing is preferably to rinse the microplastic standard in the glass culture dish into the beaker with ethanol, air dry it naturally, and then add the environmental sample.

[0028] In the present invention, the pretreatment preferably simulates the natural environmental behavior of the environmental sample, such as the accumulation of river bottom sediment at the bottom of the water, the storage of farmland soil in the natural environment, and the aerobic and anaerobic processes of municipal sludge. In the present invention, the process of the pretreatment can avoid the problem of lack of persuasiveness caused by the difference between the artificially added microplastics and the microplastics in the environmental medium.

[0029] The present invention has no special limitation on the process of the digestion treatment, and any process well-known to those skilled in the art can be used.

[0030] After obtaining the digested environmental sample, in the present invention, the digested environmental sample and the saturated salt solution are mixed and then solid-liquid separation is carried out to obtain the supernatant.

[0031] In the present invention, the saturated salt solution preferably includes one or more of a saturated zinc chloride solution, a saturated zinc bromide solution, a saturated sodium chloride solution, a saturated sodium iodide solution, and a saturated calcium chloride solution, and more preferably includes a saturated zinc chloride solution.

[0032] In the present invention, the function of the saturated salt solution is to float the microplastics with a lower density to the surface by using a solution with a high density, facilitating the collection of the microplastics on the surface of the solution.

[0033] In the present invention, the dosage ratio of the digested environmental sample to the saturated salt solution is preferably 1 g:(10 - 200) mL, and more preferably 1 g:100 mL.

[0034] In the present invention, the mixing is preferably carried out by draining the saturated salt solution into the digested environmental sample through a glass rod. In the present invention, the function of the draining is to prevent the loss of microplastics caused by splashing.

[0035] In the present invention, the solid-liquid separation is preferably carried out in a density separation beaker; a spout water outlet is provided 3 cm above the top of the side wall of the density separation beaker (as Figure 2 shown). In the present invention, the supernatant is preferably overflowed through the spout. In the present invention, the density separation beaker can be more conducive to collecting the floating solution and saving the use of the saturated salt solution.

[0036] In the present invention, the method of solid-liquid separation is preferably static settlement, the static settlement time is preferably 5 - 8 h, and more preferably 8 h. In the present invention, the number of times of solid-liquid separation is preferably 3 - 5 times.

[0037] After obtaining the supernatant, the present invention filters the supernatant with a black-bottom white-grid filter membrane, and a microplastic spike recovery sample is obtained on the surface of the black-bottom white-grid filter membrane.

[0038] In the present invention, the material of the black-bottom white-grid filter membrane is preferably cellulose acetate; the pore diameter of the black-bottom white-grid filter membrane is preferably 1.2 μm, and the diameter of the black-bottom white-grid filter membrane is preferably 25 mm or 47 mm.

[0039] In the present invention, using a black-bottom white-grid filter membrane for filtration is to facilitate the identification of microplastic particles on the filter membrane, and the white grid can be used to assist in counting to prevent missing or double-counting of plastic particles on the filter membrane.

[0040] In the present invention, the filtration method is preferably suction filtration. The present invention has no special limitation on the suction filtration process, and it can be carried out using a process well-known to those skilled in the art.

[0041] After the suction filtration is completed, the present invention preferably further includes performing suction filtration washing with a surfactant solution. In the present invention, the mass percentage concentration of the surfactant solution is preferably 1% to 10%, more preferably 2%. In the present invention, the surfactant in the surfactant solution is preferably Tween20 or Tween80, more preferably Tween20.

[0042] After obtaining the microplastic spiked recovery sample on the surface of the black-bottom white-grid filter membrane, the present invention takes a second photo of the microplastic spiked recovery sample on the surface of the black-bottom white-grid filter membrane and then performs a second counting to obtain the second counting result.

[0043] In the present invention, the process of the second photo-taking is preferably to transfer the microplastic standard particle solution to a clean petri dish using a pipette gun and take a photo using a microscope or a macro lens (as shown in the physical diagram Figure 3 ). In the present invention, the microscope is preferably a 10-180 times zoom microscope connected to a computer through a USB interface; the macro lens is preferably a 10-40 times mobile phone micro-focusing lens. In the present invention, taking a photo using the above microscope or macro lens can take the photo content in the form of 1 to 10 groups of photos, so as to avoid the time cost required for counting caused by the small field of view of a common high-power microscope in the laboratory, which requires taking photos in the form of 100 to 1000 groups of photos.

[0044] In the present invention, the process of the second counting is preferably to import the photos obtained after the second photo-taking into the Image image processing software, and then click Plugins, Analyze, Cell Counter, Cell Counter, and Initialize in sequence, and then click the mouse to count.

[0045] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0046] Example 1 Use a pipette gun to aspirate 0.2 mL of a microplastic standard PE solution with a concentration of 1 ppm (the solvent is ethanol, and the microplastic standard PE is a circular PE standard with a diameter of 75-95 μm fluorescent green and 125-150 μm red) onto a glass petri dish, and take a photo under bright field using a low-power microscope (a 10-180 times zoom microscope connected to a computer through a USB interface) (as Figure 4as shown in a) in and photographed under excitation by an ultraviolet lamp in dark field (such as Figure 4 in b)), and manually counted using Image J based on the photographed physical map (recorded as the first counting result, the counting result of red PE is 352, and the fluorescent green PE is 620), then transferred to a 50 mL glass sample bottle. After natural air drying, 2 g of anaerobic municipal wet sludge (containing microbeads of microplastics with a size of 100 microns) and 40 mL of ultrapure water were added, and then the bottle cap of the sample bottle was tightened. It was placed in a constant temperature shaker to separate and obtain the first supernatant and the remaining solid sample; 40 mL of digestion reagent (specific type: hydrogen peroxide with a mass concentration of 30%) was added to 2 g of the remaining solid sample for digestion (the temperature of the digestion was 70 °C and the time was 12 h). After the digestion was completed, it was allowed to stand for 8 h, and solid-liquid separation was carried out to obtain the second supernatant and solid sludge; 2 g of the solid sludge was transferred to a density separation beaker as shown in Figure 3 100 mL of saturated zinc chloride solution was added by guiding with a glass rod. Oscillation (the oscillation process was ultrasonic and manual shaking in sequence) and standing for 8 h were carried out in sequence, and then solid-liquid separation was carried out to obtain the third supernatant and the remaining solid sludge; the process of adding saturated zinc chloride solution, standing, and solid-liquid separation was repeated 3 times, and the supernatants of the repeated operations were collected; After mixing the above-mentioned first supernatant, second supernatant, third supernatant, and the supernatants of the repeated operations, filtration was carried out using a black-bottom white-grid cellulose acetate filter membrane to complete the recovery of the PE standard; A low-magnification microscope (a 10 - 180-fold zoom microscope connected to a computer through a USB interface) was used to photograph the PE standard on the surface of the black-bottom white-grid cellulose acetate filter membrane (such as Figure 4 c in is the physical map taken under bright field, Figure 4 d in is the physical map taken under excitation by an ultraviolet lamp in dark field; note: since the fluorescence green is affected by the environmental sample (municipal wet sludge), the physical map taken under bright field will be affected, and it needs to be taken under excitation by an ultraviolet lamp in dark field), and manual counting was carried out using Image J based on the photographed photos (the second counting result, the counting result of red PE is 176, and the fluorescent green PE is 620). The ratio of the second counting result to the first counting result was recorded as the recovery rate of microplastics (the recovery rate of red PE is 49%, and the fluorescent green PE is 36%).

[0047] Example 2 Referring to Example 1, the difference is that the microplastic standard PE was replaced with PA fibers with a thickness of 50 μm and a length of 1 - 5 mm and PBT fibers with a thickness of 65 μm and a length of 1 mm; The first counting result: the number of PA fibers is 78, and the number of PBT fibers is 113 (such as Figure 5As shown by a1 - a2 therein, a1 is a physical picture of PA fibers with a thickness of 50 μm and a length of 1 - 5 mm under bright field, and a2 is a physical picture of PBT fibers with a thickness of 65 μm and a length of 1 mm under bright field), the result of the second count is 74 for PA fibers and 95 for PBT fibers (as shown by b in Figure 5 therein); the recovery rate of PA fibers of microplastics is 95%, and the recovery rate of PBT fibers is 84%.

[0048] Example 3 Referring to Example 1, the difference is that the microplastic standard is replaced with PET fibers with a length of 1 mm and a diameter of 20 μm; The result of the first count is 82, and the result of the second count is 19; the recovery rate of microplastics is 25%.

[0049] Example 4 Referring to Example 1, the difference is that the microplastic standard is replaced with PET fibers with a length of 2 mm and a diameter of 20 μm; The result of the first count is 110, and the result of the second count is 35; the recovery rate of microplastics is 32%.

[0050] Example 5 Referring to Example 1, the difference is that the microplastic standard is replaced with PET fibers with a length of 4 mm and a diameter of 20 μm; The result of the first count is 94, and the result of the second count is 36; the recovery rate of microplastics is 38%.

[0051] Example 6 Referring to Example 1, the difference is that the microplastic standard is replaced with PET fibers with a length of 6 mm and a diameter of 20 μm; The result of the first count is 102, and the result of the second count is 46; the recovery rate of microplastics is 45%.

[0052] Example 7 Referring to Example 1, the difference is that the microplastic standard is replaced with PET fibers with a length of 8 mm and a diameter of 20 μm; The result of the first count is 85, and the result of the second count is 44; the recovery rate of microplastics is 52%.

[0053] Example 8 Referring to Example 1, the difference is that the microplastic standard is replaced with PBT fibers with a length of 1 mm and a diameter of 50 μm; The result of the first count is 110, and the result of the second count is 93; the recovery rate of microplastics is 85%.

[0054] Example 9 Referring to Example 1, the difference is that the microplastic standard is replaced with PBT fibers with a length of 1 mm and a diameter of 80 μm; The first counting result is 131, and the second counting result is 115; the recovery rate of microplastics is 88%.

[0055] Example 10 Referring to Example 1, the difference is that the microplastic standard is replaced with PBT fibers with a length of 1 mm and a diameter of 110 μm; The first counting result is 120, and the second counting result is 104; the recovery rate of microplastics is 86%.

[0056] Example 11 Referring to Example 1, the difference is that the microplastic standard is replaced with PBT fibers with a length of 1 mm and a diameter of 210 μm; The first counting result is 105, and the second counting result is 98; the recovery rate of microplastics is 93%.

[0057] Example 12 Referring to Example 1, the difference is that the microplastic standard is replaced with a PVC film with a length, width and thickness of 100*100*10 μm; The first counting result is 52, and the second counting result is 39; the recovery rate of microplastics is 74%.

[0058] Example 13 Referring to Example 1, the difference is that the microplastic standard is replaced with a PVC film with a length, width and thickness of 200*200*10 μm; The first counting result is 61, and the second counting result is 54; the recovery rate of microplastics is 89%.

[0059] Example 14 Referring to Example 1, the difference is that the microplastic standard is replaced with a PVC film with a length, width and thickness of 400*400*10 μm; The first counting result is 57, and the second counting result is 53; the recovery rate of microplastics is 92%.

[0060] Example 15 Referring to Example 1, the difference is that the microplastic standard is replaced with a PVC film with a length, width and thickness of 800*800*10 μm; The first counting result is 71, and the second counting result is 68; the recovery rate of microplastics is 96%.

[0061] The above are only the preferred embodiments of the present invention and do not impose any formal limitations on the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A visual quantitative method for the recycling of microplastics by spiked recycling, characterized in that: The following steps are involved: After taking the first photo of the microplastic standard particle solution, the first counting is performed to obtain the first counting result; The microplastic standard particles in the microplastic standard particle solution are microplastic standard particles with specific shapes and / or colors; After the above-mentioned microplastic standard particle solution and the environmental sample are mixed, pretreatment and digestion treatment are performed in sequence to obtain the environmental sample after digestion treatment; The environmental sample after the digestion treatment is mixed with a saturated salt solution and then subjected to solid-liquid separation to obtain a supernatant; The supernatant is filtered using a black-bottom white grid filter membrane, and a microplastic spiked recovery sample is obtained on the surface of the black-bottom white grid filter membrane; After taking a second photo of the microplastic spiked recovery sample on the surface of the black-bottom white grid filter membrane, a second counting is performed to obtain a second counting result; Calculate the recovery rate: Recovery rate = second counting result / first counting result; The first count and the second count were both performed using Image J image processing software in a computer.

2. The visual quantitative method according to claim 1, characterized in that: The concentration of the microplastic standard particle solution is 0.5~2ppm.

3. The visual quantification method according to claim 1 or 2, characterized in that: The particle size of the microplastic standard particles is 1~1000μm.

4. The visual quantitative method according to claim 1, characterized in that: The amount of the microplastic standard particles and environmental samples in the microplastic standard particle solution is (50~800) pieces: 1g.

5. The visual quantitative method according to claim 1, characterized in that: The solid-liquid separation is carried out in a density separation beaker; A pointed water outlet is provided above the side wall of the density separation beaker at a distance of 3 cm from the top.

6. The visual quantification method according to claim 1 or 5, characterized in that: The saturated salt solution includes one or more of a saturated zinc chloride solution, a saturated zinc bromide solution, a saturated sodium chloride solution, a saturated sodium iodide solution and a saturated calcium chloride solution; The dosage ratio of the environmental sample after the digestion treatment to the saturated salt solution is 1g: (10~200)mL.

7. The visual quantification method according to claim 6, characterized in that: The saturated zinc chloride solution is drained into the environmental sample after the digestion treatment through a glass rod.

8. The visual quantification method according to claim 7, characterized in that: The solid-liquid separation method is standing, and the standing time is 5 to 8 hours.

9. The visual quantification method according to claim 1, characterized in that: The filtering method is suction filtration; After the suction filtration is completed, the method further includes suction filtration and washing with a surfactant solution.

10. The visual quantification method according to claim 1, characterized in that: The first photographing and the second photographing are independently performed using a microscope or a macro lens; The microscope is a 10-180 times zoom microscope connected to a computer via a USB interface; The macro lens is a 10-40 times micro-focus lens for mobile phones.

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