A size-based hierarchical extraction and identification method for microplastics in bivalve molluscs in deep-sea methane seeps

Abstract

The present invention discloses a method for the hierarchical progressive extraction and identification of microplastics in bivalve molluscs in deep-sea methane seepage areas. The method proposed in the present invention freeze-dries and dehydrates biological tissues, uses a pH-phase enhanced enzyme-hydrogen peroxide mixed digestion solution, hierarchical progressive vacuum filtration, size-based advantage identification and other experimental steps to extract microplastics contained in bivalve molluscs in extreme environments non-destructively and in a classified manner, with the aim of achieving quantitative and qualitative analysis of the full-size microplastics in the bivalve molluscs within a size range. The present invention is suitable for exploring characterization methods for the enrichment of microplastics in various tissues of bivalve molluscs in deep-sea extreme ecosystems, and is combined with captured seabed shellfish beds to further deduce the time course and historical accumulation of microplastics absorbed by the entire extreme ecosystem, thereby solving the problem of large-scale sampling, extraction and analysis in deep-sea extreme environments, and providing a scientific inspection strategy for the impact of the history of microplastic pollution in specific sea areas.

Description

Technical Field

[0001] The present invention relates to the field of biological anatomy, extraction and detection technology of new pollutants in deep-sea extreme environments, and specifically to a method for size-based hierarchical progressive extraction and identification of microplastics in bivalve molluscs in deep-sea methane seepage areas. Background Art

[0002] Deep-sea extreme environments refer to areas deep in the ocean, usually at depths of more than 1,000 meters. These areas are different from conventional water environments and are characterized by high pressure, low temperature, near darkness, poor nutrients, and extreme chemical conditions (such as methane or hydrogen sulfide concentrations). This unique environment breeds various special seafloor habitats such as deep-sea plains, seamounts, hydrothermal fluids, cold springs, and abysses. However, these special extreme habitats all have different accumulation mechanisms for intercepting pollutants. They either gather pollutants contained in the bottom water through the hydrodynamic force provided by the thermohaline circulation, or increase the load gravity of pollutants through biological etching, or condense pollutants through chemical mineralization reactions to form a combined barrier, or due to the local phase pressure difference formed by strong upwelling, force and concentrate pollutants. All of these constitute the complex ocean currents and pollutant distribution control mode of the deep-sea extreme environment. The average depth of the global ocean is more than 3,600 m, and more than 90% of the sea area is located in the deep-sea extreme environment. As the main inlet of surface ocean currents, the continental shelf coastal currents and the subducting currents at the river estuaries transport the land pollutants to the edge of the ocean. When the surface ocean currents sink and converge in the continental slope area, the gravity flow converges into the ranks of the seafloor turbidity flow through the unsealing of the terrain, transporting the surface and middle seawater pollutants to the deep seabed. At the same time, the sea surface wind friction and the Coriolis force drive the surface ocean currents to the deep sea, and the Langmuir turbulence induced by the wind and waves at sea controls the rate of vertical transport of surface pollutants to the water column. On the other hand, as the near-bed shear stress increases, the surface pollutants of the sediments will be reactivated and transported as the circulating bed load. The pollutants are resuspended in the bottom seawater and participate in the water mass cycle of the ocean circulation. The ancient water masses in the deep sea have served as a bridge to connect the extreme habitats of the seabed and the material transport of the surface and middle ocean currents. The concentration of accumulated pollutant particles, such as plastic microparticles, is activated and recycled in this system for decades. Therefore, the extreme deep-sea environment is more likely to form a long-term accumulation of plastic microparticles.

[0003] Microplastics are defined as plastic particles of 0.0001 to 5 mm, which are the main carriers of marine plastic pollution. Every year, more than 10 million tons of plastic products enter the global ocean. Plastics in seawater are broken and cracked into small-sized microplastics through light radiation, mechanical stress wear, biological fouling, physical erosion, etc. However, the plastic particles accumulated on the sea surface only account for about 1% of the predicted list of marine plastics, and the remaining 13.5% enter the deep-sea extreme environment in the form of microplastics. Most of these microplastics that enter the extreme environment have sparse and porous structures on the surface, and those with a size of ≤50 μm account for more than 50% of the total abundance of microplastics in seawater. A small amount of large-sized (≥1 mm) microplastics that traverse the impact of ocean currents but rarely break are almost all made of extremely flexible polyamide. In the vast seabed, smaller-sized (<250 μm) microplastics are more easily captured by extreme environments. The bottom water microplastics in extreme environments are enriched over a long period of time and gradually form a size grade mechanism with distinct particle size ranges. The first size class is generally less than 50 μm, followed by 50-100 μm, 100-250 μm, 250-500 μm, 500-1000 μm, and 1000-5000 μm. The first three size classes almost cover 80% of the total microplastic size. However, the current instrumental methods for identifying microplastic types have their own advantages in identification areas, and it is impossible to use a single device to fully detect the collected suspected microplastics. In order to quantify small-sized microplastics in extreme deep-sea environments, the suspected microplastics can be completely extracted by size class, which has become an alternative census of microplastic extraction innovations in the full size range.

[0004] Deep-sea methane seepage areas are a special case of deep-sea extreme environments, usually found in continental margins, submarine fault zones and hydrate burial areas. The formation of methane seepage areas depends on the sudden changes in the seafloor fluid environment or geological tectonic activities, which causes the generated gaseous methane to accumulate in the sediment pores and leak upward through methane channels. At the same time, the sulfate-methane conversion zone (SMTZ) composed of methane anaerobic oxidizing bacteria and sulfate-reducing bacteria in the sediment rock layer effectively weakens the upwelling methane gas flow, accompanied by the escape of a large amount of metal elements and hydrogen sulfide gas, forming a specific cold seep fluid eruption leakage area on the seafloor. Unlike hydrothermal vents, the temperature of cold seep fluid is slightly lower than that of the surrounding seawater. The nutrients delivered by the primary producers in the rock formation provide carbon sources and element supply for the chemosynthesis of methane seep areas. The continuously leaking cold seep fluid nourishes a large class of indicator organisms in the methane seep area - bivalves, thus gradually forming a special chemosynthetic ecosystem with methane and sulfide as special carbon sources and redox energy supply. Methane seepage areas are also one of the hotspots for the accumulation of microplastics in seawater. In addition to the large number of cold spring plumes that form a vacuum layer to gather seawater microplastics around them and the support of methane bubbles that migrate seabed microplastics to the shallow layer, bivalves living in methane seepage areas also absorb microplastics in the bottom water through filter feeding and mucus capture mechanisms, and the microplastics are thus transferred into the digestive system of organisms. Therefore, when assessing the risk of deep-sea extreme ecosystems being infected by microplastics, bivalves are often used as model research organisms to measure the degree of microplastic accumulation.

[0005] In the methane seepage area where violent methane plumes continue to erupt, bivalve molluscs, as benthic organisms, engulf the bottom water flow through the villi on their gills, and continuously absorb the suspended matter (including microplastics) in the water flow into the gill filter. The cold spring ecosystem with multiple microplastic enrichment functions is a potential plastic sink for both seawater and sediments, and can play the role of reintegrating the carbon in microplastics into the marine carbon pool and storing it. At the same time, the gills and mantles of shellfish secrete mucus due to life activities to irregularly capture particulate organic matter in the filter. Most of the particulate microplastics will be retained in the gills, but a small part of the captured microplastics will enter the gastric tissue along with the organic matter and be initially biofouled. Since microplastic particles are difficult to digest and absorb, most of the microplastics in the gastric tissue are transferred to the intestine and gradually worn into smaller microplastics by peristalsis. Plastics that have reached the nanoscale will enter the blood along with organic matter through the absorption process of the digestive system, and then migrate to other tissues and organs and repolymerize into microplastics through the mediation of proteins in the tissues. Microplastics accumulated in the intestine will eventually form some pseudo-feces to be excreted from the body or continue to be adsorbed in the wrinkled intestinal cells. Tracing the migration routes of microplastics in bivalve shellfish, most studies have shown that the microplastic particles ingested into the body are mainly enriched in tissues such as gills, digestive system (stomach and intestines), mantle and hepatopancreas. The gills and digestive system are the key parts for the primary capture and transportation of microplastics, while the hepatopancreas and mantle are the sites of microplastic ablation and deposition. The accumulation and abundance of microplastics in various tissues of bivalve shellfish is affected by size. Among shellfish tissues, the hepatopancreas / intestines are considered to be the final decomposition sites of microplastics. The accumulation of microplastics in these parts can rise rapidly in a short period of time. In addition to the unconscious transportation of the digestive system, small-sized microplastic particles will also be self-decomposed and generated, forming multiple superpositions of local abundance. The distribution pattern of microplastic content in various tissues is similar to hepatopancreas / intestine>gill>mantle>gonad>lip>foot>adductor muscle, but not all types of bivalves in the methane seepage area have similar microplastic adsorption capabilities. Deep-sea mussels that can mobilize more ciliated cells on the gills to move together have the characteristics of adsorbing more benthic water microplastics due to their high filter feeding efficiency. Phoenix mussels that can secrete more viscous mucus to prey on suspended particles have the properties of more effective capture of microplastic particles due to the amount of mucus secreted. White melon mussels that can use methane strong seepage vents as their dominant ecological niche have the specific ability to gather microplastics in the surrounding water environment due to the formation of gas phase vacuum by cold spring fluid. The microplastics originally endowed in the extreme deep-sea environment are mainly small in size. After the strong fluid disturbance in the methane seepage area and the filter feeding adsorption of bivalves, the size range of microplastics in shellfish is further reduced, and most of them are mainly <250 μm. The extraction scheme of small and medium-sized microplastics in biological tissues urgently needs higher detection requirements.At present, the schemes for extracting microplastics from the biological tissues of bivalve molluscs usually only refer to molluscs in ordinary water environments or shallow sea farming, and do not involve discussions of bivalve molluscs in extreme deep-sea environments. However, bivalve molluscs bred in extreme environments have already developed highly specialized tissues and organs, such as feet that have evolved the ability to sense methane fluids and withstand shocks; the visceral mass has developed a reinforced muscle support structure to cope with the high-pressure environment of the seabed, causing the stomach and digestive glands in the tissue to twist and become indistinguishable; the density of the extracellular matrix between tissues has increased to maintain the cell morphology under high pressure, resulting in the inability to achieve a normal digestion state when the digestion scheme for ordinary bivalve molluscs is applied to molluscs in extreme environments, resulting in low efficiency in extracting microplastics, and leading to incorrect cognition and understanding of the abundance of microplastics accumulated in bivalve tissues in methane seepage areas.

[0006] By reading a large number of literature on the extraction of microplastics from bivalve molluscs bred in ordinary water environments, scholars generally use alkaline digestion to digest and filter the dissected bivalve mollusc tissues. The steps for extracting and identifying microplastics are as follows:

[0007] ① Sample preservation: Use ultrapure water to clean the surface of the captured bivalve molluscs to remove salt, place the cleaned bivalve molluscs in a clean insulated glass jar, seal it with ethanol solution and return it to the laboratory for disposal in time;

[0008] ② Sample pretreatment: Use ultrapure water to repeatedly rinse the surface of bivalve shellfish to remove adsorbed impurities and mud. Separate the viscera, gills, muscle bundles and other tissues on the dissection table, and wash all the separated soft tissues with ultrapure water for later use;

[0009] ③ Homogenate freeze-drying: Combine and mix tissues from different dissected parts, use a blender to grind them evenly to prepare tissue homogenate, and freeze-dry the homogenate to obtain a freeze-dried powder sample;

[0010] ④ Constant temperature digestion: Weigh a fixed mass of freeze-dried powder sample, add digestion solution (such as potassium hydroxide solution, sodium hydroxide solution, protease solution, hydrogen peroxide solution, nitric acid-perchloric acid solution, etc.), set the digestion temperature to 40 ~ 60℃ and shake at a constant temperature for 24 hours;

[0011] ⑤ Vacuum filtration: Filter the digestion solution with a stainless steel mesh / nitrocellulose filter membrane. After repeated filtration, use ultrapure water or organic reagents (such as carbon tetrachloride, toluene, etc.) to continuously clean the adsorbed particles on the mesh / filter membrane;

[0012] ⑥ Organic extraction: Place the sieve / filter membrane in an organic solvent for extraction, use ultrasonic vibration to elute the attached microplastics, and rinse with the corresponding solvent to collect the evenly dispersed microplastic suspension;

[0013] ⑦ Vacuum filtration: The concentrated microplastic suspension is vacuum filtered into a new nitrocellulose filter membrane and placed in an oven to dry for 3 hours to obtain a filter membrane sample enriched with microplastics;

[0014] ⑧ On-machine identification: Place the filter membrane under a magnifying glass / microscope, visually select suspicious microplastics and transfer them to a new membrane, and record the color, size, shape and other morphological information of the microplastics. Then load the suspicious microplastics into the sample chamber of the FTIR and enable the total reflection mode to identify its type and functional group information.

[0015] At present, most of the detection methods for analyzing microplastics in bivalve molluscs bred in common aquatic environments are as shown above, and have the following disadvantages: ①Use ultrapure water or organic solutions to clean or seal bivalve molluscs and their biological tissues. Bivalve molluscs living in extreme deep-sea environments have adapted to high-pressure and high-salt ecological conditions. Using ultrapure water to clean biological samples may cause a strong change in their intracellular osmotic pressure. The salts continuously precipitated from the cells will also promote the expansion of microplastic particles in the intercellular space. The intercellular matrix is ​​dredged by a large amount of water, and the tissue structure is damaged. Microplastics may escape into the external environment through the damaged tissue in advance, creating a scientific illusion of low abundance. Using organic solvents to clean and preserve will interfere more with the structure and morphology of microplastics in the tissue. Certain organic solvents (such as acetone) will not only dissolve the fluorescent additives on the surface of microplastics, but also cause severe dehydration and protein denaturation in biological tissue cells. The microplastics in between will be subjected to strong external extrusion force and will be more easily broken to form smaller microplastics or nanoplastics, resulting in a macroscopic phenomenon of high abundance; ② Other patents often improve the efficiency of digesting organic matter by grinding / mechanically crushing to obtain tissue homogenate. The biggest disadvantage of this method is that it destroys the fibrous microplastics adsorbed in biological tissues, and converts the fibrous microplastics into granules by severe abrasion, and cannot preserve their original form. Secondly, the introduction of excessive mechanical structure leads to a high recovery rate loss when collecting tissue homogenate. It may also cause the broken single type of nano-scale plastics to re-polymerize into mixed microplastics due to frequent shear stress. The morphology and composition of the microplastics after pretreatment are both very different from the in-situ environment, affecting the subsequent external characterization statistical results of microplastics in the organism; ③ Use alkaline digestion solution or single enzyme solution to digest biological tissue to obtain separated microplastics. Alkaline digestion solution alone and its combined digestion solution will dissolve specific types of microplastics. For example, 10% KOH solution will corrode PC and PET polymers, and 10%~20% NaOH solution will also degrade microplastics such as PET, PU and PC. According to the existing experimental data on the extraction of microplastics from seawater in methane seepage areas, the bottom seawater stored in cold spring fluids is rich in PU, PA, PET, PE and other difficult-to-degrade and corrosion-resistant microplastics. The strong alkaline digestion solution used to extract microplastics from biological tissues in ordinary water environments or sea areas is not applicable in extreme deep-sea environments, and also adds a serious risk of plastic loss. In addition, although the use of a single enzyme solution can selectively decompose the cell membrane and protein support structure of biological tissues, and extract microplastics in tissues almost non-destructively, the slow enzymatic reaction leads to a long time for digesting cells, which in turn increases the experimental cost in disguise and is not convenient for promotion and application. The bivalve molluscs in the methane seepage area have a harder and more complex cell protection structure, and several measures to increase the reaction rate are needed to accelerate the enzymatic reaction; ④ There is no comprehensive extraction solution for microplastics in different size ranges.In the original literature, only one or two spectroscopic instruments were used to identify the extracted suspicious microplastics. The advantage of these spectroscopic instruments in identifying microplastics is fixed. For example, the detection limit of FTIR is 200 μm, and Raman focuses on the identification of extremely small microplastics of less than 20 μm. Due to the limitation of a small number of detection methods, it is impossible to accurately obtain the comprehensive distribution range of microplastics, so there is almost no need to extract them separately by size. When facing the special habitat of methane seepage area, the distribution range of microplastics in organisms is relatively wide, mainly concentrated in 0.1~100 μm, but the remaining large-sized microplastics of more than 1 mm also exist in small quantities. If the previous single instrument identification can only clarify some of the microplastics of the advantage identification size, in order to improve the overall recovery rate of microplastics in biological tissues in extreme deep-sea environments, it is urgent to establish a multi-level extraction scheme with appropriate steps in the size range to maximize the recovery of microplastics in each size range. Summary of the invention

[0016] The present invention solves the problems existing in the prior art and provides a method for the size-based hierarchical extraction and identification of microplastics in bivalve molluscs in deep-sea methane seepage areas. The method proposed in the present invention can ignore the cell structure enhancement given by extreme environments and achieve the precision of non-destructive digestion and extraction of full-size microplastics in tissues. In addition, microplastics in various size ranges can be split out, and spectral instruments with corresponding advantages can be tailored to identify them, thereby reducing the degree of degradation and fragmentation of microplastics during the extraction and identification process. At the same time, further microplastic aging test steps in the future are taken into account to achieve the non-destructive detection requirements of biologically adsorbed microplastics in deep-sea extreme environments, and provide experimental technical method support for the identification of the interaction relationship between deep-sea environmental ecosystems and benthic indicator organisms, and between the life activities of organisms and microplastics.

[0017] The purpose of the present invention is to provide a method for the size-based hierarchical extraction and identification of microplastics in bivalve molluscs in deep-sea methane seepage areas, comprising the following steps:

[0018] S1. Cryopreservation: The captured bivalve molluscs are cryopreserved;

[0019] S2, tissue pretreatment: the bivalve molluscs frozen and stored in step S1 are thawed at a constant temperature and airtightly dissected to obtain various biological tissues of the bivalve molluscs, and the various biological tissues of the bivalve molluscs are freeze-dried and set aside for use;

[0020] S3, pre-digestion with enzyme solution: adding trypsin solution for digestion to various biological tissues of bivalve molluscs obtained in step S2 to obtain a primary digestion solution of enzyme solution with a large number of tissue filamentous coagulation bodies;

[0021] S4, pH staged enhancement: using a pH adjusting solution to adjust the pH of the primary digestion solution of the enzyme solution obtained in step S3 to 7.5, and then adding a trypsin solution to the enzyme digestion solution several times to accelerate the disintegration of the filamentous coagulants to obtain a secondary digestion solution of the enzyme solution, wherein the volume of the trypsin solution added each time is 1 / 3-1 / 4 of the volume of the trypsin solution for digestion in step S3;

[0022] S5, 30% hydrogen peroxide digestion: pick out the filamentous coagulation bodies in the secondary digestion solution of the enzyme solution obtained in step S4, add a 30% by mass fraction hydrogen peroxide solution to the filamentous coagulation bodies, and digest the residual cell adhesions at 60°C-65°C for 2-4 hours until no bubbles are generated in the digestion solution, that is, the digestion is completely completed to obtain a nearly transparent solution without obvious suspended matter, thereby obtaining a hydrogen peroxide digestion solution;

[0023] S6, progressive extraction of mixed digestion solution by size level: the enzyme solution secondary digestion solution obtained in step S4 and the hydrogen peroxide digestion solution obtained in step S5 are mixed, and a first size level extraction is performed to enrich all target objects larger than 10 μm on the filter membrane, thereby obtaining a first size level extraction filter membrane, and then the first level filtrate is subjected to a second size level extraction to enrich all target objects of 1-10 μm on the filter membrane, thereby obtaining a second size level extraction filter membrane, and then the second level filtrate is subjected to a third size level extraction to enrich all target objects of 0.1-1 μm on the filter membrane, thereby obtaining a third size level extraction filter membrane;

[0024] S7, the microplastics in the first size-level extraction filter membrane, the second size-level extraction filter membrane and the third size-level extraction filter membrane obtained in step S6 are sequentially subjected to characteristic observation, microscopic imaging and component spectral detection to obtain size-based abundance information of full-size microplastics in various biological tissues in bivalve molluscs

[0025] In order to obtain the size-grade variation characteristics of microplastics in cold spring indicator organisms in methane seepage areas, the present invention adopts a pH-phase enhanced enzyme-hydrogen peroxide mixed digestion method combined with a hierarchical progressive extraction scheme to build a set of extraction methods that are completely suitable for microplastics in bivalve molluscs in deep-sea extreme environments. This method targets the morphological characteristics of bivalve molluscs in methane seepage areas, prepares seawater close to the properties of cold spring fluids to replace the cleaning solution that changes the osmotic pressure difference, abandons the cumbersome and easy-to-lose mechanical homogenization steps, and uses a complete digestion scheme that enhances biochemical reactions to replace the traditional semi-digestion steps. The improved extraction scheme can completely release microplastics in complex organisms and reduce the microplastic degradation effect caused by chemical reagents. At the same time, the size-graded hierarchical extraction method in the filtration operation is combined with the advantages of multiple identification instruments to optimize the method for extracting full-size microplastics in bivalve molluscs in extreme environments and its matching identification scheme.

[0026] Compared with the shortcomings of existing extraction technologies based on ordinary water environments: cleaning shellfish tissues with ultrapure water / organic solvents, mechanical grinding to obtain tissue homogenate, and alkaline digestion method / single enzyme solution to digest tissue cells, the present invention starts from the physical characteristics of bivalve molluscs bred in methane seepage areas, and has completed comprehensive optimization and adjustment in terms of preservation and cleaning, tissue pretreatment, digestion process and filtration extraction. According to the advantageous identification ranges of various existing identification instruments, microplastics are extracted at different sizes to show the distribution range of full-size microplastics contained in organisms in special habitats. The purpose is to complete the basic research on the existence of microplastics in extreme deep-sea environments and being engulfed by bivalve molluscs occupying ecological niches, reduce the loss, fragmentation damage and ablation effect of microplastics caused by the extraction of microplastics from biological tissues in ordinary water environments, and ensure to the greatest extent that the extracted morphology of microplastics is consistent with the existing state in the in-situ environment. At the same time, a size-based hierarchical extraction step of microplastics is added to obtain the path characterization of the migration and digestion state of microplastics in bivalve molluscs. The microplastic size-based hierarchical extraction and identification method proposed in the present invention can effectively explore the efficiency of bivalve molluscs in feeding when cold seep fluid gushes in methane seepage areas. The abundance of microplastics in various tissue parts obtained by this method can simulate and restore the in vivo circulation route of microplastics, understand the biological damage caused by them, and further clarify the rate at which deep-sea extreme ecosystems absorb microplastics in space. This is an integrated detection-identification method for capturing and extracting microplastics in biological tissues covering the entire size range of 0.0001~5 mm.

[0027] Preferably, the specific steps of step S1 are: selecting intact bivalve molluscs, washing them multiple times until the sediment clay adsorbed on the surface is washed off, and then putting the washed bivalve molluscs into a sterile container and freezing them at -20° C. The bivalve molluscs proposed in the present invention include mussel beds, phoenix clams, white melon clams, and the like.

[0028] Further preferably, the specific steps of step S1 are:

[0029] S11. Sampling: Use a TV grab on the expedition ship to grab the bivalve shell beds (such as mussel beds, Phoenix shell beds, white melon shell beds, etc.) that have been located in the cold spring seepage area;

[0030] S12. Seawater washing: Use the onboard cruise water device to absorb the surface seawater, select intact shellfish, and wash the captured shellfish several times until the sediment clay adsorbed on the surface is washed off;

[0031] S13. Cryopreservation: Put the cleaned shellfish into sterile ziplock bags, number them, and store them in a -20℃ freezer.

[0032] Preferably, the specific steps of step S2 are: thawing the bivalve molluscs frozen and stored in step S1 at a constant temperature of 4°C for 0.5-1.5 h, and then dissecting them in a closed manner to obtain various biological tissues of the bivalve molluscs, washing the various biological tissues of the bivalve molluscs with a cleaning solution to remove seawater microplastics and various impurities attached to the outside of the shellfish tissue cells, and then freezing and storing them at -80°C for 5-7 h, and freeze-drying them at -60°C for 20-28 h for standby use.

[0033] Further preferably, the cleaning solution is prepared by the following steps: 5.04 g sodium chloride powder, 0.06 g barium chloride powder, 0.12 g ferrous sulfate powder, 0.02 g manganese sulfate powder, 1.2 g magnesium chloride powder, 0.4 g potassium chloride powder, 0.4 g calcium chloride powder, 2.6 mg sodium nitrate powder, 22 mg sodium silicate powder, 1 mg sodium dihydrogen phosphate powder and 2.5 g sodium sulfate powder are added to 800 mL of ultrapure water, 23.42 mL of concentrated hydrochloric acid is added dropwise, and the mixture is continuously shaken and stirred, and then the volume is fixed to 1 L, and filtered several times using a 0.1 μm water microporous filter membrane to obtain the cleaning solution.

[0034] Further preferably, the specific steps of step S2 are:

[0035] S21. Constant temperature thawing: Select the bivalve molluscs to be tested from a -20℃ refrigerator in advance and place them in a 4℃ refrigerator to thaw for 1 hour;

[0036] S22. Instrument preparation: Before the dissection experiment, all scissors, tweezers, aluminum boxes, and carriers must be ultrasonically vibrated in an ultrasonic cleaning machine filled with ultrapure water filtered through a 0.1 μm water system microporous filter membrane for 30 minutes. Before the dissection begins, use the filtered ultrapure water to rinse the instruments and tin foil several times and set aside.

[0037] S23, Closed dissection: Use tin foil that has been rinsed with ultrapure water several times to lay on the dissection table, and the entire dissection table is carried out in a closed ventilated room. Take the whole bivalve sample, use a scalpel to press against the inner surface of the shell, cut off the adductor muscle to fully display the internal tissue of the shell, and use spring scissors to carefully cut off various biological tissues of the shell (visceral mass, gills, lips, intestines, feet, adductor muscle, mantle), and the dissected tissues are to be cleaned. After cleaning, the tissues are placed in a 60 mm special aluminum box, wait for freeze-drying, and keep records;

[0038] S24. Prepare a cleaning solution similar to cold spring fluid: weigh 5.04 g sodium chloride powder (AR), 0.06 g barium chloride powder (AR), 0.12 g ferrous sulfate powder (AR), 0.02 g manganese sulfate powder (AR), 1.2 g magnesium chloride powder (AR), 0.4 g potassium chloride powder (AR), 0.4 g calcium chloride powder (AR), 2.6 mg sodium nitrate powder (AR), 22 mg sodium silicate powder (AR), 1 mg sodium dihydrogen phosphate powder (AR), and 2.5 g sodium sulfate powder (AR) in sequence, add them to 800 mL of ultrapure water, add 23.42 mL of concentrated hydrochloric acid (12 mol / L, AR) drop by drop, shake and stir continuously, then make the volume to 1 L, filter three times with a 0.1 μm water microporous filter membrane, and set the filtered cleaning solution aside;

[0039] S25, cleaning and sample preparation: rinse the dissected biological tissues with filtered cleaning solution to remove seawater microplastics and various impurities attached to the outer cells of shellfish tissues. After washing three times, let it stand to drain excess water and place it in a 60 mm aluminum box for later use;

[0040] S26. Weighing wet weight: Place the aluminum box containing each shellfish tissue on an electronic analytical balance, weigh the wet weight of each tissue in the tare mode, and record the data;

[0041] S27. Cryopreservation: Place each weighed aluminum box in a -80℃ medical refrigerator for 6 h and attach a wet weight label;

[0042] S28. Freeze drying: slightly unscrew each aluminum box after freezing, and place it in a freeze dryer at -60°C for 24 h;

[0043] S29. Weigh dry weight: Place each freeze-dried shellfish tissue on an analytical balance, weigh the dry weight, and record the data.

[0044] The freeze-drying pretreatment is to remove moisture from the tissue in the form of water vapor, retain its own microplastic morphology, reduce the loss of microplastics in the sample, obtain the net dry weight of the sample, and also increase the subsequent digestion rate of cell membranes and organic matter.

[0045] The existing technology is still confined to the optimization extraction steps of various reagents for bivalve molluscs that reproduce in ordinary water environments such as shallow seas or rivers and lakes. There is little explanation and discussion on the hierarchical extraction scheme of bivalve microplastics by size, and there is even less of a lack of restrictions on the digestion efficiency of bivalve molluscs in the deep-sea extreme environment on the strengthening of bivalve cell connective tissue and extracellular matrix. The preservation and adaptive cleaning of shellfish samples in deep-sea extreme environments are prerequisites for measuring the correctness of microplastic extraction experiments. As benthic indicator organisms in methane seepage areas, bivalves have been constantly devouring microplastics resuspended in surrounding seawater and sediments since the beginning of their survival. These microplastics are etched by methane gas produced by the decomposition of buried hydrates in rock formations, and are more easily cracked into microplastics or even nanoplastics within a small size range. The full-size microplastics hierarchical extraction scheme of the present invention is of great significance for understanding the long-term dynamic enrichment of microplastics in bivalve molluscs, evaluating the pollution level of microplastics in deep-sea extreme environments, and identifying the accumulation level of microplastics enriched in extreme environments within a hundred years and their migration and transformation process in the ocean.

[0046] The present invention reconstructs the properties of cold spring fluid solutions that are suitable for the survival of bivalve mollusks and uses them for storage and cleaning steps, reducing the occurrence of cell rupture and swelling when the osmotic pressure is strongly unbalanced due to improper salt solutions or ultrapure water; at the same time, it abandons the operation of mechanical grinding to prepare a homogenate used in ordinary literature, and the simple and effective freeze-drying and enhanced enzyme combination digestion solution is sufficient to ensure that the super-large-sized microplastics adsorbed by tissue cells are preserved in situ without loss; the pH-programmed enhanced enzyme solution is used to pre-digest large tissue aggregates to remove the restrictions of connective tissue and intercellular matrix filaments enhanced by the high-pressure environment, and hydrogen peroxide is used as the final disruptor of filamentous coagulants to maximize the restoration of the types and surface morphology of microplastics adsorbed by shellfish in the in-situ environment, so as to fully reproduce the original state of microplastic pollution of seabed shellfish; the pioneering biological tissue full-size microplastic size-level extraction scheme combines the advantageous recognition intervals of multiple identification instruments, cancels the connection between instruments, and drives all suspected microplastics to be classified and multi-channel at the same stage to start detection, greatly shortening the instrument time cost.

[0047] The present invention aims to explore the accumulation abundance of microplastics in organisms represented by bivalve molluscs in methane seepage areas in deep-sea extreme environments, as well as the migration routes of microplastics under the stress of life activities. By catching bivalve molluscs on the seabed in groups and coupling the survival timeline of each mollusc, the microplastic adsorption rate of extreme ecosystems over time can be further calculated, and the enrichment degree of methane seepage areas as microplastic hotspots can be clarified. The beginning of all explorations comes from the full-scale microplastic hierarchical extraction and identification integrated solution proposed by the present invention. This invention complements the lack of existing technology in the study of bivalve molluscs in extreme environments, and maintains the original cell state of the molluscs by means of self-cooling fluids. This is a field that no scholar has studied carefully yet. It provides an effective research solution for accurately quantifying the damage of human activities to deep-sea extreme systems, and has reference and technical reference for the microplastic extraction solutions of indicator organisms in other extreme waters.

[0048] Preferably, the trypsin solution described in step S3 or S4 is prepared by the following steps: weigh 6.80 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, then adjust the pH to 7.5 with 0.1 mol / L potassium hydroxide solution, add 30.00 g of trypsin, add water to dissolve it, and dilute it to 1 L to obtain the trypsin solution.

[0049] Preferably, the specific steps of step S3 are: adding trypsin solution for digestion to various biological tissues of bivalve molluscs obtained in step S2 at a ratio of 1 g biological tissue dry weight: 30-40 mL trypsin solution, digesting at 35°C-40°C for 24-30 h, and obtaining an enzyme solution primary digestion solution with a large number of tissue filamentous coagulation bodies.

[0050] Further preferably, step S3 specifically comprises the following steps:

[0051] S31. Prepare trypsin solution: weigh 6.80 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, adjust the pH to 7.5 with 0.1 mol / L potassium hydroxide solution, add 30.00 g of trypsin, dissolve it in water, dilute to 1 L, filter three times with a 0.1 μm polyvinylidene fluoride filter membrane, and place in a -20°C refrigerator for use;

[0052] S32. Constant temperature digestion: Depending on the dry weight of the tissue, each freeze-dried tissue is placed in a 50 mL or 100 mL cleaned conical bottle with a stopper, and a quantitative amount of trypsin solution for digestion is added at a ratio of 1 g tissue dry weight: 30-40 mL trypsin solution, and digested at 35℃-40℃ for 24-30 h at a constant temperature shaker at 100 rpm. Take it out every 4 hours and put it under a magnetic stirrer to accelerate mixing, and finally obtain an enzyme solution with more tissue filamentous coagulation bodies. At this time, most of the shellfish tissues are broken by the enzyme, and the microplastics are released into the digestion solution, but some microplastics adhering to the filamentous coagulation bodies have not yet been separated.

[0053] Preferably, the pH adjusting solution in step S4 is a potassium dihydrogen phosphate solution with a mass concentration of 0.0136 g / mL or a potassium hydroxide solution with a mass concentration of 0.0562 g / mL.

[0054] Further preferably, the specific steps of step S4 are:

[0055] S41. Prepare pH adjustment solution:

[0056] ① Weigh 0.68 g of potassium dihydrogen phosphate, add 50 mL of water to dissolve it, shake and bottle it for later use;

[0057] ② Weigh 2.81 g potassium hydroxide, add 50 mL water to dissolve it, shake and bottle it for later use;

[0058] All the above solutions need to be filtered three times with a 47 mm, 0.1 μm polyvinylidene fluoride filter membrane before use.

[0059] S42. Adjust the pH of the enzyme solution digestion solution: After constant temperature digestion, take out the conical flask every 12 hours, measure its pH with a pH meter, draw the pH adjustment solution with a 10 mL glass syringe, shake it drop by drop and maintain the pH of the enzyme solution digestion solution at 7.5;

[0060] S43, secondary digestion with enzyme solution: 1 / 3-1 / 4 of the volume of the trypsin solution for digestion in step S3 is added to repeatedly rinse the pH meter to accelerate the disintegration of the filamentous coagulants. Each group of samples needs to be repeated 4 times to obtain a secondary digestion solution with enzyme solution.

[0061] Preferably, in step S6, the first size level extraction uses a glass filter element with a diameter of 50 mm and a stainless steel membrane with a diameter of 47 mm and a pore size of 1200 mesh, the second size level extraction uses a filter element with a diameter of 25 mm and a glass microfiber filter membrane with a diameter of 25 mm and a pore size of 1 μm, and the third size level extraction uses a filter element with a diameter of 25 mm and an inorganic alumina membrane with a diameter of 25 mm and a pore size of 0.1 μm.

[0062] Further preferably, the specific steps of step S6 are:

[0063] S61, first size (>10 μm) level extraction: Use a 1 L glass vacuum filtration device with a 50 mm diameter glass filter element for first size level filtration, and use a self-cut stainless steel membrane with a diameter of 47 mm and a pore size of 1200. At the beginning of the experiment, the device was cleaned several times with ultrapure water filtered through a 0.1 μm water system microporous filter membrane, and then the enzyme solution secondary digestion solution and hydrogen peroxide digestion solution were added to the device together, and the vacuum filtration step was repeated three times. The eluent used each time was the filtrate obtained last time. Before completing the filtration, the inner wall of the filter and the conical flask were repeatedly rinsed with the filtered ultrapure water to enrich all the target objects >10 μm on the filter membrane. The purified filter membrane was placed in a 60 mm aluminum box and dried in an oven at 60°C for 3 h, and the first size level extraction filter membrane was stored at room temperature; the filtrate in the filter bottle was to be transferred;

[0064] S62, second size (1~10 μm) layer extraction: Use filtered ultrapure water to rinse 250 mL of micro sand core filter device, the device uses a 25 mm diameter filter element with a 25 mm, 1 μm glass microfiber filter membrane. After cleaning, pour the first layer filtrate to be transferred into the filter cup for vacuum filtration, repeat the filtration three times, and use the eluent each time to obtain the filtrate. Before completing the filtration, repeatedly use the filtered ultrapure water to rinse the inner wall of the filter cup and the inner wall of the 1 L filter bottle to ensure that all particles are collected. Place the purified filter membrane in a 30 mm aluminum box and dry it in an oven at 60°C for 3 hours, and store the dried second size layer extraction filter membrane at room temperature;

[0065] S63, third size (0.1~1 μm) level extraction: The third level extraction steps are the same as the second level, except that the glass microfiber filter membrane is replaced with an inorganic alumina membrane with a diameter of 25 mm and a pore size of 0.1 μm. When the filtration is completed, a small amount of filtered ethanol solution is used to rinse the filter bottle and the inner wall of the filter cup several times, and the cleaning liquid is filtered. The obtained filter membrane is relatively fragile due to the support ring and the membrane surface. It is placed in a 30 mm glass culture dish and dried in an oven for 3 hours and then stored at room temperature for identification;

[0066] S64, extraction: Place the first-size extraction filter membrane upright in a 50 mL beaker, add about 15 mL of anhydrous ethanol solution until the liquid surface completely submerges the filter membrane surface, seal the beaker with cleaned tin foil, fix it with a rubber band, and place it in a constant temperature shaker at room temperature and oscillate at 100 rpm for 12 hours to completely release the sediment particles and suspected microplastic particles attached to the filter membrane into the solution;

[0067] S65, ethanol washing: take out the first size layer extraction filter membrane after leaching, and repeatedly wash the front and back sides of the filter membrane and the gaps at the edge of the membrane with anhydrous ethanol until no obvious particles remain on the filter membrane;

[0068] S66, filtration membrane: vacuum filter the anhydrous ethanol rinse liquid again, and follow the same filtration steps as the first size-level extraction and filtration. Replace the filter membrane with a 47 mm, 10 μm mixed cellulose filter membrane. Repeat three times, and finally wash the cup wall and container multiple times with anhydrous ethanol to enrich all suspicious microplastics on the membrane.

[0069] S67. Constant temperature drying: Place the first-size microplastic purification filter membrane obtained in a 60 mm aluminum box and dry it in an oven at 60°C for 2 hours, preparing for the next step of microplastic identification.

[0070] All the above ultrapure water, prepared solutions, reagents and digestion solutions must be filtered through a 0.1 μm water microporous filter membrane and a 0.1 μm polyvinylidene fluoride filter membrane before use to filter out impurities in the prepared solution itself. Similarly, before using glassware, beakers, tweezers, scissors and other equipment, they must be soaked in an ultrasonic bath filled with filtered ultrapure water for 30 minutes to eliminate potential impurities as much as possible. Before using the filter membrane, use tweezers to carefully soak the filter membrane in anhydrous ethanol to clean it back and forth, and finally rinse the filter membrane with a small amount of anhydrous ethanol.

[0071] Compared with the prior art process of extracting microplastics from shallow-sea bivalve samples, the present invention avoids destroying the original microplastic morphology in the tissue by preparing a cleaning solution similar to the original living environment and a non-homogenized operation method, and innovatively uses a double mixed solution of a pH-enhanced trypsin solution and 30% hydrogen peroxide to digest biological tissues. This combined digestion solution will only produce more oil debris foam, and has no negative impact on almost all types of microplastics. It has high fidelity and can extract microplastics in the body to the greatest extent without loss. At the same time, the size-based hierarchical extraction scheme is used instead of the traditional density flotation method to separate microplastics in the concentrated solution, which can completely avoid the problems of insufficient extraction efficiency, high cost, and erroneous digestion of the flotation agent, and facilitate the subsequent combined identification instrument to scan and detect microplastics point-to-point at the same time, reducing time costs.

[0072] Preferably, step S7 specifically includes the following steps:

[0073] S71. Split the first size-level extraction filter membrane into a large-size microplastic filter membrane with a particle size greater than 500 μm and a medium-size filter membrane with a particle size of 10-500 μm, perform infrared imaging on the microplastics in the large-size microplastic filter membrane with a particle size greater than 500 μm and qualitatively analyze their distribution, obtain the uniformity of the component types of the entire suspected microplastics and determine the types of these polymers, perform microscopic infrared imaging on the microplastics in the medium-size filter membrane with a particle size of 10-500 μm and qualitatively identify their components, and calculate their technical parameters, which include particle number, particle diameter, and equivalent area;

[0074] S72. Perform morphological observation, microscopic imaging and component analysis on small-sized microplastics with a particle size of 1-10 μm extracted from the second size level filter membrane, count the size, color, shape, equivalent diameter, particle number of all small-sized microplastics, and perform Raman imaging to obtain component distribution information;

[0075] S73. Conduct morphological observation, microscopic imaging and component analysis on submicron-sized microplastics with a particle size of 0.1~1 μm extracted from the third-size layer filter membrane. Count the size, color, shape, and particle number of all submicron-sized microplastics and perform microscopic imaging to obtain component distribution information.

[0076] Further preferably, step S7 also includes S74, extracting microplastics of each size level separately, and using four spectrometers including ATR-FTIR, LDIR, Raman and micro-Raman. The total amount of microplastics measured is the full-size microplastic content in a specific tissue of a single bivalve mollusc. Combined with the abundance correction in the experimental steps, the size abundance information of full-size microplastics in each biological tissue of the bivalve mollusc is obtained.

[0077] The main purpose of step S7 is to identify all the full-size microplastics with a size of 0.0001~5 mm in the three microplastic purification filter membranes in sequence. According to the above-mentioned hierarchical progressive extraction scheme, purification filter membranes of large-size microplastics (>10μm), small-size microplastics (1~10 μm) and submicron-size microplastics (0.1~1 μm) can be obtained. In order to obtain the morphological characteristics, microscopic morphology imaging and component spectral data of microplastics in each size range after purification, the specific detection steps for the microplastic purification filter membranes in the three size ranges are as follows:

[0078] (1) Observation of morphological characteristics of large-size microplastics (>10 μm): The main purpose of this step is to separate large-size microplastics (>10 μm) in a wide detection range into two types of purification filters: large-size microplastics (>500 μm) and medium-size microplastics (10-500 μm), and record the morphological characteristics of these two types of microplastics, including macroscopic information such as color, shape, size, and quantity, to facilitate the subsequent synchronous scanning of superior identification instruments.

[0079] The first-size microplastic purification filter membrane after constant temperature drying in step S67 of the size-based hierarchical progressive extraction scheme is placed under an optical microscope for morphological observation and quantitative statistics. The observation instrument for this step is not unique. For example, a high-resolution inverted fluorescence microscope with a 4x objective lens or a stereo microscope with the same 4x objective lens can be used. Taking a stereo microscope as an example, the membrane surface can be observed in a "Z" shape, and the morphology and color of all suspected microplastics on the membrane can be recorded. Microplastics larger than 500 μm are searched in the field of view, and photos are collected, their sizes are measured, and all are picked out and transferred to a new mixed cellulose filter membrane, and marked. Thus, the obtained purification filter membrane of >500 μm realizes the re-extraction of the first-size purification filter membrane in the identification scheme.

[0080] (2) Microscopic imaging and component spectrum of the first size purification filter and then extraction filter (>500 μm): The main purpose of this step is to perform infrared imaging on the selected microplastics >500 μm and qualitatively analyze their distribution, obtain the uniformity of the component types of the entire suspected microplastics, and determine the types of these polymers. The specific steps are as follows:

[0081] Observe all selected microplastics in the purification filter membrane of >500 μm, select one of each typical microplastic with strong characteristics for infrared imaging and identify its component distribution area. The observation instrument for this step is not unique. The instrument can be FTIR, ATR-FTIR or FTIR with focal plane array detector (FPA). Taking ATR-FTIR as an example, after background correction, select the selected typical microplastics and place them on the surface of the ATR crystal. Apply pressure to ensure that the sample is in close contact with the ATR crystal, start the FTIR spectrometer, and perform spectral scanning. Select 4000 to 400 cm -1 The wave number range is 100000, covering the characteristic absorption peaks of most polymers, and each scan needs to be repeated three times. The characteristic peaks are fitted, and the spectral library similarity is greater than 70%, which means microplastics. Then the infrared imaging parameters of the FTIR microscope are set, and the imaging area matching the size of the microplastics is selected, with a spatial resolution of 5 μm and a spectral resolution of 8 cm -1 , each scan takes 30 seconds, and the generated infrared imaging image is used to construct a chemical composition distribution map.

[0082] (3) Microscopic imaging and component spectrum of the first-size purification filter (10-500 μm): The main purpose of this step is to perform microscopic infrared imaging of the suspected microplastics remaining on the first-size purification filter after re-extraction and to characterize their components, and to calculate parameters such as the number of particles, particle diameter, and equivalent area. There is no unique instrument for detecting microplastics in this range, and a laser infrared spectrometer or μFTIR can be used. Taking the laser infrared spectrometer (Agilent 8700 LDIR) as an example, the specific steps are as follows:

[0083] Place the first-size purified filter membrane after re-extraction in a 50 mL beaker, immerse the membrane surface with anhydrous ethanol again and shake it at 100 rpm on a shaker for 12 h, rinse both sides and edges of the membrane multiple times, heat the extract at 60°C on a constant temperature heating plate until it is concentrated into a 100 μL suspension, and drop the suspension onto the cleaned high-reflective glass. After the ethanol is completely evaporated, identify microplastics of 10~500 μm under a laser infrared spectrometer, with a matching degree set to >0.7. Repeat each scan three times to obtain infrared images of all microplastic particles on the filter membrane and the polymer type.

[0084] (4) Observation of morphological characteristics, microscopic imaging and component spectrum of small-sized microplastics (1-10 μm): The main purpose of this step is to perform morphological observation, microscopic imaging and component analysis on the small-sized microplastics (1-10 μm) on the second-size purification filter, count the size, color, shape, equivalent diameter, particle number of all small-sized microplastics, and perform Raman imaging to obtain component distribution information. There is no unique instrument for this step. Taking the HORIBA LabRAM Odyssey high-resolution Raman spectrometer as an example, the specific steps are as follows:

[0085] The second size purification filter was placed on the stage of the Raman spectrometer, the excitation wavelength was selected to be 785 nm, the laser power was 100 mW, and the optical resolution was 1 μm. -1 , scanning range is 500~3000 cm -1 , the integration time is 5 s, the number of integrations is set to 3, and the joystick is controlled to scan the membrane surface in a "Z" shape to identify all suspected microplastics of 1-10 μm on the membrane, and record the type, color, shape, size and other morphological information of each. Microplastics are considered to be microplastics if the matching rate in the library is greater than 70%. Each scan is repeated three times. Then use the Mapping function to perform point-to-point area scanning, with the same integration time and number as above, and the time is controlled within 30 s to obtain the component distribution imaging spectrum of microplastics on the entire membrane surface.

[0086] (5) Observation of morphological characteristics, microscopic imaging and component spectrum of submicron-sized microplastics (0.1-1 μm): The main purpose of this step is to perform morphological observation, microscopic imaging and component analysis on the submicron-sized microplastics (0.1-1 μm) of the third-size purification filter, count the size, color, shape, and number of particles of all submicron-sized microplastics, and perform microscopic imaging to obtain component distribution information. There is no unique instrument for this step. Taking the Thermo Scientific DXR 3xi Raman imaging microscope as an example, the specific steps are as follows:

[0087] Use microscopic tweezers to carefully transfer the filter membrane to the stage of the microscope, calibrate the inorganic alumina filter membrane background, and set the parameters of the Raman microscope. The laser power is set to 5 mW and the spectral range is 500~4000 cm -1 , the integration time is 1 s, and the surface scanning mode is used to automatically locate the particle imaging area. While dot-marking the spectrum, each scanning area is automatically spliced ​​into a complete Raman image approximating the filter membrane surface. Microplastic particles with a matching rate of > 70% in the spectral database are searched in sequence, and the optical module is switched to record the size, color, type, shape and other information of each microplastic particle in sequence.

[0088] (6) Abundance correction: Extracting microplastics of each size level separately and combining them with a spectroscopic instrument for dominant identification can avoid the problem of multiple identification of microplastics caused by cross-repetition of identification intervals. Therefore, the microplastic content in a single bivalve obtained by the integrated extraction-identification scheme can be converted into the detection of four dominant identification instruments for purification filter membranes of three size levels. In this scheme, the sum of the microplastics measured by the four spectrometers, ATR-FTIR, LDIR, Raman and micro-Raman, is the full-size microplastic content of a specific tissue of a single bivalve. It is also important to note that if the amount of anhydrous ethanol suspension added in the extraction step (3) of the identification scheme is less than 100 μL, the final measured content needs to be multiplied by the volume coefficient. If only half of the area is observed when observing the membrane surface, the subsequent reading of the microplastic morphological information needs to consider the area coefficient.

[0089] Compared with the prior art, the present invention has the following advantages:

[0090] 1. Compared with the existing technology of extracting microplastics from bivalve biological tissues in ordinary water environments such as shallow seas or rivers and lakes, the integrated size-based extraction and identification method proposed in the present invention modifies the ultrapure water or organic preservation fluid and cleaning fluid used to preserve shellfish tissues in ordinary water environments, and uses the cleaning and replenishing fluid with self-cooled spring fluid properties to balance the huge contrast in biological osmotic pressure to maintain the normal morphology of cells, thereby preventing microplastics from escaping prematurely under long-term storage. In addition, ordinary technology uses grinding / mechanical homogenization plus alkaline digestion solution and unenhanced enzyme solution to ensure the complete digestion of marine biological tissues. However, the extreme high-pressure environment of the deep sea has created a stronger muscle fiber structure and a membrane protein structure that is resistant to oxidative shock in seabed bivalve molluscs. The strong mechanical impeller rotation not only cuts off the intercellular filaments but also continuously crushes large-sized microplastics. The chemical damage caused by ordinary alkaline digestion solutions to microplastics when ablating cell membranes relies on the hydrolysis of ester and amide groups in the main chain. The present invention abandons the mechanical homogenization of tissues to ensure the morphology of large-sized microplastics. The enzyme solution is strengthened by pH stages to drive the freeze-dried tissue to maintain an efficient degradation rate at all times during the pre-digestion process. The hydrogen peroxide solution added completes the final elimination of the residual filamentous coagulants. This chemically enhanced catalytic cell rupture digestion method can completely retain the original morphology of microplastics, and can also prevent the degradation of specific microplastics by ordinary alkaline digestion solutions, and efficiently disintegrate the muscle fiber tissue after environmental adaptive strengthening to expose all microplastics entrained by tissue cells.

[0091] 2. Different from the single identification instrument mentioned in the prior art, the present invention uses size-based hierarchical progressive filtration to obtain three purification filter membranes and then matches them with the corresponding superior identification combination instruments, which can realize the collection of morphological multidimensional information such as size, particle diameter, color, type, shape, etc. of full-size microplastics (0.0001~5 mm), expanding the traditional abundance statistical range of microplastics and the microplastic research range limited by the instrument detection line. For the first time, the present invention incorporates bivalve molluscs from methane seepage areas into the research process of extracting microplastics from biological tissues, optimizes and adapts them in pretreatment, digestion, purification and identification, and creates an experimental scheme for biological extraction of microplastics in deep-sea extreme environments, thereby showing the final migration destination of long-term adsorbed microplastics in organisms indicating methane seepage areas on the seabed, filling the gap in this field.

[0092] 3. The present invention is simple, easy to operate and reproducible, and is suitable for exploring the characterization method of microplastics enriched in various tissues of bivalve molluscs in deep-sea extreme ecosystems. Combined with the captured seabed shellfish beds, it can further deduce the time course and historical accumulation of microplastics absorbed by the entire extreme ecosystem, solving the problem of large-scale sampling, extraction and analysis in deep-sea extreme environments, and providing a scientific inspection strategy for the impact of the history of microplastic pollution in specific sea areas. Description of the drawings:

[0093] Figure 1 It is a process flow chart of the method proposed by the present invention. Specific implementation method:

[0094] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0095] Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental materials and reagents herein are conventional commercial products in the art.

[0096] The present invention uses the deep-sea extreme environment - the methane seepage area produced by the active cold spring of the seahorse as the research area, and selects one of its benthic filter-feeding indicator organisms - the seahorse giant mussel (hereinafter referred to as mussel) as a case demonstration. The process technology flow chart for extracting microplastics from bivalve shellfish in the methane seepage area is as follows Figure 1 As shown, it includes the following steps:

[0097] ① Use a TV grab to capture the target mussel bed on the research vessel;

[0098] ② Use the ship's cruise water device to clean the clay on the surface of the mussels and seal them for storage at -20℃;

[0099] ③ Select the target mussels and thaw them at 4℃, clean all micromanipulation tools, and carefully dissect out the gills, mantle, lips, feet, intestines, visceral mass, and adductor muscles in order on a closed and ventilated dissection table, and place them in a clean 60 mm aluminum box;

[0100] ④ Prepare a cleaning solution similar to cold spring fluid, filter it, and continuously rinse the dissected biological tissues;

[0101] ⑤ Weigh the wet weight of the biological tissue, freeze-dry it in a freeze dryer after freezing at -80℃, and then weigh its dry weight;

[0102] ⑥ Prepare enzyme solution, add a certain amount of enzyme solution according to a certain mussel dry weight ratio, and digest it in a constant temperature shaker at 37℃ for 24 hours. During this period, take it out every 4 hours and stir it evenly under a magnetic stirrer;

[0103] ⑦ After constant temperature digestion, prepare pH adjustment solution, add adjustment solution every 12 hours to maintain the pH value of the digestion solution at 7.5, and add enzyme solution to digest the suspended matter for the second time. Repeat this process 4 times;

[0104] ⑧ Pick out the undissolved filamentous coagulation in the digestion solution, add it to the filtered hydrogen peroxide solution, and place the whole on an electric heating plate at 60℃ for 3 hours;

[0105] ⑨ The obtained enzyme solution and hydrogen peroxide mixed digestion solution were subjected to layered vacuum filtration in sequence, and each filtration step was repeated three times. Finally, the glassware was rinsed with anhydrous ethanol. The filter membranes were stainless steel membranes with a diameter of 47 mm and a pore size of 1200 mesh, 25 mm and 1 μm glass microfiber membranes, and inorganic alumina membranes with a pore size of 0.1 μm. After layered filtration, they were placed in 60 mm aluminum boxes, 30 mm aluminum boxes, and 30 mm glass culture dishes, respectively, and dried to obtain extraction filter membranes for three types of microplastics collection by size;

[0106] ⑩ After drying, add anhydrous ethanol to the first-size layer extraction filter membrane for extraction. After shaking on a shaker for 12 hours, rinse both sides of the filter membrane with anhydrous ethanol continuously, and perform vacuum filtration on the first-size layer again. The filter membrane is a 47 mm, 10 μm mixed cellulose filter membrane. After drying, obtain the purified filter membrane;

[0107] ⑪ Place the first-size purification filter membrane under a stereo microscope, scan the membrane surface in a "Z" shape, record the number, morphology, and color of all suspected microplastic particles on the membrane, search for suspected microplastics >500 μm in the field of view, pick them out and transfer them to the new mixed cellulose filter membrane, and mark them. Select the most characteristic suspected microplastics on the new membrane for qualitative analysis under ATR-FTIR and infrared imaging. Microplastics are obtained if the library similarity is >70%. Obtain all polymer types and single-point infrared imaging images of microplastics >500 μm in the first-size purification filter membrane;

[0108] ⑫ Immerse the first-size purification filter membrane after picking again with anhydrous ethanol, shake it on a shaker for 12 hours, rinse both sides of the filter membrane several times, then heat and concentrate it to 100 μL of suspension, add 100 μL of suspension onto high-reflective glass, dry it in a fume hood, and identify the microplastics on the glass sheet under a laser infrared spectrometer with a matching degree setting of >0.7 to obtain the polymer type of 10-500 μm microplastics in the first-size purification filter membrane and the laser infrared imaging of the membrane surface;

[0109] ⑬ Place the second-size purified filter membrane on the stage of the Raman spectrometer, set the instrument spectrum parameters, scan the membrane surface in a "Z" shape, identify all suspected microplastics of 1-10 μm on the membrane surface, and consider them as microplastics if the library matching rate is greater than 70%. Remove microplastics larger than 10 μm, record the type, color, morphology, and size data of each microplastic, and use the regional mapping function to obtain the Raman imaging distribution map of microplastics on the entire membrane surface;

[0110] ⑭ Place the third-size purified filter membrane on the stage of the Raman imaging microscope, calibrate the filter membrane background, set the Raman identification parameters and select the surface scanning mode, automatically locate the particle imaging area, identify the suspected microplastics of 0.1~1 μm on the entire membrane surface and automatically splice them into a complete Raman microscopic imaging map. The database matching rate >70% is considered as microplastic particles, and the size, color, type and shape of each microplastic particle are recorded;

[0111] ⑮ Combined with the abundance correction in the experimental steps, the size abundance information of full-size microplastics in each biological tissue of the whole mussel was obtained.

[0112] The method proposed in the present invention uses a cleaning solution to clean and preserve the dissected biological tissues, freeze-dry and dehydrate the biological tissues, use a pH-phase enhanced enzyme-hydrogen peroxide mixed digestion solution, hierarchical progressive vacuum filtration, size advantage identification and other experimental steps to non-destructively and classify the microplastics contained in bivalve molluscs in extreme environments. The purpose is to achieve quantitative and qualitative analysis of the full-size microplastics (0.0001~5 mm) in the bivalve molluscs by size range.

[0113] The following instruments and equipment are used in the following examples:

[0114] Microscissors, stainless steel tweezers, pointed tweezers, sampling needle, scalpel, surgical tray, spring scissors, medicine spoon, glass rod, 1L vacuum filtration device, 250mL micro sand core filtration device, ultrasonic cleaner, freeze dryer, oven, 1 / 10,000 electronic analytical balance, graphite electric heating plate, pH meter, magnetic stirrer, constant temperature shaker, laser infrared spectrometer, Fourier transform attenuated total reflection infrared spectrometer, stereo microscope, high-resolution Raman spectrometer, Raman imaging microscope, TV grab, cruising water device, -20℃ refrigerator, 4℃ refrigerator, -80℃ medical refrigerator, ventilation room, dissection table.

[0115] The following reagents and consumables are used in the best embodiment:

[0116] Potassium hydroxide, anhydrous ethanol solution, potassium dihydrogen phosphate, trypsin, sodium chloride, barium chloride, ferrous sulfate, manganese sulfate, magnesium chloride, potassium chloride, calcium chloride, sodium nitrate, sodium silicate, sodium dihydrogen phosphate, sodium sulfate, concentrated hydrochloric acid, 30% hydrogen peroxide solution are all commercially available, 25mm, 1 μm glass microfiber filter membrane, 47mm, 0.1 μm polyvinylidene fluoride filter membrane, 47mm, 0.1 μm water-based microporous filter membrane, 25mm, 0.1 μm inorganic alumina membrane, 1200 mesh steel membrane, 47 mm, 10 μm mixed cellulose filter membrane, 60mm aluminum storage box, 30mm aluminum storage box, 30 mm glass culture dish, 10mL glass syringe, tin foil, rubber band, 50mL beaker, 2L beaker, 1L beaker, 50mL and 100mL stoppered conical flasks, sterile ziplock bag, ultrapure water, and high reflective glass.

[0117] Example 1

[0118] This embodiment provides a method for the size-based hierarchical extraction and identification of microplastics in bivalve molluscs in a methane seepage area. The samples are taken from a mussel bed near a small plume vent in a strong methane seepage area of ​​a seahorse cold seep. Specifically, the method comprises the following steps:

[0119] (1) Sample collection and cleaning: A TV grab is lowered on the research vessel to grab the mussel bed that is bred near the central vent of the strong methane seepage area of ​​the seahorse cold seep. After being salvaged to the stern deck, the mussel shells are cleaned using the ship's onboard cruise water device to remove the attached clay;

[0120] (2) Cryopreservation: Select cleaned and intact mussels, place them individually in sterile ziplock bags, record their numbers, and store them in a -20°C refrigerator;

[0121] (3) Experimental preprocessing:

[0122] ① Constant temperature thawing: purposefully select mussels with special meanings from a -20℃ refrigerator and place them in a 4℃ refrigerator to thaw for 1 hour;

[0123] ② Instrument preparation: Soak all scissors, tweezers, aluminum boxes, tin foil, scalpels and other dissection instruments in an ultrasonic cleaner filled with ultrapure water for 30 minutes. Rinse the above dissection instruments and tin foil with filtered ultrapure water several times before the experiment.

[0124] ③Dissection and packaging: Open the ventilation system in the closed ventilation room, cover the entire dissection table with clean tin foil, take out the mussels, slightly open the shell gap, cut the adductor muscle on one side with a scalpel to completely open the entire internal tissue of the mussel, and carefully cut off each mussel tissue (gills, mantle, lip, foot, intestine, visceral mass, adductor muscle) in turn with spring scissors, and temporarily place them in the corresponding 60 mm special aluminum box;

[0125] ④ Cleaning and preservation: Prepare a cleaning solution similar to cold spring fluid and filter it three times using a 0.1 μm water-based microporous filter membrane. Use the filtered cleaning solution to rinse the dissected tissues and aluminum boxes several times, drain the water and place them in the corresponding aluminum boxes for later use;

[0126] ⑤ Weigh wet weight: Place the aluminum box corresponding to each mussel tissue on an electronic analytical balance, weigh the wet weight of each tissue in the tare mode, and record the data;

[0127] ⑥ Freeze-drying and storage: After weighing the wet weight, place each tissue aluminum box in a -80℃ medical refrigerator for 6 hours and attach a wet weight label. Then place each frozen aluminum box in a freeze dryer at -60℃ for 24 hours;

[0128] ⑦ Weigh dry weight: Place each freeze-dried mussel tissue on an electronic analytical balance, weigh the tissue dry weight after peeling, and record the dry weight data;

[0129] (4) Pre-digestion with enzyme solution: prepare trypsin solution and filter it three times through a 0.1 μm polyvinylidene fluoride filter membrane. Depending on the dry weight of the tissue, add the lyophilized tissue into a 50 mL or 100 mL cleaned ground-mouth conical flask with a stopper. Add trypsin solution at a ratio of 1 g dry weight of biological tissue: 35 mL trypsin solution. Place the flask in a sealed container at 37°C and 100 rpm for digestion for 24 h at a constant temperature shaker. Take the flask out every 4 h and place it under a magnetic stirrer to accelerate digestion.

[0130] (5) pH stage enhancement: Prepare pH adjustment solution and filter it three times through a 0.1 μm polyvinylidene fluoride filter membrane. Take out the tissue digestion solution, measure its pH with a pH meter, add pH adjustment solution drop by drop to maintain the pH of the digestion solution at 7.5, apply enzyme digestion solution again to clean the pH meter and accelerate tissue cell decomposition, and repeat this process four times;

[0131] (6) Hydrogen peroxide digestion: Use tweezers to pick out small pieces of filamentous coagulation in the enzyme solution after pH-enhanced digestion and place them in a new clean conical flask. Add a certain amount of 30% hydrogen peroxide solution, clean the tweezers, seal it, and place it on a graphite electric heating plate at 60°C for another 3 h to obtain a clear solution with almost no obvious suspended matter.

[0132] (7) Size-based progressive extraction of mixed digestion solution:

[0133] ① First size (>10 μm) level extraction: Use filtered ultrapure water to rinse the 1L glass vacuum filtration device three times, load a stainless steel membrane with a diameter of 47 mm and a pore size of 1200 mesh on a filter element with a diameter of 50 mm, pour the enzyme secondary digestion solution and hydrogen peroxide digestion solution on the filter cup, repeat the vacuum filtration three times, and the eluent used each time is the filtrate after filtration. Continuously rinse the filter cup and two conical flasks, and finally use a small amount of ultrapure water to rinse the residual digestion solution on the wall of the cup to obtain the extraction filter membrane. Place the 60 mm aluminum box carrying the extraction filter membrane in an oven at 60°C and dry for 3 hours;

[0134] ② Second size (1-10 μm) layer extraction: Use filtered ultrapure water to rinse 250 mL of micro sand core filter device three times, and load a glass microfiber filter membrane with a diameter of 25 mm and a pore size of 1 μm on a filter element with a diameter of 25 mm. Pour the above first size layer extraction filtrate slowly into the device for vacuum filtration in multiple times. Repeat the filtration three times. The rinse solution used each time is the filtrate obtained last time and is used to clean the 1L filter bottle and filter cup wall. Finally, use a small amount of ultrapure water to rinse the residual digestion solution. The obtained purification filter membrane is placed in a 30 mm aluminum box and dried in an oven at 60°C for 3 hours.

[0135] ③ The third size (0.1-1 μm) level extraction: the same as the second size level extraction steps, use ultrapure water to rinse the micro sand core filter device again, replace the filter membrane with a 25 mm, 0.1 μm inorganic alumina membrane, slowly pour the second size level extraction filtrate into the device filter cup for vacuum filtration in multiple times, and repeat the filtration three times. Before completing the filtration, use the filtered anhydrous ethanol solution to rinse the inner wall of the filter cup and the 250 mL filter bottle, transfer the residual microplastics to the membrane, and put the obtained purified filter membrane into a 30 mm glass culture dish and dry it in an oven at 60°C for 3 hours, and then store it at room temperature;

[0136] (8) Extraction and ethanol washing: Use a certain amount of anhydrous ethanol solution to immerse the membrane surface of the first-size extraction filter, seal it with tin foil, and place it in a constant temperature shaker at room temperature and oscillate at 100 rpm for 12 h. Then take out the filter membrane and continuously rinse both sides and edges of the filter membrane with anhydrous ethanol until there are no obvious particles attached.

[0137] (9) Vacuum filtration membrane preparation: 1 L of glass vacuum filtration device was continuously rinsed with ultrapure water. The filter membrane was a 47 mm, 10 μm mixed cellulose filter membrane. The anhydrous ethanol extract was filtered three times. Finally, the cup wall and container were rinsed with anhydrous ethanol for many times. The first size purification filter membrane was placed in a 60 mm aluminum box and dried in an oven at 60 °C for 2 h.

[0138] (10) Quality control: All the ultrapure water, prepared solutions, reagents and digestion solutions used above must be filtered through a 0.1 μm water microporous filter membrane and a 0.1 μm polyvinylidene fluoride filter membrane to filter out impurities in the solution itself. Similarly, before using glassware, beakers, tweezers, scissors and other equipment, they must be immersed in an ultrasonic bath filled with filtered ultrapure water for 30 minutes to eliminate potential impurities as much as possible. Before using the filter membrane, use tweezers to carefully soak the filter membrane in anhydrous ethanol to clean it back and forth, and finally rinse the filter membrane with a small amount of anhydrous ethanol. Wear cotton gloves and cotton lab coats when conducting experiments. Under the same conditions, set up a blank control group, and all operations are consistent with the experimental group;

[0139] (11) Observation of large-size microplastic characteristics, infrared imaging and component spectra (>500 μm): Place the first-size purified filter membrane obtained in step (9) on the stage of a stereo microscope, observe the membrane surface in a "Z" shape, record the morphology and color information of all suspected microplastics (>10 μm) on the membrane, search for suspected microplastics >500 μm in the field of view and pick them out and place them on another new mixed cellulose filter membrane, and mark them. Select various typical suspected microplastics with special characteristics on the new membrane, transfer the sampling needle to the ATR crystal surface, and select 4000 to 400 cm -1 The wavenumber range is set to collect spectra, and those with a similarity to the library greater than 70% are considered microplastics. Then return to the spectral image interface, set the infrared imaging parameters of the FTIR microscope, select the imaging area of ​​the corresponding size, and obtain the infrared imaging component distribution map of the entire microplastic;

[0140] (12) Laser infrared imaging and component spectrum (10-500 μm) of microplastics on the first-size purified filter membrane: Place the first-size purified filter membrane after re-extraction in step (11) in a 50 mL beaker, immerse the surface of the filter membrane with filtered anhydrous ethanol solution and shake it at 100 rpm for 12 h on a shaker, rinse both sides of the filter membrane and the edges of the gap multiple times, concentrate the extract on a constant temperature heating plate at 60°C to a 100 μL suspension, drip the suspension onto the clean surface of a high-reflective glass, transfer the high-reflective glass to the sample chamber of the laser infrared spectrometer, set the spectral parameters, capture the infrared spectra of all 10-500 μm microplastics on the membrane surface, set the library match rate to >0.7, and obtain infrared imaging images of all microplastics on the filter membrane and polymer types;

[0141] (13) Observation of small-sized microplastic characteristics, Raman imaging and component spectra (1-10 μm): Place the second-size purified filter membrane obtained in step (7) on the stage of the Raman spectrometer, select an excitation wavelength of 785 nm, and set the scanning range to 500-3000 cm -1, the integration time is set to 5 s, the number of integrations is set to 3, and the membrane surface is scanned in a "Z" shape to identify all suspected microplastics of 1-10 μm on the membrane. Only those with a matching rate in the spectral library of >70% are considered microplastics, and the type, color, shape, size and other morphological characteristics of each microplastic are recorded. The Raman accessory Mapping function is used to frame the surface scan area, and the entire membrane surface is scanned by path Raman to splice into a Raman imaging distribution map close to the membrane surface;

[0142] (14) Observation of submicron-sized microplastic characteristics, Raman microscopy imaging, and component spectra (0.1-1 μm): Place the third-size purified filter membrane obtained in step (7) on the stage of a Raman imaging microscope, calibrate the filter membrane background, and set the spectral range to 500-4000 cm -1 , the integration time was 1 s, the surface scanning mode was selected, the surface scanning area was framed, Raman microscopic identification of suspected microplastics of 0.1-1 μm on the entire membrane surface was performed and automatically spliced ​​into a complete Raman microscopic imaging spectrum near the membrane surface, and the database matching rate was set to >70% for microplastics, and the microplastics on the membrane were searched and the size, color, type, and shape information of each microplastic were recorded.

[0143] (15) Quality control scheme: This quality control scheme intends to use a standard addition recovery experiment to verify the accuracy of this method. The experimental group first follows the hierarchical progressive extraction steps (1)-(3) to obtain the freeze-dried mussel tissues after experimental pretreatment. The control group starts from the above step (4) and continues all processing operations with the experimental group. The experimental standard uses 20±10 μm, 50±20 μm, 100±20 μm, 200±50 μm and 500±50 μm standard yellow-green fluorescent PE microspheres, which are mixed and assembled according to the following deep-sea extreme environment bivalve mollusc adsorbed microplastics type component formula. The self-prepared bivalve molluscs in the present invention have a microplastic size distribution formula as shown in Table 1 below:

[0144]

[0145] The yellow-green fluorescent PE microsphere standards of 20±10 μm, 50±20 μm, 100±20 μm, 200±50 μm and 500±50 μm were screened through 800 mesh, 300 mesh, 150 mesh, 70 mesh and 32 mesh stainless steel sieves in order to remove the microplastic particles with negative particle size deviation in these five sizes. Then, 280 μg of 20-30 μm, 410 μg of 50-70 μm, 240 μg of 100-120 μm, 54 μg of 200-250 μm and 16 μg of 500-550 μm of the sieved yellow-green fluorescent PE microspheres were weighed in proportion in a one-millionth electronic analytical balance, and the five sizes of microspheres were evenly mixed in a clean beaker to prepare 1 mg of mixed fluorescent microplastic powder.

[0146] According to the dry weight of each biological tissue, each biological tissue is added to a 50 mL or 100 mL cleaned stoppered conical flask, and a quantitative trypsin solution for digestion is added at a ratio of 1 g tissue dry weight: 30 mL enzyme digestion solution, and the microplastic powder is added to each tissue enzyme digestion solution in equal proportions according to the ratio of 1 g tissue dry weight: 1 mg mixed fluorescent microplastic powder. After the system is sealed, it is digested at 37°C at 100 rpm in a constant temperature shaker for 24 h. The subsequent pH enhanced digestion, filtration, extraction, and identification steps are the same as the above steps. When using a microscope for observation in the identification step, it is necessary to deliberately select plastic microspheres that emit yellow-green fluorescence, and the remaining suspicious microplastics are not included in the identification system. The purpose of this quality control scheme is to compare the characteristic microplastic particles identified by the digestion of the present invention with the dosage of known samples, highlighting the feasibility of this method for non-destructive extraction of microplastics in biological tissues in extreme ecosystems. The experimental results are shown in Table 2 below:

[0147]

[0148] Comparative Example 1

[0149] The same as Example 1, except that: no enzyme solution digestion was performed, and an acid / alkaline digestion method was used.

[0150] Comparative Example 2

[0151] The same as Example 1, except that no stepwise enhancement of pH was performed.

[0152] Comparative Example 3

[0153] The same as Example 1, except that hydrogen peroxide digestion was not performed.

[0154] Comparing Comparative Examples 1-3 with Example 1, ① no enzyme solution was used for digestion. The method of Comparative Example 1 will cause microplastic types such as PC, PET, and PA in shellfish tissues to be chemically degraded, and the apparent morphology will be eroded, resulting in a false positive result of in-situ degradation of microplastics in deep-sea shellfish; ② no pH tuning solution was used to enhance the enzymatic reaction. Deep-sea shellfish cells have evolved a tough protein support structure, and heat-stable proteins with stronger resistance to denaturation and degradation can weaken the decomposition of proteases, and different enhancement methods must be adopted from offshore; ③ no hydrogen peroxide solution was used to digest the remaining tissue condensate. In the process of protease separation after strengthening, a part of the tough protein complex still remains in the bivalve tissue. Only by applying a stronger oxidation reaction can the remaining complex organic matter be completely removed, and the microplastics contained therein are exposed without loss to ensure purity. These three digestion steps complement each other and are indispensable, and they synergistically improve the digestion efficiency of organic matter in shellfish tissues. The protein barrier is initially decomposed by trypsin, the pH-enhanced enzyme solution further decomposes the complex organic matter, and finally the remaining stubborn organic matter is completely oxidized by hydrogen peroxide. This multi-step combined progressive digestion method can effectively remove resistant organic matter in tissue cells, allowing full-size microplastics to be extracted one by one in a nearly non-destructive state, completing the mission of size identification.

[0155] In addition, the prior art often uses mechanical pulverizers to homogenize all tissues, resulting in the microplastics adsorbed or degraded in the tissues being cut into particles with uniform texture and small particle size. And most technologies use a single identification method to obtain information on microplastics in a fixed size range. Compared with the prior art, this embodiment 1 does not need to expand the enzymatic reaction area of ​​shellfish tissues by cutting with a pulverizer. The digestion efficiency of the three digestion methods is sufficient to achieve the same effect, and the proposed advantageous instrument combination identification scheme can realize the identification of full-size microplastics, broadening the detection range of previous technologies.

[0156] Example 2

[0157] Same as Example 1, except that:

[0158] Pre-digestion with enzyme solution: add trypsin solution to various biological tissues at a ratio of 1 g dry weight of biological tissue: 30 mL trypsin solution, and digest at 35°C for 30 h;

[0159] pH staged enhancement: Use pH adjusting solution to adjust pH to 7.5, then add trypsin solution to the enzyme digestion solution several times to accelerate the disintegration of filamentous coagulants to obtain enzyme solution secondary digestion solution, the volume of trypsin solution added each time is 1 / 3 of the volume of trypsin solution for digestion in step S2;

[0160] 30% hydrogen peroxide digestion: Pick out the filamentous aggregates, add 30% hydrogen peroxide solution by mass to the filamentous aggregates, and digest the residual cell adhesions at 65°C for 2 h.

[0161] Example 3

[0162] Same as Example 1, except that:

[0163] Pre-digestion with enzyme solution: add trypsin solution to various biological tissues at a ratio of 1 g dry weight of biological tissue: 40 mL trypsin solution, and digest at 40°C for 24 h;

[0164] pH staged enhancement: Use pH adjusting solution to adjust pH to 7.5, then add trypsin solution to the enzyme digestion solution several times to accelerate the disintegration of filamentous coagulants to obtain enzyme solution secondary digestion solution, the volume of trypsin solution added each time is 1 / 4 of the volume of trypsin solution for digestion in step S2;

[0165] 30% hydrogen peroxide digestion: Pick out the filamentous aggregates, add 30% hydrogen peroxide solution by mass to the filamentous aggregates, and digest the residual cell adhesions at 60°C for 4 hours.

[0166] The description of the above embodiments is only used to help understand the technical solution and core ideas of the present invention. It should be pointed out that for technicians in this technical field, several improvements and modifications can be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for the size-based hierarchical extraction and identification of microplastics in bivalve molluscs in deep-sea methane seepage areas, characterized in that: The steps include: S1. Cryopreservation: The captured bivalve molluscs are cryopreserved; S2, tissue pretreatment: the bivalve molluscs frozen and stored in step S1 are thawed at a constant temperature and airtightly dissected to obtain various biological tissues of the bivalve molluscs, and the various biological tissues of the bivalve molluscs are freeze-dried and set aside for use; S3, pre-digestion with enzyme solution: add the trypsin solution for digestion to various biological tissues of bivalve molluscs obtained in step S2 to obtain a primary digestion solution of enzyme solution with a large number of tissue filamentous coagulation bodies, wherein the trypsin solution is prepared by the following steps: weigh 6.80 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, then adjust the pH to 7.5 with 0.1 mol / L potassium hydroxide solution, add 30.00 g of trypsin, add water to dissolve it, and dilute it to 1 L to obtain the trypsin solution; S4, pH stage-by-stage enhancement: Use pH adjusting liquid to adjust the pH of the primary digestion liquid of the enzyme solution obtained in step S3 to 7.5, then add trypsin solution to the enzyme digestion liquid several times to accelerate the disintegration of the filamentous coagulant to obtain a secondary digestion liquid of the enzyme solution, and the volume of the trypsin solution added each time is 1 / 3-1 / 4 of the volume of the trypsin solution for digestion in step S3; the pH adjusting liquid is a potassium dihydrogen phosphate solution with a mass concentration of 0.0136 g / mL or a potassium hydroxide solution with a mass concentration of 0.0562 g / mL; the trypsin solution is prepared by the following steps: weigh 6.80 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, then adjust the pH to 7.5 with 0.1 mol / L potassium hydroxide solution, add 30.00 g of trypsin, add water to dissolve it, and dilute it to 1 L to obtain the trypsin solution; S5, 30% hydrogen peroxide digestion: pick out the filamentous coagulation bodies in the secondary digestion solution of the enzyme solution obtained in step S4, add a 30% by mass fraction hydrogen peroxide solution to the filamentous coagulation bodies, and digest the residual cell adhesions at 60°C-65°C for 2-4 hours until no bubbles are generated in the digestion solution, that is, the digestion is completely completed to obtain a nearly transparent solution without obvious suspended matter, thereby obtaining a hydrogen peroxide digestion solution; S6, progressive extraction of mixed digestion solution by size level: the enzyme solution secondary digestion solution obtained in step S4 and the hydrogen peroxide digestion solution obtained in step S5 are mixed, and a first size level extraction is performed to enrich all target objects of >10 μm on the filter membrane, thereby obtaining a first size level extraction filter membrane, and then the first level filtrate is subjected to a second size level extraction to enrich all target objects of 1-10 μm on the filter membrane, thereby obtaining a second size level extraction filter membrane, and then the second level filtrate is subjected to a third size level extraction to enrich all target objects of 0.1-1 μm on the filter membrane, thereby obtaining a third size level extraction filter membrane; S7, the microplastics in the first size level extraction filter membrane, the second size level extraction filter membrane and the third size level extraction filter membrane obtained in step S6 are sequentially subjected to characteristic observation, microscopic imaging and component spectral detection to obtain size abundance information of full-size microplastics in various biological tissues in bivalve molluscs, specifically comprising the following steps: S71. Split the first size-level extraction filter membrane into a large-size microplastic filter membrane with a particle size greater than 500 μm and a medium-size filter membrane with a particle size of 10-500 μm, perform infrared imaging on the microplastics in the large-size microplastic filter membrane with a particle size greater than 500 μm and qualitatively analyze their distribution, obtain the uniformity of the component types of the entire suspected microplastics and determine the type of polymer of the suspected microplastics, perform microscopic infrared imaging on the microplastics in the medium-size filter membrane with a particle size of 10-500 μm and qualitatively identify their components, and calculate their technical parameters, which include particle number, particle diameter, and equivalent area; S72. Perform morphological observation, microscopic imaging and component analysis on small-sized microplastics with a particle size of 1-10 μm extracted from the second size level filter membrane, count the size, color, shape, equivalent diameter, particle number of all small-sized microplastics, and perform Raman imaging to obtain component distribution information; S73. Conduct morphological observation, microscopic imaging and component analysis on submicron-sized microplastics with a particle size of 0.1~1 μm extracted from the third-size layer filter membrane. Count the size, color, shape, and particle number of all submicron-sized microplastics and perform microscopic imaging to obtain component distribution information.

2. The method according to claim 1, characterized in that The specific steps of step S1 are: selecting intact bivalve molluscs, washing them multiple times until the sediment clay adsorbed on the surface is washed off, and then putting the washed bivalve molluscs into a sterile container and freezing them at -20°C.

3. The method according to claim 1, characterized in that The specific steps of step S2 are: the bivalve molluscs cryopreserved in step S1 are thawed at a constant temperature of 4°C for 0.5-1.5 h, and then the various biological tissues of the bivalve molluscs are obtained after closed dissection. The various biological tissues of the bivalve molluscs are cleaned with a cleaning solution to remove seawater microplastics and various impurities attached to the outside of the shellfish tissue cells, and then frozen at -80°C for 5-7 h, and freeze-dried at -60°C for 20-28 h for standby use.

4. The method according to claim 3, characterized in that: The cleaning solution is prepared by the following steps: 5.04 g sodium chloride powder, 0.06 g barium chloride powder, 0.12 g ferrous sulfate powder, 0.02 g manganese sulfate powder, 1.2 g magnesium chloride powder, 0.4 g potassium chloride powder, 0.4 g calcium chloride powder, 2.6 mg sodium nitrate powder, 22 mg sodium silicate powder, 1 mg sodium dihydrogen phosphate powder and 2.5 g sodium sulfate powder are added to 800 mL of ultrapure water, 23.42 mL of concentrated hydrochloric acid is added dropwise, and the mixture is continuously shaken and stirred, and then the volume is adjusted to 1 L, and filtered several times using a 0.1 μm water system microporous filter membrane to obtain the cleaning solution.

5. The method according to claim 1, characterized in that The specific steps of step S3 are: adding the trypsin solution for digestion to the various biological tissues of the bivalve molluscs obtained in step S2 at a ratio of 1 g of biological tissue dry weight: 30-40 mL of trypsin solution, digesting at 35°C-40°C for 24-30 h, and obtaining a primary digestion solution of the enzyme solution with a large number of tissue filamentous coagulation bodies.

6. The method according to claim 1, characterized in that In step S6, the first size level extraction uses a glass filter element with a diameter of 50 mm and a stainless steel membrane with a diameter of 47 mm and a pore size of 1200 mesh, the second size level extraction uses a filter element with a diameter of 25 mm and a glass microfiber filter membrane with a diameter of 25 mm and a pore size of 1 μm, and the third size level extraction uses a filter element with a diameter of 25 mm and an inorganic alumina membrane with a diameter of 25 mm and a pore size of 0.1 μm.

7. The method according to claim 1, characterized in that Step S7 also includes S74, extracting microplastics of each size level separately, and using four spectrometers, namely ATR-FTIR, LDIR, Raman and micro-Raman, the total amount of microplastics measured is the full-size microplastic content in each biological tissue of a single bivalve mollusc. Combined with the abundance correction in the experimental steps, the size-based abundance information of full-size microplastics in each biological tissue of the bivalve mollusc is obtained.

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