WO2020253036A1 - 一种人畜粪便中提取微塑料的方法 - Google Patents

一种人畜粪便中提取微塑料的方法 Download PDF

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WO2020253036A1
WO2020253036A1 PCT/CN2019/116644 CN2019116644W WO2020253036A1 WO 2020253036 A1 WO2020253036 A1 WO 2020253036A1 CN 2019116644 W CN2019116644 W CN 2019116644W WO 2020253036 A1 WO2020253036 A1 WO 2020253036A1
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filter membrane
feces
beaker
microplastics
human
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PCT/CN2019/116644
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French (fr)
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张宴
闫泽华
任洪强
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南京大学
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Priority to US16/935,241 priority Critical patent/US11360006B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/08—Polysaccharides
    • B01D71/12—Cellulose derivatives
    • B01D71/14—Esters of organic acids
    • B01D71/16—Cellulose acetate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/08—Polysaccharides
    • B01D71/12—Cellulose derivatives
    • B01D71/20—Esters of inorganic acids, e.g. cellulose nitrate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/30—Polyalkenyl halides
    • B01D71/32—Polyalkenyl halides containing fluorine atoms
    • B01D71/36—Polytetrafluoroethene
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4094—Concentrating samples by other techniques involving separation of suspended solids using ultrasound
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

Definitions

  • the invention relates to a method for extracting microplastics from human and animal feces, which can extract microplastics in human and animal feces quickly, efficiently and with high fidelity under the condition of minimizing the impact on microplastics, and is convenient to accurately analyze the microplastics in biological feces
  • Types and abundances provide strong technical support and data support for studying the accumulation and excretion of microplastics in organisms, as well as health risk assessment.
  • the invention belongs to the field of environmental health risk assessment.
  • Microplastics refer to plastic particles with a diameter of less than 5mm. On the one hand, they come from industrial raw materials, personal care products (such as toothpaste, facial cleanser) and the primary source of small particles of plastic microbeads in medical products; on the other hand, they can come from large blocks in the environment. Plastic waste is a secondary source formed by the division of physical, chemical and biological processes. Microplastics can be ingested by organisms and produce toxic effects. As the global production and use of plastics continue to increase, environmental hazards and health risks caused by microplastic pollution have become the focus of attention from all walks of life. Studies at home and abroad have confirmed that microplastics enter organisms through accidental ingestion and other ways, and are mainly concentrated in the intestinal tract with potential health risks.
  • Existing microplastic extraction methods mainly target biological tissue and sludge samples.
  • the main steps include: direct digestion of the sample with strong acid and alkali, and density separation of the digested sample to separate the microplastic.
  • Fecal samples are not only different from biological tissues which are almost all biological organic matter, but also different from sludge which contains a lot of sediment.
  • the invention provides a method for extracting microplastics from human and animal feces, which can accurately analyze the species and abundance of microplastics in biological feces in a simple, fast and high-efficiency manner, and has little effect on the properties of the microplastics themselves, and is convenient to use a microscope -Raman spectroscopy to observe and identify microplastics.
  • the present invention provides a method for extracting microplastics from human and animal feces, which can quickly, conveniently and efficiently separate microplastics in feces.
  • the present invention provides the following technical solutions:
  • a method for extracting microplastics from human and animal feces which is characterized in that it comprises the following steps:
  • Feces freeze-drying freeze-dry the collected organism feces in a freeze dryer, and take N g of the freeze-dried sample;
  • the number of filters in each beaker does not exceed 3.
  • the 30% H 2 O 2 added when the reaction is completed in step (2) is N ⁇ 100 mL, and the volume ratio of addition is 20 g/L FeSO 4 ⁇ 7H 2 O catalyst and 30% H 2 O 2 1:2.5.
  • the pore size of the water-based CN-CA filter membrane and the PTFE hydrophilic filter membrane in step (2) is 1 ⁇ m.
  • the ultrasonic treatment in step (3) is ultrasonicated for 10-15 minutes under the condition of 100KHz.
  • the biological feces in step (1) include human feces and livestock feces, and each sample of biological feces is not less than 3 parallel samples.
  • microplastics in the biological feces digested and extracted by the above digestion method are within the protection scope of the present invention.
  • the method disclosed in the present invention can extract microplastics in human and animal feces quickly, efficiently and with high fidelity under the condition that the impact on microplastics is minimized, and can detect the types and abundance of microplastics contained in feces within a certain quality. degree.
  • This method is based on the Fenton reaction to digest the easily digestible organic matter in the feces, and then use concentrated nitric acid to digest the water system CN-CA membrane and the remaining organic matter under the condition of 50°C water bath, and finally use concentrated nitric acid at 70°C water bath condition
  • the next step is to digest difficult organic matter. After the digestion is completed, anhydrous ethanol is used to dissolve part of the remaining organic substances covering the surface of the microplastics under ultrasonic conditions, and the microplastics are extracted to improve the efficiency of micro-Raman spectroscopy to identify microplastics.
  • the experimental results show that the digestion method provided by the present invention can effectively digest the feces of the organism without significant impact on the physical and chemical characteristics of the plastic.
  • the traditional single strong acid and strong alkali are used for digestion, and the digestion time is longer.
  • Digestion is not complete, and the process of digestion has a greater impact on the optical characteristics and morphology of the microplastics.
  • the present invention solves these problems and can quickly, efficiently, and high-fidelity extract human and animal feces under the condition of minimizing the impact on microplastics.
  • the microplastics are convenient to accurately analyze the types and abundance of microplastics in biological feces, and can be widely used in the extraction and detection of microplastics in biological feces.
  • Figure 1 is the process flow of the method of the present invention
  • Figure 2 is an image of fecal digestion efficiency of the present invention
  • Fig. 3 is a comparison image of Raman spectra of PE plastic before and after the fecal digestion experiment of the present invention
  • Fig. 4 is a comparison image of the Raman spectrum of PS plastic before and after the fecal digestion experiment of the present invention
  • Fig. 5 is a comparison image of Raman spectra of PVC plastic before and after the fecal digestion experiment of the present invention
  • Figure 6 is a microscopic image of the microplastics extracted after the fecal digestion experiment of the present invention.
  • Figure 7 is an image of the remaining organic matter on the PTFE hydrophilic filter membrane after the fecal digestion experiment of the present invention.
  • Figure 8 is an image of the recovery rate of plastic particles after the fecal digestion test of the present invention.
  • the present invention relates to a method for extracting microplastics from human and animal feces.
  • the following examples of preferred embodiments are given. The following examples should never be interpreted as limiting or limiting the scope of the present invention.
  • the invention discloses a method for extracting microplastics from human body and poultry feces.
  • the method mainly includes three steps. Firstly, the feces are retrieved and put into a freeze dryer for freeze-drying, and then digested by adding a digestion reagent, and vacuum filtered. , The method of vacuum filtration after ultrasonic treatment to extract microplastics.
  • the invention can ensure that the microplastics can be conveniently and quickly extracted from human and animal feces under the condition of minimizing the impact on the microplastics, which facilitates accurate analysis of the types and abundance of microplastics in biological feces, and is used for studying the enrichment of microplastics in organisms. And excretion rules, as well as health risk assessment to provide strong technical support and data support.
  • the method of the present invention includes the following steps:
  • Feces freeze-drying freeze-dry the collected organism feces in a freeze dryer, and take N g of the freeze-dried sample;
  • the number of filters in each beaker does not exceed 3.
  • the method for extracting microplastics from human and animal feces is characterized in that the weight of the freeze-dried sample taken in step (1) is 1-5 g.
  • the method for extracting microplastics from human and animal feces is characterized in that, when the reaction in step (2) is completed, the total amount of 30% H 2 O 2 added is N ⁇ 100 mL, and the volume ratio of addition is 20 g/L FeSO 4. 7H 2 O catalyst and 30% H 2 O 2 1:2.5.
  • the water-based CN-CA filter membrane and the PTFE hydrophilic filter membrane have a pore size of 1 ⁇ m.
  • the method for extracting microplastics from human and animal feces is characterized in that the ultrasonic treatment in step (3) is ultrasonic treatment under 100KHz conditions for 10-15 minutes.
  • the organism feces include human feces and livestock feces.
  • step (1) there are no less than 3 parallel samples per sample of biological stool.
  • Feces freeze-drying The human feces taken are freeze-dried in a freeze-drying apparatus, and 5 g dry weight of the freeze-dried sample is taken, and 3 parallel samples are taken.
  • a 500mL beaker add 100mL 65% concentrated nitric acid to each beaker, digest for 30 minutes under 50°C water bath, then transfer the beaker to 70°C water bath for 15 minutes, cool in ice bath after digestion, and use circulation
  • the water vacuum pump sucks and filters the remaining solution onto the PTFE hydrophilic filter membrane.
  • Feces freeze-drying The human feces taken are freeze-dried in a freeze-drying apparatus, and 2 g dry weight of the freeze-dried sample is taken, and 3 parallel samples are taken.
  • the water vacuum pump sucks and filters the remaining solution onto the PTFE hydrophilic filter membrane.
  • Feces freeze-drying freeze-dry the fetched chicken manure in a freeze dryer, take 5g dry weight of the freeze-dried sample, and 3 parallel samples.
  • a 500mL beaker add 100mL 65% concentrated nitric acid to each beaker, digest for 30 minutes under 50°C water bath, then transfer the beaker to 70°C water bath for 15 minutes, cool in ice bath after digestion, and use circulation
  • the water vacuum pump sucks and filters the remaining solution onto the PTFE hydrophilic filter membrane.
  • Feces freeze-drying freeze-dry the fetched chicken manure in a freeze dryer, take 2g dry weight of the freeze-dried sample, and 3 parallel samples.
  • Feces freeze-drying freeze-dry the zebrafish feces taken in a freeze-drying apparatus, take 2g dry weight of the freeze-dried sample, and 3 parallel samples.
  • the number of water-based CN-CA filters is 5, and transfer the water-based CN-CA filter membrane after suction to a 500mL beaker.
  • the number of filter membranes in each beaker is not more than 3, add 100mL 65% concentrated nitric acid to each beaker, digest in a water bath at 50°C for 30 minutes, then transfer the beaker to a water bath at 70°C for digestion for 15 minutes, after completion It is cooled in an ice bath, and the remaining solution is filtered by a circulating water vacuum pump onto the PTFE hydrophilic membrane.
  • Feces freeze-drying freeze-dry the collected human feces in a freeze dryer, take 5g dry weight of the freeze-dried sample, add PS plastic particles with an average particle size of 250 ⁇ m to each of the 3 parallel samples 10 10 pellets, 10 PE plastic pellets with an average particle size of 150 ⁇ m, and 10 PVC plastic pellets with an average particle size of 75 ⁇ m, add distilled water and mix well and freeze-dry again.
  • a 500mL beaker add 100mL 65% concentrated nitric acid to each beaker, digest for 30 minutes under 50°C water bath, then transfer the beaker to 70°C water bath for 15 minutes, cool in ice bath after digestion, and use circulation
  • the water vacuum pump sucks and filters the remaining solution onto the PTFE hydrophilic filter membrane.
  • the retentate on the PTFE hydrophilic filter membrane is dried and microscopically inspected, and the suspected plastic particles are selected for microscope-Raman spectroscopy to obtain the detection pattern, and the microplastics are identified according to the identification standard.
  • Feces freeze-drying freeze-dry the fetched chicken feces in a freeze dryer, take 5g dry weight of the freeze-dried sample, add PS plastic particles with an average particle size of 250 ⁇ m to each of the 3 parallel samples 10 10 pellets, 10 PE plastic pellets with an average particle size of 150 ⁇ m, and 10 PVC plastic pellets with an average particle size of 75 ⁇ m, add distilled water and mix well and freeze-dry again.
  • a 500mL beaker add 100mL 65% concentrated nitric acid to each beaker, digest for 30 minutes under 50°C water bath, then transfer the beaker to 70°C water bath for 15 minutes, cool in ice bath after digestion, and use circulation
  • the water vacuum pump sucks and filters the remaining solution onto the PTFE hydrophilic filter membrane.
  • the retentate on the PTFE hydrophilic filter membrane is dried and microscopically inspected, and the suspected plastic particles are selected for microscope-Raman spectroscopy to obtain the detection pattern, and the microplastics are identified according to the identification standard. Dry and weigh the PTFE hydrophilic filter membrane.

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Abstract

一种从人体和家禽粪便中提取微塑料的方法，属于环境健康风险评价领域。方法包括粪便冻干、样品消解及样品提取，其中消解过程采用芬顿试剂处理，浓硝酸消解，无水乙醇提取。可保障对微塑料影响最小化的条件下方便、快捷地从人畜粪便中提取微塑料，便于精准分析生物粪便中微塑料的种类和丰度，为研究微塑料在生物体内的富集和排泄规律，以及健康风险评价提供强有力的技术保障和数据支撑。

Description

一种人畜粪便中提取微塑料的方法 技术领域
本发明涉及一种人畜粪便中提取微塑料的方法，在保障对微塑料影响最小化的条件下可快速、高效、高保真地提取人畜粪便中的微塑料，便于精准分析生物粪便中微塑料的种类和丰度，为研究微塑料在生物体内的富集和排泄规律，以及健康风险评价提供强有力的技术保障和数据支撑。本发明属于环境健康风险评估领域。
背景技术
微塑料是指直径小于5mm的塑料颗粒，一方面来自于工业原料、个人护理品(如牙膏、洗面奶)和医药制品中小颗粒塑料微珠的一次源；另一方面可来自于环境中大块塑料垃圾经过物理、化学和生物过程分裂而形成的二次源。微塑料可被生物体摄入，产生毒害作用。随着全球塑料的产量和使用量不断增加，微塑料污染带来的环境危害和健康风险成为各界关注的焦点。国内外研究证实，微塑料通过误食等途径进入生物体内，主要在富集在肠道内潜在健康风险。但是目前有关存在于肠道中的微塑料的种类(材质、粒径、形状等)、富集丰度、残留时间以及其对肠道组织和肠道微生物的毒性效应尚不明确。分析粪便中的微塑料是研究解决上述问题的必要手段。但是目前缺少快速、高效、高保真的粪便微塑料提取方法，严重制约了微塑料健康风险评价的相关研究。
现有的微塑料提取方法主要针对生物组织和污泥样品，主要步骤包括:利用强酸、强碱对样品直接进行消解，对消解过后的样品进行密度分离将微塑料分离出来。粪便样品既不同于生物组织几乎全是生物有机质，也不同于污泥含有大量泥沙。现有的生物组织和污泥微塑料提取方法并不适用于粪便样品，主要存在以下不足:(1)密度分离不适用于分离粪便消解液与微塑料；(2)消解完的有机物会覆盖在微塑料的表面上，影响后续的微塑料的显微镜-拉曼光谱鉴别；(3)现有的消解条件过于激烈，对微塑料的损害较大，不利于真实准确地评估微塑料的健康风险。
因此，在研究微塑料在生物体内的富集和排泄规律以及微塑料的健康风险评价时，现有的方法对微塑料本身的性质影响较大、不能高效的从粪便中分离微塑料，亟需一种快速、方便、高保真的方法来提取粪便中的微塑料。本发明提供了一种人畜粪便中提取微塑料的方法，能够简单、快捷、高效率的精准分析生物粪便中微塑料的种和丰度，且对微塑料本身的性质影响较小，便于利用显微镜-拉曼光谱对微塑料进行观察和鉴别。
发明内容
针对现在普遍缺乏从生物体粪便中提取微塑料的方法，本发明提供一种人畜粪便中提取微塑料的方法，能够快速、方便、高效的分离粪便中的微塑料。为了解决上述问题以及实现发明目的，本发明提供以下技术方案:
一种人畜粪便中提取微塑料的方法，其特征在于，包括如下步骤:
(1)粪便冻干:将取来的生物体粪便在冷冻干燥仪中冻干，取冻干后的样品N g；
(2)样品消解:将步骤(1)中冻干的粪便转移到烧杯中，依次向其中添加20g/L的FeSO 4·7H 2O催化剂和30％H 2O 2试剂，其中添加体积比为20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5，在搅拌下充分接触反应，直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的体积比继续添加芬顿试剂，每次添加30％H 2O 2不超过50mL，直至反应完成，反应过程中控制温度不超过40℃；然后将剩余有机物抽滤到水系CN-CA滤膜上，将抽滤后的水系CN-CA滤膜转移到500mL的烧杯中，每个烧杯的滤膜张数不超过3张，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，将剩余溶液抽滤到PTFE亲水型滤膜上；
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到500mL烧杯并向烧杯中添加200mL无水乙醇，超声处理后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，将烧杯中溶液再次抽滤到PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和镜检，挑选疑似为塑料颗粒进行显微镜-拉曼光谱检测，得到检测图谱，按照鉴定标准鉴定微塑料。
优选的，步骤(1)所述取冻干的样品的重量N＝1-5g。
优选的，步骤(2)所述FeSO 4·7H 2O催化剂制备是按每10g  FeSO 4·7H 2O溶于500mL蒸馏水的比例溶解，并用浓硫酸调节pH＝3。
优选的，步骤(2)中反应完成时添加的30％H 2O 2为N×100mL，添加体积比为20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5。
优选的，步骤(2)所述使用水系CN-CA滤膜的数量为M＝2N+1张，其中M取整数。
优选的，步骤(2)所述水系CN-CA滤膜和PTFE亲水型滤膜孔径为1μm。
优选的，步骤(3)所述超声处理在100KHz条件下超声10-15分钟。
优选的，步骤(1)中生物体粪括人体粪便、畜禽粪便，生物粪便每个样本不少于3个平行样。
采用以上消解方法所消解提取生物粪便中的微塑料在本发明的保护范围中。
本发明所公开的方法，在保障对微塑料影响最小化的条件下可快速、高效、高保真地提取人畜粪便中的微塑料，并可以检测出一定质量内粪便中含有微塑料的种类和丰度。本方法基于芬顿反应将粪便中的易消解的有机物质消解，再利用浓硝酸在50℃水浴条件下对水系CN-CA滤膜和剩余有机物质进行消解，最后利用浓硝酸在70℃水浴条件下对难消解的有机物质消解。消解完成后利用无水乙醇在超声条件下溶解部分剩余覆盖在微塑料表面的有机物质并提取微塑料，提高显微-拉曼光谱鉴别微塑料的效率。
实验结果表明:采用本发明所提供的消解方法能够高效的消解生物体的粪便，并不会对塑料的物化特征产生明显的影响，而采用传统的单一的强酸强碱进行消解，消解时间较长、消解不完全、消解过程中对微塑料的光学特征和形态影响较大，本发明解决了这些问题，在保障对微塑料影响最小化的条件下可快速、高效、高保真地提取人畜粪便中的微塑料，便于精准分析生物粪便中微塑料的种类和丰度，可以广泛应用于生物体粪便中微塑料的提取和检测。
附图说明
图1为本发明所述方法的工艺流程；
图2位本发明粪便消解效率图像；
图3为本发明粪便消解实验前后的PE塑料的拉曼光谱对比图像；
图4为本发明粪便消解实验前后的PS塑料的拉曼光谱对比图像；
图5为本发明粪便消解实验前后的PVC塑料的拉曼光谱对比图像；
图6位本发明粪便消解实验后在提取到的微塑料的显微图像；
图7为本发明粪便消解实验后的PTFE亲水型滤膜上剩余有机物图像；
图8位本发明粪便消解试验后的塑料颗粒回收率图像。
具体实施方式
本发明涉及一种人畜粪便中提取微塑料的方法，为了更好的应用本发明，给出了优选实施方案的以下案例。下面的实施例绝不应当解 读为限制或者限定本发明的范围。
本发明公开一种从人体和家禽粪便中提取微塑料的方法，该方法主要包括三个步骤，首先将粪便取回后放入冷冻干燥仪冻干，然后通过添加消解试剂进行消解、真空抽滤、超声处理后真空抽滤的方法提取微塑料。通过本发明可保障对微塑料影响最小化的条件下方便、快捷地从人畜粪便中提取微塑料，便于精准分析生物粪便中微塑料的种类和丰度，为研究微塑料在生物体内的富集和排泄规律，以及健康风险评价提供强有力的技术保障和数据支撑。
具体来说，本发明的方法包括以下步骤:
(1)粪便冻干:将取来的生物体粪便在冷冻干燥仪中冻干，取冻干后的样品N g；
(2)样品消解:将步骤(1)中冻干的粪便转移到烧杯中，依次向其中添加20g/L的FeSO 4·7H 2O催化剂和30％H 2O 2试剂，其中添加体积比为20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5，在搅拌下充分接触反应，直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的体积比继续添加芬顿试剂，每次添加30％H 2O 2不超过50mL，直至反应完成，反应过程中控制温度不超过40℃；然后将剩余有机物抽滤到水系CN-CA滤膜上，将抽滤后的水系CN-CA滤膜转移到500mL的烧杯中，每个烧杯的滤膜张数不超过3张，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，将剩余溶液抽滤到PTFE亲水型滤膜上；
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到500mL烧杯并向烧杯中添加200mL无水乙醇，超声处理后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，将烧杯中溶液再次抽滤到PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和镜检，挑选疑似为塑料颗粒进行显微镜-拉曼光谱检测，得到检测图谱，按照鉴定标准鉴定微塑料。
所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(1)所述取冻干的样品的重量1-5g。
所述的消解生物体粪便并从中提取微塑料的方法，其特征在于，步骤(2)所述FeSO 4·7H 2O催化剂制备将10g FeSO 4·7H 2O溶于500mL蒸馏水中，并用浓硫酸调节pH＝3。
所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(2)中反应完成时，30％H 2O 2的添加总量为N×100mL，添加体积比为20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5。
所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(2)所述使用水系CN-CA滤膜的数量为M＝2N+1张(M取整数)。
所述的一种人畜粪便中提取微塑料的方法，步骤(2)所述水系CN-CA滤膜和PTFE亲水型滤膜孔径为1μm。
在本发明中，所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(3)所述超声处理为在100KHz条件下超声处理10-15分钟。
所述的方法，所述步骤(1)中生物体粪括人体粪便、畜禽粪便。
所述的方法，所述步骤(1)生物粪便每个样本不少于3个平行样。
实施例1
(1)粪便冻干:将取来的人体粪便在冷冻干燥仪中冻干，取冻干后的样品5g干重，3个平行样。
(2)样品消解:将步骤(1)中冻干的粪便转移到2L的烧杯中，向其中添加20mL的20g/L FeSO 4·7H 2O催化剂，再向其中加入50mL 30％H 2O 2试剂，利用磁力搅拌器充分接触反应直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的比例继续添加芬顿试剂，每次添加30％H 2O 2不得超过50mL，直至添加200mL 30％H 2O 2反应完成，反应过程中利用冰浴控制温度不超过40℃。利用循环水式真空泵将剩余有机物抽滤到水系CN-CA滤膜上，使用水系CN-CA滤膜的数量为M＝11张，将抽滤后的水系CN-CA滤膜小于等于3张转移到500mL的烧杯中，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，利用循环水式真空泵将剩余溶液抽滤到PTFE亲水型滤膜上。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到(2)中500mL烧杯并向烧杯中添加200mL无水乙醇，在超声波清洗仪中100KHz超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯中溶液再次抽滤到(2)中所述的PTFE亲水型滤膜上，对 PTFE亲水型滤膜上的截留物进行干燥和称量。
将PTFE亲水型滤膜进行烘干、称重。消解效率见图2。
实施例2
(1)粪便冻干:将取来的人体粪便在冷冻干燥仪中冻干，取冻干后的样品2g干重，3个平行样。
(2)样品消解:将步骤(1)中冻干的粪便转移到2L的烧杯中，向其中添加20mL的20g/L FeSO 4·7H 2O催化剂，再向其中加入50mL 30％H 2O 2试剂，利用磁力搅拌器充分接触反应直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的比例继续添加芬顿试剂，每次添加30％H 2O 2不得超过50mL，直至添加200mL 30％H 2O 2反应完成，反应过程中利用冰浴控制温度不超过40℃。利用循环水式真空泵将剩余有机物抽滤到水系CN-CA滤膜上，使用水系CN-CA滤膜的数量为M＝5张，将抽滤后的水系CN-CA滤膜小于等于3张转移到500mL的烧杯中，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，利用循环水式真空泵将剩余溶液抽滤到PTFE亲水型滤膜上。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到(2)中500mL烧杯并向烧杯中添加200ml无水乙醇，在超声波清洗仪中超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯 中溶液再次抽滤到(2)中所述的PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和称量。
将PTFE亲水型滤膜进行烘干、称重。消解效率见图2。
实施例3
(1)粪便冻干:将取来的鸡粪在冷冻干燥仪中冻干，取冻干后的样品5g干重，3个平行样。
(2)样品消解:将步骤(1)中冻干的粪便转移到2L的烧杯中，向其中添加20mL的20g/L FeSO 4·7H 2O催化剂，再向其中加入50mL 30％H 2O 2试剂，利用磁力搅拌器充分接触反应直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的比例继续添加芬顿试剂，每次添加30％H 2O 2不得超过50mL，直至添加500mL 30％H 2O 2反应完成，反应过程中利用冰浴控制温度不超过40℃。利用循环水式真空泵将剩余有机物抽滤到水系CN-CA滤膜上，使用水系CN-CA滤膜的数量为M＝11张，将抽滤后的水系CN-CA滤膜小于等于3张转移到500mL的烧杯中，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，利用循环水式真空泵将剩余溶液抽滤到PTFE亲水型滤膜上。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到(2)中500mL烧杯并向烧杯中添加200mL无水乙醇，在超声波清洗仪中超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型 滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯中溶液再次抽滤到(2)中所述的PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和称量。
将PTFE亲水型滤膜进行烘干、称重。消解效率见图2。
实施例4
(1)粪便冻干:将取来的鸡粪在冷冻干燥仪中冻干，取冻干后的样品2g干重，3个平行样。
(2)样品消解:将步骤(1)中冻干的粪便转移到2L的烧杯中，向其中添加20mL的20g/L FeSO 4·7H 2O催化剂，再向其中加入50mL 30％H 2O 2试剂，利用磁力搅拌器充分接触反应直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的比例继续添加芬顿试剂，每次添加30％H 2O 2不得超过50mL，直至添加200mL 30％H 2O 2反应完成，反应过程中利用冰浴控制温度不超过40℃。利用循环水式真空泵将剩余有机物抽滤到水系CN-CA滤膜上，使用水系CN-CA滤膜的数量为M＝5张，将抽滤后的水系CN-CA滤膜小于等于3张转移到500mL的烧杯中，每个烧杯加入100mlL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，利用循环水式真空泵将剩余溶液抽滤到PTFE亲水型滤膜上。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到(2)中500mL烧杯并向烧杯中添加200mL无水乙醇，在超声波 清洗仪中超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯中溶液再次抽滤到(2)中所述的PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和称量。
将PTFE亲水型滤膜进行烘干、称重。
实施例5
(1)粪便冻干:将取来的斑马鱼粪便在冷冻干燥仪中冻干，取冻干后的样品2g干重，3个平行样。
(2)样品消解:将步骤(1)中冻干的粪便转移到2L的烧杯中，依次向其中添加体积比为20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5，在搅拌下充分接触反应，直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的体积比继续添加芬顿试剂，每次添加30％H 2O 2不得超过50mL，直至添加200mL 30％H 2O 2且反应完成，反应过程中利用冰浴控制温度不超过40℃。利用循环水式真空泵将剩余有机物抽滤到水系CN-CA滤膜上，用水系CN-CA滤膜的数量为5张，将抽滤后的水系CN-CA滤膜转移到500mL的烧杯中，每个烧杯的滤膜张数不超过3张，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，完成后冰浴冷却，利用循环水式真空泵将剩余溶液抽滤到PTFE亲水型滤膜上。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移 到(2)中500mL烧杯并向烧杯中添加200mL无水乙醇，在超声波清洗仪中超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯中溶液再次抽滤到PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和称量。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到(2)中500mL烧杯并向烧杯中添加200mL无水乙醇，在超声波清洗仪中超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯中溶液再次抽滤到(2)中所述的PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和称量。
将PTFE亲水型滤膜进行烘干、称重。消解效率见图2。
实施例6
(1)粪便冻干:将取来的人体粪便在冷冻干燥仪中冻干，取冻干后的样品5g干重，3个平行样中每个平行样加入平均粒径250μm的PS塑料颗粒10粒、平均粒径150μm的PE塑料颗粒10粒、平均粒径75μm的PVC塑料10粒，加蒸馏水混匀后再次冻干。
(2)样品消解:将步骤(1)中冻干的粪便转移到2L的烧杯中，向其中添加20mL的20g/L FeSO 4·7H 2O催化剂，再向其中加入50mL 30％H 2O 2试剂，利用磁力搅拌器充分接触反应直至没有气泡产生后，按照FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的比例继续添加芬 顿试剂，每次添加30％H 2O 2不得超过50mL，直至添加200mL 30％H 2O 2反应完成，反应过程中利用冰浴控制温度不超过40℃。利用循环水式真空泵将剩余有机物抽滤到水系CN-CA滤膜上，使用水系CN-CA滤膜的数量为M＝11张，将抽滤后的水系CN-CA滤膜小于等于3张转移到500mL的烧杯中，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，利用循环水式真空泵将剩余溶液抽滤到PTFE亲水型滤膜上。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到(2)中500mL烧杯并向烧杯中添加200mL无水乙醇，在超声波清洗仪中超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯中溶液再次抽滤到(2)中所述的PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和镜检，挑选疑似为塑料颗粒进行显微镜-拉曼光谱检测，得到检测图谱，按照鉴定标准鉴定微塑料。
将PTFE亲水型滤膜上疑似微塑料的颗粒进行拉曼光谱对比、显微镜图像观察分析，结果见图3-4。
实施例7
(1)粪便冻干:将取来的鸡粪便在冷冻干燥仪中冻干，取冻干后的样品5g干重，3个平行样中每个平行样加入平均粒径250μm的PS塑料颗粒10粒、平均粒径150μm的PE塑料颗粒10粒、平均粒径 75μm的PVC塑料10粒，加蒸馏水混匀后再次冻干。
(2)样品消解:将步骤(1)中冻干的粪便转移到2L的烧杯中，向其中添加20mL的20g/L FeSO 4·7H 2O催化剂，再向其中加入50mL 30％H 2O 2试剂，利用磁力搅拌器充分接触反应直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的比例继续添加芬顿试剂，每次添加30％H 2O 2不得超过50mL，直至添加200mL 30％H 2O 2反应完成，反应过程中利用冰浴控制温度不超过40℃。利用循环水式真空泵将剩余有机物抽滤到水系CN-CA滤膜上，使用水系CN-CA滤膜的数量为M＝11张，将抽滤后的水系CN-CA滤膜小于等于3张转移到500mL的烧杯中，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，利用循环水式真空泵将剩余溶液抽滤到PTFE亲水型滤膜上。
(3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到(2)中500mL烧杯并向烧杯中添加200mL无水乙醇，在超声波清洗仪中超声处理10-15分钟后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，利用循环水式真空泵将烧杯中溶液再次抽滤到(2)中所述的PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和镜检，挑选疑似为塑料颗粒进行显微镜-拉曼光谱检测，得到检测图谱，按照鉴定标准鉴定微塑料。将PTFE亲水型滤膜进行烘干、称重。
将PTFE亲水型滤膜上疑似微塑料的颗粒进行拉曼光谱对比、显 微镜图像观察分析，结果见图3-4。
上面结合附图和具体实施例对本发明的实施方式做了详细的说明，但是本发明不限于上述实施方式，在所述技术领域的普通技术人员所具备的指示范围内，还可以在不脱离本发明宗旨的前提下做出各种变化。

Claims (9)

  1. 一种人畜粪便中提取微塑料的方法，其特征在于，包括如下步骤:
    (1)粪便冻干:将取来的生物体粪便在冷冻干燥仪中冻干，取冻干后的样品Ng；
    (2)样品消解:将步骤(1)中冻干的粪便转移到烧杯中，依次向其中添加20g/L的FeSO 4·7H 2O催化剂和30％H 2O 2试剂，其中添加体积比为20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5，在搅拌下充分接触反应，直至没有气泡产生后，按照20g/L FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5的体积比继续添加芬顿试剂，每次添加30％H 2O 2不超过50mL，直至反应完成，反应过程中控制温度不超过40℃；然后将剩余有机物抽滤到水系CN-CA滤膜上，将抽滤后的水系CN-CA滤膜转移到500mL的烧杯中，每个烧杯的滤膜张数不超过3张，每个烧杯加入100mL 65％的浓硝酸，在50℃水浴条件下消解30分钟，然后将烧杯转移至70℃水浴条件下消解15分钟，消解完成后冰浴冷却，将剩余溶液抽滤到PTFE亲水型滤膜上；
    (3)样品提取:将步骤(2)中得到的PTFE亲水型滤膜再次转移到500mL烧杯并向烧杯中添加200mL无水乙醇，超声处理后，用无水乙醇反复冲洗PTFE亲水型滤膜三次，将PTFE亲水型滤膜依次取出，将烧杯中溶液再次抽滤到PTFE亲水型滤膜上，对PTFE亲水型滤膜上的截留物进行干燥和镜检，挑选疑似为塑料颗粒进行显微镜-拉曼光谱检测，得到检测图谱，按照鉴定标准鉴定微塑料。
  2. 根据权利要求1所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(1)所述取冻干的样品的重量N＝1-5g。
  3. 根据权利要求1所述的消解生物体粪便并从中提取微塑料的方法，其特征在于，步骤(2)所述FeSO 4·7H 2O催化剂制备是按每10g FeSO 4·7H 2O溶于500mL蒸馏水的比例溶解，并用浓硫酸调节pH＝3。
  4. 根据权利要求1所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(2)中反应完成时添加30％H 2O 2为N×100mL，添加体积比为FeSO 4·7H 2O催化剂和30％H 2O 2 1:2.5。
  5. 根据权利要求1所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(2)所述使用水系CN-CA滤膜的数量为M＝2N+1张，其中M取整数。
  6. 根据权利要求1所述的一种人畜粪便中提取微塑料的方法，步骤(2)所述水系CN-CA滤膜和PTFE亲水型滤膜孔径为1μm。
  7. 根据权利要求1所述的一种人畜粪便中提取微塑料的方法，其特征在于，步骤(3)所述超声处理在100KHz条件下超声10-15分钟。
  8. 根据权利要求1中所述的方法，其特征在于，所述步骤(1)中生物体粪包括人体粪便、畜禽粪便。
  9. 根据权利要求1中所述的方法，其特征在于，所述步骤(1)生物粪便每个样本不少于3个平行样。
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