Soil ecological environment damage confirmation method suitable for illegally dumping waste slag area and application of soil ecological environment damage confirmation method

Abstract

The invention discloses a soil ecological environment damage confirmation method suitable for an illegally dumping waste slag area, which comprises the following steps: carrying out characteristic pollution index screening analysis on soil samples in an evaluation area and a control area; performing high-throughput sequencing and data processing to obtain bacterial community composition structures of the soil samples in the evaluation area and the control area, and comparing and analyzing changes of bacterial communities in the evaluation area to judge whether the soil environment of the evaluation area is obviously changed or not; the correlation between the change of the bacterial community and the characteristic pollution index is analyzed to confirm whether the waste slag has influence on the soil environment and the influence degree of the waste slag, the confirmation and evaluation method is scientific and reasonable, and a plurality of indexes such as the increase and decrease amplitude of the relative abundance of the dominant group can be used for quantifying the influence degree of the waste slag on the soil environment; the method can assist in confirming damage facts of damage identification and evaluation cases of illegal dumping of waste slag.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental monitoring, and in particular relates to a method for confirming soil ecological environment damage in an area where waste slag is illegally dumped, and an application thereof. Background Art

[0003] Cases involving soil and groundwater ecological environmental damage identification and assessment account for the largest proportion of such cases, and scientific and quantifiable assessment of environmental damage has become a focus and problem that needs to be solved in the field of environmental justice. Therefore, building a sound ecological environmental damage identification and assessment technology system is not only an inevitable requirement for implementing the reform of ecological environmental damage compensation, but also a necessary guarantee for the refined management of the ecological environment and the maintenance of environmental judicial justice, which has far-reaching significance for promoting the construction of ecological civilization in my country.

[0004] For example, the patent document with application number CN202211589012.3 discloses an ecological environmental damage assessment method and system applied to underground engineering water gushing events. The method includes a damage causal relationship judgment method, including the following steps: determining the source and receptor; judging homology; judging correlation; judging connectivity.

[0005] The above-mentioned existing technology combines the characteristics of the underground engineering industry, combines the legal theory of causal relationship determination with hydrogeological surveys, online monitoring, mathematical statistics and other methods, and develops a targeted method for evaluating the value of ecological environmental damage caused by underground engineering water gushing incidents, providing technical and normative guarantees for the evaluation of ecological environmental damage caused by underground engineering water gushing incidents. However, this existing technology was developed for underground engineering water gushing incidents and is not suitable for soil environmental damage identification and assessment cases.

[0006] On the other hand, the illegal dumping and landfill of waste slag is a typical type of environmental violation case, and the current ecological environmental damage identification work mainly focuses on the investigation of conventional physical and chemical indicators and heavy metal indicators. However, compared with other types of environmental pollution, the pollution of soil environment by waste slag has obvious hidden and cumulative characteristics. It often happens that when conducting environmental impact assessments, the conventional physical and chemical indicators and heavy metal indicators do not exceed the national standards, but the facts of illegal dumping of waste slag and soil pollution do exist, which aggravates the difficulty of tracing the causes of pollution and defining responsibilities, and thus makes the process of confirming the facts of damage more complicated.

[0007] The present invention aims to provide an assessment method that can further quantify the damage of waste slag to the soil ecological environment when the conventional physical and chemical indicators and heavy metal indicators of the soil in the area where waste slag is illegally dumped do not exceed the national standards. Summary of the invention

[0008] In response to the problems in the related technology, the present invention proposes a method for confirming soil ecological environment damage in areas where waste slag is illegally dumped and its application, so as to overcome the above-mentioned technical problems existing in the existing related technology.

[0009] The technical solution of the present invention is achieved in this way:

[0010] A method for confirming soil ecological environment damage in areas where waste slag is illegally dumped, comprising at least the following steps:

[0011] 1) Screening characteristic pollution indicators and determining characteristic pollution indicators;

[0012] 2) Determine the content of characteristic pollution indicators in soil samples from the control area and the assessment area where waste slag is illegally dumped, and analyze and determine whether the content of characteristic pollution indicators in the assessment area exceeds the soil baseline level in the control area;

[0013] 3) Perform high-throughput sequencing on soil samples from the evaluation area and the control area, perform OTU cluster analysis based on the sequence information, and at least use PCA analysis to determine whether the bacterial community in the evaluation area has changed significantly relative to the control area;

[0014] 4) The Pearson correlation test combined with the correlation heat map was used to conduct a correlation test on the changes in the bacterial community in the assessment area and the characteristic pollution indicators, to indirectly confirm whether the waste slag has caused damage to the soil ecological environment, and to quantify the impact of the waste slag on the soil ecological environment through changes in various indexes of the bacterial community.

[0015] Preferably, since the present invention is aimed at monitoring the soil environment, i.e., solid waste, the screening of characteristic pollution indicators in step 1) adopts the following determination method:

[0016] Heavy metals were determined by inductively coupled plasma mass spectrometry, and semivolatile organic compounds and volatile organic compounds were determined by gas chromatography-mass spectrometry.

[0017] Preferably, step 2) comprises the following steps:

[0018] 2.1) The soil samples from the assessment area and the control area were respectively tested for the content of characteristic pollution indicators;

[0019] 2.2) Determine soil baseline level:

[0020] The specific operation is to perform statistical calculations on the characteristic pollution index content data measured in the control area to check whether it belongs to a normal distribution;

[0021] If it is a normal distribution, the 90% reference upper limit of the control point data is used as the soil baseline, and the arithmetic mean + 1.65 times the standard deviation; if it is not a normal distribution, the 90th percentile of the control point data is used as the soil baseline;

[0022] 2.3) Compare with the soil baseline level to determine whether the content of characteristic pollution indicators in the assessment area exceeds the soil baseline level.

[0023] Preferably, in step 3), OTU clustering analysis is performed on the sequences obtained by high-throughput sequencing at a similarity level of 97%.

[0024] Preferably, in step 3), 0.25-0.5 g of soil sample is taken for high-throughput sequencing, stored in a refrigerator at ≤80°C, and high-throughput sequencing is performed within 24 hours. The sample nucleic acid is extracted using a magnetic bead method, and then PCR amplification and sequencing are performed to obtain raw sequencing data.

[0025] Preferably, the PCR amplification program is: initial denaturation at 95°C for 4-6 min, followed by denaturation at 95°C for 25-35 s and 25 cycles, annealing at 45-55°C for 20-35 s, and extension at 72°C for 35-45 s.

[0026] Preferably, DNA is extracted from soil samples using a TS96 Magnetic Soil DNA Kit.

[0027] Preferably, in step 3), at least the following steps are included:

[0028] 3.1) High-throughput sequencing of soil samples from the evaluation area and the control area;

[0029] 3.2) Perform PCA analysis at least at the phylum level to confirm whether there are differences in the microbial community structure and composition between the evaluation area and the control area;

[0030] 3.3) Perform taxonomic analysis on the sequence results obtained by high-throughput sequencing at least at the phylum level, and then determine the top 10 dominant bacterial phyla and their relative abundance in the evaluation area, and determine the relative abundance and increase / decrease range of the dominant bacterial phyla corresponding to the control area.

[0031] Preferably, step 4) comprises at least the following steps:

[0032] 4.1) The Pearson correlation test was combined with the correlation heat map to test the correlation between the top 10 dominant bacterial phyla in relative abundance determined in step 3) and the characteristic pollution index, to indirectly confirm whether the waste slag has damaged the soil ecological environment;

[0033] 4.2) Use the changes in various bacterial community indexes to quantitatively evaluate the impact of waste slag on the soil ecological environment.

[0034] As a core component of the soil ecosystem, soil microbial communities are directly involved in the decomposition of substances and nutrient cycling in the soil. About 80 to 90% of soil functions are closely related to soil microorganisms, including the effective transformation of soil nutrients, the maintenance of ecosystem stability, and resistance to external interference. From a biological perspective, microorganisms are recognized as the most sensitive soil quality indicators. The present invention uses highly sensitive microorganisms as key indicators for assessing whether the soil environment has changed significantly, which can directly reflect the toxicity intensity and potential ecological impact of waste slag.

[0035] Based on the above ideas, the present invention innovatively introduces the analytical framework of the damaged soil microbial community structure and its dynamic changes into the practice of ecological environmental damage identification and assessment, and uses high-throughput sequencing technology, OTU cluster analysis and other means to analyze the characteristics of bacterial communities, providing a scientific and quick method for evaluating the impact of waste slag on microbial communities.

[0036] The method for confirming soil ecological environmental damage in waste slag areas proposed in the present invention is comprehensive and reasonable. Specifically, the soil environment of the area where waste slag is landfilled is evaluated at multiple levels from the perspectives of the difference in characteristic pollutant indicators relative to the baseline, the increase or decrease in the relative abundance of dominant bacterial phyla in the evaluation area relative to the control area, etc., and the correlation between pollution indicators and bacterial community changes is analyzed to ensure that the evaluation model is scientific and reasonable. At the same time, the difference in characteristic pollutant indicators relative to the baseline and the increase or decrease in the relative abundance of dominant bacterial phyla in the evaluation area relative to the control area introduced in the present invention can be used to quantify the impact of waste slag on the soil environment, which can effectively assist in confirming the damage facts in ecological environmental damage identification and evaluation cases involving illegal dumping of waste slag.

[0037] The present invention also discloses the application of the soil ecological environment damage confirmation method to conduct preliminary source tracing in a soil area suspected of being dumped with waste slag. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A specific geographical location map of the present invention;

[0039] Figure 2 A relative abundance bar graph of the dominant groups at the phylum level in the evaluation area of ​​the present invention compared to the control area;

[0040] Figure 3 It is the PCA analysis diagram of the evaluation area and the control area of ​​the present invention;

[0041] Figure 4 It is a heat map of the correlation between the dominant groups and characteristic pollutant indicators at the phylum level in the assessment area of ​​the present invention. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention will be described clearly and completely below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] This example is an illegal dumping of waste slag about 20 years ago in the Pearl River Delta region of Guangdong Province in southern China. The area is flat, the soil types are ferroalloy and anthropogenic soil, the climate type is subtropical monsoon climate, the annual average temperature is 22.6℃, the annual average precipitation is 1920.4mm, and the specific geographical location is as follows: Figure 1 The evaluation area involved in this embodiment is approximately 42,000 m 2 From 2002 to 2015, there was illegal dumping of waste slag in the area, with an area of ​​about 20,000 square meters and a weight of about 62,000 tons.

[0044] Relevant personnel from the environmental department arrived at the scene to carry out relevant work.

[0045] A. Soil Sampling

[0046] A-1) The soil sampling operation in the assessment area is as follows: randomly select 3 sample plots with an area of ​​1m×1m in the assessment area, drill 5cm of soil below the waste slag in the center of the sample plot, take 3 soil samples in this section and mix them in equal proportions, which is a soil sample of the assessment area. Three soil samples are taken in the assessment area and are numbered SL1~SL3.

[0047] A-2) The surrounding area that is obviously uncontaminated serves as a control area for soil sampling;

[0048] Five soil control points at the same depth as the assessment area were set up in areas around the area to be assessed that were obviously not contaminated by waste slag. One soil sample was collected from each point, and the soil samples were numbered NC1 to NC5.

[0049] B. Conduct physical and chemical property analysis and heavy metal content determination on soil samples from the evaluation area and control area.

[0050] The results showed that none of them exceeded China's soil environmental quality standards.

[0051] C. Using the soil ecological environment damage confirmation method applicable to the illegal dumping of waste slag of the present invention to evaluate the area, comprising the following steps:

[0052] 1) Screen soil samples from the assessment area and the control area for characteristic pollution indicators and determine the characteristic pollution indicators.

[0053] Heavy metals were determined by inductively coupled plasma mass spectrometry (7900), and semivolatile organic compounds and volatile organic compounds were determined by gas chromatography-mass spectrometry (8860-5977B).

[0054] According to the screening results, the characteristic pollution indicators were determined to be Cu, Ni and Zn.

[0055] 2) The content of characteristic pollution indicators was measured in soil samples from the evaluation area and the control area.

[0056] 2.1) The contents of heavy metals Cu, Ni and Zn in soil samples SL1-SL3 and NC1-NC5 were determined by flame atomic absorption spectrometry (240FS AA).

[0057] 2.2) Statistically calculate the content data of heavy metals Cu, Ni and Zn measured in the control area to check whether they belong to normal distribution;

[0058] If it belongs to normal distribution, the 90% reference upper limit (arithmetic mean + 1.65 times standard deviation) of the control point data is used as the soil baseline. If it does not belong to normal distribution, the 90th percentile of the control point data is used as the soil baseline.

[0059] The results are shown in the following table.

[0060]

[0061] It can be seen from the above table that the Cu, Ni, and Zn detection data of soil samples NC1 to NC5 in the control area and the Cu, Ni, and Zn detection data of soil samples SL1 to SL3 in the evaluation area all obey the normal distribution.

[0062] 2.3) Compare with the soil baseline level to determine whether the content of characteristic pollution indicators in the assessment area exceeds the soil baseline level.

[0063] The heavy metals Cu, Ni and Zn in the soil samples SL1 to SL3 in the assessment area all exceeded the baseline values ​​to varying degrees. In other words, by comparing with the baseline values, it was proved that the waste slag caused the formation of mild heavy metal composite pollution of Cu, Ni and Zn in the soil in the assessment area.

[0064] 3) High-throughput sequencing

[0065] 3.1) Perform 16S rRNA high-throughput sequencing on soil samples from the evaluation area and the control area.

[0066] All soil samples collected this time were stored in a refrigerator at ≤80℃ and sent to the laboratory for relevant sequencing work within 24 hours after sampling.

[0067] 3.1a) 350 mg of soil samples were collected from SL1-SL3 and NC1-NC5, respectively, and DNA was extracted using TS96 Magnetic Soil DNA Kit. The DNA concentration was measured using Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer.

[0068] 3.1b) The V3-V4 region of the 16S rRNA gene was amplified from the genomic DNA of each soil sample using universal primers 338F (50-ANCCNCACGGGAGGCAGCA-30) and 806R (50-GGANCACHVGGGGTWTNCAAT-30).

[0069] Both forward and reverse 16S primers carry sample-specific Illumina index sequences to facilitate deep sequencing.

[0070] The total reaction volume of the polymerase chain reaction is 10 μl: DNA template 5-50 ng, *Vn F (10 μM) 0.3 μl, *Vn R (10 μM) 0.3 μl, KOD FX Neo Buffer 5 μL, dNTP (2 mM each) 2 μl, KOD FX Neo 0.2 μl, ddH2O to 10 μl, VN F and VN R are selected according to the amplification region.

[0071] Subsequently, an initial denaturation at 95 °C for 5 min was performed, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 40 s, and finally denaturation at 72 °C for 7 min.

[0072] PCR amplification products were purified using Agencourt AMPure XP Beads and quantified using the Qubit dsDNA HS AssayKit and the Qubit 4.0 Fluorometer; after the individual quantification steps, amplification products were pooled in equal amounts;

[0073] 3.1c) Performing library detection by Qsep-400 method, if the detection is qualified, proceed to the next step;

[0074] 3.1d) High-throughput sequencing was performed using Illumina NovaSeq 6000 to obtain raw sequencing data.

[0075] 3.1e) The data obtained by high-throughput sequencing were processed. The raw sequencing data were quality filtered using Trimmomatic 0.33, and the primer sequences were identified and removed using Cutadapt 1.9.1. The double-end reads were then spliced ​​using USEARCH 10.0, and chimeras were removed using UCHIME (version 8.1) to obtain high-quality sample sequence data.

[0076] 3.1f) OTU clustering analysis was performed on the sequences using USEARCH 10.0 at the 97% similarity level, and the quality-controlled data were denoised using the DADA2 method in QIIME2.

[0077] 3.2) Verify the comprehensiveness of the test data volume;

[0078] When the number of tested sequences reached 50,000, the sequencing coverage reached 99.97% and above, indicating that the sequencing depth was sufficient to cover most microorganisms.

[0079] 3.3) PCA analysis was performed to confirm whether there were significant differences in the microbial community structure and composition between the evaluation area and the control area.

[0080] The separation between the evaluation area and the control area is obvious, with high reliability. PC1 and PC2 can explain 72.06% and 16.21% of the total variation, respectively. PC1 and PC2 obtained by PCA analysis are selected for plotting. The results are shown in Figure 2 As shown, the distribution within the control area group is relatively concentrated, indicating that the difference in soil microbial community structure within the control area is small, and the distribution within the slag area group is discrete to a certain extent, indicating that there are obvious differences in the microbial community structure composition of soil habitat types in the evaluation area and the control area.

[0081] 3.4) Perform taxonomic analysis on the sequence results obtained by high-throughput sequencing at the phylum level, and then determine the top 10 dominant bacterial phyla and their relative abundance in the evaluation area, and determine the relative abundance and increase / decrease range of the dominant bacterial phyla corresponding to the control area.

[0082] The results are shown in the following table.

[0083]

[0084] 3.5) Make a distribution bar graph of the top 10 dominant bacterial phyla in relative abundance in the evaluation area and compare it with the control area to intuitively display the changes in the relative abundance of dominant groups at the phylum level.

[0085] Figure 3 The figure is a bar graph showing the relative abundance of the top 10 dominant bacterial phyla in soil samples from the evaluation area and the control area.

[0086] 4) The Pearson correlation test was combined with the correlation heat map to test the correlation between the changes in the bacterial community in the assessment area and the characteristic pollution indicators.

[0087] 4.1) Pearson correlation test was performed between the top 10 dominant bacterial phyla in relative abundance and Cu, Ni, and Zn characteristic heavy metal indicators, and the correlation heat map (Heatmap) was used to further display the correlation between Cu, Ni, and Zn characteristic heavy metal indicators and dominant groups. "*" indicates the significance level (where "*" is P < 0.05, "**" is P < 0.01, and "***" is P < 0.001). The analysis results are as follows: Figure 4 As shown, Cu, Ni and Zn have significant effects on 5 of the top 10 dominant bacterial phyla in relative abundance.

[0088] 4.2) There is a certain correlation between the changes in the dominant groups at the phylum level in the assessment area relative to the control area and the characteristic heavy metal indicators, indicating that some changes in the soil environment in the assessment area are caused by waste slag. The difference in characteristic pollutant indicators in the assessment area relative to the baseline, the increase or decrease in relative abundance of the dominant bacterial phylum in the assessment area relative to the control area, etc. can be used as quantitative indicators to evaluate the impact of waste slag on the soil environment, which can assist in the confirmation of damage facts in damage identification and assessment cases involving illegal dumping of waste slag.

[0089] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also change and modify the above implementation modes. Therefore, the present invention is not limited to the specific implementation modes disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the present invention.

Claims

1. A method for confirming soil ecological environment damage in areas where waste slag is illegally dumped, characterized in that: At least the following steps are included: 1) Screening characteristic pollution indicators and determining characteristic pollution indicators; 2) Determine the content of characteristic pollution indicators in soil samples from the control area and the assessment area where waste slag is illegally dumped, and analyze and determine whether the content of characteristic pollution indicators in the assessment area exceeds the soil baseline level in the control area; 3) Perform high-throughput sequencing on soil samples from the evaluation area and the control area, perform OTU cluster analysis based on the sequence information, and at least use PCA analysis to determine whether the bacterial community in the evaluation area has changed significantly relative to the control area; 4) The Pearson correlation test combined with the correlation heat map was used to conduct a correlation test on the changes in the bacterial community in the assessment area and the characteristic pollution indicators, to indirectly confirm whether the waste slag has caused damage to the soil ecological environment, and to quantify the impact of the waste slag on the soil ecological environment through changes in various indexes of the bacterial community.

2. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 1 is characterized in that: In step 3), OTU clustering analysis was performed on the sequences obtained by high-throughput sequencing at a similarity level of 97%.

3. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 1 or 2, characterized in that: In step 3), at least the following steps are included: 3.1) High-throughput sequencing of soil samples from the evaluation area and the control area; 3.2) Perform PCA analysis at least at the phylum level to confirm whether there are differences in the microbial community structure and composition between the evaluation area and the control area; 3.3) Perform taxonomic analysis on the sequence results obtained by high-throughput sequencing at least at the phylum level, and then determine the top 10 dominant bacterial phyla and their relative abundance in the evaluation area, and determine the relative abundance and increase / decrease range of the dominant bacterial phyla corresponding to the control area.

4. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 3 is characterized in that: Step 4) at least includes the following steps: 4.1) The Pearson correlation test was combined with the correlation heat map to test the correlation between the top 10 dominant bacterial phyla in relative abundance determined in step 3) and the characteristic pollution index, to indirectly confirm whether the waste slag has damaged the soil ecological environment; 4.2) Use the changes in various bacterial community indexes to quantitatively evaluate the impact of waste slag on the soil ecological environment.

5. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 1 is characterized in that: The screening of characteristic pollution indicators in step 1) adopts the following determination method: Heavy metals were determined by inductively coupled plasma mass spectrometry, and semivolatile organic compounds and volatile organic compounds were determined by gas chromatography-mass spectrometry.

6. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 5 is characterized in that: Step 2) includes the following steps: 2.1) The soil samples from the assessment area and the control area were respectively tested for the content of characteristic pollution indicators; 2.2) Determine the soil baseline level: The specific operation is to perform statistical calculations on the characteristic pollution index content data measured in the control area to check whether it belongs to a normal distribution; If it is a normal distribution, the 90% reference upper limit of the control point data is used as the soil baseline, and the arithmetic mean + 1.65 times the standard deviation; if it is not a normal distribution, the 90th percentile of the control point data is used as the soil baseline; 2.3) Compare with the soil baseline level to determine whether the content of characteristic pollution indicators in the assessment area exceeds the soil baseline level.

7. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 1 is characterized in that: In step 3), 0.25-0.5 g of soil sample was taken for high-throughput sequencing and stored in a refrigerator at ≤80°C. High-throughput sequencing was performed within 24 h. The sample nucleic acid was extracted using the magnetic bead method, and then PCR amplification and sequencing were performed to obtain the original sequencing data.

8. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 7 is characterized in that: The PCR amplification program was as follows: initial denaturation at 95°C for 4-6 min, followed by denaturation at 95°C for 25-35 s and 25 cycles, annealing at 45-55°C for 20-35 s, and extension at 72°C for 35-45 s.

9. The method for confirming soil ecological environment damage applicable to the area where waste slag is illegally dumped according to claim 7 or 8, characterized in that: DNA was extracted from soil samples using the TS96 Magnetic Soil DNA Kit.

10. Application of the soil ecological environment damage confirmation method described in any one of claims 1 to 9 to conduct preliminary tracing of soil areas suspected of being dumped with waste slag.

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