A method for evaluating biological effects of microplastics based on multi-level response indicators of typical soil animals

Abstract

The application discloses a kind of microplastic biological effect evaluation methods based on typical soil animal multilevel response index, it is related to environmental toxicology evaluation and ecological technical field, by setting microplastic treatment group and control group, the multilevel response index of soil animal individual, organ, tissue, molecule level is determined, and the integrated biomarker response index method is used to calculate comprehensive effect index, and constructs microplastic effect comprehensive evaluation model.The application can realize the comprehensive evaluation of microplastic biological effect and early warning of low concentration pollution, improves the comprehensiveness and accuracy of ecological risk assessment, provides reliable technical support for soil microplastic pollution control.

Description

Technical Field

[0001] This invention relates to the fields of environmental toxicology evaluation and ecological technology, specifically to a method for assessing the biological effects of microplastics based on multi-level response indicators of typical soil animals. Background Technology

[0002] With the mass production and widespread use of plastic products, the continuous accumulation of microplastics in the soil environment has become a global environmental problem of great concern. The ecological hazards of microplastics stem not only from the chemical release of their additives, but also from the unique stresses posed to soil organisms by the physical properties of the particles themselves, such as the combined effects of intestinal blockage, interference with feeding behavior, and energy metabolism disorders. my country's Soil Pollution Prevention and Control Law and related action plans have explicitly included microplastics and other emerging pollutants in the scope of key risk management, necessitating the establishment of a scientific and systematic biological effect assessment technology system to support precise management decisions.

[0003] Currently, ecotoxicological studies of soil microplastics largely focus on single model species and the measurement of their phenotypic indicators, lacking comprehensive assessment methods that link individual phenotypes with deeper biological mechanisms. Due to the limited assessment dimensions, existing technologies struggle to capture sensitive early warning signals of microplastic stress in the early stages of its effects, and cannot deeply analyze the transmission process of biological effects. In fact, the biological response induced by microplastics follows a bottom-up hierarchical cascade: it begins with abnormal gene expression and metabolic pathway dysregulation at the molecular level, progresses through histopathological damage, further evolves into organ physiological dysfunction, and ultimately manifests as changes in feeding and behavior at the individual level. However, current research protocols rarely systematically construct response indicator systems encompassing multidimensional effect characteristics, resulting in assessment results that neither fully elucidate the intrinsic mechanisms of stress effects nor provide forward-looking early warning evidence for soil ecological risks.

[0004] In agricultural production, polybutylene terephthalate (PBAT) and polyhydroxyalkanoates (PHA), as high-performance biodegradable polymers, have been widely used in agricultural products such as biodegradable mulch films, seedling containers, slow-release fertilizer / pesticide carriers, and biodegradable vine ropes. After fulfilling their functions of heat preservation, moisture retention, crop cultivation, and controlled nutrient release, these materials can gradually degrade in the soil environment, and are considered an important green alternative to alleviate the residual pollution of traditional plastic mulch films. However, their incomplete degradation and aging process in farmland soil still generates a large amount of PBAT / PHA microplastics, which continue to accumulate in the topsoil, becoming a typical new type of pollutant in farmland soil.

[0005] Therefore, there is an urgent need in this field to develop a novel comprehensive assessment method for the biological effects of microplastics that can rely on typical soil animals, systematically integrate multi-level response indicators at the individual, organ, tissue, and molecular levels, and possess early warning capabilities. This invention addresses this technological gap by constructing a multi-factor weighted assessment model based on the multi-level biological responses of typical soil animals. The aim is to deeply analyze the cascading transmission mechanism of microplastic stress effects, providing a more solid scientific basis and decision support for the ecological risk identification, early warning, and precise control of soil microplastic pollution. Summary of the Invention

[0006] In view of this, the present invention provides a method and application for assessing the biological effects of microplastics based on multi-level response indicators of typical soil animals. By conducting exposure experiments on typical soil animals with different concentrations and types of microplastics, multi-level biological effect indicators from the individual organism level to the molecular level are systematically measured. Taking into account the multi-level response characteristics of typical soil animals under microplastic stress, a multi-factor weighted assessment model covering the above-mentioned multi-level indicators is constructed, thereby achieving a comprehensive evaluation and early warning of the entire chain of microplastic biological effects, from macroscopic phenotypes to intrinsic mechanisms.

[0007] The microplastic biological effect assessment model based on the multi-level response index of soil animals further integrates multi-dimensional effect indicators at the individual, organ, tissue, and molecular levels. It normalizes and weights the biological effects of each treatment group based on the comprehensive biomarker response index method, thereby achieving unified quantification and comparison of ecological risk indices under different microplastic exposure conditions.

[0008] The technical solution of the present invention is as follows: <First Aspect> This invention provides a method for assessing the biological effects of microplastics based on the multi-level response of soil animals, comprising the following steps: S1. Set up a treatment group and a control group, wherein the treatment group is the test soil and the control group is the soil with a microplastic background value lower than the detection limit or not higher than 0.05‰. Soil animals are placed in the soil of the treatment group and the control group respectively for exposure culture. S2. Collect soil animal individuals after exposure and culture, and determine the multi-level response indicators of the soil animals, including one or more of the following: individual-level response indicators, organ-level response indicators, tissue-level response indicators, and molecular-level response indicators. S3. Based on the aforementioned response indicators, the comprehensive effect index IBRv2 is calculated using the comprehensive biomarker response index method. S4. Based on the comprehensive effect index, construct a comprehensive evaluation model for microplastic effects and obtain the ecological risk assessment results of microplastics.

[0009] As one implementation, the soil animal is an earthworm.

[0010] As one implementation scheme, the conditions for the exposure culture are: temperature 20±1℃, photocycle 16 h light / 8 h dark, light intensity 600 Lux, relative humidity 75%, no feed is given during the experiment, and water is replenished once every 2 days.

[0011] As one implementation, deionized water is added to the soil before the exposure culture to adjust the water content to 50% to 60% of the maximum field capacity, and the soil is pre-equilibrated for 7 days under the exposure culture conditions.

[0012] As one embodiment, the microplastics include polyhydroxyalkanoates and / or polybutylene terephthalate.

[0013] As one implementation, the individual-level response indicators include one or more of the following: survival rate, mortality rate, and systemic oxidative stress indicators.

[0014] As one implementation, the systemic oxidative stress indicators include one or more of the following: antioxidant enzyme activity, oxidative damage product content, and non-enzymatic antioxidant content.

[0015] In some embodiments, the antioxidant enzymes include superoxide dismutase (SOD) and / or peroxidase (POD).

[0016] In some embodiments, the oxidative damage products include malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG).

[0017] In some embodiments, the non-enzymatic antioxidant includes reduced glutathione (GSH).

[0018] As one implementation, the organ-level response indicators include neurotoxicity-related enzyme activity and / or reproductive toxicity-related enzyme activity.

[0019] In some embodiments, the neurotoxicity-associated enzyme includes brain-derived Ca2+. 2+ -ATPase and / or brain calcium 2+ -Mg 2+ -ATPase.

[0020] In some embodiments, the reproductive toxicity-related enzyme includes Ca 2+ -ATPase, Ca 2+ -Mg 2+ -One or more of ATPase, sorbitol dehydrogenase SDH, and lactate dehydrogenase LDH.

[0021] As one implementation, the tissue-level response indicators target brain neural tissue and / or reproductive tissue.

[0022] As one implementation, the tissue-level response indicators include HE section observation of tissue damage and / or immunohistochemical analysis.

[0023] As one implementation, the immunohistochemical analysis includes the expression levels of one or more proteins among Caspase-3, Bax, and Bcl-2.

[0024] As one implementation, the molecular-level response indicators are applied to brain neural tissue and / or reproductive tissue.

[0025] As one implementation, the molecular-level response indicators include inflammatory cytokine gene expression and / or transcriptomics.

[0026] In some embodiments, the expression of the inflammatory genes includes IL-1β and / or TNF-α.

[0027] As one implementation scheme, based on the aforementioned multi-level response indicators, the comprehensive effect index of soil animals is calculated using the comprehensive biomarker response index method, a dose-response relationship model between the multi-level biological response indicators and microplastic exposure levels is established, and the ecological risk level of microplastic pollution is comprehensively evaluated.

[0028] As one implementation scheme, the algorithm for the comprehensive effect index IBRv2 is as follows:

[0029]

[0030]

[0031]

[0032] in, The average value of biomarker i in the treatment group. The mean value of biomarker i in the control group is given by denominator, and µ is the mean value of biomarker i. The overall mean of the values, σ is the biomarker i. The standard deviation of the value A represents the degree to which the control group deviates from the global mean, and A is the reference deviation index.

[0033] As one implementation, the biomarker is one or more of the response indicators.

[0034] As an implementation scheme, between steps S2 and S3, add the step: screening for statistically significant concentration dependence ( pA response index ≤0.05) and exhibiting a monotonically increasing or monotonically decreasing dose-response relationship is used as a sensitive index, which serves as a biomarker in IBRv2 calculation.

[0035] <Second aspect> This invention provides a microplastic biological effect assessment system based on multi-level response indicators of soil animals, including an exposure experiment module, a response indicator detection module, and a model evaluation module. The response indicator detection module includes individual effect detection, organ effect detection, tissue effect detection, and molecular effect detection modules. The exposure experiment module is used to establish and manage soil animal exposure experiment systems with different concentrations and types of microplastics; The individual effect detection module is used to determine the individual-level response indicators of typical soil animals. The organ effect detection module is used to determine organ-level response indicators of typical soil animals. The tissue effect detection module is used to determine tissue-level response indicators of typical soil animals; The molecular effect detection module is used to determine the molecular-level response indicators of typical soil animals. The model evaluation module has a built-in multi-level response index comprehensive evaluation model. Based on the biological multi-level response index data measured by the response index detection module, it calculates and outputs the ecological risk assessment results of microplastic pollution.

[0036] <Third aspect> This invention provides a method for assessing the biological effects of microplastics based on multi-level responses of soil animals, and its application in assessing and predicting the toxic risks of microplastics to soil ecosystems.

[0037] As one implementation, the method is used in microplastic exposure systems to assess and predict PBAT and / or PHA microplastic stress.

[0038] As one implementation, the method is used to assess and test the concentration of added microplastics in soil ranging from 0.1% to 1.0% (w / w dry soil).

[0039] As one implementation, the method is used for early warning of microplastic soil pollution risks.

[0040] As one implementation method, the method is applied in the ecological risk assessment of biodegradable microplastics in farmland soil.

[0041] As one implementation scheme, for the PBAT microplastic soil system, the indicators are superoxide dismutase (SOD) activity, non-enzymatic antioxidant GSH content, malondialdehyde (MDA) content (an oxidative damage product), and brain calcium content.2+ -ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity, brain Caspase-3 protein expression level, brain Bax protein expression level, seminal vesicle Caspase-3 protein expression level, brain IL-1β expression level, brain TNF-α expression level, and seminal vesicle TNF-α expression level.

[0042] As one implementation scheme, for a PHA microplastic soil system, the indicators are peroxidase (POD) activity, superoxide dismutase (SOD) activity, and brain calcium (Ca). 2+ -Mg 2+ -ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity, brain Bax protein expression level, brain IL-1β expression level, brain TNF-α expression level, and seminal vesicle TNF-α expression level.

[0043] <Fourth Aspect> This invention provides a combination of early warning markers for soil microplastic pollution.

[0044] As one implementation scheme, for PBAT, the early warning biomarker combination consists of superoxide dismutase (SOD) activity, non-enzymatic antioxidant GSH content, oxidative damage product malondialdehyde (MDA) content, and brain calcium levels. 2+ - This consists of ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity, brain Caspase-3 protein expression level, brain Bax protein expression level, seminal vesicle Caspase-3 protein expression level, brain IL-1β expression level, brain TNF-α expression level, and seminal vesicle TNF-α expression level. As one implementation scheme, for PHA, the early warning biomarker combination consists of peroxidase POD activity, superoxide dismutase SOD activity, and brain calcium... 2+ -Mg 2+ - It consists of ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity, brain Bax protein expression level, brain IL-1β expression level, brain TNF-α expression level, and seminal vesicle TNF-α expression level.

[0045] As one implementation scheme, when calculating the modified IBRv2 using a combination of early warning markers for PBAT microplastics, a modified IBRv2 index of 9.8–10.9 indicates that the soil has reached a PBAT pollution concentration of approximately 0.1%; a modified IBRv2 index of 20.5–22.7 indicates that the soil has reached a PBAT pollution concentration of approximately 1%. As one implementation scheme, when the modified IBRv2 is calculated using a combination of early warning markers for PHA microplastics, a modified IBRv2 index of 9.8 to 10.8 indicates that the soil has reached a PHA pollution concentration of approximately 0.1%; a modified IBRv2 index of 18.3 to 20.3 indicates that the soil has reached a PHA pollution concentration of approximately 1%.

[0046] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects: (1) Based on typical soil animals, this invention constructs a multi-factor exposure experimental system covering different microplastic concentration gradients and polymer types, and systematically measures multi-level biological response indicators from the individual level to the molecular level. Unlike existing technologies that only target a single microplastic type or a single species and are limited to a single endpoint at the individual level, this invention analyzes multi-level effect indicators of soil animals, identifies key characteristic factors affecting the biological effects of microplastics, and establishes a comprehensive evaluation model based on dose-response relationships, thereby realizing the quantitative assessment and comparison of soil ecological risks under different microplastic exposure scenarios.

[0047] (2) By measuring the multi-level response indicators of typical soil animals at the individual, organ, tissue and molecular levels, this invention can systematically reflect the degree of harm of different concentrations and types of microplastic exposure to soil ecosystems, and the assessment conclusions are more comprehensive and hierarchically correlated.

[0048] (3) This invention clarifies the key characteristic factors affecting the multi-level biological effects of microplastics and constructs a comprehensive evaluation model that integrates microplastic concentration, polymer type and biological response indicators, which effectively improves the rationality and accuracy of ecological risk prediction.

[0049] (4) This invention provides a standardized assessment method based on multi-level response indicators, which can quantitatively assess and predict the ecological risks under different microplastic pollution scenarios, and provides a reliable technical tool for early warning and precise control of soil microplastic pollution.

[0050] (5) This invention further screens and clarifies common early warning sensitive indicators for PHA and / or PBAT microplastics, which can identify the potential biological effects of low concentration microplastics in advance before organisms show obvious damage or death. This breaks through the limitations of traditional detection that only focuses on individual lethal effects and cannot achieve early warning, and significantly improves the sensitivity and foresight of microplastic ecological risk assessment.

[0051] (6) The early warning sensitivity index determined by the present invention can show a significant response in the detection of low concentration microplastic soil. It has high sensitivity and can provide repeatable detection markers for risk warning of low concentration microplastics in the environment. It realizes the comprehensive evaluation of the biological effects of microplastics and early warning of low concentration pollution, improves the comprehensiveness and accuracy of ecological risk assessment, and provides reliable technical support for soil microplastic pollution control. Attached Figure Description

[0052] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The study investigated the mortality rate of earthworms exposed to PBAT and PHA microplastics and their effects on the levels of 8-hydroxydeoxyguanosine, glutathione, malondialdehyde, peroxidase, and superoxide dismutase. Figure 2 Exposure to PBAT and PHA microplastics on earthworm brain nerve Ca 2+ -ATPase and Ca 2+ -Mg 2+ -The effect on ATPase activity; Figure 3 The effects of PBAT and PHA microplastic exposure on sorbitol dehydrogenase activity, lactate dehydrogenase activity, and Ca2+ activity in earthworm seminal vesicles. 2+ -ATPase and Ca 2+ -Mg 2+ -The effect on ATPase activity; Figure 4 Tissue sections and immunohistochemistry of earthworm brain nerves exposed to PBAT and PHA microplastics are shown. (a) Histopathological sections stained with HE are shown, including control groups (2.0X and 20.0X magnification) and low-dose PBAT group (PBAT-L), high-dose PBAT group (PBAT-H), low-dose PHA group (PHA-L), and high-dose PHA group (PHA-H) at 20.0X magnification. Yellow arrows indicate neuronal necrosis / degeneration, green arrows indicate nuclear fragmentation or dissolution, and blue arrows indicate congestion. (b) Immunohistochemical staining results are shown, including the expression of Caspase-3, Bax, and Bcl-2 proteins in the control group (CK), PBAT-H treatment group, and PHA-H treatment group. The inset is a magnified view of the local area. (c) Immunohistochemical quantitative statistical graphs of the control group and the high-dose treatment group are shown. Figure 5Tissue sections and immunohistochemistry of earthworm seminal vesicles exposed to PBAT and PHA microplastics are shown. (a) Histopathological sections stained with HE include control (2.0X and 20.0X magnification) and low-dose PBAT (PBAT-L), high-dose PBAT (PBAT-H), low-dose PHA (PHA-L), and high-dose PHA (PHA-H) groups (20.0X magnification). In the figure, yellow arrows indicate a decrease in the number of germ cells, green arrows indicate deep-stained nuclear condensation, blue arrows indicate congestion, and cyan arrows indicate pigmentation. (b) Immunohistochemical staining results include the expression of Caspase-3, Bax, and Bcl-2 proteins in the control (CK), PBAT-H, and PHA-H treatment groups. The inset is a magnified view of a local area. (c) Immunohistochemical quantitative statistical graphs of the control and high-dose treatment groups are shown. Figure 6 Inflammatory damage to the earthworm brain caused by exposure to PBAT and PHA microplastics; Figure 7 Inflammatory damage to earthworm seminal vesicles caused by exposure to PBAT and PHA microplastics; Figure 8 Transcriptomic analysis of the effects of PBAT and PHA microplastic exposure on the earthworm brain, among which, Figure 8 (a) to (c) show PLS-DA (partial least squares discriminant analysis) and volcano plot analysis of brain neural tissue. Figure 8 (d) and (e) show the KEGG / metabolic pathway enrichment analysis of the PBAT-H and PHA-H treatment groups; Figure 9 Transcriptomic analysis of the effects of PBAT and PHA microplastic exposure on earthworm seminal vesicles, among which, Figure 9 (a) to (c) show the PLS-DA (partial least squares discriminant analysis) and volcano plot analysis of seminal vesicle tissue. Figure 9 (d) and (e) show the KEGG / metabolic pathway enrichment analysis of the PBAT-H and PHA-H treatment groups; Figure 10 A star plot of the deviation index of earthworm multilevel response indices under PBAT and PHA microplastic exposure. Detailed Implementation

[0053] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0054] This invention provides a method for assessing the biological effects of microplastics based on multi-level response indicators of typical soil animals, comprising the following steps: Step 1, establish a microplastic exposure experimental system: select a typical soil animal, set up multiple experimental treatment groups with different concentration gradients and different types of microplastics, and record the exposure condition parameters of each treatment group; Step 2: After the exposure experiment, collect soil animal individuals from each experimental treatment group and measure individual-level response indicators, including at least one of survival rate, mortality rate, antioxidant enzyme activity, and oxidative damage product content. Step 3: Dissect the soil animal individual from Step 2, obtain the target organ, and measure the organ-level response index. The organ-level response index includes at least one of neurotoxicity-related enzyme activity and reproductive toxicity-related enzyme activity. Step 4: Perform tissue sectioning on the target organ from Step 3 and measure tissue-level response indicators. The tissue-level response indicators include at least one of tissue damage HE section observation and immunohistochemical analysis. Step 5: Extract brain and seminal vesicle tissues from the soil animals in Step 2 and determine molecular-level response indicators, including at least one of the expression levels of inflammatory factors and transcriptomic changes. Step 6: Based on the biological multi-level response indicators measured in Steps 2 to 5, construct a comprehensive evaluation model for the microplastic effect and obtain the ecological risk assessment results of microplastic pollution.

[0055] The steps of the microplastic biological effect assessment method based on multi-level response indicators of typical soil animals provided by this invention are described in detail below.

[0056] Step 1: Establishment of the microplastic exposure experimental system The test soil was collected from areas uncontaminated by microplastics. After natural air drying, removal of stones and plant debris, and grinding through a 2 mm nylon sieve, it was prepared for use. Before use, the background microplastic content of the soil was measured to ensure that the background value was below the detection limit or not higher than 5% of the lowest experimental concentration, thus ensuring the accuracy of the exposure experiment. A blank control group was set up with test soil without added microplastics.

[0057] Soil animals were selected from *Pheretima wielderii* (William ringed worm). Pheretima guillelmi The earthworms were purchased from Wangjun Earthworm Farm in Jurong, Jiangsu Province, and healthy earthworms of similar size with reproductive clitellum were selected as test materials.

[0058] The microplastics used were polybutylene terephthalate (PBAT) and polyhydroxyalkanoate (PHA). The concentration gradient was set at 0.1% and 1.0% (w / w dry soil).

[0059] Five treatment groups were set up: 1) blank control + *Verticillium wiltii* (CK); 2) 0.1% polybutylene terephthalate (PBAT-L) microplastic + *Verticillium wiltii* (PBAT-L); 3) 0.1% polyhydroxyalkanoate (PHA-L) microplastic + *Verticillium wiltii* (PHA-L); 4) 1% polybutylene terephthalate (PBAT-H) microplastic + *Verticillium wiltii* (PBAT-H); 5) 1% polyhydroxyalkanoate (PHA-H) microplastic + *Verticillium wiltii* (PHA-H). Each treatment group was replicated three times.

[0060] The culture conditions were: temperature 20±1℃, photoperiod of 16 h light / 8 h darkness, light intensity of 600 Lux, and relative humidity of 75%. No feed was provided during the experiment, and water was added every 2 days. Before exposure, deionized water was added to the soil to adjust the moisture content to 50%–60% of maximum field capacity, and the soil was pre-equilibrated under the experimental conditions for 7 days. Healthy, undamaged soil animals with normal activity were selected for subsequent exposure experiments, with an exposure period of 21 days.

[0061] Subsequent test data were subjected to multiple comparisons using Duncan's method, with significance levels determined. p =0.05 is represented by a lowercase letter, and n=3.

[0062] Step 2: Survival and mortality rate determination and systemic oxidative stress index determination Step 2.1 Survival and mortality rate determination: After the experimental exposure period, the earthworms were completely separated from the soil using a manual collection method. After removing any adhering substances from their bodies with clean filter paper, the earthworms were placed in a petri dish lined with moist filter paper and observed statically at room temperature. The earthworm body wall was gently touched with blunt forceps; those exhibiting obvious contraction or peristaltic response to mechanical stimulation were considered alive, while those showing no response and exhibiting loose, discolored body walls were considered dead. At least three biological replicates were set up for each indicator measurement. Survival rate and mortality rate were calculated using the following formulas: Survival rate (%) = (Number of surviving individuals / Initial total number of subjects) × 100% Mortality rate (%) = (Number of deaths / Total number of initial subjects) × 100%.

[0063] Step 2.2 Measurement of systemic oxidative stress indicators: Weigh an appropriate amount of whole tissue sample from surviving earthworms and add pre-cooled phosphate buffer (0.05 mol / L, pH 7.4) at a weight-to-volume ratio of 1:9 (W / V). Homogenize the homogenate under ice bath conditions. After low-temperature high-speed centrifugation (4℃, 10000×g, 10 min), collect the supernatant as the enzyme solution to be tested. Commercially available assay kits (Hangzhou Zhenyoupin Biotechnology Co., Ltd.: catalog number YPFG0477-48 (SOD); YPFG0111-48 (POD); YPWB0005-48 (MDA); YPFG0113-48 (GSH); Jiangsu Enzyme Immunoassay Co., Ltd.: catalog number MM-92537801 (8-OHdG)) were used to determine the activities of antioxidant enzymes (superoxide dismutase SOD, peroxidase POD), the content of oxidative damage products (malondialdehyde MDA, 8-hydroxydeoxyguanosine 8-OHdG), and the content of non-enzymatic antioxidants (reduced glutathione GSH). At least three biological replicates were set up for each assay.

[0064] The test results for mortality rate, antioxidant enzyme activity, oxidative damage product content, and non-enzymatic antioxidant content are as follows: Figure 1 As shown.

[0065] Step 3: Assay of neural and reproductive enzyme activities After the experimental exposure period ended, the surviving earthworm samples were placed on an ice tray for rapid dissection. The nerve tissue was obtained by separating the area where the head ganglion was located, and the body cavity was opened to precisely cut the seminal vesicle tissue at the location of the seminal vesicle.

[0066] The collected tissues were rinsed with pre-cooled physiological saline to remove blood and body cavity fluid, blotted dry with filter paper, and weighed. Pre-cooled homogenizing medium (0.25 mol / L sucrose solution containing 1 mmol / L EDTA, pH 7.4) was added at a weight-to-volume ratio of 1:9, and mechanical homogenization was performed using a glass homogenizer under ice bath conditions. The homogenate was centrifuged at low temperature (4℃, 3500×g, 10 min), and the supernatant was used as the enzyme solution to be tested. After aliquoting, the solution was stored at -80℃ for later use. A commercially available reagent kit (Hangzhou Zhenyoupin Biotechnology Co., Ltd.: Catalog No. YPFG0032-24 (Ca)) was used. 2+ -ATP); YPFG0031-24 (Ca 2+ -Mg 2+ -ATP); YPZY0404-48 (SDH); YPFG0173-48 (LDH)) to determine the activity of nerve-related enzymes (Ca) 2+ -ATP, Ca 2+ -Mg 2+ -ATP) and reproductive toxicity-related enzyme activities (Ca) 2+ -ATP, Ca 2+ -Mg 2+-ATP, sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH). At least three biological replicates were set up for each indicator measurement.

[0067] The test results of neurotoxicity-related enzyme activities are as follows: Figure 2 As shown.

[0068] The test results of reproductive toxicity-related enzyme activities are as follows: Figure 3 As shown.

[0069] Step 4: Histopathological and Immunohistochemical Examination Step 4.1 Histopathological examination: After cleaning and rinsing the intestines of surviving earthworm samples, they were placed in 4% paraformaldehyde fixative (1:20 by weight) and fixed at room temperature for 48 h. The fixed earthworm tissue samples were then embedded in paraffin to prepare continuous sections with a thickness of 3 μm. Brain nerve tissue and seminal vesicle tissue sections were stained with hematoxylin and eosin (HE) and then microscopic images were acquired using an optical microscope.

[0070] Tissue sections of brain nerve tissue, such as Figure 4 As shown in (a), a tissue section of the seminal vesicle is as follows. Figure 5 As shown in (a).

[0071] Step 4.2 Immunohistochemical detection: Tissue sections (unstained with hematoxylin and eosin) were dewaxed with xylene, then rehydrated stepwise with a series of ethanol solutions for 5 minutes each. After rinsing with distilled water, the sections were transferred to PBS for equilibration. Antigen retrieval was performed using citrate buffer (0.01 mol / L, pH 6.0) via autoclaving, followed by three PBS rinses for 5 minutes each. The sections were then incubated with 3% hydrogen peroxide solution at room temperature in the dark for 25 minutes to eliminate endogenous peroxidase activity. After PBS rinsing, 3% bovine serum albumin (BSA) was added, and the sections were incubated in a humidified chamber at 37°C for 30 minutes to block non-specific protein binding sites. The blocking solution was gently removed, and primary antibody diluted in PBS according to the manufacturer's instructions was added. The sections were then placed flat in a humidified chamber and incubated overnight at 4°C. The next day, the slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each. After slightly drying, HRP-labeled secondary antibody of the appropriate species was added around the tissue, and the sections were incubated at room temperature for 50 minutes. Wash three times with PBS (5 minutes each time), shake off excess water, and add freshly prepared DAB chromogenic solution. Control the staining time under a microscope; positive results show a brownish-yellow color. Rinse with water to stop the staining process. Counterstain cell nuclei with hematoxylin for 3 minutes, then rinse with running water to achieve a blue stain. Dehydrate sections with graded ethanol, clear with xylene, mount with neutral resin, dry, and observe and image under an optical microscope. Determine the expression levels of Caspase-3, Bax, and Bcl-2 proteins. At least three biological replicates were set up for each assay.

[0072] Immunohistochemical test results of brain nerve tissue as follows Figure 4 (a) and Figure 4 As shown in (b), the immunohistochemistry of the seminal vesicle tissue is as follows: Figure 5 (a) and Figure 5 As shown in (b).

[0073] Step 5: qPCR detection and transcriptomics sequencing analysis of inflammatory factor gene expression Step 5.1 qPCR detection of inflammatory factor gene expression: First, 5-20 mg (nerve or seminal vesicle) tissue was added to a grinding tube containing RNA extraction buffer (1 mL) and 3 grinding beads. After thorough grinding, the mixture was centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected, and 100 μL of chloroform substitute was added. The mixture was allowed to stand for 3 min, and after centrifugation again, 400 μL of the supernatant was collected and 550 μL of isopropanol was added. The mixture was placed at -20°C for 15 min and centrifuged to obtain RNA precipitate. The precipitate was washed twice with 75% ethanol, dried, and dissolved in 15 μL of RNA lysis buffer. The concentration was then measured and adjusted to 200 ng / μl. Subsequently, reverse transcription was performed: 4 μL of 5×SweScript All-in-One SuperMix for qPCR and 1 μL of gDNA Remover were added to the total RNA, and Nuclease-Free Water was added to a final volume of 20 μL. After mixing and centrifugation, cDNA was synthesized on a conventional PCR instrument according to the program of 25°C for 5 min, 42°C for 30 min, and 85°C for 5 sec. Finally, quantitative PCR was performed: A reaction mixture containing 7.5 μL of 2×Universal Blue SYBR Green qPCR Master Mix, 1.5 μL of gene primers, 2.0 μL of cDNA, and Nuclease-Free Water was prepared in a PCR reaction plate, and the mixture was sealed, centrifuged, and then run on a real-time PCR instrument with the amplification program (95℃ pre-denaturation for 30 s; 40 cycles: 95℃ for 15 s, 60℃ for 30 s; melting curve: fluorescence signal was collected every 0.5℃ increase from 65℃ to 95℃). The fold change in the expression of the target gene was calculated using the ΔΔCT method. The expression levels of the two pro-inflammatory cytokines, IL-1β and TNF-α, were measured. At least three biological replicates were set up for each indicator measurement.

[0074] Results of tests for inflammation and damage to brain nerve tissue, such as Figure 6 As shown; the results of the inflammatory damage test of the seminal vesicle tissue are as follows. Figure 7 As shown.

[0075] Step 5.2 Transcriptomics sequencing analysis: Extraction of *Vibrio Williamii* exposed to microplastics was performed using Trizol reagent (Ambion, USA). Pheretima guillelmi Total RNA from nerve or seminal vesicle tissue, after passing quality control using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), was used to construct sequencing libraries for the Illumina NovaSeq 6000 platform (Illumina Scientific, USA). pGenes with differentially expressed expression (DEGs) were selected based on a criterion of <0.05 and |Log2(FC)|>1.5, including both upregulated and downregulated genes. DEGs were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and subjected to pathway enrichment analysis using the free online Majorbio cloud platform (www.majorbio.com).

[0076] The results of transcriptomic differential analysis of brain neural tissues are as follows: Figure 8 As shown in (a) to (c), the KEGG / metabolic pathway enrichment analysis is as follows: Figure 8 As shown in (d) and (e).

[0077] The results of transcriptomic differential analysis of seminal vesicle tissue are as follows: Figure 9 As shown in (a) to (c), the KEGG / metabolic pathway enrichment analysis is as follows: Figure 9 As shown in (d) and (e).

[0078] Step 6: Construct a comprehensive evaluation model for the microplastics effect. Based on the multi-level response index data of individuals, organs, tissues, and molecules measured above, the comprehensive biological effect index IBRv2 of soil animals under different microplastic exposure conditions was calculated using the comprehensive biomarker response index method.

[0079] The IBRv2 index was calculated using the total response index as a biomarker.

[0080] Furthermore, based on the monotonically increasing or decreasing trend of dose-effect, key indicators that are sensitive to microplastic exposure and have stable responses are selected as sensitive indicators. Then, the improved IBRv2 index is calculated using the sensitive indicators to construct a comprehensive evaluation model of microplastic effects, thereby obtaining the ecological risk assessment results of microplastic pollution.

[0081] The formula for calculating IBRv2 is:

[0082]

[0083]

[0084]

[0085] in: The average value of biomarker i in the treatment group. This represents the average value of biomarker i in the control group. µ is a biomarker i The overall mean of the values, σ is the biomarker i The standard deviation of the value This represents the degree to which the control group deviates from the global mean. A represents the reference deviation index.

[0086] The deviation index star diagram of the multi-level response index of earthworms under microplastic exposure is shown below. Figure 10 As shown.

[0087] Results Analysis according to Figure 1 It was found that all microplastic treatments significantly increased earthworm mortality, with the PHA group showing a clear concentration-dependent effect, where mortality increased with increasing exposure concentration. The DNA oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) significantly increased in most treatment groups, with the most pronounced increase in the PHA group; except for PBAT, PHA induced stronger DNA damage at low concentrations. Malondialdehyde (MDA) levels generally increased, indicating that all treatments induced membrane lipid peroxidation, particularly pronounced in the PHA group. Glutathione (GSH) levels generally decreased, with the largest decrease in the PHA group, reflecting severe depletion of antioxidant reserves. Peroxidase (POD) activity decreased significantly with increasing concentration, suggesting impaired function of the antioxidant enzyme system; while superoxide dismutase (SOD) activity increased, which can be considered a compensatory response to oxidative stress. Overall, PBAT-induced oxidative damage was relatively mild. These results indicate that microplastic exposure can induce oxidative stress in earthworms, weaken their antioxidant defense capabilities, lead to DNA damage, and ultimately increase mortality.

[0088] according to Figure 2 It can be known that Ca2+ exists both inside and outside the nerve cell membrane. 2+ It exhibits a gradient distribution that is high on the outside and low on the inside, and its steady state is mainly determined by Ca. 2+ -ATPase and Ca 2+ -Mg 2+ -ATPase synergistically maintains [the condition]. Results showed that different types and concentrations of agricultural film-derived microplastics significantly interfered with Ca2+ in earthworm brain tissue. 2+ Homeostatic regulatory systems. Microplastic exposure generally caused an increase in the activity of two ATPases, suggesting that the body initiates a compensatory stress response. The interference effect was concentration-dependent, with enzyme activity at high concentrations (1%, w / w) being higher than at low concentrations (0.1%, w / w). Furthermore, there were differences in the type of interference: bio-based (PHA) and non-bio-based (PBAT) microplastics elicited different enzyme activity responses, reflecting that their toxic mechanisms are closely related to the chemical structure of the materials. In summary, microplastics may promote the release of calcium phosphate (calcium phosphate) by disrupting the structure of nerve cell membranes. 2+ Influx and disruption of intracellular Ca2+ 2+ Homeostasis ultimately leads to neurological dysfunction.

[0089] according to Figure 3It is known that sorbitol dehydrogenase (SDH) and lactate dehydrogenase (LDH) are key enzymes involved in energy metabolism and germ cell development. The results showed that LDH activity decreased significantly with increasing microplastic concentration; while SDH activity only decreased slightly or remained the same as the control group, without significant damage. Intracellular Ca 2+ Transmembrane transport mainly depends on Ca 2+ -ATPase and Ca 2+ -Mg 2+ -ATPase regulation. Inhibition of the activity of these two enzymes leads to Ca2+. 2+ Transport barriers disrupt intracellular calcium homeostasis and affect calcium levels across the axial contractile membrane. 2+ Concentration gradients may inhibit the acrosome reaction. Compared to the control group, the microplastic-exposed group had significantly higher levels of Ca in earthworm sperm. 2+ -ATPase and Ca 2+ -Mg 2+ -ATPase activity was significantly reduced in all groups, with the inhibitory effect being particularly pronounced in the PHA group.

[0090] according to Figure 4 It was found that at concentrations of 0.1% and 1%, both types of agricultural film-derived microplastics (PBAT and PHA) caused various pathological damages to the brain nerve tissue of earthworms, including neuronal necrosis or degeneration, nuclear fragmentation and dissolution, and congestion. The degree of damage increased with increasing exposure concentration, showing a clear concentration-dependent effect. Immunohistochemical staining results showed that, compared with the control group, the expression levels of Caspase-3, Bax, and Bcl-2 proteins in the brain tissue of the microplastic-exposed group were all increased. Neurons were more sensitive to microplastic-induced oxidative stress, as evidenced by the upregulation of the pro-apoptotic protein Bax and the activation of Caspase-3; while glial cells tended to compensatorily upregulate the anti-apoptotic protein Bcl-2 to maintain self-protection. Furthermore, Caspase-3 is also involved in non-apoptotic processes such as synaptic pruning and neuroinflammatory regulation in the nervous system; its increased expression does not directly equate to cell death. Therefore, the simultaneous increase of the three proteins is not a typical manifestation of mitochondrial apoptosis pathway activation, but rather reflects the complex adaptive state of brain tissue under neurotoxic stress caused by microplastics, where apoptosis and anti-apoptosis, damage and compensation coexist.

[0091] according to Figure 5It was found that at concentrations of 0.1% and 1% (w / w), both types of agricultural film-derived microplastics (PBAT and PHA) induced various pathological damages in earthworm seminal vesicle tissue, including nuclear condensation, congestion, reduction of germ cells, and pigmentation. The severity of the lesions increased with increasing exposure concentration, showing a clear concentration-dependent effect. Furthermore, the lesion patterns induced by bio-based (PHA) and non-bio-based (PBAT) microplastics differed: for example, nuclear condensation and dissolution were more pronounced at high concentrations of PHA. Immunohistochemical staining results showed that, compared with the control group, the expression of Caspase-3, Bax, and Bcl-2 proteins was increased in the seminal vesicle tissue of the microplastic exposure group. This phenomenon stems from the unique cellular composition and stress response of the seminal vesicle as a reproductive tissue. The seminal vesicle contains germ cells at different developmental stages, as well as supporting cells and interstitial cells. Microplastics can induce apoptosis in germ cells through oxidative stress and endocrine disruption (upregulating Bax and Caspase-3). Simultaneously, supporting cells and some surviving germ cells compensatorily upregulate the anti-apoptotic protein Bcl-2 to maintain seminal vesicle microenvironment homeostasis and protect immature germ cells. Furthermore, Caspase-3 participates in non-apoptotic physiological processes such as cytoplasmic removal and mature sperm release during spermatogenesis; microplastic exposure may interfere with this regulatory mechanism, leading to abnormally elevated Caspase-3 expression. Therefore, the co-elevation of these three factors does not represent a unidirectional activation of the classic mitochondrial apoptosis pathway, but rather reflects a complex toxic response in the seminal vesicle tissue under microplastic stress, characterized by both germ cell apoptosis and protective responses from supporting cells.

[0092] according to Figure 6 It is known that after microplastics enter earthworms, they mainly induce oxidative stress and cell damage, leading to the upregulation of pro-inflammatory cytokines IL-1β and TNF-α. Specific mechanisms include: the physical stimulation of microplastic particles can disrupt the stability of lysosomes in brain tissue cells, releasing reactive oxygen species and inducing local inflammatory responses; simultaneously, as soil invertebrates, earthworms' innate immune system excessively releases inflammatory mediators during the process of engulfing and clearing foreign particles. These effects ultimately increase the levels of IL-1β and TNF-α, and this effect intensifies with increasing microplastic concentration.

[0093] according to Figure 7 It is known that when microplastics enter the seminal vesicles of earthworms, the sharp edges of the particles directly scratch the tissue cells, causing internal cellular damage and stress responses. This damage triggers the earthworm's immune alarm system, forcing cells to release two "distress signals," IL-1β and TNF-α, to fight the foreign substance. Simultaneously, the cells repeatedly fail to engulf these indigestible particles, further exacerbating the release of inflammatory signals, and this effect is enhanced with increasing microplastic concentration in the PHA treatment group.

[0094] according to Figure 8Transcriptomic analysis further revealed the differential effects of different microplastic exposures on gene expression in the brain of earthworms, a typical soil animal. Principal component analysis showed that both microplastic exposure groups exhibited obvious clustering characteristics, with clear separation between the control and exposure groups. Specifically, PBAT microplastic exposure led to the upregulation of 1354 genes and the downregulation of 1928 genes in *Pheretima wiltani*, while PHA microplastic exposure led to the upregulation of 1154 genes and the downregulation of 1848 genes. KEGG enrichment analysis systematically elucidated the functional significance of these differentially expressed genes and revealed the intrinsic molecular mechanisms of earthworm responses. PBAT microplastics regulated energy metabolism-related pathways, including protein digestion and absorption, and glycolysis / gluconeogenesis. In addition to regulating energy metabolism pathways, PHA microplastics also regulated immune and oxidative stress-related pathways, such as apoptosis.

[0095] according to Figure 9 Transcriptomic analysis further revealed the differential effects of different microplastic exposures on the seminal vesicle gene expression of earthworms, a typical soil animal. Principal component analysis showed that both microplastic exposure groups exhibited obvious clustering characteristics, and the boundary between the control and exposed groups was clear. Specifically, PBAT microplastic exposure led to the upregulation of 682 genes and the downregulation of 1102 genes in *Pheretima wiltani*, while PHA microplastic exposure caused the upregulation of 328 genes and the downregulation of 4048 genes. KEGG enrichment analysis systematically elucidated the functional significance of these differentially expressed genes and provided an in-depth analysis of the intrinsic molecular mechanisms of earthworm responses. PBAT microplastics mainly regulate energy metabolism-related pathways (such as protein digestion and absorption) and immune and oxidative stress-related pathways (such as apoptosis); PHA microplastics regulate energy metabolism pathways such as the TCA cycle and glycolysis / gluconeogenesis.

[0096] according to Figure 10 As can be seen, based on the concept of reference bias, the IBRv2 method was used to comprehensively evaluate the multi-level biological responses of the two microplastics at different concentrations. The star plot visually presents the bias patterns of each biomarker: positive deviations (inducible effects) mainly occur at the individual level in most treatment groups, affecting mortality, MDA levels, and SOD activity, while at the organ level, brain calcium levels... 2+ -ATPase and Ca 2+ -Mg 2+ -ATPase, expression of Caspase-3, Bax, and Bcl-2 proteins in the brain and seminal vesicle at the tissue level, and pro-inflammatory cytokines IL-1β and TNF-α in the brain and seminal vesicle at the molecular level; negative deviations (inhibitory effects) are seen in POD activity, GSH activity, and 8-OHdG content at the individual level, and Ca2+ in the seminal vesicle at the organ level. 2+ -ATPase, Ca 2+ -Mg 2+-ATPase, SDH activity and LDH activity, and the deviation increases with increasing exposure concentration.

[0097] Table 1 shows the reference deviation index A for the multilevel response indicators of earthworms to PBAT and PHA microplastic exposure, and Table 2 shows the comprehensive evaluation index IBRv2 for the multilevel response indicators of earthworms to PBAT and PHA microplastic exposure.

[0098] Table 1

[0099] A higher IBRv2 (Integrated Biomarker Response Index) value indicates a more severe environmental stress or toxic damage to the organism. Table 2 shows that all biological response indicators were included in the IBRv2 calculation, including individual-level mortality, MDA content, SOD activity, POD activity, GSH activity, and 8-OHdG content; and organ-level brain Ca2+ levels. 2+ -ATPase, brain calcium 2+ -Mg 2+ -ATPase, seminal vesicle Ca 2+ -ATPase, seminal vesicle Ca 2+ -Mg 2+ -ATPase, seminal vesicle SDH and LDH activities; tissue-level expression of Caspase-3, Bax, and Bcl-2 proteins in the brain and seminal vesicles; molecular-level expression of pro-inflammatory cytokines IL-1β and TNF-α in the brain and seminal vesicles.

[0100] Table 2 shows that the IBRv2 composite index of each treatment group, from highest to lowest, is as follows: 1% PHA (40.50) > 0.1% PHA (39.54) > 1% PBAT (33.55) > 0.1% PBAT (28.03). This indicates that both microplastics induced dose-dependent enhancement of overall stress, and at the same concentration, PHA had a stronger overall toxic effect than PBAT. The specific response patterns of each biomarker revealed by the star plot corroborate the quantitative ranking of IBRv2, jointly reflecting the differences in stress intensity and toxicity characteristics among different treatments.

[0101] Table 2

[0102] Preliminary thresholds for IBRv2-based pollution level diagnosis: For PBAT: When IBRv2 ≥ 28, it can be inferred that the PBAT concentration in the soil is no less than 0.1%; If IBRv2 increases further to above 33, it may reach a concentration level of 1%.

[0103] For PHA: When IBRv2 ≥ 40, it can be inferred that the PHA concentration in the soil is no less than 0.1%; It is also worth noting that PHA is close to its maximum effect at 0.1% (40.50 for 1% PHA), suggesting that even low concentrations of this microplastic can cause strong stress. Therefore, IBRv2 should be considered as a warning of PHA contamination once it approaches 40.

[0104] In Table 2, the gradient difference between the low and high concentration groups of PBAT and PHA in the IBRv2 index is relatively weak. Therefore, this invention further proposes a sensitive index with a significant concentration-dependent monotonic response for the calculation of the IBRv2 index, and the calculation result is named the modified IBRv2 index.

[0105] Screening method for sensitive indicators: Screening is based on the principle of consistency of dose-response trend. Based on the measured data of the control group, low-dose treatment group and high-dose treatment group, the indicators that show a significant monotonically increasing or significantly monotonically decreasing trend with increasing microplastic exposure concentration are identified as sensitive indicators.

[0106] Combination Figures 1 to 10 Based on the measured data, the following sensitive indicators were finally determined: For PBAT microplastic exposure, the sensitive indicators are: Individual level: Superoxide dismutase (SOD) activity, non-enzymatic antioxidant GSH content, and malondialdehyde (MDA) content (oxidative damage product). Organ level: Brain Ca 2+ -ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity; Tissue level: expression levels of Caspase-3 and Bax proteins in the brain, and expression levels of Caspase-3 protein in the seminal vesicle; At the molecular level: expression levels of IL-1β and TNF-α in the brain, and expression level of TNF-α in the seminal vesicles.

[0107] For PHA microplastic exposure, the sensitive indicators are: Individual level: Peroxidase (POD) activity, superoxide dismutase (SOD) activity; Organ level: Brain Ca 2+ -Mg 2+ -ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity; Tissue level: Expression level of Bax protein in the brain; At the molecular level: expression levels of IL-1β and TNF-α in the brain, and expression level of TNF-α in the seminal vesicles.

[0108] Table 3 shows the modified IBRv2 comprehensive index calculated based on the selected sensitive indicators. Since non-monotonic and fluctuating indicators were excluded, the modified IBRv2 value showed significantly improved sensitivity to concentration gradients. It can more clearly reflect the early stress signals under low concentration exposure, avoid the problem of insignificant differences between low and high concentrations, and greatly improve the dose response sensitivity and early warning evaluation capability of soil environmental microplastics of the modified IBRv2 model.

[0109] Table 3

[0110] In summary, this invention significantly improves the early identification ability and concentration discrimination of low-concentration microplastic stress by reconstructing the improved IBRv2 index through screening multi-level sensitive indicators with significant concentration-dependent monotonic responses.

[0111] The modified IBRv2 index can be used to determine the degree of soil microplastic (PBAT or PHA) pollution, specifically: For PBAT microplastic pollution: When calculating modified IBRv2 using the sensitive indicator i as the biomarker, a modified IBRv2 index of 9.8 to 10.9 can indicate that the soil has reached a PBAT pollution concentration of about 0.1%; a modified IBRv2 index of 20.5 to 22.7 can indicate that the soil has reached a PBAT pollution concentration of about 1%. For PHA microplastic pollution: When calculating modified IBRv2 using the sensitive indicator as biomarker i, a modified IBRv2 index of 9.8 to 10.8 can indicate that the soil has reached a PHA pollution concentration of about 0.1%; a modified IBRv2 index of 18.3 to 20.3 can indicate that the soil has reached a PHA pollution concentration of about 1%.

Claims

1. A method for assessing the biological effects of microplastics based on multi-level responses of soil animals, characterized in that, Includes the following steps: S1. Set up a treatment group and a control group, wherein the treatment group is the test soil and the control group is the soil with a microplastic background value lower than the detection limit or not higher than 0.05‰. Soil animals are placed in the soil of the treatment group and the control group respectively for exposure culture. S2. Collect soil animal individuals after exposure and culture, and determine the multi-level response indicators of the soil animals, including one or more of the following: individual-level response indicators, organ-level response indicators, tissue-level response indicators, and molecular-level response indicators. S3. Based on the aforementioned response indicators, the comprehensive effect index is calculated using the comprehensive biomarker response index method; S4. Based on the comprehensive effect index, construct a comprehensive evaluation model for microplastic effects and obtain the ecological risk assessment results of microplastics.

2. The method according to claim 1, characterized in that, Between steps S2 and S3, add a response indicator screening step: Screening showed statistically significant concentration dependence ( p Response indices ≤0.05 and showing a monotonically increasing or decreasing dose-response relationship are used to calculate the comprehensive effect index.

3. The method according to claim 1, characterized in that, Includes at least one of the following technical features: A1. The individual-level response indicators include one or more of the following: survival rate, mortality rate, antioxidant enzyme activity, content of oxidative damage products, and content of non-enzymatic antioxidants; B1. The organ-level response indicators include neurotoxicity-related enzyme activity and / or reproductive toxicity-related enzyme activity; C1. The tissue-level response indicators include HE section observation and / or immunohistochemical analysis of tissue damage; D1. The molecular-level response indicators include inflammatory factor gene expression and / or transcriptomics.

4. The method according to claim 1, characterized in that, In A1, the antioxidant enzymes include superoxide dismutase (SOD) and / or peroxidase (POD), the oxidative damage products include malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG), and the non-enzymatic antioxidants include reduced glutathione (GSH). In B1, the neurotoxicity-related enzyme includes Ca 2+ -ATPase and / or Ca 2+ -Mg 2+ -ATPase, the reproductive toxicity-related enzymes include Ca 2+ -ATPase, Ca 2+ -Mg 2+ -One or more of ATPase, sorbitol dehydrogenase SDH, and lactate dehydrogenase LDH; In C1, the immunohistochemical analysis includes the expression levels of one or more of the proteins Caspase-3, Bax, and Bcl-2; In D1, the expression of inflammatory genes includes IL-1β and / or TNF-α.

5. The method according to claim 1, characterized in that, The microplastics are polybutylene terephthalate and / or polyhydroxy fatty acid esters.

6. The method according to any one of claims 1 to 5, characterized in that, The algorithm for the comprehensive effect index IBRv2 is as follows: in, The average value of biomarker i in the treatment group. The mean value of biomarker i in the control group is given by denominator, and µ is the mean value of biomarker i. The overall mean of the values, σ is the biomarker i. The standard deviation of the value The degree to which the control group deviates from the global mean is represented by A, which is the reference deviation index. The biomarker is one or more of the response indicators.

7. A microplastic bioeffect assessment system based on multi-level response indices of soil animals for implementing the method of any one of claims 1 to 6, characterized in that, It includes an exposure experiment module, a response index detection module, and a model evaluation module. The response index detection module includes an individual effect detection module, an organ effect detection module, a tissue effect detection module, and a molecular effect detection module. The exposure experiment module is used to establish and manage soil animal exposure experiment systems with different concentrations and types of microplastics; The individual effect detection module is used to determine the individual-level response indicators of typical soil animals. The organ effect detection module is used to determine organ-level response indicators of typical soil animals. The tissue effect detection module is used to determine tissue-level response indicators of typical soil animals; The molecular effect detection module is used to determine the molecular-level response indicators of typical soil animals. The model evaluation module has a built-in multi-level response index comprehensive evaluation model. Based on the biological multi-level response index data measured by the response index detection module, it calculates and outputs the ecological risk assessment results of microplastic pollution.

8. The application of the method according to any one of claims 1 to 6 in assessing and predicting the toxic risks of microplastics to soil ecosystems.

9. The application according to claim 8, characterized in that, The method is used for early warning of microplastic soil pollution risks.

10. A combination of early warning biomarkers for soil microplastic pollution, characterized in that: Includes any of the following technical features: A2. Regarding PBAT: Factors include superoxide dismutase (SOD) activity, non-enzymatic antioxidant GSH content, malondialdehyde (MDA) content (an oxidative damage product), and brain calcium levels. 2+ - This consists of ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity, brain Caspase-3 protein expression level, brain Bax protein expression level, seminal vesicle Caspase-3 protein expression level, brain IL-1β expression level, brain TNF-α expression level, and seminal vesicle TNF-α expression level. B2. Targeting PHA: This is due to peroxidase (POD) activity, superoxide dismutase (SOD) activity, and brain calcium... 2+ -Mg 2+ - It consists of ATPase activity, seminal vesicle lactate dehydrogenase (LDH) activity, brain Bax protein expression level, brain IL-1β expression level, brain TNF-α expression level, and seminal vesicle TNF-α expression level.

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