Marine benthic ecological environment quality assessment method based on environment DNA

Abstract

The invention belongs to the field of ecological environment evaluation and treatment, and particularly relates to a marine benthic ecological environment quality evaluation method based on an environmental DNA technology. Comprising the following steps: 1) collecting an environmental DNA sediment sample; 2) extracting sediment environment DNA from the environment DNA sediment sample; (3) amplifying macro bar code fragments by taking the environmental DNA extracted in the step (2) as a template, and sequencing; (4) analyzing the species category of the sequencing result of the macro bar code in the step (3), and defining the macrobenthonic animals; and 5) evaluating the quality of the marine benthic ecological environment by combining the environment DNA macro bar code with AMBI. The environment DNA macro bar code technology is applied to marine benthonic habitat quality evaluation, and environment DNA PCR amplification, screening of large benthonic animals in sequence species, AMBI index data calculation and habitat quality evaluation grade technologies are defined; wherein screening of large benthonic animals in an environment DNA macro bar code sequence and introduction of sequence absolute abundance into AMBI calculation are key technologies for influencing a final marine benthonic habitat quality evaluation result.

Description

Technical Field

[0001] The present invention belongs to the field of ecological environment evaluation and governance, and particularly relates to a method for evaluating the quality of marine benthic ecological environment based on environmental DNA technology. Background Art

[0002] Species diversity monitoring is the basis for scientifically carrying out marine ecological environment protection and management. Benthic organisms occupy the core position of the marine ecosystem, and the distribution of their species and quantities has a certain indicative effect on the health status of the marine ecosystem. Therefore, it is often used for ecosystem monitoring and quality evaluation. At present, marine benthic organism monitoring mainly involves collecting biological samples and identifying them based on morphological methods. However, offshore operations are greatly affected by environmental factors such as weather and waves. The sample sorting and identification are difficult, time-consuming, and costly in terms of manpower and time. The loss of key characteristic parts and sample fragmentation are very common during the sample screening process. Moreover, there are many categories of benthic organisms, and the morphological characteristics of some closely related species are very similar and difficult to distinguish with the naked eye, which requires high professional qualities and experience judgment abilities of the identification personnel. All these problems make it difficult to implement large-scale, multi-station, and high-frequency benthic organism monitoring in China's marine ecological environment monitoring work based on morphological identification.

[0003] Environmental DNA metabarcoding technology is a new method for detecting biodiversity internationally. Its technology originated from DNA barcoding technology and has great application potential in biological monitoring. The DNA substances containing species diversity information released into the surrounding environment by organisms during their activities in the environment are called environmental DNA. Environmental DNA metabarcoding technology uses different primers to perform polymerase chain reaction (PCR) amplification on environmental DNA samples, and combines second- and third-generation high-throughput sequencing technologies to generate millions of sequences reflecting species diversity. By analyzing the sequences, the diversity information such as species composition and community structure in the environment can be identified. Compared with traditional monitoring methods, environmental DNA metabarcoding technology can identify the subtle changes in biological communities in aquatic ecosystems more quickly, accurately, and economically.

[0004] As an intuitive and simple environmental quality assessment factor, the biological index can reflect problems such as toxic accumulation, pollution degree, eutrophication, habitat loss or overexploitation in the sediment environment, and can effectively avoid the influence of species with strong motility. The biological index based on benthic organisms has become an effective tool for the health assessment of aquatic ecosystems and relevant administrative decisions. In particular, the Marine Biotic Index (AMBI) has been very successful in different geographical regions and different environments around the world, from the intertidal zone to the deep sea, or from intertidal fresh water to nearshore habitats. Due to the difficulties of morphological identification, as well as the constraints of time and economic costs, AMBI has some limitations in monitoring and assessment, which can be alleviated by biological DNA barcoding and metabarcoding. Summary of the Invention

[0005] The technical problem to be solved by the present invention is as follows: Currently, the identification of marine benthic organisms is based on morphological methods, which are greatly affected by environmental factors such as weather and waves during offshore operations. The sorting and identification of samples are difficult, time-consuming, and costly in terms of manpower and time. The loss of key characteristic parts and sample fragmentation are very common during the sieving process. Moreover, there are many benthic organism categories, and the morphological characteristics of some closely related species are very similar and difficult to distinguish with the naked eye, requiring high professional qualities and experience judgment abilities of the identification personnel. These problems make it difficult to implement large-scale, multi-station, and high-frequency benthic organism monitoring in the marine ecological environment monitoring work in China.

[0006] To solve the above technical problems, the present invention assesses the quality of the marine benthic ecological environment through the monitoring of benthic animal diversity based on environmental DNA, using benthic organism environmental DNA metabarcoding combined with AMBI. The method includes the following steps:

[0007] 1) Collection of environmental DNA sediment samples;

[0008] 2) Extraction of sediment environmental DNA from the environmental DNA sediment samples;

[0009] 3) Using the environmental DNA extracted in step (2) as a template, amplifying the metabarcoding fragment and sequencing,

[0010] The primer sequences for amplifying the environmental DNA template are 5'-GCGGTAATTCCAGCTCCAA-3' and 5'-AATCCRAGAATTTCACCTCT-3';

[0011] 4) Analyzing the species category to which the metabarcoding sequencing result in step (3) belongs and defining macrobenthos;

[0012] 5) Using benthic organism environmental DNA metabarcoding combined with AMBI to assess the quality of the marine benthic ecological environment.

[0013] Macrozoobenthos species are classified into different biological types (GⅠ - GⅤ) according to the AMBI species list. For species not included in the species list, species of the same genus in the list are used instead. The absolute abundance of environmental DNA metabarcoding and the abundance of morphological species are imported into the AMBI 5.0 software for AMBI value calculation. AMBI = [(0×%GⅠ) + (1.5×%GⅡ) + (3×%GⅢ) + (4.5×%GⅣ) + (6×%GⅤ)] / 100. Among them, an AMBI value ≤ 1.2 indicates no disturbance, 1.2 - 3.3 indicates mild disturbance, 3.3 - 5 indicates moderate disturbance, 5 - 6 indicates severe disturbance, and < 6 indicates extreme disturbance.

[0014] Specifically, step (1) of the present invention is as follows: Use a sterilized plastic spoon to collect an environmental DNA sediment sample from the surface layer (0 - 3 cm) of the sea area, remove impurities, and store it at ultra - low temperature until environmental DNA extraction.

[0015] In step (2) of the present invention, CTAB method is used to extract sediment DNA.

[0016] Specifically, step (3) of the present invention is as follows: After using the CTAB method to extract sediment DNA, agarose gel electrophoresis is used to detect the purity and concentration of DNA. The purified environmental DNA sample uses the 18S V4 region primer with a Barcode - specific primer to perform PCR amplification on the environmental DNA template. The primer sequences are 528F: 5'-GCGGTAATTCCAGCTCCAA - 3', 706R: 5'-AATCCRAGAATTTCACCTCT - 3'. The obtained target bands are used for library construction and sequencing.

[0017] Specifically, step (4) of the present invention is as follows: The raw data of each sample is obtained by splitting according to the barcode. After removing the barcode and primer, the sequence data is spliced by the FLASH software. Subsequently, the fastp software is used to perform quality control on the spliced RawTags to obtain Clean Tags. Then, chimera filtering is performed to obtain the effective data (Effective Tags) that can be used for subsequent analysis. The DADA2 module in the QIIME2 software is used to perform sequence quality control and noise reduction processing on the effective data, and sequences with an abundance less than 5 are filtered out to obtain the final ASVs. The Naive Bayes classifier pre - trained in the classify - sklearn algorithm of QIIME2 is used to compare the nucleic acid sequence database in NCBI, and species annotation of the sequencing results is carried out based on the best value of BLAST in the NT database. The biological functional group and body size of the ASVs sequence species are queried through the WoRMS official website. Organisms with a benthic functional group and a body size greater than 0.5 mm are defined as macrozoobenthos.

[0018] In step (3), the reaction system includes 15 μL of Phusion Master Mix, 0.2 μL of PrimerF (1 μM), 0.2 μL of PrimerR (1 μM), 10 μL of gDNA (about 10 ng), and ddH2O is added to make up a 30 μL system. The PCR reaction program is pre-denaturation at 98°C for 1 min, denaturation at 98°C for 10 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, for 30 cycles, and finally incubation at 72°C for 5 min.

[0019] The present invention also includes the synchronous calculation of the Shannon-Wiener diversity index (H'): H' = –ΣP i log2(P i ), where S is the total number of species in the sample, N is the total number of individuals in the sample, and P i is the ratio of the number of individuals of the i-th species in the sample to the total number of individuals. Among them, when the H' value is less than 1, it is heavily polluted; when the H' value is between 1 and 2, it is moderately polluted; when the H' value is between 2 and 3, it is lightly polluted; when the H' value is greater than 3, it is clean.

[0020] The beneficial effects of the present invention are as follows: The environmental DNA metabarcoding technology is applied to the evaluation of the quality of marine benthic habitats, and the technologies of environmental DNA PCR amplification, screening of macrobenthos in sequence species, calculation of AMBI index data, and habitat quality assessment level are clarified. Among them, the screening of macrobenthos in environmental DNA metabarcoding sequences and the introduction of sequence absolute abundance into AMBI calculation are the key technologies affecting the final evaluation results of marine benthic habitat quality. Description of the Drawings

[0021] Figure 1 Sampling station map of the waters of the Miaodao Archipelago;

[0022] Figure 2 Process of querying Hydroides panamensis on the Worms official website and main retrieved entries diagram. Detailed Embodiments

[0023] Example 1 Sample Collection

[0024] An ecological survey was carried out on the waters of the Miaodao Archipelago. A total of stations were set up to collect water quality, sediment, environmental DNA sediment samples, and morphological classification macrobenthos samples ( Figure 1 ). When collecting morphological classification samples, 0.05 m was used at each station 2The box corer was sampled 4 times repeatedly and combined into 1 sample. After screening and sorting through a 0.5-mm aperture sieve, it was fixed and preserved with 5% formaldehyde solution. On-site, a sterilized plastic spoon was used to collect the environmental DNA sediment sample from the surface layer of 0 - 3 cm, and impurities (shells, gravel) were removed. Approximately 20 g of the sample was placed in a 50-mL sterile and enzyme-free cryogenic tube. Three parallel samples were taken at each station and stored in a -20°C in-vehicle refrigerator and transported to the laboratory. In the laboratory, the sediment samples were homogenized and transferred to an -80°C ultra-low temperature refrigerator for storage until environmental DNA extraction. Meanwhile, bottom seawater was collected and bottled, and its determination was carried out in the laboratory within 24 hours. A certain amount of surface sediment was collected and stored frozen in a -20°C freezer.

[0025] Example 2 Indoor Analysis

[0026] Environmental factor determination: sediment elements including grain size, petroleum, organic carbon, sulfide; water quality elements including water temperature, water depth, salinity, pH, nutrients, dissolved oxygen, chemical oxygen demand, chlorophyll a, heavy metals. For morphological classification, samples were classified, identified, and counted for macrozoobenthos under a stereomicroscope, and weighed using an electronic balance with a sensitivity of 0.001 g. The standard species names were from the WoRMS official website. The collection, processing, preservation, and detection of all samples were carried out in accordance with the "Marine Monitoring Specifications".

[0027] Example 3 Environmental DNA Extraction, PCR Amplification, and Sequencing

[0028] The DNA of sediment was extracted by the CTAB method. After that, the purity and concentration of DNA were detected by agarose gel electrophoresis. The purified environmental DNA sample was used as a template for PCR amplification of the 18S V4 region with specific primers with Barcode. The primer sequences were 528F: 5'-GCGGTAATTCCAGCTCCAA-3' and 706R: 5'-AATCCRAGAATTTCACCTCT-3'. The reaction system was 30 μL, including 15 μL of Phusion Master Mix, 0.2 μL of PrimerF (1 μM), 0.2 μL of PrimerR (1 μM), 10 μL of gDNA (about 10 ng), and ddH2O was added to make up the 30 μL system. The PCR reaction procedure was pre-denaturation at 98 °C for 1 min, denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, for 30 cycles, and finally incubation at 72 °C for 5 min. ddH2O was used as a template for each PCR reaction as a PCR negative control. Each sample was amplified 3 times. After mixing the PCR products of the same sample, 2% agar gel electrophoresis was used for detection. The qualified PCR products were purified by magnetic beads, quantified by enzyme-linked immunosorbent assay, and mixed in equal amounts according to the concentration of PCR products. After thorough mixing, 2% agarose gel electrophoresis was used to detect the PCR products, and the target bands were recovered using the gel recovery kit provided by Qiagen. The library was constructed using the Illumina Truseq DNA PCR-Free Library Preparation Kit. The constructed library was quantified by Qubit and detected. The PCR products of each sample were individually library-constructed. After the library was qualified, the second-generation high-throughput sequencing was performed using the Illumina NovaSeq 6000 sequencing platform.

[0029] Example 4 Data Analysis

[0030] The raw data of each sample was obtained by splitting according to the barcode. After removing the barcode and primer, the sequence data was spliced ​​using FLASH software. Then, the spliced ​​Raw Tags were quality controlled using fastp software to obtain Clean Tags, and then chimeras were filtered to obtain effective data (Effective Tags) that can be used for subsequent analysis. The DADA2 module in the QIIME2 software was used to perform sequence quality control and noise reduction on the effective data, and sequences with an abundance of less than 5 were filtered out to obtain the final ASVs. The pre-trained NaiveBayes classifier in the classify-sklearn algorithm of QIIME2 was used to align the nucleic acid sequence database in NCBI, and the sequencing results were annotated for species based on the NT database BLAST best value. The ASVs sequence species were queried for biological functional groups and body sizes through the WoRMS official website. Those with benthic functional groups and body sizes greater than 0.5 mm were defined as large benthic animals. Figure 2 As shown, Figure 2 A diagram showing the search process and main search terms for Hydroides panamensis on the Worms website.

[0031] Environmental DNA analysis results

[0032] High-throughput sequencing was performed on 48 environmental DNA sediment samples from 16 stations in the Miaodao Archipelago. The number of valid sequences for each sample ranged from 54,425 to 86,565, with an average length of 308 bp and a valid sequence percentage of more than 90%. After the data were denoised by the DADA2 method (equivalent to 100% similarity clustering), 10,102 ASVs were obtained. After removing bacteria, fungi, plankton, terrestrial organisms and small benthic animal-related groups, 223 large benthic animal ASVs were finally obtained and included in the subsequent analysis, belonging to 12 phyla, 19 classes, 43 orders, 88 families and 110 genera of biological groups, as shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] Morphological classification results

[0041] A total of 73 species of macrozoobenthos were identified, including 68 genera in 7 major categories: Cnidaria, Nemertea, Annelida, Mollusca, Arthropoda, Brachiopoda, and Echinodermata, as shown in Table 2.

[0042] Table 2

[0043]

[0044]

[0045] The macrozoobenthos species obtained from morphological classification and environmental DNA were distinguished into different biological types (GⅠ - GⅤ) according to the AMBI species list. For species not included in the species list, species in the same genus in the list were used instead. The absolute abundances of environmental DNA metabarcodes (Table 3) and morphological species abundances (Table 4) were imported into AMBI 5.0 software for AMBI value calculation. AMBI = [(0×%GⅠ)+(1.5×%GⅡ)+(3×%GⅢ)+(4.5×%GⅣ)+(6×%GⅤ)] / 100. Among them, an AMBI value ≤ 1.2 indicates no disturbance, 1.2 - 3.3 indicates mild disturbance, 3.3 - 5 indicates moderate disturbance, 5 - 6 indicates severe disturbance, and < 6 indicates extreme disturbance.

[0046] Table 3 AMBI - Environmental DNA Abundance Analysis Bottom Table

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Table 4 Bottom table of AMBI morphological abundance analysis

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] The present invention synchronously calculates the Shannon-Wiener diversity index (H'): H' = -ΣP i log2(P i ), where S is the total number of species in the sample, N is the total number of individuals in the sample, and P i is the ratio of the number of individuals of the i-th species in the sample to the total number of individuals. Among them, when the H' value is less than 1, it is heavily polluted; when the H' value is between 1 and 2, it is moderately polluted; when the H' value is between 2 and 3, it is lightly polluted; when the H' value is greater than 3, it is clean. By comparing the numerical differences and evaluation conclusions of the two methods AMBI and H', and combining the results of environmental factors such as seawater quality and marine sediment quality, it is determined that the environmental DNA technology is feasible and effective in the assessment of the quality of marine benthic habitats.

[0068] According to the Seawater Quality Standard and Marine Sediment Quality, the quality of marine sediments in the surveyed sea area meets the first-class standard. Only the heavy metal zinc at one station exceeds the second-class water quality standard, and other elements meet the seawater quality requirements of the marine functional zoning in the area. The marine environmental status of the sea area is good, as shown in Table 5.

[0069] Table 5

[0070]

[0072] The AMBI values obtained from environmental DNA ranged from 0.125 to 2.566, with a mean of 1.429; the Shannon-Weaver diversity index (H') ranged from 1.41 to 4.42, with a mean of 2.64. The AMBI values obtained from morphological classification ranged from 0.714 to 3.000, with a mean of 1.551; H' ranged from 1.19 to 4.41, with a mean of 3.49. The results of AMBI from environmental DNA and morphological classification were basically consistent when evaluating the overall habitat quality of the surveyed sea area, but there were differences in the evaluation among stations. The overall pollution degree of the surveyed sea area obtained from the Shannon-Weaver diversity index (H') showed differences, and the morphological results were better than those of environmental DNA, with a highly significant difference (p < 0.01). Combining the evaluation results of seawater quality and sediment quality in the sea area, the AMBI index was more suitable for the assessment of marine benthic habitat quality based on environmental DNA metabarcoding technology. The obtained results were in good agreement with the morphological related evaluation results and the habitat chemical parameters of the sea area, with high feasibility and effectiveness. The results are shown in Table 6.

[0073] Table 6

[0074] Station AMBI Habitat quality assessment AMBI (eDNA) Habitat quality assessment H' Pollution level H'(eDNA) Pollution level 1 0.714 Undisturbed 1.385 Slightly disturbed 2.51 Lightly polluted 2.90 Lightly polluted 2 0.804 Undisturbed 1.661 Slightly disturbed 3.30 Clean 1.41 Moderately polluted 3 1.500 Slightly disturbed 1.353 Slightly disturbed 3.38 Clean 3.59 Clean 4 1.714 Slightly disturbed 2.442 Slightly disturbed 2.36 Lightly polluted 1.52 Moderately polluted 5 1.759 Slightly disturbed 0.886 Undisturbed 4.18 Clean 1.98 Moderately polluted 6 2.375 Slightly disturbed 2.524 Slightly disturbed 3.02 Clean 2.47 Lightly polluted 7 2.100 Slightly disturbed 0.125 Undisturbed 1.91 Moderately polluted 1.70 Moderately polluted 8 1.406 Slightly disturbed 0.986 Undisturbed 3.62 Clean 3.75 Clean 9 1.306 Slightly disturbed 1.827 Slightly disturbed 4.41 Clean 2.60 Lightly polluted 10 0.729 Undisturbed 1.932 Slightly disturbed 3.88 Clean 3.05 Clean 11 1.645 Slightly disturbed 0.440 Undisturbed 4.22 Clean 4.27 Clean 12 3.000 Slightly disturbed 1.680 Slightly disturbed - - 2.45 Lightly polluted 13 1.087 Undisturbed 1.267 Slightly disturbed 3.77 Clean 4.42 Clean 14 1.531 Slightly disturbed 2.566 Slightly disturbed 4.25 Clean 2.44 Lightly polluted 15 1.750 Slightly disturbed 0.266 Undisturbed 3.96 Clean 2.12 Lightly polluted 16 1.397 Slightly disturbed 1.525 Slightly disturbed 3.65 Clean 1.63 Moderately polluted

Claims

1. A method for evaluating the quality of marine benthic ecological environment based on environmental DNA, comprising the following steps: 1) Collection of environmental DNA sediment samples; 2) Extraction of sediment environmental DNA from environmental DNA sediment samples; 3) Using the environmental DNA extracted in step (2) as a template, amplifying the metabarcoding fragment and sequencing; The primer sequences for amplifying the environmental DNA template are 5'-GCGGTAATTCCAGCTCCAA-3' and 5'-AATCCRAGAATTTCACCTCT-3'; 4) Analyzing the species categories of the sequencing results of the metabarcoding in step (3) to define macrobenthos; 5) Evaluating the quality of the marine benthic ecological environment by combining environmental DNA metabarcoding with AMBI; The macrobenthos species are distinguished into different biological types (GⅠ-GⅤ) according to the AMBI species list. For species not included in the species list, the species of the same genus in the list are used instead. The absolute abundance of environmental DNA metabarcoding and the morphological species abundance are imported into the AMBI 5.0 software for calculating the AMBI value. AMBI = [(0×%GⅠ)+(1.5×%GⅡ)+(3×%GⅢ)+(4.5×%GⅣ)+(6×%GⅤ)] / 100, where the AMBI value ≤ 1.2 is undisturbed, 1.2 - 3.3 is slightly disturbed, 3.3 - 5 is moderately disturbed, 5 - 6 is severely disturbed, and < 6 is extremely disturbed.

2. The method for evaluating the quality of marine benthic ecological environment based on environmental DNA according to claim 1, characterized in that: The specific operation of step (1) is as follows: Use a sterilized plastic spoon to collect environmental DNA sediment samples from the surface layer (0 - 3 cm) of the sea area, remove impurities, and store them at ultra-low temperature until environmental DNA extraction.

3. The method for evaluating the quality of marine benthic ecological environment based on environmental DNA according to claim 1, characterized in that: In step (2), the CTAB method is used to extract sediment DNA.

4. The marine benthic ecological environment quality assessment method based on environmental DNA according to claim 1, characterized in that: The specific operation of step (3) is as follows: After using the CTAB method to extract sediment DNA, use agarose gel electrophoresis to detect the purity and concentration of DNA. The purified environmental DNA sample uses the 18S V4 region primers with Barcode specific primers to perform PCR amplification on the environmental DNA template. The primer sequences are 528F: 5'-GCGGTAATTCCAGCTCCAA-3' and 706R: 5'-AATCCRAGAATTTCACCTCT-3'. Construct a library for the obtained target band and sequence it.

5. The method for evaluating the quality of marine benthic ecological environment based on environmental DNA according to claim 1, characterized in that: The specific steps of step (4) are as follows: the original data of each sample is obtained by splitting according to the barcode. After removing the barcode and primers, the sequence data is spliced by the FLASH software. Subsequently, the fastp software is used to perform quality control on the spliced Raw Tags to obtain Clean Tags. Then, chimera filtering is performed to obtain effective data (EffectiveTags) that can be used for subsequent analysis. The DADA2 module in the QIIME2 software is used to perform sequence quality control and noise reduction processing on the effective data, and sequences with an abundance less than 5 are filtered out, thereby obtaining the final ASVs. The Naive Bayes classifier pre-trained in the classify-sklearn algorithm of QIIME2 is used to compare the nucleic acid sequence database in NCBI, and species annotation of the sequencing results is carried out based on the best value of NT database BLAST. The biological functional groups and body sizes of the ASVs sequences are queried through the WoRMS official website. Those with a benthic functional group and a body size greater than 0.5 mm are defined as macrobenthos.

6. The method for evaluating the quality of marine benthic ecological environment based on environmental DNA according to claim 1, wherein: In step (3), the reaction system includes 15 μL of Phusion Master Mix, 0.2 μL of PrimerF (1 μM), 0.2 μL of PrimerR (1 μM), 10 μL of gDNA (about 10 ng), and ddH2O is added to make up a 30 μL system. The PCR reaction program is pre-denaturation at 98 °C for 1 min, denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and finally incubation at 72 °C for 5 min.

7. The method for evaluating the quality of marine benthic ecological environment based on environmental DNA according to claim 1, wherein: It also includes the synchronous calculation of the Shannon-Wiener diversity index (H'): H' = –ΣP i log2(P i ), where S is the total number of species in the sample, N is the total number of individuals in the sample, and P i is the ratio of the number of individuals of the i-th species in the sample to the total number of individuals. Among them, when the H' value is less than 1, it is heavily polluted; when the H' value is between 1 and 2, it is moderately polluted; when the H' value is between 2 and 3, it is lightly polluted; when the H' value is greater than 3, it is clean.

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