Method for detecting microplastics from animal tissues
By combining KOH digestion and ultrasonic treatment with centrifuge separation, the problems of long extraction time and difficulty in separating microplastics from earthworm tissues were solved, achieving rapid and economical microplastic detection.
Patent Information
- Application Number
- CN202510908465.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-31
- Publication Date
- 2025-09-26
AI Technical Summary
Existing technologies make it difficult to effectively separate and detect microplastics from earthworm tissues, as they present problems such as long digestion time, difficulty in separation, and high cost.
KOH digestion solution combined with ultrasonic treatment was used, a centrifuge was used for separation, and microplastics were extracted by fluorescent staining and counting. ZnCl2 solution was used for separation to reduce costs.
It significantly shortens the digestion time, improves the extraction efficiency of microplastics, reduces the separation cost, and achieves fast and accurate microplastic detection.
Smart Images
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Abstract
Description
[0001] This invention is a divisional application. The name of the original application is "A method for detecting microplastics from earthworm tissues", the application number is 202410133484.0, and the application date is January 31, 2024. Technical Field
[0002] The present invention relates to the field of detection technology, and specifically to a method for detecting microplastics from earthworm tissue. Background Art
[0003] Since the 1950s, the production of plastics has grown rapidly. It is estimated that the global annual production of plastics is as high as 368 million tons. Currently, only 9% of plastic waste is recycled, and the rest enters the natural environment in various forms. These plastic wastes that enter the natural environment are broken / degraded into microplastics (less than 5 mm in diameter) through sunlight, mechanical wear and biological interactions. Soil, as an important gathering place for various pollutants, contains a large amount of microplastics. Microplastics widely distributed in the soil are easily ingested by earthworms, affecting their survival and health. Nowadays, many researchers have begun to study microplastic pollution, aiming to find ways to control microplastic pollution. However, due to the characteristics of microplastics such as small particle size, many types, and strong adsorption, it is difficult to directly observe and quantify them from biological tissues, which increases the difficulty of studying microplastic pollution. The current detection methods of microplastics in earthworm tissues mainly face the following three problems:
[0004] (1) Earthworms feed on various substances in the soil for a long time. Their intestines contain soil particles, undigested microplastics, etc., making it difficult to separate these substances from microplastics.
[0005] (2) The extraction time of microplastics is relatively long. Currently, the digestion time of earthworm tissue requires at least 24 hours.
[0006] (3) Difficulty in separating microplastics: There are various organic and inorganic substances in organisms, and it is difficult to accurately identify and separate microplastics. Currently, the more accurate method is to use fluorescent microplastics, but the cost is too high.
[0007] Therefore, it is urgent to find an effective method to extract microplastics from earthworm tissue to facilitate the detection of microplastics in biological samples and promote the research progress of microplastic pollution in soil environment. Summary of the Invention
[0008] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a method for detecting microplastics from earthworm tissues, which has a good digestion effect, greatly accelerates the process of tissue fragmentation, and greatly shortens the digestion time.
[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0010] A method for detecting microplastics from earthworm tissue, comprising:
[0011] S1: Obtain earthworm samples without excrement in the intestine;
[0012] S2: Digestion: Take 1.8-2.2 g of the earthworm sample treated in step S1, chop it into small pieces, then soak it in KOH and digest it in a shaker for 2.5-3.5 hours at a temperature of 55-65°C. During the digestion process, take out the digestion solution and ultrasonicate it for 3 minutes every half an hour to obtain the digestion solution;
[0013] S3: Filtration: Vacuum filter the digestion solution onto a polycarbonate nuclear track etched filter membrane;
[0014] S4: Separation: Place the filter membrane in step S3 into a centrifuge tube, add saturated ZnCl2 solution and sonicate for 8-12 minutes; remove the filter membrane and rinse it with saturated salt solution. Collect the rinse solution into the same centrifuge tube and centrifuge for 8-12 minutes to obtain the supernatant;
[0015] S5: The supernatant was vacuum filtered onto a new polycarbonate nuclear track etched filter membrane;
[0016] S6: Drying: Place the new polycarbonate nuclear track etched filter membrane from step S5 in an oven to dry overnight;
[0017] S7: Fluorescent staining and counting: Add Nile red solution to the dried polycarbonate nuclear track etched filter membrane to evenly soak it, let it stand in the dark for 8 to 12 minutes, and then observe it under a fluorescence microscope.
[0018] Furthermore, the operation method of step S1 is: after rinsing the surface of the earthworm to be tested with ultrapure water, place it on a clean moistened filter paper to empty the intestine, and change the filter paper every 8 hours. This process continues for 2 days until no new excrement appears on the filter paper.
[0019] Furthermore, in step S2, the earthworm sample is cut into small segments of 0.8 to 1.2 cm.
[0020] Furthermore, in step S2, the concentration of KOH is 10%, the amount added is 20 mL, and the rotation speed of the shaking table is 200 rpm.
[0021] Furthermore, in step S4, the centrifuge tube is 50 mL, the amount of saturated ZnCl2 solution added is 20 mL, the filter membrane is ultrasonically cleaned for 10 minutes, and then the filter membrane is rinsed with 20 mL of saturated salt solution.
[0022] Furthermore, the temperature of the oven in step S6 is 40°C.
[0023] Furthermore, in step S7, the concentration of the Nile red solution is 1 ug / mL, and the amount of the solution added is 2 to 3 drops.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] (1) The present invention adopts reasonable design, selects KOH as the digestion solution, and adds ultrasonic treatment during the digestion process, which greatly accelerates the process of tissue fragmentation and greatly shortens the digestion time. The digestion effect is good and no large amount of foam is generated. The digestion rate can reach 98.1±1.1% in 3 hours.
[0026] (2) In the separation process of the present invention, conventional static separation is not selected. Instead, centrifugation is used, so that microplastics can be separated from other substances more quickly and concentrated in the upper liquid layer; this separation process is combined with the digestion process, which speeds up the entire microplastic extraction process by at least twice.
[0027] (3) The present invention selects a saturated ZnCl2 solution when performing separation, thereby reducing costs while ensuring the separation effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a diagram of the earthworm's intestinal emptying in the present invention;
[0029] Figure 2 This is the fluorescence microscopic observation when NaCl is selected as the separation solution;
[0030] Figure 3 This is the fluorescence microscopic observation picture when NaI is selected as the separation solution;
[0031] Figure 4 This is a fluorescence microscopic observation image when ZnCl2 is selected as the separation solution. DETAILED DESCRIPTION
[0032] The present invention will be further described below with reference to the accompanying drawings and examples. The embodiments of the present invention include but are not limited to the following examples.
[0033] This embodiment provides a method for detecting microplastics from earthworm tissue, comprising:
[0034] (1) Rinse the surface of the earthworms to be tested with ultrapure water and place them on clean, moistened filter paper to empty their intestines. Change the filter paper every 8 hours to prevent the earthworms from ingesting excrement again. This process continues for 2 days until no new excrement appears on the filter paper. Expelling the excrement from the earthworms to be tested ensures that the extracted microplastics come from biological tissues.
[0035] (2) Digestion: Cut 2 g of the earthworm sample treated in step (1) into small pieces of about 1 cm, place them in a 50 mL Erlenmeyer flask, add 20 mL of 10% KOH digestion solution, and place them in a shaker at 60°C and 200 rpm for 3 h. During this period, take out the Erlenmeyer flask every half an hour and ultrasonicate for 3 min.
[0036] (3) Filtration: The solution obtained in step (2) was vacuum filtered onto a polycarbonate nuclear track etched filter (PCTE);
[0037] (4) Separation: Place the above filter membrane in a 50 mL centrifuge tube, add 20 mL of saturated ZnCl2 solution, sonicate for 10 min, remove the filter membrane, and rinse the filter membrane with 20 mL of saturated salt solution. Collect the rinse solution in the same centrifuge tube and centrifuge for 10 min.
[0038] (5) Re-filtration: The upper layer of the centrifuged solution was vacuum filtered onto a new PCTE membrane;
[0039] (6) Drying: Dry the PCTE membrane in step (5) in an oven at 40°C overnight;
[0040] (7) Fluorescence staining and counting: Add 2-3 drops of 1 μg / mL Nile red solution to the dried PCTE membrane to evenly wet the membrane. After standing in the dark for 10 min, observe under a fluorescence microscope.
[0041] In the above method, ultrasonic treatment is added to the digestion process of organic matter in step (2), which greatly accelerates the process of tissue fragmentation and greatly shortens the digestion time. In the separation process of step (4), conventional static separation is not adopted, but centrifugation is used, so that microplastics can be separated from other substances more quickly and concentrated in the upper liquid. These two steps speed up the entire tissue microplastic extraction process by at least twice. During density separation, unlike traditional saturated NaCl and NaI solutions, the above method uses saturated ZnCl2 solution, which reduces costs while ensuring separation effect.
[0042] The specific contents of the above method are:
[0043] (1) Preparation of earthworm samples containing microplastics in tissues
[0044] 20g of microplastics (PE, PP or PET) with a diameter of 50μm were mixed into 180g of soil to prepare a soil sample with a microplastic concentration of 10%. The soil sample was then cultured with earthworms for 15 days to allow them to fully ingest the microplastics in the soil.
[0045] (2) Earthworm sample cleaning
[0046] After the surface of the earthworms to be tested is rinsed with ultrapure water, they are placed on clean, moistened filter paper. The filter paper is changed every 8 hours to prevent the earthworms from ingesting excrement again. After this process continues for 2 days, the clean filter paper no longer has particles, indicating that the earthworm intestines have been basically emptied (e.g. Figure 1 ).
[0047] (3) Dissolution
[0048] Three digestion solutions were tested in this step: a. 30% H₂O₂ solution; b. 10% KOH solution; and c. 69% HNO₃ solution. The effects of different digestion times were also tested. The specific conditions are shown in Table 1.
[0049] Table 1 Digestion experiment design
[0050] Experimental plan digestion solution Digestion time Experiments a1-a4 <![CDATA[30%H2O2]]> 1h,3h,6h,24h Experiments b1-b4 10% KOH 1h,3h,6h,24h Experiments c1-c4 <![CDATA[69%HNO3]]> 1h,3h,6h,24h
[0051] 3.1 H2O2 digestion
[0052] Cut four clean earthworm samples (total weight approximately 2g) into small pieces approximately 1cm in size and place them in a 50mL Erlenmeyer flask. Add 20mL of 30% H₂O₂. Because H₂O₂ digestion is accompanied by significant foaming, allow the sample to digest for 1 hour after addition. Then, place the sample in a shaker at 60°C and 200 rpm for digestion. Remove the flask every half hour and sonicate for 3 minutes. After digestion times a1-a4 have been reached, vacuum filter the digested solution onto a PCTE membrane, dry the membrane, and weigh it. Calculate the digestion rate according to Equation 1.
[0053]
[0054] W1 is the initial earthworm sample weight, W2 is the weight of the filter membrane after drying, and W3 is the weight of the original filter membrane.
[0055] Each experiment was repeated three times, and the results are shown in Table 2.
[0056] Table 2 Hydrogen peroxide digestion efficiency
[0057]
[0058] Experiments a1-a4 showed that H2O2 digestion took a long time to complete, reaching a stable state after 24 hours, with a digestion rate of 99%. Although H2O2 can effectively digest biological tissue, the digestion process is long and accompanied by a large amount of foam. Excessive foam can overflow the container or adhere to the container surface, resulting in the loss of the plastic sample.
[0059] 3.2 KOH digestion
[0060] Four clean earthworm samples (total weight approximately 2g) were cut into approximately 1cm segments and placed in a 50mL Erlenmeyer flask. 20mL of 10% KOH digestion solution was added. Since KOH digestion is relatively stable, the samples were directly placed in a shaker at 60°C and 200rpm for digestion. Every half hour, the flask was removed and sonicated for 3 minutes. After reaching the digestion time for experiments b1-b4, the digestion solution was vacuum filtered onto a PCTE membrane, dried, and weighed. The digestion rate was calculated according to Equation 1, as shown in Table 3.
[0061] Table 3 KOH digestion efficiency
[0062]
[0063] Experiments b1-b4 showed that the KOH digestion effect was also relatively good, and the digestion time was short. It was basically stable after 3 hours, and the digestion rate could reach 98%.
[0064] 3.3 HNO3 digestion
[0065] Four clean earthworm samples (total weight approximately 2g) were cut into approximately 1cm segments and placed in a 50mL Erlenmeyer flask. 20mL of 69% HNO3 digestion solution was added. Since HNO3 digestion is relatively stable, the samples were placed directly on a shaker at 60°C and 200rpm for digestion. Every half hour, the flask was removed and sonicated for 3 minutes. After reaching the digestion times c1-c4 of the experiment, the digestion solution was vacuum filtered onto a PCTE membrane, dried, and weighed. The digestion rate was calculated according to Equation 1, as shown in Table 4.
[0066] Table 4 KOH digestion efficiency
[0067] Experiment number Digestion time digestion solution Digestion rate c1 1h <![CDATA[69%HNO3]]> 79.8±7.1％ c2 3h <![CDATA[69%HNO3]]> 88.2±3.3％ c3 6h <![CDATA[69%HNO3]]> 96.2±1.1％ c4 24h <![CDATA[69%HNO3]]> 96.3±0.5％
[0068] Experiments c1-c4 showed that compared with H2O2 and KOH, HNO3 had the worst digestion effect, was basically stable after 6 hours, and the digestion rate could only reach 96%.
[0069] 3.4. Selection of digestion conditions
[0070] Based on comprehensive consideration of the above experimental results, this embodiment selects a 10% KOH digestion solution for 3 hours in the digestion stage, which can ensure both the digestion efficiency and the accuracy of the experimental data.
[0071] (IV) Separation + Fluorescence Staining Counting
[0072] After the digestion experiment, the organic matter in the earthworm samples was largely removed. However, to further purify the microplastics in the digested samples, we will utilize the low density of microplastics to further separate them from other substances. In this step, we tested three different salt solutions: saturated NaCl, NaI, and ZnCl₂ solutions. The specific experimental design is shown in Table 5.
[0073] Table 5 Separation experiment design
[0074]
[0075] Ultrasonicate the centrifuge tube containing the PCTE membrane and 20 mL of saturated NaCl solution for 10 minutes, remove the filter membrane, and rinse the filter membrane with 20 mL of the saturated salt solution. Collect the rinse liquid in the same centrifuge tube and centrifuge for 10 minutes. Vacuum filter the upper solution onto a new PCTE membrane. Place the PCTE membrane in a 40°C oven to dry overnight, then drop 2-3 drops of 1 ug / mL Nile red solution on the filter paper to evenly soak the filter membrane. After standing for 10 minutes in the dark for staining, observe and count the fluorescent particles on the filter membrane using a fluorescence microscope at an excitation wavelength of 515-561 nm (such as Figure 2 ).
[0076] The microplastic content in a single earthworm was calculated based on the observed number of microplastics. The calculation formula 2 is as follows:
[0077] Microplastic content = n / m (pieces / item) (Formula 2)
[0078] Where n is the number of microplastics (pieces), m is the number of earthworms, and the results are shown in Table 6
[0079] Table 6 NaCl separation experimental results
[0080] Experimental plan Types of microplastics Microplastics (pieces) Experiment d1 PE 21 Experiment d2 PP 19.75 Experiment d3 PET 1.25
[0081] Ultrasonicate the centrifuge tube containing the PCTE membrane and 20 mL of saturated NaI solution for 10 minutes, remove the filter membrane, and rinse the filter membrane with 20 mL of the saturated salt solution. Collect the rinse liquid in the same centrifuge tube and centrifuge for 10 minutes. Vacuum filter the upper solution onto a new PCTE membrane. Place the PCTE membrane in a 40°C oven to dry overnight, then drop 2-3 drops of 1 ug / mL Nile red solution on the filter paper to evenly soak the filter membrane. After standing for 10 minutes in the dark for staining, observe and count the fluorescent particles on the filter membrane using a fluorescence microscope at an excitation wavelength of 515-561 nm, as shown in the following example. Figure 3 shown.
[0082] The microplastic content in a single earthworm was calculated based on the observed number of microplastics. The calculation formula 2 is as follows:
[0083] Microplastic content = n / m (pieces / item) (Formula 2)
[0084] Where n is the number of microplastics (pieces), and m is the number of earthworms. The results are shown in Table 7.
[0085] Table 7 NaI separation experimental results
[0086]
[0087] Ultrasonicate the centrifuge tube containing the PCTE membrane and 20 mL of saturated ZnCl2 solution for 10 minutes, remove the filter membrane, and rinse the filter membrane with 20 mL of the saturated salt solution. Collect the rinse liquid in the same centrifuge tube and centrifuge for 10 minutes. Vacuum filter the upper solution onto a new PCTE membrane. Place the PCTE membrane in a 40°C oven to dry overnight, then drop 2-3 drops of 1 ug / mL Nile red solution on the filter paper to evenly soak the filter membrane. After standing for 10 minutes in the dark for staining, observe and count the fluorescent particles on the filter membrane using a fluorescence microscope at an excitation wavelength of 515-561 nm, as shown in the following example. Figure 4 shown.
[0088] The microplastic content in a single earthworm was calculated based on the observed number of microplastics. The calculation formula 2 is as follows:
[0089] Microplastic content = n / m (pieces / item) (Formula 2)
[0090] Where n is the number of microplastics (pieces), and m is the number of earthworms. The results are shown in Table 8.
[0091] Table 8 ZnCl2 separation experimental results
[0092] Experimental plan Types of microplastics Microplastic content (pieces) Experiment d7 PE 19.75 Experiment d8 PP 19 Experiment d9 PET 17.25
[0093] 4.4 Selection of separation conditions
[0094] Based on the experimental results above, NaCl was found to be effective in separating PE and PP microplastics. However, the separation effect was poor for high-density microplastics, such as PET. Both NaI and ZnCl2 were effective in suspending and separating different types of microplastics. However, the price of NaI reagent is significantly higher than that of ZnCl2. Therefore, ZnCl2 was selected as the separation and purification solution in this patent.
[0095] The above embodiment is only one of the preferred implementation methods of the present invention and should not be used to limit the scope of protection of the present invention. Any changes or modifications that have no substantive meaning made to the main design concept and spirit of the present invention, as long as the technical problems solved are still consistent with the present invention, should be included in the scope of protection of the present invention.
Claims
1. A method for detecting microplastics from animal tissues, characterized in that: The animal tissue includes earthworms, comprising the following steps: S1: Obtain earthworm samples without excrement in the intestine; S2: Digestion: Take 1.8-2.2 g of the earthworm sample treated in step S1, chop it into small pieces, then soak it in KOH and digest it in a shaker for 2.5-3.5 hours at a temperature of 55-65°C. During the digestion process, take out the digestion solution and ultrasonicate it for 3 minutes every half an hour to obtain the digestion solution; S3: Filtration: Vacuum filter the digestion solution onto a polycarbonate nuclear track etched filter membrane; S4: Separation: Place the filter membrane in step S3 into a centrifuge tube, add saturated ZnCl2 solution and sonicate for 8-12 minutes; remove the filter membrane and rinse it with saturated salt solution. Collect the rinse solution into the same centrifuge tube and centrifuge for 8-12 minutes to obtain the supernatant; S5: The supernatant was vacuum filtered onto a new polycarbonate nuclear track etched filter membrane; S6: Drying: Place the new polycarbonate nuclear track etched filter membrane from step S5 in an oven to dry overnight; S7: Fluorescent staining and counting: Add Nile red solution to the dried polycarbonate nuclear track etched filter to evenly wet it. Let it stand in the dark for 8-12 minutes and then observe it under a fluorescence microscope. The operation method of step S1 is as follows: after rinsing the surface of the earthworm to be tested with ultrapure water, place it on clean moistened filter paper to empty the intestine, and change the filter paper every 8 hours. This process continues for 2 days until no new excrement appears on the filter paper. In step S2, the earthworm sample is cut into small segments of 0.8 to 1.2 cm; In step S2, the concentration of KOH is 10%, the amount added is 20 mL, and the shaking speed is 200 rpm; In step S4, the centrifuge tube is 50 mL, the amount of saturated ZnCl2 solution added is 20 mL, the filter membrane is ultrasonically cleaned for 10 min, and then the filter membrane is rinsed with 20 mL of saturated salt solution; The concentration of the Nile red solution in step S7 is 1 ug / mL, and the amount of the solution added is 2 to 3 drops.