A method for extracting degradable microplastics from biological tissues

Abstract

The present invention discloses a method for extracting degradable microplastics from biological tissues, characterized in that the method comprises: (1) washing the biological tissues, cutting and crushing them for standby use; (2) taking an appropriate amount of the crushed biological tissues, adding a hydrochloric acid solution containing pepsin, adding H2O2 after the first oscillation, and vacuum filtering with a PTFE hydrophilic filter membrane after the second oscillation; (3) immersing the above-mentioned PTFE hydrophilic filter membrane in a NaI solution for ultrasonic treatment and then rinsing with a NaI solution, letting the solution stand, and vacuum filtering the upper solution to obtain a retentate; (4) Raman spectroscopy identification and statistics of the retentate. The method of the present invention can ensure that degradable microplastics can be extracted from biological tissues conveniently and quickly under the premise of minimizing the impact on plastics, and is convenient for accurately analyzing the types and abundance of degradable microplastics in organisms, and providing technical support for further studying the enrichment and migration transformation rules of degradable plastics in organisms and health risk assessment.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental health risk assessment, and specifically relates to a method for extracting degradable microplastics from biological tissues. Background Art

[0002] Microplastics refer to plastic particles with a diameter of less than 5mm, and their sources can be divided into primary microplastics and secondary microplastics. Primary microplastics refer to plastic particles directly added to products, including plastic microbeads and granular plastic products in cosmetics and household products; secondary microplastics refer to large pieces of plastic that enter the environment and are formed into smaller plastic particles through mechanical wear, light aging, chemical reactions and biological fragmentation. The potential risks of microplastics to organisms mainly come from direct ingestion and toxin release. On the one hand, once microplastics are accidentally ingested by organisms, they may block the digestive tract and even cause pathological damage to organ tissues; on the other hand, the plasticizers carried by the microplastics themselves and the persistent organic pollutants adsorbed from the environment may be released in the organism and cause compound toxic effects.

[0003] Biodegradable plastics have become the most promising alternative to traditional non-degradable plastics due to their environmental friendliness. They are currently widely used in agricultural films, food packaging, medical devices and sanitary products. However, the degradation rate of so-called "biodegradable plastics" is evaluated under ideal conditions in industrial composting systems. Due to differences in temperature, humidity and microorganisms in actual environments, the actual life cycle of degradable plastics may be greatly extended or even comparable to that of traditional plastics. Therefore, the degradation of biodegradable plastics in natural environments and their impact on ecosystems are largely unknown. Analyzing degradable microplastics in organisms is a necessary means to study and solve the above problems. However, there is currently a lack of methods to quickly, efficiently and non-destructively extract degradable microplastics from organisms, which seriously restricts related research on the health risk assessment of degradable microplastics.

[0004] At present, the methods for extracting microplastics from biological tissues are mainly chemical reagent digestion methods, including acid digestion (HNO3, HClO4), alkaline digestion (KOH, NaOH), strong oxidant digestion (H2O2, NaClO), enzyme digestion (pepsin, pancreatin, proteinase K) and combinations of the above schemes. However, these methods were originally developed for non-degradable microplastics, and the violent chemical reactions at high temperatures may destroy degradable plastics.

[0005] Therefore, when studying the enrichment, migration and transformation patterns of degradable microplastics in organisms and their health risks, the existing methods have a great impact on the properties of the degradable microplastics themselves and cannot efficiently and non-destructively separate degradable microplastics from biological tissues. There is an urgent need for an efficient and gentle digestion and separation method for extracting degradable microplastics from biological tissues. Summary of the invention

[0006] The technical problem to be solved by the present invention is: in view of the general lack of methods for extracting degradable microplastics from organisms, a method for extracting degradable microplastics from biological tissues is provided. The method of the present invention can simply, quickly and efficiently accurately analyze the types and abundance of microplastics enriched in organisms, and has little effect on the plastics themselves, which is convenient for identification and statistics using micro-Raman spectroscopy.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions:

[0008] One aspect of the present invention provides a method for extracting degradable microplastics from biological tissues, the method comprising the following steps:

[0009] (1) washing the biological tissue, cutting it into pieces and crushing it to obtain the crushed biological tissue for later use;

[0010] (2) taking an appropriate amount of the disrupted biological tissue obtained above, then adding a 0.063 mol / L hydrochloric acid solution containing 0.5% pepsin (w / v), adding 30% H2O2 (w / v) after the first oscillation, and vacuum filtering with a PTFE hydrophilic filter membrane after the second oscillation;

[0011] (3) The PTFE hydrophilic filter membrane obtained above was immersed in a filter having a density of 1.5 g / cm 3 The PTFE hydrophilic filter membrane is then rinsed with the NaI solution, the obtained solution is allowed to stand, and the upper layer solution is vacuum filtered through the PTFE hydrophilic filter membrane to obtain a retentate;

[0012] (4) The intercepted material obtained above is dried and examined under a microscope, and suspected plastic particles are selected for Raman spectroscopy detection to obtain an identification spectrum, and the microplastics are identified according to the identification standards, and then the microplastics of different materials are counted and statistically analyzed separately.

[0013] Preferably, the biological tissue in step (1) includes soft tissue, muscle and visceral tissue of fish, shrimp, crab, bivalves and squid.

[0014] Preferably, the first oscillation conditions in step (2) are: temperature 37° C., time 3 h.

[0015] Preferably, the second oscillation conditions in step (2) are: temperature 40° C., time 12 h.

[0016] Preferably, the pore size of the PTFE hydrophilic filter membrane in step (2) is 1 μm.

[0017] Preferably, the density in step (3) is 1.5 g / cm 3The NaI solution is prepared as follows: dissolve 178 g of NaI in 180 mL of ultrapure water at 20°C.

[0018] Preferably, the ultrasonic treatment conditions in step (3) are: frequency 40 KHz, time 10 to 15 min.

[0019] The beneficial effects brought by the present invention are:

[0020] 1. The method disclosed in the present invention can quickly, efficiently and accurately extract degradable microplastics from biological tissues while minimizing the impact on degradable microplastics. The method of the present invention is based on the combined digestion method of pepsin and hydrogen peroxide to remove easily degradable organic substances in tissues, and at the same time, density separation is performed on difficult-to-digest organic and inorganic substances such as fish bones and sand in shellfish. Degradable plastics with a density less than that of NaI solution float on the surface of the salt solution, while inorganic substances with higher density and difficult-to-digest organic substances are deposited at the bottom of the solution. First, the solution of the lower layer (about 90% of the total volume) is released from the lower port, and the filtrate can be recycled after filtration; then the solution of the upper layer (about 10% of the total volume) is poured out from the upper port and vacuum filtered to extract degradable microplastics, and finally identified and counted by micro-Raman spectroscopy.

[0021] 2. The digestion and density separation method provided by the present invention can efficiently digest biological tissues without significantly affecting the physicochemical characteristics of degradable plastics. However, the traditional digestion using a single strong acid or alkali takes a long time, is incomplete, and has a significant impact on the physicochemical characteristics of plastics during the digestion process. The present invention solves the above problems and can quickly, efficiently and accurately extract degradable microplastics from biological tissues while minimizing the impact on degradable microplastics. This method facilitates the accurate analysis of the types and abundance of degradable plastics in biological tissues and is easy to use and promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a flow chart of the preparation method of the present invention;

[0023] Figure 2 It is the digestion efficiency diagram of Examples 1-4 of the present invention;

[0024] Figure 3 It is the digestion efficiency diagram of comparative examples 1 and 2;

[0025] Figure 4 This is a comparison of the Raman spectra of PBS plastics before and after the extraction of degradable microplastics in biological tissues of the present invention;

[0026] Figure 5 This is a comparison of PCL plastic Raman spectra before and after the extraction of degradable microplastics in biological tissues of the present invention;

[0027] Figure 6 This is a comparison of the Raman spectra of PHA plastics before and after the extraction of degradable microplastics in biological tissues of the present invention;

[0028] Figure 7 This is a comparison of the Raman spectra of PLA plastics before and after the extraction of degradable microplastics in biological tissues of the present invention;

[0029] Figure 8 This is a comparison diagram of the Raman spectra of starch-based plastics before and after the extraction of degradable microplastics in biological tissues of the present invention;

[0030] Fig. 9 This is a diagram of the residual organic matter on the PTFE hydrophilic filter membrane after the biological tissue digestion test of the present invention;

[0031] Fig.10 This is a microscopic picture of degradable microplastics extracted after the biological tissue digestion experiment of the present invention;

[0032] Fig.11 This is a microscopic picture of degradable microplastics extracted after the biological tissue digestion experiment of the present invention;

[0033] Fig.12 This is a microscopic picture of the degradable microplastics extracted after the biological tissue digestion experiment of the present invention. DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] (1) Collection and homogenization of biological tissues: Clam samples collected from the wild or purchased from the market were placed in an ice box and brought back to the laboratory and stored in a -20°C refrigerator. The surface of the clams was washed with ultrapure water to remove any adhering particles. The soft tissue of the clam was cut with scissors and crushed with a homogenizer, and then 2 g of the crushed tissue was placed in a 250 mL glass beaker, and three parallel samples were taken.

[0037] (2) Sample digestion: Add 30 mL of 0.063 mol / L hydrochloric acid solution containing 0.5% pepsin (w / v) to the beaker of step (1), shake at 37°C for 3 hours, then add 20 mL of 30% H2O2 (w / v) solution, shake at 40°C for 12 hours, and then vacuum filter with a PTFE hydrophilic filter membrane.

[0038] (3) Density separation: The PTFE hydrophilic filter membrane obtained in step (2) was transferred to a 250 mL beaker and 30 mL of a 1.5 g / cm 3 NaI solution (178g NaI is dissolved in 180mL ultrapure water at 20°C), after ultrasonic treatment at 40KHz in an ultrasonic cleaner for 15 minutes, the PTFE hydrophilic filter membrane is repeatedly rinsed three times with the above NaI solution while controlling the total solution volume not to exceed 50mL and the PTFE hydrophilic filter membranes are taken out one by one, the solution in the beaker is transferred to a 60mL separatory funnel and allowed to stand for 30 minutes, the lower layer solution (accounting for about 90% of the total volume) is vacuum filtered through the PTFE hydrophilic filter membrane, and the filtrate can be recycled; the upper layer solution (accounting for about 10% of the total volume) is poured out from the upper opening, vacuum filtered through the PTFE hydrophilic filter membrane, and the retentate on the PTFE hydrophilic filter membrane is dried and weighed.

[0039] The PTFE hydrophilic filter membrane was dried and weighed. Figure 2 , corresponding to the clam in the figure.

[0040] Example 2

[0041] (1) Collection and homogenization of biological tissues: Squid samples collected from the wild or purchased from the market were placed in an ice box and brought back to the laboratory and stored in a -20°C refrigerator. The surface of the squid was washed with ultrapure water to remove any adhering particles. The squid soft tissue was cut with scissors and crushed with a homogenizer, and then 2 g of the crushed tissue was placed in a 250 mL glass beaker, and three parallel samples were taken.

[0042] (2) Sample digestion: Add 30 mL of 0.063 mol / L hydrochloric acid solution containing 0.5% pepsin (w / v) to the beaker of step (1), shake at 37°C for 3 hours, then add 20 mL of 30% H2O2 (w / v) solution, shake at 40°C for 12 hours, and then vacuum filter with a PTFE hydrophilic filter membrane.

[0043] (3) Density separation: The PTFE hydrophilic filter membrane obtained in step (2) was transferred to a 250 mL beaker and 30 mL of a 1.5 g / cm 3NaI solution (178g NaI is dissolved in 180mL ultrapure water at 20°C), after ultrasonic treatment at 40KHz in an ultrasonic cleaner for 15 minutes, the PTFE hydrophilic filter membrane is repeatedly rinsed three times with the above NaI solution while controlling the total solution volume not to exceed 50mL and the PTFE hydrophilic filter membranes are taken out one by one, the solution in the beaker is transferred to a 60mL separatory funnel and allowed to stand for 30 minutes, the lower layer solution (accounting for about 90% of the total volume) is vacuum filtered through the PTFE hydrophilic filter membrane, and the filtrate can be recycled; the upper layer solution (accounting for about 10% of the total volume) is poured out from the upper opening, vacuum filtered through the PTFE hydrophilic filter membrane, and the retentate on the PTFE hydrophilic filter membrane is dried and weighed.

[0044] The PTFE hydrophilic filter membrane was dried and weighed. Figure 2 , corresponding to the squid in the figure.

[0045] Example 3

[0046] (1) Collection and homogenization of biological tissues: Swimming crab samples collected from the wild or purchased from the market were placed in an ice box and brought back to the laboratory and stored in a -20°C refrigerator. The surface of the swimming crab was washed with ultrapure water to remove any adhering particles. The soft tissue of the swimming crab was cut with scissors and crushed with a homogenizer, and then 2 g of the crushed tissue was placed in a 250 mL glass beaker, and three parallel samples were taken.

[0047] (2) Sample digestion: Add 30 mL of 0.063 mol / L hydrochloric acid solution containing 0.5% pepsin (w / v) to the beaker of step (1), shake at 37°C for 3 hours, then add 20 mL of 30% H2O2 (w / v) solution, shake at 40°C for 12 hours, and then vacuum filter with a PTFE hydrophilic filter membrane.

[0048] (3) Density separation: The PTFE hydrophilic filter membrane obtained in step (2) was transferred to a 250 mL beaker and 30 mL of a 1.5 g / cm 3 NaI solution (178g NaI is dissolved in 180mL ultrapure water at 20°C), after ultrasonic treatment at 40KHz in an ultrasonic cleaner for 15 minutes, the PTFE hydrophilic filter membrane is repeatedly rinsed three times with the above NaI solution while controlling the total solution volume not to exceed 50mL and the PTFE hydrophilic filter membranes are taken out one by one, the solution in the beaker is transferred to a 60mL separatory funnel and allowed to stand for 30 minutes, the lower layer solution (accounting for about 90% of the total volume) is vacuum filtered through the PTFE hydrophilic filter membrane, and the filtrate can be recycled; the upper layer solution (accounting for about 10% of the total volume) is poured out from the upper opening, vacuum filtered through the PTFE hydrophilic filter membrane, and the retentate on the PTFE hydrophilic filter membrane is dried and weighed.

[0049] The PTFE hydrophilic filter membrane was dried and weighed. Figure 2 , corresponding to the swimming crab in the figure.

[0050] Example 4

[0051] (1) Collection and homogenization of biological tissues: Crayfish samples collected from the wild or purchased from the market were placed in an ice box and brought back to the laboratory and stored in a -20°C refrigerator. The surface of the crayfish was washed with ultrapure water to remove any adhering particles. The crayfish soft tissue was cut with scissors and crushed with a homogenizer, and then 2 g of the crushed tissue was placed in a 250 mL glass beaker, and three parallel samples were taken.

[0052] (2) Sample digestion: Add 30 mL of 0.063 mol / L hydrochloric acid solution containing 0.5% pepsin (w / v) to the beaker of step (1), shake at 37°C for 3 hours, then add 20 mL of 30% H2O2 (w / v) solution, shake at 40°C for 12 hours, and then vacuum filter with a PTFE hydrophilic filter membrane.

[0053] (3) Density separation: The PTFE hydrophilic filter membrane obtained in step (2) was transferred to a 250 mL beaker and 30 mL of a 1.5 g / cm 3 NaI solution (178g NaI is dissolved in 180mL ultrapure water at 20°C), after ultrasonic treatment at 40KHz in an ultrasonic cleaner for 15 minutes, the PTFE hydrophilic filter membrane is repeatedly rinsed three times with the above NaI solution while controlling the total solution volume not to exceed 50mL and the PTFE hydrophilic filter membranes are taken out one by one, the solution in the beaker is transferred to a 60mL separatory funnel and allowed to stand for 30 minutes, the lower layer solution (accounting for about 90% of the total volume) is vacuum filtered through the PTFE hydrophilic filter membrane, and the filtrate can be recycled; the upper layer solution (accounting for about 10% of the total volume) is poured out from the upper opening, vacuum filtered through the PTFE hydrophilic filter membrane, and the retentate on the PTFE hydrophilic filter membrane is dried and weighed.

[0054] The PTFE hydrophilic filter membrane was dried and weighed. Figure 2 , corresponding to the crayfish in the figure.

[0055] Comparative Example 1

[0056] (1) Collection and homogenization of biological tissues: Clam samples collected from the wild or purchased from the market were placed in an ice box and brought back to the laboratory and stored in a -20°C refrigerator. The surface of the clams was washed with ultrapure water to remove any adhering particles. The soft tissue of the clam was cut with scissors and crushed with a homogenizer, and then 2 g of the crushed tissue was placed in a 250 mL glass beaker, and three parallel samples were taken.

[0057] (2) Sample digestion: Add 40 mL of 30% (w / v) HNO3 solution to the beaker in step (1), shake at a constant temperature of 40°C for 12 hours, and then perform vacuum filtration using a PTFE hydrophilic filter membrane. The retentate on the PTFE hydrophilic filter membrane is dried and weighed.

[0058] The PTFE hydrophilic filter membrane was dried and weighed. Figure 3 , corresponding to HNO3 in the figure.

[0059] Comparative Example 2

[0060] (1) Collection and homogenization of biological tissues: Clam samples collected from the wild or purchased from the market were placed in an ice box and brought back to the laboratory and stored in a -20°C refrigerator. The surface of the clams was washed with ultrapure water to remove any adhering particles. The soft tissue of the clam was cut with scissors and crushed with a homogenizer, and then 2 g of the crushed tissue was placed in a 250 mL glass beaker, and three parallel samples were taken.

[0061] (2) Sample digestion: Add 40 mL of 30% (v / v) H2O2 solution to the beaker in step (1), shake at a constant temperature of 40°C for 12 hours, and then perform vacuum filtration using a PTFE hydrophilic filter membrane. The retentate on the PTFE hydrophilic filter membrane is dried and weighed.

[0062] The PTFE hydrophilic filter membrane was dried and weighed. Figure 3 , corresponding to H2O2 in the figure.

[0063] Application Example 1 Raman Spectrum Identification and Statistics

[0064] The intercepted material obtained by the method of Example 1 was subjected to Raman spectroscopy identification and statistics, and the specific steps were as follows:

[0065] (1) Collection and homogenization of biological tissues: Clams collected from the wild or purchased from the market were placed in an ice box and brought back to the laboratory and stored in a -20°C refrigerator. The surface of the clams was washed with ultrapure water to remove any adhering particles. The muscles or intestines were cut with scissors and the tissues were crushed with a homogenizer. Then 2 g of the crushed tissue was placed in a 250 mL glass beaker for the subsequent plastic extraction step.

[0066] (2) Sample digestion: Add 30 mL of 0.063 mol / L hydrochloric acid solution containing 0.5% pepsin (w / v) to the beaker of step (1), shake at 37°C for 3 hours, then add 20 mL of 30% H2O2 (w / v) solution, shake at 40°C for 12 hours, and then vacuum filter with a PTFE hydrophilic filter membrane.

[0067] (3) Density separation: The PTFE hydrophilic filter membrane obtained in step (2) was transferred to a 250 mL beaker and 30 mL of a 1.5 g / cm 3 NaI solution (178g NaI is dissolved in 180mL ultrapure water at 20°C), after ultrasonic treatment in an ultrasonic cleaner for 15 minutes, the PTFE hydrophilic filter membrane is repeatedly rinsed three times with the above NaI solution while controlling the total solution volume not to exceed 50mL and the PTFE hydrophilic filter membranes are taken out one by one, the solution in the beaker is transferred to a 60mL separatory funnel and allowed to stand for 30 minutes, the lower layer solution (accounting for about 90% of the total volume) is vacuum filtered through the PTFE hydrophilic filter membrane, and the filtrate can be recycled; the upper layer solution (accounting for about 10% of the total volume) is poured out from the upper opening, vacuum filtered through the PTFE hydrophilic filter membrane, and the retentate is detected by Raman spectroscopy.

[0068] (4) Raman spectroscopy identification and statistics: The retentate of the upper layer solution vacuum filtered onto the PTFE hydrophilic filter membrane is dried and examined under a microscope. Suspected plastic particles are selected for Raman spectroscopy identification and the Raman spectra before and after digestion are compared (see Figures 4 to 8 ) and microscopic image analysis (see Figures 9 to 12 ).

[0069] From the above results, it can be seen that the digestion method provided by the present invention can efficiently digest biological tissues and will not have a significant impact on the physicochemical characteristics of degradable plastics. However, the use of traditional single strong acid or hydrogen peroxide for digestion will result in incomplete digestion and a significant impact on the optical characteristics and morphology of degradable microplastics during the digestion process. The present invention solves these problems and can quickly, efficiently and with high fidelity extract degradable microplastics from biological tissues while minimizing the impact on plastics. It is convenient for accurately analyzing the types and abundance of degradable microplastics in biological tissues, and has broad application and promotion prospects.

[0070] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for extracting degradable microplastics from biological tissues, characterized in that: The method comprises the following steps: (1) Washing the biological tissue, cutting it into pieces and crushing it to obtain the crushed biological tissue for later use; (2) Take an appropriate amount of the above-obtained disrupted biological tissue, then add a 0.063 mol / L hydrochloric acid solution containing 0.5% pepsin by mass volume, add a 30% H2O2 solution by mass volume after the first oscillation, and after the second oscillation, vacuum filter with a PTFE hydrophilic filter membrane; (3) The PTFE hydrophilic filter membrane obtained above was immersed in a 3 The PTFE hydrophilic filter membrane is then rinsed with the NaI solution, the obtained solution is allowed to stand, and the upper layer solution is vacuum filtered through the PTFE hydrophilic filter membrane to obtain a retentate; (4) Drying and microscopically inspecting the intercepted material obtained above, selecting suspected plastic particles for Raman spectroscopy detection, obtaining an identification spectrum, identifying microplastics according to the identification standards, and then counting and statistically analyzing microplastics of different materials.

2. The method according to claim 1, characterized in that The biological tissues in step (1) include soft tissues, muscles and visceral tissues of fish, crustaceans, bivalves and molluscs.

3. The method according to claim 1, characterized in that The first shaking conditions in step (2) are: temperature 37° C., time 3 h.

4. The method according to claim 1, characterized in that: The second oscillation conditions in step (2) are: temperature 40°C, time 12h.

5. The method according to claim 1, characterized in that The pore size of the PTFE hydrophilic filter membrane in step (2) is 1 μm.

6. The method according to claim 1, characterized in that The density in step (3) is 1.5 g / cm 3 The NaI solution was prepared as follows: 178 g NaI was dissolved in 180 mL ultrapure water at 20 °C.

7. The method according to claim 1, characterized in that The ultrasonic treatment conditions in step (3) are: frequency 40 KHz, time 10-15 min.

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