Size-based hierarchical extraction and identification method for microplastics in bivalves from deep-sea methane seeps

A size-based hierarchical extraction and identification method for microplastics in bivalves from deep-sea methane seeps, comprising the following steps:

• S1, frozen preservation:
• freezing retrieved bivalves for preservation;
• S2, tissue pretreatment:
• after the bivalves frozen for preservation in step S1 undergo constant temperature thawing and closed dissection, obtaining various biological tissues of the bivalves, and freeze-drying the various biological tissues of the bivalves for later use;
• S3, enzyme solution pre-digestion:
• adding trypsin solution for digestion to the various biological tissues of the bivalves obtained in step S2 to perform digestion, to obtain a primary enzyme digestion solution with numerous tissue filamentous condensates;
• S4, pH phased enhancement:
• using pH adjustment solution to adjust a pH of the primary enzyme digestion solution obtained in step S3 to 7.5, then adding trypsin solution to the enzyme digestion solution multiple times to accelerate a breakdown of filamentous condensates, to obtain a secondary enzyme digestion solution, a volume of trypsin solution added each time being ⅓ to ¼ of a volume of the trypsin solution for digestion in step S3;
• wherein the pH adjustment solution in step S4 is potassium dihydrogen phosphate solution with a mass concentration of 0.0136 g/mL or potassium hydroxide solution with a mass concentration of 0.0562 g/mL;
• the trypsin solution is prepared by the following steps:
• weighing 6.80 g potassium dihydrogen phosphate, adding 500 mL water to dissolve it, adjusting pH to 7.5 with 0.1 mol/L potassium hydroxide solution, adding 30.00 g trypsin, dissolving with water, then diluting to 1 L to obtain the trypsin solution;
• S5, 30% hydrogen peroxide digestion:
• picking out the filamentous condensates from the secondary enzyme digestion solution obtained in step S4, adding 30% hydrogen peroxide solution by mass percentage to the filamentous condensates, and digesting remaining cellular adhesive materials at 60-65° C. for 2-4 hours until no bubbles are generated in a digestion solution, wherein complete digestion is achieved to obtain a nearly transparent solution with no obvious suspended matter, to obtain a hydrogen peroxide digestion solution;
• S6, mixed digestion solution size-based extraction:
• mixing the secondary enzyme digestion solution obtained in step S4 and the hydrogen peroxide digestion solution obtained in step S5, performing first-size extraction to enrich all target objects>10 μm onto a filter membrane, to obtain a first-size extraction filter membrane;
• then performing second-size extraction on a first-size filtrate to enrich all target objects of 1-10 μm onto a filter membrane, to obtain a second-size extraction filter membrane;
• then performing third-size extraction on a second-level filtrate to enrich all target objects of 0.1-1 μm onto a filter membrane, to obtain a third-size extraction filter membrane;
• S7, subjecting microplastics on the first-size extraction filter membrane, the second-size extraction filter membrane, and the third-size extraction filter membrane obtained in step S6 to characteristic observation, microscopic imaging, and compositional spectral detection in sequence to obtain size-based abundance information of full-scale microplastics in the various biological tissues in the bivalves, wherein specific steps are as follows:
• S71, separating the first-size extraction filter membrane into a large-size microplastic filter membrane with particle size greater than 500 μm and a medium-size filter membrane with particle size of 10-500 μm;
• performing infrared imaging on microplastics in the large-size microplastic filter membrane with particle size greater than 500 μm and qualitatively analyzing their distribution to obtain a compositional type uniformity of all suspected microplastics and determine types of these polymers;
• performing microscopic infrared imaging on microplastics in the medium-size filter membrane with particle size of 10-500 μm and qualitatively analyzing their composition, and calculating their technical parameters comprising particle count, particle diameter, and equivalent area;
• S72, performing morphological observation, microscopic imaging, and compositional analysis on small-size microplastics with particle size of 1-10 μm in the second-size extraction filter membrane, counting size, color, shape, equivalent diameter, and particle count of all small-size microplastics, and performing Raman imaging to obtain compositional distribution information;
• S73, performing morphological observation, microscopic imaging, and compositional analysis on submicron-size microplastics with particle size of 0.1-1 μm in the third-size extraction filter membrane, counting size, color, shape, and particle count of all submicron-size microplastics, and performing microscopic imaging to obtain compositional distribution information.