Method for labeling micro / nano plastic based on stable isotope and application

Abstract

The invention belongs to the field of environmental analytical chemistry, and provides a method for preparing micro / nano plastic based on stable isotope labeling and application of the micro / nano plastic. Specifically, an initiator, a pH buffer solution, ultrapure water, an emulsifier and a deuterated styrene monomer are mixed and emulsified through an emulsion polymerization method, a polymerization reaction is performed after emulsification, and then the deuterated polystyrene microspheres are obtained through cleaning and drying. According to the method, high sensitivity, high accuracy and safety of deuterium labeling are utilized, and a new research thought is provided for deeply researching the migration and transformation rule of the micro / nano plastic in the environment and long-term influence of the micro / nano plastic on organisms. The method is expected to promote the wide application of a hydrogen stable isotope labeling quantitative technology in the field of environmental science, and provides a scientific basis for evaluating the influence of the micro / nano plastic on the ecological environment and human health.

Description

Technical Field

[0001] The invention belongs to the field of environmental analytical chemistry and proposes a method and application of micro / nano plastics labeled with stable isotopes. Background Art

[0002] With the mass production, use and disposal of plastic products, plastic pollution has become one of the most serious threats to the global ecosystem (Barnes et al., 2009). Among them, microplastics (particle size less than 5 mm) as an emerging pollutant have attracted much attention due to their wide distribution in the environment, large quantity and potential ecological and health risks. Microplastics are small in size and easily ingested by organisms, which may have adverse effects on ecosystems and human health. In recent years, studies have found that microplastics are widely present in aquatic organisms and food chains (von Moos et al., 2012). Not only nano-scale, but also submicron-scale and even micron-scale plastic particles can be accumulated and transported in higher plants such as crops (Dong et al., 2021; Jiang et al., 2019; Li et al., 2019; Sun et al., 2020), and studies have revealed the pathways and mechanisms of plant absorption of microplastics (Li et al., 2020). However, due to the small size and complex properties of microplastics, stable, reliable and highly sensitive quantitative detection of microplastics in complex environments and biological systems still faces huge technical challenges, becoming an international problem in the field of microplastic research. This technical bottleneck limits our ability to accurately quantify the accumulation of microplastics in plants, their transport to edible parts, and their transfer in the food chain.

[0003] At present, the tracking of microplastics in complex environmental biological systems mainly relies on various labeling methods. Although the fluorescent labeling method is easy to operate, it has problems such as interference from spontaneous fluorescence of biological tissues and dye leakage (Catarino et al., 2019). Although the metal ion labeling method can quantify microplastics, metal ions may dissolve or transform, affecting the detection results (Mitrano et al., 2019). These traditional labeling methods have problems such as marker shedding and biological toxicity, and may change the intrinsic properties of microplastics.

[0004] As a highly sensitive quantitative analysis method, isotope labeling plays an irreplaceable role in biological effects and toxicology research. This method is mainly divided into radioactive isotope labeling and stable isotope labeling. Although radioactive isotope labeling has advantages such as high sensitivity, its radioactivity, high cost, and the need for special equipment and special treatment limit its widespread application. In contrast, stable isotope labeling has the advantages of being non-radioactive, environmentally friendly, and able to distinguish between endogenous and exogenous elements, and has become an important tool in nanotoxicology research. Stable isotope labeling, especially 13C labeling, has been successfully used in the study of the environmental behavior and biological effects of carbon nanomaterials and metal nanomaterials. However, in the study of microplastics, stable isotope labeling is still in its infancy. On the one hand, the 13C signal of natural organic matter in environmental samples may interfere with the detection of microplastics; on the other hand, different instruments have different detection sensitivities to isotope abundance, and some labeled elements (such as 109 Ag) is expensive or toxic to organisms. Therefore, it is urgent to develop a more efficient, reliable and environmentally friendly microplastic labeling method to achieve multi-scale tracking of microplastics in the environment and organisms, and deeply reveal their migration and transformation laws and biological effects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method and application of micro / nano plastics based on stable isotope labeling.

[0006] The technical solution of the present invention to solve the above problems is as follows:

[0007] A method based on stable isotope labeled micro / nano plastics, wherein an initiator, a pH buffer solution, ultrapure water, an emulsifier and deuterated styrene monomers are mixed and emulsified by an emulsion polymerization method, and then subjected to polymerization reaction after emulsification, and then washed and dried to obtain deuterated polystyrene microspheres.

[0008] Specifically, under an inert gas environment, an emulsifier and a pH buffer solution are added to water, and the mixture is stirred and emulsified at room temperature. Then, deuterated styrene monomer is added to the system, and the temperature is raised to 60-65°C, and the mixture is further stirred and mixed. After mixing, an initiator is added to the system, and the polymerization reaction is carried out at 60-85°C under closed conditions for 24±2h; after the reaction, the polymer emulsion is successively centrifuged and washed with ethanol and ultrapure water, and the precipitate is collected and dried to obtain deuterated polystyrene microspheres.

[0009] The mass ratio of the initiator, pH buffer, ultrapure water, emulsifier and deuterated styrene monomer in the system is (1-10):1:(1-3):(1-3):1; wherein the initiator is water-soluble potassium persulfate (KPS), the pH buffer is sodium bicarbonate (NaHCO 3), and the emulsifier was sodium dodecyl sulfate (SDS).

[0010] An application of the stable isotope-labeled micro / nano plastics prepared by the method described, and application of the microspheres in quantitative tracing detection of microplastics in complex biological systems.

[0011] A method for detecting deuterated polystyrene microspheres:

[0012] S1 thoroughly washes the biological sample that absorbs the prepared deuterated polystyrene nanoplastic;

[0013] S2 separates the biological samples by parts, dries and grinds them separately to obtain powder samples with uniform particle size;

[0014] S3 sends the sample prepared in S2 to an element analyzer and an isotope ratio mass spectrometer for analysis, and the H% is measured by the element analyzer. sample , the isotope ratio mass spectrometer measured δ sample , δ control , the sample mass m is measured by analytical balance sample；

[0015] S4 calculates the content of deuterated polystyrene in the tracking sample using the measured H isotope ratio according to a pre-established mathematical formula model.

[0016] The mathematical model in S4 is:

[0017]

[0018] Where R s and R std Respectively 2 H / 1 The abundance ratio of H in the sample and the international standard (Vienna-Standard Mean Ocean Water, V-SMOW), the H isotope ratio (δ 2 H,‰) were expressed as normalized values ​​(detection accuracy <3‰), and the deuterated polystyrene concentration (C 2 H-PS ) is calculated as follows

[0019]

[0020]

[0021]

[0022] According to formula (1), based on the measured δ value, it can be calculated back to 2 H / 1 The abundance ratio of H to RS ; R obtained in formula (1) S Substituting into formula (2) to calculate the sample 2 The mass fraction of H in the total H, i.e., ω 2 H ; Then calculate the sample according to formula (3) 2 The mass of H isotope labeled polystyrene particles m 2 H-PS , H% in formula (3) sample is the mass percentage of H in the sample dry weight, ω 2 H(sample) yes 2 The mass fraction of H in the total H in the sample, ω 2 H(control) yes 2 The mass fraction of H in the total H in the control sample, m sample is the dry weight of the sample, 16 / 112 is the 2 The mass fraction of H in the sample 2 The concentration of H isotope labeled polystyrene microspheres C 2 H-PS It can be obtained according to formula (4).

[0023] In step S1, the biological sample is ultrasonically cleaned with ultrapure water for 3-5 minutes;

[0024] The drying in step S2 is performed at a high temperature of 85-90°C for 2 hours, and then at a medium temperature of 55-60°C for 24 hours, until the plant body completely loses moisture;

[0025] Grinding is done in two steps. First, it is machine-ground using a low-temperature biological tissue preparation machine at a speed of 6.5 m / s and a temperature of 23°C. Then it is manually ground using a mortar. The final powder must be able to pass a 60-mesh sieve.

[0026] The advantages of the present invention are:

[0027] The present invention uses 2 The H labeling technology labels polystyrene plastic microspheres in situ during the synthesis stage, and constructs a highly sensitive quantitative tracking technology method for plastic particles based on stable isotope labeling. This labeling method not only does not change the chemical structure and properties of the microplastics themselves, but also does not cause the detachment or breakage of the tracer atoms in the body. In addition, the stable isotopes are non-radioactive and safe and harmless to organisms. This direct labeling method allows polystyrene to be labeled with H. 1 H by 2 H substitution, 2 H / 1H ratio changes, and elemental analyzer (Vario EL Cube, Elementar, Germany) and isotope ratio mass spectrometer (MAT 253plus, ThermoFisher Scientific, Germany) can be used for high-precision and ultra-sensitive quantitative detection, objectively reflecting the biological behavior of the intrinsic properties of polystyrene particles, and providing a stable and reliable quantitative technical means for quantitatively evaluating the accumulation and transfer behavior of microplastic particles in organisms and food chains; thereby solving the lack of accurate quantitative tracing methods for microplastic particles in complex environmental biological media and the lack of research in terrestrial food chains. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the structural formula of deuterated polystyrene (D is 2 H marker site);

[0029] Figure 2 This is the infrared spectrum analysis spectrum of deuterated polystyrene microspheres;

[0030] Figure 3 is a scanning electron microscope image of deuterated polystyrene microspheres;

[0031] Figure 4 Scanning electron micrographs of deuterated polystyrene microspheres in plant root tissues (global image (a), local image (b)). DETAILED DESCRIPTION

[0032] The principles and features of the present invention are described below in conjunction with specific embodiments. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative achievements belong to the protection scope of the present invention.

[0033] The present invention uses the preparation of polystyrene microspheres labeled with stable isotope 2H for quantitative tracing, which is used to study the migration, transformation, bioaccumulation and enrichment process of microplastics in complex biological systems. By deuterium labeling the polystyrene microspheres, highly sensitive and accurate quantitative tracking of the behavior of micro / nano plastics in the environment is achieved. This method provides a reliable technical means for in-depth understanding of the environmental behavior and ecological effects of micro / nano plastics, and has broad application prospects in the fields of environmental monitoring, ecological risk assessment, etc.

[0034] The present invention obtains microspheres using 2 H-labeled, the obtained material has stable properties, sensitive detection, safe and pollution-free use, and the obtained microspheres can help trace their transport and absorption pathways when studying water metabolism and transpiration in plants or animals. In addition, in mass spectrometry analysis 2H labeling can significantly change the molecular mass, thus providing a degree of differentiation, and 2 The introduction of H does not easily change the original properties of the molecule and can reduce the interference of the label on the molecular behavior.

[0035] The method of the present invention utilizes the high sensitivity, high accuracy and safety of deuterium labeling to provide a new research idea for in-depth study of the migration and transformation rules of micro / nano plastics in the environment and the long-term effects on organisms. Specifically, the present invention prepares deuterated polystyrene nanoplastic particles by emulsion polymerization, and conducts plant absorption and animal ingestion experiments. The H isotope ratio (δ2H) and the H element mass fraction in the biological sample are accurately determined by using an element analyzer and a stable isotope ratio mass spectrometer (IRMS). Combined with the established mathematical formula model, the concentration of deuterated polystyrene nanoplastics in the organism can be accurately calculated. The method provides a reliable tool for quantitative analysis of the absorption, transport and enrichment process of nanoplastics by organisms, thereby deeply revealing the behavior process and mechanism of nanoplastics in organisms. The present invention is expected to promote the widespread application of H stable isotope labeling quantitative technology in the field of environmental science, and provide a scientific basis for evaluating the impact of micro / nanoplastics on the ecological environment and human health.

[0036] Example 1: A nanostructured ... 2 Preparation method of H stable isotope labeled polystyrene microspheres;

[0037] This embodiment provides a 2 The preparation method and characterization of H stable isotope labeled polystyrene microspheres include the following steps:

[0038] 1. 2 Preparation of H stable isotope labeled polystyrene microspheres

[0039] (1) Before the reaction starts, all media are quenched with N 2 The method involves mixing 100 mg KPS (potassium persulfate) along with 50 mg Na 2 CO 3 Add to a three-necked flask containing 100 mL of ultrapure water and continue to flow N at 25 °C. 2 , and stirred vigorously at 350r / min for 30min. Subsequently, 4mL of deuterated styrene monomer was added, while the temperature was raised to 60°C and stirred for another 30min. After this step, 7.5mL of a heated solution containing 0.031g of SDS was added and further reacted at 75°C for 24h.

[0040] (2) After the prepared emulsion is polymerized, the obtained precipitate is centrifuged multiple times with ethanol and ultrapure water (12,000 rpm / 60 min), and vacuum dried at 60° C. for 12 h to obtain deuterated polystyrene microsphere powder, which is stored in a constant temperature drying oven before analysis.

[0041] 2. 2 Characterization of H stable isotope labeled polystyrene microspheres

[0042] (1) The total H content in the sample was measured using an elemental analyzer (Vario EL Cube, Elementar, Germany), and the H isotope ratio (δ2H) of the deuterated polystyrene microspheres was accurately determined using an isotope ratio mass spectrometer (MAT 253plus, Thermo Fisher Scientific, Germany).

[0043] In order to accurately determine R S The present invention uses synthetic deuterated polystyrene microspheres ( 2 H-PSNPs) and unlabeled polystyrene microspheres (PSNPs) were measured using 2 The deuterium rate DR (%) was calculated based on the H mass conservation principle;

[0044]

[0045] In formula (5), we mix the synthesized deuterated polystyrene and unlabeled polystyrene and use 2 H mass conservation principle to calculate ω 2 H-PS ,ω 2 H-PS For the synthesis of deuterated polystyrene 2 The mass fraction of H in the total H, ω PS for 2 H in unlabeled polystyrene 2 The mass fraction of H in the total H, ω mix A mixture of deuterated polystyrene and unlabeled polystyrene 2 The mass fraction of H in the total H, m 2 H-PS is the mass of deuterated polystyrene, m PS is the mass of unlabeled polystyrene; 2 H-PS Substituting into formula (6) we can obtain the synthesized deuterated polystyrene 2 The atomic percentage of H in the total H, i.e., the deuterium rate DR (%);

[0046] The results show that the deuterium rate of the prepared deuterated polystyrene microspheres calculated by mass conservation formula (5-6) is 82.2%.

[0047] (2) Infrared spectroscopy was used to characterize the chemical structure of deuterated polystyrene microspheres. Figure 2 As shown, characteristic absorption peaks of benzene ring skeleton vibration appear in the range of 1400-1700cm-1, confirming the existence of benzene ring. In addition, the absorption peaks at 2193cm-1 and 2272cm-1 are characteristic absorption peaks of CD bonds, indicating that deuterium has been introduced into the prepared microspheres.

[0048] (3) The morphology and particle size distribution of deuterated polystyrene microspheres were characterized using scanning electron microscopy and laser particle size analyzer. Figure 3 ) showed that the prepared microspheres had a regular morphology, uniform particle size distribution, and an average particle size of about 80nm. The hydrated particle size of the synthesized microspheres measured by dynamic light scattering was 172.6nm, and the Zeta potential was -43.4mV (Table 1), indicating that the surface of the microspheres had a certain negative charge.

[0049] Table 1. Hydrated particle size and Zeta potential of deuterated polystyrene microspheres

[0050]

[0051] Example 2: Based on stable isotopes 2 Quantitative study of nanoplastic absorption by plants using H-labeled polystyrene microspheres

[0052] In this example, lettuce was used as a model plant, and the 2H-labeled polystyrene microspheres prepared as described above were used as tracers. The absorption and distribution of the polystyrene microspheres by lettuce seedlings were studied through a hydroponic experiment.

[0053] 1. Preparation of exposure test solution: Disperse the 2H-labeled polystyrene microsphere powder prepared above into ultrapure water and ultrasonically disperse for 10 min to ensure uniform dispersion of the microspheres to obtain a stock solution with a concentration of 55 mg mL -1 The stock solution was mixed with 1 / 5 concentration of Hoagland nutrient solution and the exposure test solution with a concentration of 10 mg·mL-1 was obtained after ultrasonic dispersion.

[0054] 2. Preparation of experimental materials: Select 3-week-old lettuce seedlings as experimental materials, and select seedlings with uniform growth.

[0055] 3. Exposure culture: Set up a blank control group and an experimental group, with 3 replicates in each group. Transfer the lettuce seedlings to blank ultrapure water and exposure test solution, respectively, and culture them in a greenhouse for 14 days. The greenhouse conditions are: temperature 25±2℃, light-dark cycle 14:10h, relative humidity 50%. Replace the test solution every 7 days.

[0056] 4. Scanning electron microscopy observation: After the exposure culture, the washed lettuce root base was manually sliced. The aggregation of deuterated polystyrene microplastics in the roots was observed using high-resolution images taken with a scanning electron microscope (S-4800, Hitachi, Japan). Figure 4 ), the image shows that the microspheres are aggregated in the shape of grape chains in the middle column of the lettuce root.

[0057] 5. Sample processing: After the cultivation is completed, the lettuce plants are taken out, rinsed with ultrapure water and ultrasonically cleaned to remove surface attachments. The aboveground and underground parts of the plants are separated and dried to constant weight. The dried plant samples are ground and passed through a 60-mesh sieve.

[0058] 6. Instrumental analysis: The percentage of H in root and stem and leaf samples was measured by elemental analyzer (Vario EL Cube, Elementar, Germany): H% 2 H-root 、H% 2 H-shoot , H% control-root 、H% control-shoot The H isotope ratio (δ2H) in the sample was accurately determined by isotope ratio mass spectrometry (MAT 253plus, Thermo Fisher Scientific, Germany): δ 2 H-root , δ 2 H-shoot , δ control-root , δ control-shoot , the sample mass m is measured by analytical balance 2 H-shoot ;m 2 H-root ;m control-root 、m control-shoot The concentration of deuterated polystyrene microspheres in the sample was calculated using the following formula.

[0059]

[0060]

[0061]

[0062]

[0063] The results showed (Table 2) that the average concentrations of deuterated polystyrene microspheres in the roots and stems and leaves of lettuce were 27.53 μg / g and 6010.78 μg / g, respectively.

[0064] Table 2. Quantitative detection data of deuterated polystyrene microspheres in lettuce in the example, 3 sets of parallel tests were set for the aboveground part (stems and leaves) and the underground part (roots)

[0065]

[0066] Example 3: Based on stable isotopes 2 Quantitative study of animal absorption of nanoplastics using H-labeled polystyrene microspheres

[0067] In this example, quail was used as a model animal, and the 2H-labeled polystyrene microspheres prepared as described above were used as tracers. The absorption and distribution of deuterated polystyrene microspheres in quail eggs were studied by gavage experiments.

[0068] 1. Experimental animal preparation: 8 adult female Chinese white quails weighing between 140-170 g were randomly divided into an experimental group and a control group. The quails were raised in an indoor environment simulating natural light and provided with sufficient food and drinking water.

[0069] 2. Preparation of exposure test solution: After ultrasonic dispersion of 2H-labeled polystyrene microspheres, make a suspension with ultrapure water for standby use, with a concentration of 55 mg mL -1 .

[0070] 3. Oral gavage exposure: After 14 days of feeding, the experimental group quails were gavaged with normal saline, and the control group was gavaged with normal saline. The quails were fasted for 4 hours before and after gavage. The heads of the quails were fixed during gavage to ensure that the microspheres completely entered the digestive tract. The weight of the quails used in the experiment and the gavage dose of deuterated polystyrene microspheres are shown in Table 2. The experimental protocols used were in accordance with the requirements of Qingdao University for experimental animals.

[0071] 4. Collection and processing of quail eggs: After intragastric administration, quail eggs were collected daily and stored at 4°C. The quail eggs were separated into yolk, egg white and eggshell, freeze-dried until constant weight, ground and sieved through a 60-mesh sieve.

[0072] 5. Isotope analysis and quantification: Representative samples of quail egg yolk, egg white and eggshell from the experimental group and the control group on the second and fifth days were selected, and the percentage of H in the samples was measured by an element analyzer (Vario EL Cube, Elementar, Germany): H% 2 H-PS-yolk 、H% 2 H-PS-white , H% control-yolk 、H% control-white The δ in the sample was measured by isotope ratio mass spectrometer (MAT 253plus, Thermo Fisher Scientific, Germany). 2 H-PS-yolk , δ 2H-PS-white , δ control-yolk , δ control-white , the mass of the sample measured by the analytical balance is m 2 H-PS-yolk ;m 2 H-PS-white ;m control-yolk 、m control-white The concentrations of deuterated polystyrene microspheres in egg yolk, egg white and eggshell in the sample were calculated by the following formulas.

[0073]

[0074]

[0075]

[0076]

[0077] 6. The results show (Table 4) that 2H-labeled polystyrene microspheres can be delivered to quail egg yolk and egg white, but not detected in eggshell. 2 The concentrations of H-labeled polystyrene microspheres in egg white and egg yolk on the second and fifth days were 41.24 μg / g, 31.14 μg / g and 13.38 μg / g, 30.41 μg / g, respectively.

[0078] Table 3 Experimental quail weight and oral dose of deuterated polystyrene microspheres

[0079]

[0080] Table 4 Concentration of deuterated polystyrene microspheres in different tissues of quail eggs in the sample dry weight (μg / g)

[0081]

[0082] The above descriptions illustrate the embodiments of the present invention, but the present invention is not limited to the above embodiments. Those skilled in the art may make various modifications and variations without departing from the spirit and principles of the present invention, and such modifications and variations shall be included in the protection scope of the present invention.

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[0085] Dong,Y.,Gao,M.,Qiu,W.and Song,Z.2021.Uptake of microplastics bycarrots in presence of As(III):Combined toxic effects.Journal of HazardousMaterials 411.

[0086] Jiang,X.,Chen,H.,Liao,Y.,Ye,Z.,Li,M.and G.2019.Ecotoxicity andgenotoxicity of polystyrene microplastics on higher plant Viciafaba.Environmental Pollution 250,831-838.

[0087] Li,L.,Zhou,Q.,Yin,N.,Tu,C.and Luo,Y.2019.Uptake and accumulation ofmicroplastics in an edible plant.Chinese Science Bulletin 64(9),928-934.

[0088] Li,Z.,Li,Q.,Li,R.,Zhou,J.and Wang,G.2020.The distribution and impactof polystyrene nanoplastics on cucumber plants.Environmental Science andPollution Research 28(13),16042-16053.

[0089] Mitrano,D.M.,Beltzung,A.,Frehland,S.,Schmiedgruber,M.,Cingolani,A.andSchmidt,F.2019.Synthesis of metal-doped nanoplastics and their utility toinvestigate fate and behaviour in complex environmental systems.NatNanotechnol 14(4),362-368.

[0090] Sun,X.-D.,Yuan,X.-Z.,Jia,Y.,Feng,L.-J.,Zhu,F.-P.,Dong,S.-S.,Liu,J.,Kong,X.,Tian,H.,Duan,J.-L.,Ding,Z.,Wang,S.-G.and Xing,B.2020.Differentiallycharged nanoplastics demonstrate distinct accumulation in Arabidopsisthaliana.Nature Nanotechnology 15(9),755-760.

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Claims

1. A method based on stable isotope labeled micro / nano plastics, characterized in that: By emulsion polymerization, an initiator, a pH buffer solution, ultrapure water, an emulsifier and a deuterated styrene monomer are mixed and emulsified, and then polymerization reaction is carried out after emulsification, and then washing and drying are carried out to obtain deuterated polystyrene microspheres.

2. The method based on stable isotope labeling of micro / nano plastics according to claim 1, characterized in that: Under an inert gas environment, an emulsifier and a pH buffer solution are added to water, stirred and mixed at room temperature for emulsification, and then deuterated styrene monomer is added to the system, and the temperature is raised to 60-65°C, and further stirred and mixed. After mixing, an initiator is added to the system, and a polymerization reaction is carried out at 60-85°C under closed conditions for 24±2h; after the reaction, the polymer emulsion is centrifugally washed with ethanol and ultrapure water in turn, and the precipitate is collected and dried to obtain deuterated polystyrene microspheres.

3. The method based on stable isotope labeling of micro / nano plastics according to claim 1 or 2, characterized in that: The mass ratio of the initiator, pH buffer, ultrapure water, emulsifier and deuterated styrene monomer in the system is (1-10):1:(1-3):(1-3):1; wherein the initiator is water-soluble potassium persulfate (KPS), the pH buffer is sodium bicarbonate (NaHCO3), and the emulsifier is sodium dodecyl sulfate (SDS).

4. An application of the stable isotope labeled micro / nano plastics prepared by the method according to claim 1, characterized in that: The microspheres are used in the quantitative tracing detection of microplastics in complex biological systems.

5. A method for detecting deuterated polystyrene microspheres, characterized in that: S1. thoroughly washing the biological sample that has absorbed the deuterated polystyrene nanoplastic prepared in claim 1; S2 separates the biological samples by parts, dries and grinds them separately to obtain powder samples with uniform particle size; S3 sends the sample prepared in S2 to an element analyzer and an isotope ratio mass spectrometer for analysis, and the H% is measured by the element analyzer. sample , the isotope ratio mass spectrometer measured δ sample , δ control , the sample weight m is measured by analytical balance sample； S4 calculates the content of deuterated polystyrene microspheres in the sample using the measured H isotope ratio according to a pre-established mathematical formula model.

6. The method for detecting deuterated polystyrene microspheres according to claim 5, characterized in that: The mathematical model in S4 is: Where R s and R std Respectively 2 H / 1 The abundance ratio of H in the sample and the international standard (Vienna-Standard Mean Ocean Water, V-SMOW), the H isotope ratio (δ 2 H,‰) was expressed as a normalized value (detection accuracy <3‰), and the deuterated polystyrene concentration in the sample (C 2 H-PS ) is calculated as follows 7. The method for detecting deuterated polystyrene microspheres according to claim 5, characterized in that: In step S1, the biological sample is ultrasonically cleaned with ultrapure water for 3-5 minutes.

8. The method for detecting deuterated polystyrene microspheres according to claim 5, characterized in that: The drying in step S2 is performed at a high temperature of 85-90°C for 2 hours, and then at a medium temperature of 55-60°C for 24-48 hours, until the plant body completely loses moisture; Grinding is done in two steps. First, it is machine-ground using a low-temperature biological tissue preparation machine at a speed of 6.5 m / s and a temperature of 23°C. Then it is manually ground using a mortar. The final powder must be able to pass a 60-mesh sieve.

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