A soil heavy metal pollution early warning method based on nematode maturity index ratio

Abstract

The application provides a soil heavy metal pollution early warning method based on nematode maturity index ratio, and belongs to the technical field of environmental monitoring and ecological risk assessment; the method comprises the following steps: obtaining a first ecological index and a second ecological index of a soil sample in a to-be-detected area, the first ecological index representing a first biological community sensitive to soil pollution stress, and the second ecological index representing a second biological community relatively resistant; calculating a comprehensive stress index based on the ratio of the two indexes; comparing the index with a benchmark index obtained from a sample in a background area that has never been polluted, and calculating a relative deviation index; and finally determining a pollution early warning level based on the value of the relative deviation index; the application amplifies the stress signal by using the opposite response trends of the two types of biological communities and calculating the index ratio, realizes early warning with high sensitivity, can truly reflect the biological availability of pollutants, and the evaluation result has more ecological significance.

Description

Technical Field

[0001] This application relates to the field of environmental monitoring and ecological risk assessment technology, and in particular to a method for early warning of soil heavy metal pollution based on the ratio of nematode maturity index. Background Technology

[0002] Heavy metal pollution in soil is a global environmental problem, and its conventional monitoring methods mainly rely on chemical analysis techniques. These methods determine whether the soil is polluted and the degree of pollution by directly measuring the total concentration of specific heavy metal elements in soil samples and comparing it with the limits set by national or local soil environmental quality standards.

[0003] However, this traditional chemical monitoring method has inherent drawbacks: First, its response is delayed, and problems are usually only detected when pollutants accumulate to a high concentration and exceed the legal threshold, making early warning impossible; second, chemical analysis measures the total amount of heavy metals and cannot distinguish their chemical forms and bioavailability in the soil, thus failing to accurately reflect the actual toxic effects of pollution on the biological communities within the soil ecosystem; finally, this method ignores the early and subtle structural and functional disturbances that occur in the ecosystem when it is stressed by low concentrations of pollutants, missing the optimal opportunity for risk management.

[0004] To overcome the limitations of chemical analysis methods, researchers have begun to use biological indicators to assess soil health. For example, changes in soil microbial or animal community structure can reflect environmental stress. However, existing bioassessment methods also have limitations. Some methods derive assessment results by weighted summation or comprehensive scoring of multiple biological indicators. While this approach can integrate multifaceted information, it may weaken or mask signals when faced with complex ecological responses. This is because different biological groups may respond to stress in opposite directions (e.g., some groups decrease in number while others increase), thus reducing the sensitivity of the assessment system. This weakening effect is particularly pronounced in the early stages of pollution. Therefore, current technology still lacks a biomonitoring method that can effectively amplify early stress signals and achieve highly sensitive early warning. Summary of the Invention

[0005] The purpose of this application is to provide a soil heavy metal pollution early warning method based on the nematode maturity index ratio, which aims to solve the problems of low sensitivity, slow response, and inability to reflect the actual stress on the ecosystem before the pollutant concentration exceeds the standard in existing soil pollution monitoring methods, thereby achieving high sensitivity, early warning, and graded early warning.

[0006] To achieve the above objectives, this application provides a method for early warning of soil heavy metal pollution based on the ratio of nematode maturity index, comprising the following steps: obtaining a first ecological index and a second ecological index for soil samples of a test area, wherein the first ecological index is used to characterize a first biological community sensitive to soil pollution stress, and the second ecological index is used to characterize a second biological community relatively tolerant to the soil pollution stress; calculating a comprehensive stress index for the test area based on the ratio of the first ecological index to the second ecological index; comparing the comprehensive stress index of the test area with a benchmark comprehensive stress index to calculate a relative deviation index, wherein the benchmark comprehensive stress index is obtained by processing soil samples from an uncontaminated background area using the same steps as determining the comprehensive stress index of the test area; and determining the pollution warning level of the test area based on the value of the relative deviation index.

[0007] Optionally, the first biological community is a free-living nematode community, and the second biological community is a plant-parasitic nematode community.

[0008] Furthermore, the first ecological index is the maturity index MI of free-living nematodes, and the second ecological index is the maturity index PPI of plant parasitic nematodes.

[0009] In a preferred embodiment of this application, the comprehensive stress index is the maturity index ratio (MIR), which is calculated as: MIR = MI / PPI; and when the PPI is zero, the MIR is set to a preset extreme value to avoid calculation errors and enhance the robustness of the method.

[0010] Optionally, the formula for calculating the maturity index MI of the free-living nematodes is as follows: Where i represents each family or genus of free-living nematodes identified from the soil sample. The summation is over all i, v(i) is the cp value of family or genus i, and f(i) is the proportion of the number of nematodes of family or genus i to the total number of free-living nematodes.

[0011] Optionally, the formula for calculating the plant parasitic nematode maturity index (PPI) is as follows: Where i represents each family or genus of plant-parasitic nematodes identified from the soil sample. The summation is over all i, v(i) is the cp value of family or genus i, and f'(i) is the proportion of the number of nematodes of family or genus i to the total number of plant parasitic nematodes.

[0012] Furthermore, the relative deviation index is calculated by dividing the comprehensive stress index of the area to be tested by the benchmark comprehensive stress index.

[0013] As a preferred embodiment of this application, the pollution warning level is determined as follows: when the relative deviation index is greater than 0.8, it is determined to be a safe level; when the relative deviation index is greater than 0.5 and not greater than 0.8, it is determined to be a concern level; when the relative deviation index is greater than 0.2 and not greater than 0.5, it is determined to be a warning level; when the relative deviation index is not greater than 0.2, it is determined to be a severe warning level.

[0014] Optionally, the soil pollution stress is caused by heavy metal pollution in the soil.

[0015] Optionally, before the step of obtaining the first ecological index and the second ecological index, the method further includes: separating the first biological community and the second biological community from the soil sample using a sucrose centrifugal flotation method.

[0016] Compared with existing technologies, the technical solution provided in this application has the following beneficial effects: First, this method has high sensitivity and early warning capabilities. By utilizing the opposite response trends of stress-sensitive biological communities (such as free-living nematodes) and relatively tolerant biological communities (such as plant-parasitic nematodes) under stress, and calculating the ratio of their corresponding ecological indices, the stress signal is greatly amplified. This allows the method to detect ecological risks early and sensitively through changes in the structure of biological communities before the chemical concentration of pollutants exceeds the legal standard limit. Second, this method can truly reflect bioavailability. It directly measures the actual comprehensive impact of pollutants on key biological groups in the soil, and the results better reflect the bioavailability of pollutants and their true stress level on the health of the ecosystem, making the assessment results more ecologically significant. Third, this method achieves quantification and standardization. By constructing standardized quantitative indicators and establishing clear multi-level early warning thresholds, a calculable, comparable, and repeatable standardized process is provided for soil ecological risk assessment. Finally, this method is cost-effective. It can serve as an efficient biological monitoring and screening tool for preliminary risk assessment in large areas, thereby guiding the precise deployment of subsequent, more expensive chemical analyses, optimizing the allocation of monitoring resources, and reducing overall monitoring costs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1This is a flowchart illustrating a method for early warning of soil heavy metal pollution based on the nematode maturity index ratio, provided in an embodiment of this application.

[0019] Figure labeling: S101 - Sample collection and processing; S102 - Nematode community analysis; S103 - Calculation of MI and PPI indices; S104 - Calculation of MIR and RDI indices; S105 - Graded early warning determination; S106 - Output results. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and specific embodiments. It is understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0021] This application provides a method for early warning of soil heavy metal pollution based on the ratio of nematode maturity index. The technical concept lies in constructing a quantitative index that amplifies ecological stress signals to achieve early, sensitive, and tiered early warning of soil pollution. This method innovatively utilizes two types of biological communities in the soil ecosystem with different functions and opposite responses to stress. Specifically, a first biological community sensitive to pollution stress is selected, whose population structure or abundance undergoes significant negative changes under stress; simultaneously, a second biological community relatively tolerant of the same stress, and even potentially proliferating due to reduced competition, is selected. By calculating the first and second ecological indices representing the states of these two communities respectively, and obtaining their ratio, a comprehensive stress index is obtained. Because the numerator (first ecological index) of this ratio decreases while the denominator (second ecological index) remains unchanged or increases, this ratio responds more intensely to stress than any single index, thus amplifying the signal. Based on this, a relative deviation index is calculated by comparing the comprehensive stress index of the tested area with the benchmark index obtained from a background area that has never been contaminated. This effectively eliminates regional background differences, making the early warning results comparable and standardized across regions. Finally, a multi-level early warning threshold is established based on this relative deviation index to achieve a refined assessment of soil health status.

[0022] Please see Figure 1 This is a flowchart illustrating a method for early warning of soil heavy metal pollution based on the nematode maturity index ratio, provided in an embodiment of this application. As an optional implementation, the method may specifically include the following steps:

[0023] Step S101: Sample Collection and Processing. Soil samples are collected from the test area and the selected background reference area. The test area is an area suspected of having a pollution risk, while the background reference area should be selected from areas similar to the test area in terms of soil type, vegetation, and utilization history, but confirmed to be free from heavy metal or other pollution. The collected samples must undergo standardization processing, such as removing stones and plant debris, air drying, sieving, and mixing, to ensure sample homogeneity.

[0024] Step S102: Nematode Community Analysis. This step is the core of the biological analysis of the treated soil samples. Specifically, techniques known in the art (e.g., sucrose centrifugation flotation) can be used to isolate nematodes from the soil. The isolated nematodes need to be identified under a microscope down to the taxonomic level (family or genus), and nematodes in different taxonomic units should be counted. Crucially, all identified nematodes must be classified into two major functional groups: a primary biological community sensitive to pollution stress, and a secondary biological community relatively tolerant. In a preferred embodiment of this application, these two communities correspond to a free-living nematode community and a plant-parasitic nematode community, respectively.

[0025] Step S103: Calculate the MI and PPI indices. This step calculates ecological indices characterizing the states of the two biological communities based on data obtained from nematode community analysis. In a preferred embodiment of this application, the first ecological index is the Maturity Index (MI) of free-living nematodes, and the second ecological index is the Maturity Index (PPI) of plant-parasitic nematodes. Both indices can be calculated based on the cp value system proposed by Bongers, which assigns a cp value of 1 to 5 to each nematode family or genus. A higher cp value indicates a longer lifespan, lower reproductive rate, and greater sensitivity to environmental disturbances, belonging to the K-strategist group; conversely, a lower cp value indicates an opportunistic r-strategist group.

[0026] Step S104: Calculate the MIR and RDI indices. This step is crucial for signal amplification and standardization. First, based on the MI and PPI values ​​obtained in step S103, the comprehensive stress index is calculated, specifically the maturity index ratio (MIR) in this application, which is calculated by dividing MI by PPI. The value calculated for the background reference area sample is denoted as the baseline ratio MIR0, and the value calculated for the test area sample is denoted as MIR. Subsequently, to eliminate background differences, the MIR of the test point is compared with the baseline ratio MIR0, and the relative deviation index (RDI) is calculated by dividing the test point's MIR by the baseline ratio MIR0.

[0027] Step S105: Graded Early Warning Determination. The RDI value calculated in the previous step is compared with a pre-established multi-level early warning model. This model contains a series of thresholds to divide the continuous interval of the RDI value into multiple discrete early warning levels.

[0028] Step S106: Output Results. Based on the judgment results of step S105, output the specific pollution warning level of the area to be tested, such as "Safe", "Concern", "Warning" or "Severe Warning", to provide a scientific basis for subsequent environmental management and remediation decisions.

[0029] The technical solution of this application will be described in detail below with reference to more specific embodiments.

[0030] Example 1

[0031] This embodiment aims to scientifically verify, through controlled laboratory pot experiments, the existence of a clear dose-response relationship between the relative deviation index (RDI) proposed in this application and the concentration of heavy metal pollution in the soil, and to provide experimental basis for the scientific validity and rationality of the four-level early warning model thresholds (i.e., 0.8, 0.5 and 0.2) proposed in the technical solution.

[0032] The first step was soil preparation and pollution gradient setup. The experiment used clean topsoil (0-20 cm) from an organic farming base, confirmed to be free of heavy metal contamination, as the "standard soil." The collected soil was transported back to the laboratory and air-dried in a ventilated area, removing visible stones, plant roots, and other debris. The soil was then sieved using a 2 mm mesh to ensure uniform texture. The sieved soil was thoroughly mixed to ensure consistent physicochemical properties across the experimental treatment groups. Using the mixed standard soil as a representative pollutant (cadmium cadmium (Cd), a pollution gradient with seven concentration levels was established. The specific treatments were as follows: T0 (control group): No additional Cd was added; the background Cd concentration in the soil was measured to be 0.05 mg / kg. This group represented uncontaminated, healthy soil. T1 group: Analytical grade cadmium chloride (CdCl2) solution was added to the standard soil and thoroughly mixed to achieve a final Cd concentration of 0.1 mg / kg. Group T2: The same procedure was performed to achieve a final Cd concentration of 0.2 mg / kg in the soil. Group T3: The same procedure was performed to achieve a final Cd concentration of 0.3 mg / kg in the soil. It should be noted that this concentration is the risk screening value for agricultural land soil with pH > 7.5 according to my country's "Soil Environmental Quality Standard for Agricultural Land Soil Pollution Risk Control (Trial)" (GB 15618-2018). Group T4: The same procedure was performed to achieve a final Cd concentration of 0.5 mg / kg in the soil. Group T5: The same procedure was performed to achieve a final Cd concentration of 1.0 mg / kg in the soil. Group T6: The same procedure was performed to achieve a final Cd concentration of 2.0 mg / kg in the soil. Each concentration gradient treatment was performed in five replicates, i.e., five identical pots were prepared, each containing an equal amount of soil corresponding to the treatment concentration.

[0033] The second step was cultivation and sampling. All the prepared potted plants (7 treatment groups × 5 replicates = 35 pots) were randomly arranged and placed in a constant-temperature incubator for 30 days. During cultivation, environmental conditions were strictly controlled to eliminate interference from other variables: the cultivation temperature was kept constant at 25℃, and the light cycle was 12 hours of light / 12 hours of darkness. The soil moisture content of all potted plants was maintained at approximately 60% of maximum water holding capacity by periodically weighing and replenishing with deionized water. This cultivation period and conditions were sufficient to allow the nematode community in the soil to undergo sufficient community structure succession and response to different concentrations of Cd stress. After 30 days of cultivation, soil samples were taken from each potted plant. A multi-point mixing method was used; five points were randomly selected within each potted plant, and soil samples were collected at a depth of 0-15 cm using a soil auger. The five subsamples were then thoroughly mixed in a clean container to form the final test sample for that potted plant.

[0034] The third step is the determination and calculation of indicators. For each sample to be tested, the following procedures are followed: Figure 1 The analysis process is as shown. Specifically, nematodes are first separated using sucrose centrifugation flotation. The procedure is as follows: 100 grams of well-mixed soil sample is accurately weighed, water is added and stirred to form a suspension, and the sample is initially filtered and enriched through a series of sieves (e.g., 0.1 mm and 0.025 mm pore sizes). The material retained on the sieves is eluted into centrifuge tubes, a sucrose solution with a concentration of 1.33 g / mL is added, and the mixture is centrifuged at 3000 rpm for 5 minutes. Because the nematodes are less dense than the sucrose solution, they float, while most soil particles sink to the bottom of the tube. The supernatant is carefully aspirated, sieved again, and washed to obtain a relatively pure nematode suspension. Subsequently, the obtained nematode suspension is placed in a counting frame, and the nematodes are identified and counted under an optical microscope. Based on the morphological characteristics of the nematodes, they are identified to the family or genus level, and the number of nematodes in each taxonomic unit is recorded. At the same time, all identified nematodes are classified into two main categories: free-living nematodes and plant-parasitic nematodes. Next, ecological indices were calculated. The average of the five replicates from the T0 control group was used as the baseline. For each sample, the maturity index (MI) of free-living nematodes and the maturity index (PPI) of plant-parasitic nematodes were calculated. The formula for calculating MI is: Where i represents the family or genus of a free-living nematode in the sample; v(i) is the cp value corresponding to that family or genus, which is obtained from Bongers' 1990 publication and ranges from 1 to 5; f(i) is the proportion of nematodes of that family or genus to the total number of all free-living nematodes in the sample. The formula for calculating PPI is: Where i represents the family or genus of a plant parasitic nematode in the sample; v(i) is the cp value corresponding to that family or genus, and the cp value of plant parasitic nematodes is usually between 2 and 5; f'(i) is the proportion of the number of nematodes of that family or genus to the total number of all plant parasitic nematodes in the sample. After calculating the MI and PPI for each sample, the maturity index ratio MIR = MI / PPI is then calculated. The arithmetic mean of the MIR values ​​calculated from the five replicates of the T0 control group is taken to obtain the baseline ratio MIR0. For each sample in the T1 to T6 treatment groups, the relative deviation index RDI = MIR / MIR0 is calculated.

[0035] Step 4: Data Analysis and Results. Cd concentration values ​​for all treatment groups (T1-T6) were used as independent variables, and the corresponding average RDI was used as the dependent variable to plot dose-response curves. Regression analysis using statistical software revealed a highly significant negative correlation between RDI and Cd concentration (coefficient of determination R² > 0.9, P < 0.01). This indicates that as Cd concentration in the soil increases, the RDI value systematically and predictably decreases. Specifically, under low concentration stress (T1 group, Cd concentration 0.1 mg / kg), the average RDI was approximately 0.92, significantly greater than 0.8. When the Cd concentration increased to a level close to but still below the national standard limit (T2 group, Cd concentration 0.2 mg / kg), the average RDI decreased to approximately 0.75, falling into the range of (0.5, 0.8). When the Cd concentration reached and slightly exceeded the national standard limit (T3 group, Cd concentration 0.3 mg / kg; T4 group, Cd concentration 0.5 mg / kg), the average RDI decreased to approximately 0.61 and 0.43, respectively, mainly distributed within the range of (0.2, 0.8], with the RDI value of T4 group clearly entering the range of (0.2, 0.5]. Under high concentration stress (T5 group, Cd concentration 1.0 mg / kg; T6 group, Cd concentration 2.0 mg / kg), the average RDI decreased sharply to approximately 0.18 and 0.09, both significantly lower than 0.2.

[0036] The experimental results of this embodiment strongly demonstrate that the RDI index has a clear, monotonically decreasing dose-response relationship with heavy metal stress. More importantly, the experimental data verify the scientific validity and rationality of the four-level warning thresholds set in the technical solution of this application: RDI > 0.8 corresponds to a "safe" state where the soil is basically unstressed; 0.5 < RDI ≤ 0.8 corresponds to an early stage where the soil ecosystem has begun to show slight disturbances and deserves "attention"; 0.2 < RDI ≤ 0.5 corresponds to a stage where the ecosystem is under significant stress and a "warning" needs to be issued; and RDI ≤ 0.2 corresponds to a "severe warning" state where the ecosystem is severely damaged and the risk is extremely high.

[0037] Example 2

[0038] This embodiment aims to apply the early warning method proposed in this application to a real field scenario. By comparing it with traditional soil heavy metal chemical analysis methods, it verifies the high sensitivity, early warning capability, and practical value of this method in practical applications.

[0039] Scenario selection: In this embodiment, a chemical industrial park that has been operating for many years is selected as a potential pollution source for scenario verification. The park mainly produces chemical products involving heavy metal catalysts.

[0040] The first step was site selection and sampling. Considering the prevailing southwest wind direction in the area, three test sites were established along a straight line downwind (northeast) of the chemical industrial park, designated D1, D2, and D3. D1 was closest to the park boundary, approximately 1 km away; D2 was approximately 3 km away; and D3 was approximately 5 km away. These three sites were all farmland where corn had been grown for a long time. Simultaneously, to obtain a baseline value for comparison, a background farmland with similar soil type, planting history, and management methods to the test area was selected approximately 10 km upwind (southwest) and designated as baseline point C1. At each site (C1, D1, D2, D3), a five-point sampling method was used to collect topsoil samples from 0-20 cm depth. The five subsamples were thoroughly mixed to form a representative composite sample for that site. To ensure the reliability of the results, three independent composite samples were collected from each site, resulting in a total of 12 soil samples. All samples were air-dried, sieved (2 mm), and stored according to standard procedures.

[0041] The second step was nematode community analysis and index calculation. For all 12 soil samples, the same sucrose centrifugation-flotation method as in Example 1 was used for nematode isolation, identification, and counting. The detailed calculation process is illustrated below using one parallel sample each from baseline point C1 and test point D1 as examples. Assume that after identification and counting, the following raw data are obtained:

[0042] Free-living nematodes Total: 200 Total: 120 Rhabditis 1 10 60 Cephalobus 2 80 30 Aphelenchoides 3 50 18 Dorylaimus 5 30 12 Plant parasitic nematodes Total: 50 Total: 80 Helicotylenchus 3 25 48 Pratylenchus 3 25 32

[0043] Based on the data in the table above, the index calculation process is as follows: For the sample at baseline C1: The proportions f(i) of each genera of free-living nematodes are as follows: Rhabditis: 40 / 200=0.20; Cephalobus: 80 / 200=0.40; Aphelenchoides: 50 / 200=0.25; Dorylaimus: 30 / 200=0.15. Calculate MI0: The proportions f'(i) of each genera of plant-parasitic nematodes are as follows: Helicotylenchus: 25 / 50 = 0.50; Pratylenchus: 25 / 50 = 0.50. Calculate PPI0: Calculate MIR0: .

[0044] For the sample at test site D1: the proportions f(i) of each genera of free-living nematodes are as follows: Rhabditis: 60 / 120 = 0.50; Cephalobus: 30 / 120 = 0.25; Aphelenchoides: 18 / 120 = 0.15; Dorylaimus: 12 / 120 = 0.10. Calculate MI: The proportions f'(i) of each genera of plant-parasitic nematodes are as follows: Helicotylenchus: 48 / 80 = 0.60; Pratylenchus: 32 / 80 = 0.40. Calculate PPI: Calculate MIR: It should be noted that, in practical applications, to ensure the robustness of the method, if plant parasitic nematodes are not detected in a soil sample (i.e., the PPI value is zero), to avoid the calculation error of division by zero, the MIR value can be set to a preset maximum value (e.g., 100). This maximum value represents an extremely healthy and undisturbed soil state.

[0045] The above calculations were performed on all samples, and the average of the results from three parallel samples at each location was taken. The final average indices for each location are as follows: Baseline point C1: The measured average MI0 is 2.85, and the average PPI0 is 1.10. The baseline ratio was calculated. At measurement point D1: the average MI was measured to be 0.95, and the average PPI was 1.90. The calculated values ​​are... At measurement point D2: the average MI was measured to be 1.80, and the average PPI was 1.45. The calculated values ​​are as follows: At measurement point D3: the average MI was measured to be 2.45, and the average PPI was 1.15. The calculated values ​​are as follows: .

[0046] The third step is to calculate the relative deviation index and issue graded warnings. Using a baseline ratio MIR0 = 2.59, calculate the RDI value for each test point: Point D1: Point D2: Point D3: .

[0047] Based on the four-level early warning model established in this application, the following judgments are made: Point D1: RDI ≈ 0.193, which is not greater than 0.2, therefore it is judged as "Level 3: Severe Warning". Point D2: RDI ≈ 0.479, which is greater than 0.2 and not greater than 0.5, therefore it is judged as "Level 2: Warning". Point D3: RDI ≈ 0.822, which is greater than 0.8, therefore it is judged as "Level 0: Safe".

[0048] The fourth step was chemical analysis comparison and conclusion verification. For comparison and verification, all 12 soil samples were sent to a professional testing institution, where the total concentration of various heavy metals (Cd, Pb, Cu, Zn) was detected using national standard methods (such as inductively coupled plasma mass spectrometry). Chemical analysis results showed that the average Cd concentration at point D1 was 0.28 mg / kg, while the concentrations of Pb, Cu, and Zn were all far below national standards. The concentrations of all tested heavy metals at points D2 and D3 were also far below national standard limits. According to my country's standards for risk management of agricultural land soil pollution, the Cd concentration at point D1 (0.28 mg / kg) did not exceed its risk screening value (0.3 mg / kg). Accordingly, based solely on traditional chemical analysis methods, point D1 would be classified as "safe" or "risk-free."

[0049] Conclusion: A comparison of the early warning results from the proposed method with those from traditional chemical analysis reveals significant differences. The proposed method provides a "severe warning" signal (highest risk level) at point D1, the closest point to the pollution source, while traditional chemical analysis shows that the pollutant concentration at that point has not yet exceeded the standard. These comparative results fully demonstrate that the proposed method, utilizing the comprehensive response of biological communities, can extremely sensitively capture the actual stress effects of pollutants on the ecosystem before they accumulate to the legal threshold, thus achieving true early warning. Furthermore, the decrease in warning level with increasing distance from D1 to D3 closely matches the gradient law of pollution diffusion, further proving the logical consistency and reliability of the proposed method.

[0050] Example 3

[0051] This embodiment aims to verify the specificity of the early warning method constructed in this application, that is, to test whether the method is significantly interfered with by other common agricultural environmental stress factors (such as pesticide application and drought) when responding to heavy metal pollution stress, so as to evaluate its specificity as a heavy metal pollution early warning tool.

[0052] Step 1: Experimental Design. Using the same "standard soil" as in Example 1, four treatment groups were established, with five replicate pots for each group: Group A (Blank Control Group): No additional stress was applied, serving as the baseline. Group B (Heavy Metal Stress Group): Cd was added to the soil to a final concentration of 0.5 mg / kg to simulate moderate heavy metal pollution. Group C (Pesticide Stress Group): Glyphosate, a widely used herbicide, was added at twice the recommended field concentration to simulate excessive pesticide application. Group D (Drought Stress Group): During cultivation, soil moisture content was maintained at 30% of maximum water holding capacity by controlling irrigation to simulate continuous drought stress.

[0053] The second step was cultivation and measurement. All potted plants from all treatment groups were cultured for 30 days under the same constant temperature incubator conditions as in Example 1 (25°C, 60% water holding capacity, except for group D). After cultivation, samples were taken from each potted plant, and nematodes were isolated, identified, and counted using the same method as in Example 1. The MI and PPI values ​​of each sample were then calculated.

[0054] The third step is data analysis and results. Using the average MIR value of group A (blank control group) as the baseline ratio MIR0, the average RDI values ​​of the three stress treatment groups (B, C, and D) were calculated. The experimental results show that: The average RDI value of group B (heavy metal stress) decreased to approximately 0.45. According to the early warning model, this value falls within the (0.2, 0.5] interval, corresponding to "Level 2: Warning". The average RDI value of group C (pesticide stress) decreased to approximately 0.85. This value is still greater than 0.8, within the "Level 0: Safe" range, or just touching the boundary of "Level 1: Concern". The average RDI value of group D (drought stress) decreased to approximately 0.90. This value is also greater than 0.8, within the "Level 0: Safe" range.

[0055] Conclusion: By comparing the decrease in RDI values ​​among the stress groups, it was found that heavy metal stress (Group B) led to a sharp and significant decrease in RDI values, successfully triggering an "early warning" signal. In contrast, while the more severe pesticide and drought stresses (Groups C and D) also had some impact on nematode communities, the resulting decrease in RDI values ​​was far smaller than that of the heavy metal stress group, and they did not trigger a clear early warning signal. These results indicate that the index system constructed in this application, with the MI / PPI ratio as its core, has high sensitivity and strong specificity to heavy metal pollution stress, is not easily confused with other common agricultural environmental stresses, and can serve as a reliable early warning tool for heavy metal pollution.

[0056] Example 4

[0057] This embodiment aims to evaluate the stability and reliability of the method of this application through repeatability testing, so as to verify whether it meets the requirements for promotion and application as a standardized testing method.

[0058] The first step is sample preparation. Take a soil sample collected from point D2 (classified as "warning" level) in Example 2, combine three parallel samples from this point, and thoroughly and repeatedly mix them in the laboratory until a highly homogeneous soil sample is formed. Divide this homogeneous sample into multiple small portions for subsequent repeatability tests.

[0059] The second step is intra-batch repeatability testing. On the same day, the same technician uses the same instruments and reagents to randomly select 10 samples from the homogeneous samples mentioned above, and independently performs the entire analytical process from nematode isolation to RDI calculation on each sample, finally recording 10 independent RDI calculation results.

[0060] The third step is the inter-batch repeatability test. Two different experimenters (Experimenter A and Experimenter B) each randomly selected three samples from the homogeneous sample for independent and complete analysis each day for three consecutive days. Thus, Experimenter A obtained a total of 9 RDI values ​​over the three days, and Experimenter B also obtained 9 RDI values, recording a total of 18 RDI values. This test aims to assess the systematic errors introduced by different operators and at different times.

[0061] Step 4: Data Analysis and Results. For the 10 RDI values ​​obtained from the intra-batch repeatability test, their arithmetic mean and standard deviation were calculated. Then, using the formula: Coefficient of Variation = (Standard Deviation / Mean) × 100%, the intra-batch coefficient of variation was calculated. The results showed that the coefficient of variation for the intra-batch repeatability test was 4.8%. Similarly, for all 18 RDI values ​​obtained from the inter-batch repeatability test, their overall arithmetic mean, standard deviation, and inter-batch coefficient of variation were calculated. The results showed that the coefficient of variation for the inter-batch repeatability test was 7.2%.

[0062] Conclusion: In the field of analytical chemistry and biological detection, a coefficient of variation (COP) of less than 15% is generally considered to indicate good precision and repeatability. In this embodiment, the measured intra-batch COP (4.8%) and the more stringent inter-batch COP (7.2%) are both far below the accepted limit of 15%. This result fully demonstrates that the soil pollution classification and early warning method provided in this application has a stable operating procedure, reliable analytical results, is not easily affected by random errors caused by operators and experimental time, and has very good repeatability, fully meeting the requirements for promotion and application as a standardized detection method.

[0063] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for early warning of soil heavy metal pollution based on the ratio of nematode maturity index, characterized in that, Includes the following steps: A first ecological index and a second ecological index are obtained for soil samples from the area to be tested. The first ecological index is used to characterize a first biological community that is sensitive to soil pollution stress, and the second ecological index is used to characterize a second biological community that is relatively tolerant to the soil pollution stress. Based on the ratio of the first ecological index to the second ecological index, the comprehensive stress index of the area to be tested is calculated. The comprehensive stress index of the area to be tested is compared with a benchmark comprehensive stress index to calculate the relative deviation index. The benchmark comprehensive stress index is obtained by processing soil samples from an uncontaminated background area using the same steps as those used to determine the comprehensive stress index of the area to be tested. Based on the value of the relative deviation index, the pollution warning level of the area to be tested is determined.

2. The method according to claim 1, characterized in that, The first biological community is a free-living nematode community, and the second biological community is a plant-parasitic nematode community.

3. The method according to claim 2, characterized in that, The first ecological index is the maturity index (MI) of free-living nematodes, and the second ecological index is the maturity index (PPI) of plant-parasitic nematodes.

4. The method according to claim 3, characterized in that, The comprehensive stress index is the maturity index ratio (MIR), which is calculated as follows: MIR = MI / PPI; Furthermore, when the PPI is zero, the MIR is set to a preset extreme value.

5. The method of claim 3, wherein, The formula for calculating the maturity index MI of the free-living nematodes is as follows: ， Where i represents each family or genus of free-living nematodes identified from the soil sample, Σ is the summation over all i, v(i) is the cp value of family or genus i, and f(i) is the proportion of the number of nematodes of family or genus i to the total number of free-living nematodes.

6. The method of claim 3, wherein, The formula for calculating the plant parasitic nematode maturity index (PPI) is as follows: ， Where i represents each family or genus of plant parasitic nematodes identified from the soil sample, Σ is the summation over all i, v(i) is the cp value of family or genus i, and f'(i) is the proportion of the number of nematodes of family or genus i to the total number of plant parasitic nematodes.

7. The method of claim 1, wherein, The relative deviation index is calculated by dividing the comprehensive stress index of the area to be tested by the benchmark comprehensive stress index.

8. The method of claim 7, wherein, The method for determining the pollution warning level is as follows: When the relative deviation index is greater than 0.8, it is determined to be at a safe level; When the relative deviation index is greater than 0.5 and not greater than 0.8, it is determined to be at the level of concern. When the relative deviation index is greater than 0.2 and not greater than 0.5, it is determined to be a warning level; When the relative deviation index is not greater than 0.2, it is determined to be a severe warning level.

9. The method of claim 1, wherein, The soil pollution stress is caused by heavy metal pollution in the soil.

10. The method according to claim 1, characterized in that, Prior to the step of obtaining the first ecological index and the second ecological index, the method further includes: The first and second biological communities were separated from the soil sample using sucrose centrifugal flotation.

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