CN116482313A - Water ecology monitoring and comprehensive evaluation method based on environment DNA technology - Google Patents

Abstract

The invention discloses a water ecology monitoring and comprehensive evaluation method based on an environment DNA technology, which comprises the following steps of collecting and arranging basin basic data, and initially surveying to determine monitoring points; providing an evaluation index for special investigation, measuring by an environment DNA technology, and calculating by an index formula to obtain an accurate evaluation result; assigning points to the index item evaluation; and determining the final score and grade of each index weight calculation target river water ecology evaluation. Compared with the prior art, the invention comprehensively considers the water ecology evaluation requirement and the environmental DNA technical characteristics, builds a set of brand-new water ecology comprehensive evaluation method based on the environmental DNA technology, not only comprises biological indexes which are closely related with the surrounding environment and are sensitive to the reaction after interference, but also comprises physicochemical indexes which respond to the environmental change quickly, and monitors and comprehensively evaluates from multiple directions, so that the comprehensive water ecology evaluation by using the environmental DNA technology has operability, effectiveness and reliability.

Description

Water ecology monitoring and comprehensive evaluation method based on environment DNA technology

Technical Field

The invention relates to the technical field of ecological environment protection, in particular to a water ecological monitoring and comprehensive evaluating method based on an environment DNA technology.

Background

The water ecology is the most important component of the earth ecology, can be said to be a source spring of everything, and is one of important natural resources for human survival. Although the freshwater ecosystem covers less than 1% of the earth's surface area, more than 10% of the human-perceived organisms inhabit, and about 40% of the fish and more than 30% of the vertebrates on earth live in the freshwater ecosystem. However, with the development of human socioeconomic and industrialization, the freshwater ecosystem is more and more severely damaged, resulting in a decrease in the watershed biodiversity and a deterioration of the freshwater ecological function. Therefore, the enhancement of aquatic organism monitoring and management of an aquatic ecology system has important significance for maintaining biodiversity and ecological balance and promoting ecological civilization construction. In the ecological environment planning of each province, a water ecological monitoring and evaluating system is also explicitly proposed to be established, and water ecological protection is emphasized.

The common water ecology evaluation method mainly monitors three monitoring indexes of water environment, aquatic environment and aquatic organism, and then briefly analyzes the water ecology condition of the evaluation area according to the monitoring results. The water environment monitoring is to monitor the concentration and the change trend of various pollutants in the water body from the dimension of the water quality, so as to objectively evaluate the water environment quality condition. The aquatic environment is an essential key part of the watershed ecosystem, can provide material guarantee and environment basis for living and development of organisms, and has important practical significance for watershed health monitoring. Aquatic organism monitoring is to monitor environmental pollution from a biological point of view by observing changes in aquatic organism population, number, community composition and structure, biological habit, growth and propagation, and even genetic characteristics. The advantages are that: 1) Sensitivity, indicating that organisms are more sensitive to low concentrations of contaminants, some organisms may exhibit a corresponding effect on even trace amounts of contaminants; 2) Stability, the living environment of the indicator organism is relatively fixed, and compared with a physical and chemical monitoring means, continuous monitoring can be conveniently realized in the same area; 3) The variety of the organisms in the water environment is various, the comprehensive effects of antagonism, synergy and the like of the pollutants can be reflected, and the reactions of the same organisms to different pollutants are different; 4) The biological monitoring result can reflect the accumulated pollution condition for a long time, and the effect on chronic toxicity is more obvious. Therefore, accurately monitoring aquatic organisms is a precondition for effective assessment of the ecological environmental quality of the river basin water.

In the current water ecology monitoring process, each monitoring index is investigated by manually sampling and detecting on site. For example, plankton needs to collect water samples and return the water samples to a laboratory, and a special taxonomy technician performs microscopic examination, so that fishes are more required to use fishermen and leasing ships to obtain investigation samples in a net capturing mode, an electric capturing mode and the like. The monitoring method has limitations, firstly, human factors in the investigation process have great influence on results, the method is difficult to standardize, and data errors can be large; secondly, the biological group with lower density is difficult to obtain in the field sampling, and the biological group obtained by identifying according to the morphological method in the traditional monitoring has high technical requirements on personnel engaged in species identification; finally, the on-site capturing mode has a great influence on fishes and rare endangered species, and the capturing of partial natural water areas is forbidden. The monitoring method for on-site sampling and detection causes that the water ecology investigation needs to consume a large amount of manpower and material resources, has long investigation period, and can not meet the water ecology evaluation requirements of the current-stage normalized monitoring and quick response.

With the development of molecular biology technology, environmental DNA technology is gradually applied to water ecological monitoring. The environment DNA (eDNA) technology is to extract environment microorganism from water, living cell DNA fallen from organism and extracellular DNA fragments released by cell disruption after death of organism, conduct gene sequencing on sample, then compare species gene library to obtain analysis conclusion, can realize accurate analysis of species composition and abundance of each living state group of ecological system, subvert traditional species identification monitoring mode based on morphological characteristics, break through limitation of large amount of manpower and material resources required by traditional biological monitoring, greatly improve efficiency of biological monitoring, have small interference degree on environment, and provide new direction for aquatic ecology monitoring and evaluation. The ecological environment monitoring planning clearly provides a river basin development environment DNA monitoring test point for encouraging the development of small river basin water ecological investigation monitoring in various places, such as Taihu lake, liaohe river, sea river and the like. However, the current environmental DNA technology can only measure the number of environmental DNA sequences of each species, but the number of environmental DNA sequences has a certain positive correlation with the biomass of the species, but the relationship between the number of environmental DNA sequences and the number of individuals of the organism is not clear, so that the technology cannot accurately obtain the biomass and the number of individuals of each species of the aquatic organism, and the data are required for calculating most of the aquatic organism diversity evaluation indexes at present, which results in that part of the aquatic organism indexes in the current aquatic ecology evaluation method cannot be obtained through the environmental DNA technology, so that the aquatic ecology comprehensive evaluation method suitable for the environmental DNA technology is missing, and the application and development of the environmental DNA technology in the field of aquatic ecology evaluation are limited.

Chinese patent application 202210074149.9 discloses a comprehensive evaluation method for environmental quality of water ecosystem. The method mainly comprises the following steps: the comprehensive scoring comprises the following steps of: the nutrient salt index, habitat quality, phytoplankton index, microorganism index, fish index, and then calculated by the following formula to obtain a comprehensive score: composite score = (nutrient salt index + habitat mass + phytoplankton index + microorganism index + fish index)/5; and evaluating the comprehensive score corresponding to the evaluation standard, thereby obtaining the environmental quality condition of the water ecological system. The method has the beneficial effects that five standardized indexes which can be used for the method are screened, and a comprehensive and comprehensive water ecological environment quality evaluation method is provided for the first time, so that the result obtained by the method is more similar to the real situation of the water ecological environment. The defects are that: 1) The patent is mainly an innovation aiming at the traditional morphological evaluation method, and does not relate to the evaluation of an environmental DNA monitoring method; 2) The traditional manual sampling detection method is adopted to obtain monitoring data, so that the problems of complex operation, low efficiency, long investigation period, higher requirement on the professional degree of investigation personnel and the like exist, and the requirements of rapid and intelligent water ecological monitoring at the present stage cannot be met; 3) In the monitoring process, sample data is derived from biological individuals captured by traditional investigation means, and biological species are morphologically identified through the knowledge and experience of taxonomy staff, so that the accuracy caused by human errors and systematic errors is low.

Chinese patent application 202110797533.7 discloses a method for evaluating the health of a freshwater ecological system, which comprises the steps of firstly calculating the number of classification unit species, shannon-wiener diversity index, relative abundance of pollution sensitive species, relative abundance of anaerobic species and modular coefficient of a species network based on a high-throughput sequencing technology, calculating plankton integrity index through standardized parameters, and evaluating based on calculation results. The defects are that: although the patent adopts the environmental DNA technology to carry out biological integrity monitoring, the invention only carries out integrity evaluation on one plankton group, and can not objectively and truly reflect the aquatic organism condition when a plurality of biological groups of different grades survive; and the evaluation of water resources, water environment and other three layers is not involved, the biological information can only reflect the condition of river biological groups, and the quality of the water ecological environment cannot be comprehensively and accurately indicated.

Disclosure of Invention

The invention aims to solve the problem that the existing environmental DNA technology monitoring result is not matched with the existing biological monitoring index based on the morphological evaluation method, so that the water ecology comprehensive evaluation method suitable for the environmental DNA technology is absent, and provides the water ecology monitoring and comprehensive evaluation method based on the environmental DNA technology.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a water ecology monitoring and comprehensive evaluation method based on the environmental DNA technology comprises the following steps,

step one, collecting basic data and basic drawing related to the tidying watershed;

step two, performing initial investigation on the target river to determine monitoring points;

thirdly, comprehensively considering the water ecological evaluation requirement and the technical characteristics of environmental DNA from the viewpoint of the integrity of the evaluation of the water ecological system according to the early-stage data collection and the on-site initial investigation condition, providing evaluation indexes covering four aspects of water resources, water environment, water habitat and aquatic organisms, performing special investigation on each item index, measuring by the environmental DNA technology according to the special investigation result, and calculating by an index formula to obtain an accurate evaluation result;

step four, scoring the index items;

and fifthly, determining the weight of each index, and calculating the final score and grade of the target river water ecology evaluation according to the evaluation result of each index.

In the second step, the river hydrologic characteristics, habitat conditions, water environment quality and biological community characteristics in the monitored area are divided into different river segments, and monitoring points are determined according to the point location setting principle in the different river segments.

Preferably, in the third step, the evaluation index is a water resource-ecological flow/water level satisfaction degree, a water environment-water quality degree, an aquatic environment-habitat condition index, an aquatic organism-fish conservation index, an important protection species conservation index, a fish dominance index, an alien invasive species index, a benthonic animal BMWP index, a benthonic animal hilsingoff index, a Shannon-Wiener (Shannon-Wiener) diversity index, and a uniformity index.

Preferably, the method comprises: the method for carrying out special investigation on various indexes of water resources, water environment and aquatic organisms comprises the following steps of,

A. water resource-ecological flow/water level satisfaction degree

Collecting the hydrological data of each evaluation river section of the target river in the past year, or installing a vertical rod type hydrological automatic monitoring device at the shore position of the adjacent water area at the selected water ecological monitoring point, and selecting 4 hydrological indexes of liquid level, flow rate and rainfall for automatic real-time monitoring;

B. water environment-quality of water

Combining the existing water quality monitoring station or installing a water quality automatic monitoring station at the monitoring station, selecting conventional five-parameter, permanganate index, ammonia nitrogen, total phosphorus, total nitrogen and other water quality indexes to automatically monitor in real time, evaluating the water quality category of the monitored section by adopting a single factor evaluation method, and calculating the proportion of the river length of each water quality category to the total length of the target river to represent the water quality degree;

C. Aquatic environment-habitat condition index

The method comprises the steps of obtaining the base quality category, the habitat complexity, the flow speed/depth combination characteristic, the river bank stability, the river channel change, the river water volume condition, the river bank vegetation coverage rate, the water quality condition, the human activity intensity, the river bank land utilization type and other habitat condition indexes of a bank slope by adopting manners of investigation, measurement, aviation flight, remote sensing image interpretation and the like for each evaluation river segment of a target river in the field;

D. aquatic organism index

I, on-site sampling

According to the actual situation, constructing an environment DNA automatic sampling station or manually collecting an environment DNA sample on a monitoring section, and sending the sample to a molecular laboratory for detection as soon as possible after the sampling is finished;

II, laboratory detection

Carrying out DNA extraction, PCR amplification, library construction and high-throughput sequencing on the collected sample, wherein the high-throughput sequencing data analysis flow mainly comprises sequencing data quality control, sample sequence analysis, repeated sequence removal, sequence clustering, sequence annotation and OTUs table generation, and the related original data and biological informatics analysis conclusion are contained in the finally submitted aquatic organism detection report;

III, obtaining results

And acquiring the aquatic organism species composition, dominant species and the relative abundance of each species in the monitored water area according to a detection report of the molecular laboratory on the environmental DNA sample.

Preferably, the specific investigation method of the aquatic environment-habitat condition index is that,

the substrate category adopts a field investigation mode, sampling points are defined according to river channel conditions, sampling is carried out at the sampling points, and the substrate category (silt, clay, sediment, coarse sand, gravel, pebble, rock or other) and the occurrence proportion thereof are described and recorded;

the method for combining the field investigation and the aerial image interpretation is adopted for investigating the habitat on both sides of the river bank zone, and the habitat is classified into various small habitats such as aquatic vegetation, dead branches and fallen leaves, inverted woods, inverted concave river banks, large stones, broken stones and the like;

the flow rate/depth combination characteristic records the presence and relative proportion of the slow-deep, slow-shallow, fast-deep and fast-shallow 4 types by field investigation and field measurement;

the stability of the river bank is obtained by means of site investigation, remote sensing interpretation, unmanned aerial vehicle detection and the like, and the river bank erosion proportion is calculated by the river bank length, erosion degree, erosion length and the like;

the river channel change is combined with field investigation according to river channel planning, whether the river channel is in canal or not is described and recorded, and whether the river channel has influence on organisms or not is judged according to the scope of the river channel;

the river water quantity condition is measured on site to submerge the river water to the river channel height proportion on two sides of the river bank, and if the river water quantity condition is different, an average value is adopted;

Vegetation coverage of the river bank zone: the method combining the field investigation and the aerial image interpretation is used for investigating vegetation areas, total areas, plant types, vegetation types and vegetation related characteristics within a range of 15 meters extended outside the two sides of the river bank zone and calculating the vegetation coverage rate of the bank slope;

water quality condition: from the sense angle, the characteristics of water color, smell, surface floaters, surface oil stains, water suspended matters and the like are observed and recorded;

intensity of human activity: on-site investigation, investigation region and surrounding human interference or human activities, whether pedestrians and vehicles pass or not;

type of land use on river bank: the area proportion of cultivated soil and construction land is investigated by means of field investigation, unmanned plane river channel inspection, remote sensing interpretation and the like.

Preferably, the index is calculated as follows,

a. index of fish preservation

Collecting the condition of fish before 2000 years in a target river basin by adopting modes of historical data investigation, expert consultation and the like, carrying out differential analysis by combining the existing fish condition detected by the environmental DNA, and calculating according to the following formula

FOEI＝FO/FE×100

Wherein: foei-fish conservation index (%)

FO-data of fish species (excluding foreign species) obtained by environmental DNA investigation

FE-evaluation of the number of fish species in river and lake before 2000

b. Index of degree of deviation of dominant fish

Detecting the ratio of the highest and second highest species of fishes in the fish community of the target river by the environmental DNA, reflecting the stability of the composition of the species of fishes, respectively calculating the ratio of the highest and second highest species of fishes according to the composition data of the species of fishes obtained by the investigation of the aquatic organism resource, comparing the deviation degree of the current value and the reference value,

dominant species ratio = number of fish dominant species/total number of fish species

Deviation index of fish dominance family= (fish dominance family species ratio-fish dominance family species ratio reference value)/fish dominance family species ratio reference value

c. Conservation index of important protective species

In the water body, the biological species detected by the environmental DNA sample are evaluated, and the biological species are subject to national class 1 and class 2 protection aquatic organisms, local protection species and aquatic germplasm resource protection zone protection species, and reflect the protection species conditions, and the index calculation method comprises the following steps:

important protective species conservation index=important protective species number present value/important protective species number reference value

d. Foreign invasive species index

Investigation of the condition of wading foreign invasive species through historical data collection, field investigation and environmental DNA sample detection results;

e. Benthonic animal BMWP index

The BM WP index was calculated from the environmental DNA samples detected from the zoobenthos species belonging to the subject according to the following formula

BMWP＝∑Fi

Wherein F is _i For the sensitivity value of Kei

f. Benthonic Hilsenhoff index (BI biological index)

The Hilsenhoff index (BI biological index) was calculated from the phylum of the zoozoological species detected from the environmental DNA sample according to the following formula

Wherein: pi-relative abundance of species i

Contamination resistance value of Bi-species i corresponding family

S-number of species

Shannon-Wiener diversity index (H')

Based on the species composition and abundance ratio detected by the environmental DNA sample, the shannon-wiener diversity index was calculated in combination with the following formula:

H'＝-∑(piln pi)

wherein: h' -shannon-wiener diversity index;

pi-the ratio of the number of i-th fish individuals or biomass to the total fish individuals or total biomass (biomass ratio is in positive correlation with the ratio of ambient DNA abundance).

h. Uniformity index (Evenness)

J'＝H'/lnS

Wherein J' is the uniformity index

H' is shannon-wiener diversity index

S is the number of species.

Preferably, in the fourth step, the method of assigning scores to the index scores is as follows,

a. ecological flow/water level satisfaction

Calculating the percentage of the minimum daily average flow of 4-9 months and 10-3 months to the average flow of the same period for years respectively, calculating the scoring values according to scoring criteria respectively, and taking the lowest scoring of the two to be scoring for the satisfaction degree of the river ecological water;

b. Quality of water

Assigning points according to the proportion of the water quality class river length of the investigation and monitoring to the total length of the target river;

c. habitat condition index

Obtaining habitat monitoring data according to a habitat investigation method, respectively scoring 10 parameters of the habitat according to the habitat scoring data, wherein the score range of each parameter is 0-20, dividing the habitat into four evaluation grades, accumulating and calculating the total score (H) of the habitat of each monitoring point by 10 parameter scores, and assigning scores according to grading evaluation standards;

d. index of fish preservation

The index monitors the current value of the fish species number through the environmental DNA, calculates the fish preservation index, and carries out scoring by adopting a linear interpolation method according to a fish preservation index scoring table;

e. index of degree of deviation of dominant fish

The index monitors the deviation degree of the current value and the reference value of the dominant fish through the environmental DNA and assigns according to the deviation degree index of the dominant fish;

f. conservation index of important protective species

The index is classified by monitoring the ratio of the current value of the number of species of the important protection fish to the standard value through the environmental DNA.

g. Foreign invasive species index

The index is assigned by collecting or investigating a historical and current wading foreign invasive species situation assignment table;

h. Benthonic animal BMWP index

The index is calculated according to the environmental DNA monitoring result and then assigned according to the benthonic animal BMWP index assignment table.

i. Benthonic Hilsenhoff index (BI biological index)

The index is assigned according to a BI biological index assignment table after being calculated according to an environmental DNA monitoring result;

j. Shannon-Wiener (Shannon-Wiener) diversity index

The index is assigned according to a shannon-wiener diversity index assignment table after being calculated according to an environmental DNA monitoring result;

k. uniformity index (Evenness)

The index is assigned according to a uniformity index assignment table after being calculated according to the environmental DNA monitoring result.

Preferably, in the fifth step, a method for calculating a final score and grade of the target river water ecology evaluation comprises the steps of:

the comprehensive index method is adopted to carry out comprehensive evaluation on the quality of the water ecology environment, the water resource, the water environment, the water habitat and the water biology indexes are weighted and summed to construct a comprehensive evaluation index WEQI of the quality of the water ecology environment, the index is used for representing the overall quality condition of each evaluation unit and the water environment, and the calculation formula of the comprehensive evaluation index WEQI of the quality of the water ecology environment is as follows:

wherein: WEQI-comprehensive evaluation index of water ecological environment quality;

xi-an evaluation index score;

wi-evaluation index weight

In the initial stage of the operation of the evaluation system, the weights of all indexes can be tentatively equal, and along with the application of the evaluation system, the weights of all indexes are actually and properly adjusted by combining all evaluation water areas in the later stage.

The water ecological environment quality grade is classified into five grades according to the value of the water ecological environment quality comprehensive evaluation index (WEQI).

Compared with the prior art, the invention has the advantages that:

(1) The invention constructs a set of brand new comprehensive water ecology evaluation method based on the environment DNA technology, comprehensively considers the water ecology evaluation requirement and the environmental DNA technical characteristics, creatively proposes 11 evaluation indexes covering four aspects of water resources, water environment, aquatic environment and aquatic organisms and the scoring and evaluation methods thereof, and the method not only comprises a plurality of biological indexes which are closely related with the surrounding environment and are sensitive to reaction after interference, but also comprises physicochemical indexes which are fast in response to environmental change, and monitors and comprehensively evaluates the ecological health condition of river water from multiple directions and multiple angles, so the method is a more effective and reliable water ecology evaluation method compared with the single-aspect evaluation method.

(2) The biological indexes of the indexes provided by the invention can be all determined by the environmental DNA technology, and an accurate evaluation result is obtained through calculation, so that the defects that the environmental DNA technology cannot determine the number of aquatic organisms, cannot calculate partial biological indexes and the like are overcome, the environmental DNA technology is applied to comprehensively evaluate the water ecology, the operability is realized, and the popularization and the use of the environmental DNA technology in the field of water ecology evaluation are facilitated.

(3) The water ecology comprehensive evaluation method based on the environment DNA technology has the advantages of comprehensive indexes, wide application range, simplicity in operation, high accuracy, environmental friendliness and the like, and has important popularization and application values in the field of water ecology evaluation.

(4) The evaluation scoring standard disclosed by the invention can completely fit the river water ecological evaluation technical guideline issued by the current ecological environment department, and simultaneously combines the rapid and accurate monitoring advantages of the environmental DNA technology, so that a plurality of indexes which are not provided in the guideline but need to be focused and have great value in the actual monitoring work are brought into comprehensive evaluation scoring, and the water ecological monitoring and evaluation result prepared by the method is more valuable and convincing.

(5) The water ecological monitoring and evaluating method provided by the invention applies the environmental DNA technology to the field of aquatic organisms, solves the problems of higher requirements on taxonomic identification experience, lower accuracy caused by human errors and systematic errors, complex operation process, low efficiency and the like in the morphological method, and identifies the biological community information of the ecological system from the gene angle, thereby achieving the effects of no need of taxonomic identification experience, higher accuracy, simple operation process, high efficiency, standardization and small interference degree to the environment.

Drawings

FIG. 1 is a flow chart of the method of the present invention.

Detailed Description

The invention will be further described below, namely, a water ecology monitoring and comprehensive evaluation method based on the environmental DNA technology, which is shown in the following steps of the method in FIG. 1:

step one, basic data are collected,

collecting basic data such as hydrological water resources, weather, water quality, aquatic organisms, water resource development and utilization, flood control planning and the like related to the tidying river basin and basic drawing pieces;

step two, preliminary investigation;

the method comprises the steps of initially surveying a target river, dividing a region with obvious differences of river hydrologic characteristics, habitat conditions, water environment quality and biological community characteristics in a monitored region into different river segments, and determining monitoring points in the different river segments according to a point location setting principle;

thirdly, each index special investigation and index calculation;

step four, evaluating and scoring each index item;

and fifthly, determining the weight of each index, and calculating the final score and grade of the target river water ecology evaluation according to the evaluation result of each index.

Step three, performing special investigation and index calculation of each index, wherein the specific investigation and index calculation are as follows:

according to the early-stage data collection and the on-site initial investigation condition, special investigation is carried out, and the method comprehensively considers the water ecological evaluation requirement and the technical characteristics of environmental DNA from the viewpoint of the integrity of the water ecological system evaluation, creatively provides 11 evaluation indexes covering four aspects of water resources, water environment, water habitat and aquatic organisms, wherein the indexes comprise a plurality of biological indexes which are closely related with the surrounding environment and are sensitive to post-interference reaction, and physicochemical indexes which are fast in response to environmental change, and monitors and comprehensively evaluates the ecological health condition of river water from multiple directions and multiple angles.

The 11 evaluation indexes are respectively: water resource-ecological flow/water level satisfaction, water environment-quality of water, aquatic environment-habitat condition index, aquatic organism-fish preservation index, important protection species preservation index, fish dominance index, foreign invasive species index, benthonic animal BMWP index, benthonic animal Hilsenhoff index, shannon-Wiener diversity index and uniformity index.

(1) The monitoring method of each index is specifically as follows:

A. water resource-ecological flow/water level satisfaction degree

The method comprises the steps of collecting hydrological data of each evaluation river section of a target river in the past year, or installing a vertical rod type hydrological automatic monitoring device at a selected water ecology monitoring point position close to the shore position of a water area, and selecting 4 hydrological indexes such as liquid level, flow rate, flow quantity and rainfall to carry out automatic real-time monitoring. And calculating the percentage of the minimum daily average flow in the dead water period (10-3 months) and the rich water period (4-9 months) to the average flow of the same period for years according to the flow data.

B. Water environment-quality of water

The existing water quality monitoring station or an automatic water quality monitoring station installed at the monitoring station is combined, 9 water quality indexes such as conventional five parameters (pH, dissolved oxygen, turbidity, temperature and conductivity), permanganate index, ammonia nitrogen, total phosphorus and total nitrogen are selected for automatic real-time monitoring, the water quality category of the monitored section is evaluated by adopting a single factor evaluation method, the water quality category is divided according to the standard GB3838-200 of the quality standard of the surface water environment, and the water quality degree is represented by calculating the proportion of the river length of each water quality category to the total length of the target river.

C. Aquatic environment-habitat condition index

And obtaining the base quality category, the habitat complexity, the flow speed/depth combination characteristic, the river bank stability, the river channel change, the river water volume condition, the river bank vegetation coverage, the water quality condition, the human activity intensity, the river bank land utilization type and other habitat condition indexes of the bank slope by adopting the modes of investigation, measurement, aviation flight, remote sensing image interpretation and the like for each evaluation river segment of the target river in the field. Note that: the habitat is investigated 1 time per year in principle, if the vegetation in the riparian zone obviously suggests that the investigation is carried out synchronously during each monitoring along with the seasonal variation, the investigation is tracked in time when the habitat is found to be influenced by artificial interference or special natural environment variation (construction, storm flushing, etc.).

Substrate category: and (3) defining sampling points according to river channel conditions by adopting a field investigation mode, sampling at the sampling points, and describing and recording substrate categories (silt, clay, sediment, coarse sand, gravel, pebbles, rocks or the like) and occurrence proportions thereof.

Habitat complexity: the method of combining the field investigation and the aerial image interpretation is adopted to investigate the habitat on both sides of the river bank zone, and the habitat is classified into various small habitats such as aquatic vegetation, dead branches and fallen leaves, inverted wood, inverted concave river bank, large stones, broken stones and the like.

Flow rate/depth combination characteristics: the presence and relative proportions of the 4 types of slow-deep, slow-shallow, fast-deep and fast-shallow were recorded by field investigation, field measurements.

River bank stability: and obtaining the river bank length, erosion degree, erosion length and the like through the modes of site investigation, remote sensing interpretation, unmanned aerial vehicle detection and the like, and calculating the river bank erosion proportion.

River course change: describing and recording whether the river channel is channelized according to the river channel planning and combining with field investigation, and judging whether the river channel has an influence on organisms according to the river channel range.

River water volume status: and (5) measuring the height proportion of river water submerged into the river channels at two sides of the river bank on site, and adopting an average value if the river channel height proportion is different.

Vegetation coverage of the river bank zone: the method combines field investigation and aerial image interpretation, and is used for investigating vegetation areas, total areas, plant types, vegetation type and vegetation related characteristics within a range of 15 meters extended outside the two sides of the river bank zone and calculating the coverage rate of the vegetation on the bank slope.

Water quality condition: from the sense point of view, the characteristics of water color, smell, surface floating matters, surface oil stains, water suspended matters and the like are observed and recorded.

Intensity of human activity: on-site investigation is performed to investigate whether pedestrians and vehicles pass through the area and surrounding human interference or human activities.

Type of land use on river bank: the area proportion of cultivated soil and construction land is investigated by means of field investigation, unmanned plane river channel inspection, remote sensing interpretation and the like.

D. Aquatic organism index

I, on-site sampling

According to the actual conditions, an environment DNA automatic sampling station is constructed on a monitoring section or an environment DNA sample is manually collected, after sampling is completed, the sample is sent to a molecular laboratory for detection as soon as possible, and the accuracy of data is required to be ensured in the sampling process.

The sampling mode of the automatic sampling station of the environment DNA is as follows: and installing an automatic sampling and enriching device for the environmental DNA at the position of the monitored section close to the water area, and setting any 1 week as a sampling period, wherein 1 sample is acquired every day during the period, and 7 samples are acquired in total. After the sampling is finished, the investigator goes to the site to take out the filter membrane sample after the suction filtration enrichment, and sends the filter membrane sample to a molecular laboratory for detecting the environmental DNA sample.

The manual sampling mode is as follows: and collecting a certain volume of water sample at each monitoring point, and carrying out on-site suction filtration on the water sample to a sterile microporous filter membrane with the thickness of 0.45 mu m. After the suction filtration is finished, the filter membrane is put into a 5mL sterile tube, preservation solution is added, the mixture is uniformly mixed, then the mixture is preserved at normal temperature, 3 biological repetitions are collected at each sampling point, and the mixture is brought back to a laboratory for detecting the environmental DNA sample after the investigation is finished.

II, laboratory detection

After the collected samples were handed over to laboratory personnel, the samples were subjected to DNA extraction, PCR amplification, library construction, and high throughput sequencing by laboratory professionals. The high-throughput sequencing data analysis flow mainly comprises sequencing data quality control, sample sequence analysis, repeated sequence removal, sequence clustering, sequence annotation and OTUs table generation. Both the relevant raw data and the bioinformatic analysis conclusions will be included in the final submitted aquatic organism detection report.

III, obtaining results

According to the detection report of the molecular laboratory on the environmental DNA sample, the aquatic organism species composition, dominant species and the relative abundance of each species in the monitored water area can be accurately obtained.

(2) The calculation formula of the index is as follows:

a. index of fish preservation

And collecting the condition of the fish before 2000 years in the target river basin by adopting the modes of historical data investigation, expert consultation and the like, and carrying out differential analysis by combining the condition of the existing fish detected by the environmental DNA. Investigation of fish species excludes extraneous fish species. The calculation is carried out according to the following formula:

FOEI＝FO/FE×100

wherein: FOEI-fish retention index (%);

fo—fish species data (knockout of foreign species) (species) obtained from environmental DNA investigation;

FE-the number of fish species in rivers and lakes was evaluated before 2000.

b. Index of degree of deviation of dominant fish

The environmental DNA detects the ratio of the highest number of fish species to the next highest family in the fish community of the target river, reflecting the stability of the fish species composition. Based on the fish species composition data obtained by the aquatic organism resource investigation, the ratio of the number of species of the highest species to the number of species of the next highest species is calculated, and the degree of deviation of the present value from the reference value is compared.

Dominant species ratio = number of fish dominant species/total number of fish species.

Deviation index of fish dominance family= (fish dominance family species ratio-fish dominance family species ratio reference value)/fish dominance family species ratio reference value

c. Conservation index of important protective species

And (3) evaluating the biological species detected by the environmental DNA sample in the water body, wherein the biological species belong to the national class 1 and class 2 protective aquatic organisms, local protective species and aquatic germplasm resource protective zone protective species, and reflecting the conditions of the protective species. The index calculation method comprises the following steps:

important protective species conservation index=important protective species number present value/important protective species number reference value

d. Foreign invasive species index

The condition (the number of types and whether common) of the wading foreign invasive species is investigated by taking the Chinese foreign invasive species list issued by the government of the people's republic of China as a basis and through historical data collection, field investigation and environmental DNA sample detection results.

e. Benthonic animal BMWP index

The BM WP index was calculated from the environmental DNA samples detected zoobenthos species belonging to the subjects according to the following formula:

BMWP＝∑Fi

wherein F is _i Is the sensitive value of the Kei.

f. Benthonic Hilsenhoff index (BI biological index)

The zoobenthos species genus subject detected from the environmental DNA sample was calculated according to the following formula, hil senhoff index (BI biological index):

wherein: pi-the relative abundance of species i;

contamination resistance value of Bi-species i corresponding family

S-number of species.

Shannon-Wiener diversity index (H')

Based on the species composition and abundance ratio detected by the environmental DNA sample, the shannon-wiener diversity index was calculated in combination with the following formula:

H'＝-∑(pilnpi)

wherein: h' -shannon-wiener diversity index;

pi-the ratio of the number of i-th fish individuals or biomass to the total fish individuals or total biomass (biomass ratio is in positive correlation with the ratio of ambient DNA abundance).

h. Uniformity index (Evenness)

J'＝H'/lnS

Wherein J' is the uniformity index

H' is shannon-wiener diversity index

S is the number of species.

The core biological indexes are all determined by the environment DNA technology, and an accurate evaluation result is obtained through formula calculation, so that the defect that the indexes related to the biological quantity cannot be determined by the environment DNA technology is overcome, the environment DNA technology is applied to comprehensively evaluate the water ecology, the operability is realized, and the popularization and the use of the environment DNA technology in the field of water ecology evaluation are facilitated.

The specific method for evaluating and assigning the index items is as follows:

the evaluation and grading of the monitoring indexes are carried out according to the index calculation result in the step three, the evaluation and grading standard of the method can completely fit the river water ecological evaluation technical guideline issued by the current ecological environment department, meanwhile, the method combines the rapid and accurate monitoring advantages of the environment DNA technology, and integrates a plurality of indexes which are not contained in the guideline but need to be focused and have great value in the actual monitoring work to carry out comprehensive evaluation and grading, so that the water ecological monitoring and evaluation results made according to the method are more valuable and convincing.

The method for evaluating the score of each index is as follows,

a. ecological flow/water level satisfaction

The percentage of the minimum daily average flow of 4-9 months and 10-3 months to the average flow of the same period for years is calculated respectively, the scoring values are calculated respectively according to the scoring criteria in the following table, and the lowest scoring of the two is taken as the scoring of the satisfaction degree of the river ecological water.

River ecological water satisfaction degree evaluation scoring standard table

b. Quality of water

The proportions of the water quality class river length to the total length of the target river are classified according to the following table.

Water quality degree evaluation and grading standard table

c. Habitat condition index

And (3) obtaining habitat monitoring data according to a habitat investigation method, respectively scoring 10 parameters of the habitat, wherein the score range of each parameter is 0-20, and dividing the parameters into four evaluation grades, and the scoring standard is seen in a habitat evaluation data table. The overall habitat score (H) of each monitoring point is calculated by accumulating 10 parameter scores, and the grading evaluation standard is the grading evaluation standard of the habitat condition index.

Habitat evaluation data table

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Hierarchical evaluation criteria for habitat situation index

Habitat grade	Excellent and excellent properties	Good quality	Medium and medium	Poor quality	Very poor
						Evaluation criteria	H>150	120＜H≤150	90＜H≤120	60＜H≤90	H≤60
Score assignment	100	80	60	40	20

d. Index of fish preservation

The index is a fish preservation index calculated by monitoring the current value of the fish species number through the environmental DNA, and the index is assigned by adopting a linear interpolation method according to the following table.

Index score table for fish preservation

e. Index of degree of deviation of dominant fish

The index is classified according to the following table by monitoring the deviation degree of the current value and the reference value of the dominant fish through the environmental DNA.

Index scoring table for deviation degree of dominant fish

f. Conservation index of important protective species

The index is characterized in that the ratio of the current value of the number of species of the important protection fish to the standard value is calculated according to the following table through environmental DNA monitoring.

Important protection species conservation index assignment table

g. Foreign invasive species index

The index is assigned according to the following table after collecting or investigating history and current wading foreign invasive species.

Foreign invasive species index scoring standard table

h. Benthonic animal BMWP index

The index was calculated from the environmental DNA monitoring results and assigned according to the following table.

Benthonic animal BMWP index scoring table

BMWP index (river without wading)	BMWP index (wading river)	Score assignment
			≥86	≥146	100
65≤BMWP≤85	110≤BMWP≤145	80
			43≤BMWP≤64	73≤BMWP≤109	60
22≤BMWP≤42	37≤BMWP≤72	40
			≤21	≤36	20

i. Benthonic Hilsenhoff index (BI biological index)

The index was calculated from the environmental DNA monitoring results and assigned according to the following table.

BI biological index assignment table

j. Shannon-Wiener (Shannon-Wiener) diversity index

The index was calculated from the environmental DNA monitoring results and assigned according to the following table.

Shannon-wiener diversity index scoring table

k. Uniformity index (Evenness)

The index was calculated from the environmental DNA monitoring results and assigned according to the following table.

Uniformity index assignment table

In the fifth step, the method for determining the weight of each index and calculating the final score and grade of the target river water ecology evaluation is as follows:

and (3) comprehensively evaluating the quality of the water ecological environment by adopting a comprehensive index method, constructing a water ecological environment quality comprehensive evaluation index WEQI by weighting and summing the water resources, the water environment, the aquatic environment and the aquatic organism indexes, and expressing the overall quality condition of each evaluation unit and the water environment by the index. In the initial stage of the operation of the evaluation system, the weights of all indexes can be tentatively equal, and along with the application of the evaluation system, the weights of all indexes are actually and properly adjusted by combining all evaluation water areas in the later stage.

The water ecological environment quality comprehensive evaluation index WEQI has the following calculation formula:

wherein: WEQI-comprehensive evaluation index of water ecological environment quality;

x _i -evaluating the index score;

w _i -evaluating the index weight.

The water ecological environment quality class is classified into five classes according to the value of the water ecological environment quality comprehensive evaluation index (WEQI) score, and the details are shown in the following table.

Water ecological environment quality grading standard table

Quality of aquatic ecology environment	Excellent and excellent properties	Good quality	Medium and medium	Poor quality	Very poor
						Comprehensive index (WEQI)	WEQI＝100	100>WEQI≥80	80>WEQI≥60	60>WEQI≥40	WEQI<40
Characterization of color	Blue color	Green colour	Yellow colour	Orange color	Red color

The water ecology monitoring and comprehensive evaluation method based on the environment DNA technology is described in detail, and specific examples are applied to illustrate the principle and the implementation mode of the method, and the description of the examples is only used for helping to understand the method and the core idea of the method; also, it will be apparent to those skilled in the art from this disclosure that various changes and modifications can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (8)

1. A water ecology monitoring and comprehensive evaluation method based on an environment DNA technology is characterized in that: the method comprises the steps of,

step one, collecting basic data and basic drawing related to the tidying watershed;

step two, performing initial investigation on the target river to determine monitoring points;

thirdly, comprehensively considering the water ecological evaluation requirement and the technical characteristics of environmental DNA from the viewpoint of the integrity of the evaluation of the water ecological system according to the early-stage data collection and the on-site initial investigation condition, providing evaluation indexes covering four aspects of water resources, water environment, water habitat and aquatic organisms, performing special investigation on each item index, measuring by the environmental DNA technology according to the special investigation result, and calculating by an index formula to obtain an accurate evaluation result;

step four, scoring the index items;

and fifthly, determining the weight of each index, and calculating the final score and grade of the target river water ecology evaluation according to the evaluation result of each index.

2. The method for water ecology monitoring and comprehensive evaluation based on the environmental DNA technology of claim 1, wherein the method comprises the following steps: in the second step, dividing the area with obvious differences of the river hydrologic characteristics, the habitat conditions, the water environment quality and the biological community characteristics in the monitored area into different river segments, and determining the monitored points in the different river segments according to the point location setting principle.

3. The method for water ecology monitoring and comprehensive evaluation based on the environmental DNA technology of claim 1, wherein the method comprises the following steps: in the third step, the evaluation index is water resource-ecological flow/water level satisfaction degree, water environment-water quality degree, aquatic environment-habitat condition index, aquatic organism-fish preservation index, important protection species preservation index, fish dominance degree index, foreign invasive species index, benthonic animal BMWP index, benthonic animal Hilsenhoff index, shannon-Wiener diversity index and uniformity index.

4. The method for water ecology monitoring and comprehensive evaluation based on the environmental DNA technology of claim 3, wherein the method comprises the following steps: the method for carrying out special investigation on various indexes of water resources, water environment and aquatic organisms comprises the following steps of,

A. water resource-ecological flow/water level satisfaction degree

Collecting the hydrological data of each evaluation river section of the target river in the past year, or installing a vertical rod type hydrological automatic monitoring device at the shore position of the adjacent water area at the selected water ecological monitoring point, and selecting 4 hydrological indexes of liquid level, flow rate and rainfall for automatic real-time monitoring;

B. Water environment-quality of water

Combining the existing water quality monitoring station or installing a water quality automatic monitoring station at the monitoring station, selecting conventional five-parameter, permanganate index, ammonia nitrogen, total phosphorus and total nitrogen water quality indexes for automatic real-time monitoring, evaluating the water quality category of the monitored section by adopting a single factor evaluation method, and calculating the proportion of the river length of each water quality category to the total length of the target river to represent the water quality degree;

C. aquatic environment-habitat condition index

Investigation, measurement, aviation and remote sensing image interpretation modes are adopted for each evaluation river segment of the target river on site, and the base quality category, the habitat complexity, the flow speed/depth combination characteristic, the river bank stability, the river channel change, the river water volume condition, the river bank vegetation coverage rate, the water quality condition, the human activity intensity and the river bank land utilization type habitat condition index of the bank slope are obtained;

D. aquatic organism index

I, on-site sampling

Constructing an environment DNA automatic sampling station or manually collecting an environment DNA sample on a monitoring section according to actual conditions, and sending the sample to a molecular laboratory for detection after the sampling is completed;

II, laboratory detection

Carrying out DNA extraction, PCR amplification, library construction and high-throughput sequencing on the collected sample, wherein the high-throughput sequencing data analysis flow comprises sequencing data quality control, sample sequence analysis, repeated sequence removal, sequence clustering, sequence annotation and OTUs table generation, and relevant original data and biological information analysis conclusion are contained in a finally submitted aquatic organism detection report;

III, obtaining results

And acquiring the aquatic organism species composition, dominant species and the relative abundance of each species in the monitored water area according to a detection report of the molecular laboratory on the environmental DNA sample.

5. The method for water ecology monitoring and comprehensive evaluation based on the environmental DNA technology of claim 4, wherein the method comprises the following steps: the specific investigation method of the aquatic environment-habitat condition index is that,

the substrate category adopts a field investigation mode, sampling points are defined according to river channel conditions, sampling is carried out at the sampling points, and the substrate category and the occurrence proportion thereof are described and recorded;

the method for combining the field investigation and the aerial image interpretation is adopted for investigating the habitat on both sides of the river bank zone, and the habitat is classified into aquatic vegetation, dead branches and fallen leaves, inverted wood, inverted concave river bank and various small habitats;

the flow rate/depth combination characteristic records the presence and relative proportion of the slow-deep, slow-shallow, fast-deep and fast-shallow 4 types by field investigation and field measurement;

the river bank stability is obtained by site investigation, remote sensing interpretation and unmanned aerial vehicle detection modes, and the river bank erosion proportion is calculated by the river bank length, erosion degree and erosion length;

the river channel change is combined with field investigation according to river channel planning, whether the river channel is in canal or not is described and recorded, and whether the river channel has influence on organisms or not is judged according to the scope of the river channel;

The river water quantity condition is measured on site to submerge the river water to the river channel height proportion on two sides of the river bank, and if the river water quantity condition is different, an average value is adopted;

the vegetation coverage rate of the river bank zone is investigated by a method combining field investigation and aerial image interpretation, vegetation areas, total areas, vegetation types and vegetation related characteristics within a range of 15 meters of the two sides of the river bank zone are investigated, and the vegetation coverage rate of a bank slope is calculated;

the water quality condition, from the sense angle, the water color, smell, surface floaters, surface oil stains and water suspension characteristics are observed and recorded;

human activity intensity, on-site investigation, investigation region and surrounding human interference or human activity, whether pedestrians and vehicles pass or not;

the land utilization type of the river bank is that the land type of the two sides of the river bank and the area ratio of cultivated soil and construction land are investigated by a field investigation, unmanned plane river channel inspection and remote sensing interpretation mode.

6. The method for water ecology monitoring and comprehensive evaluation based on the environmental DNA technology of claim 3, wherein the method comprises the following steps: the calculation formula of the index is as follows,

a. index of fish preservation

Collecting fish conditions before 2000 years in a target river basin by adopting a historical data investigation and expert consultation mode, carrying out differential analysis by combining the existing fish conditions detected by the environmental DNA, and calculating FOEI=FO/FE×100 according to the following formula

Wherein: foei-fish conservation index (%)

FO-data (species) of fish species obtained by environmental DNA investigation, excluding foreign species

FE-evaluation of the number of fish species in river and lake before 2000

b. Index of degree of deviation of dominant fish

Detecting the ratio of the highest and second highest species of fishes in the fish community of the target river by the environmental DNA, reflecting the stability of the composition of the species of fishes, respectively calculating the ratio of the highest and second highest species of fishes according to the composition data of the species of fishes obtained by the investigation of the aquatic organism resource, comparing the deviation degree of the current value and the reference value,

dominant species ratio = number of fish dominant species/total number of fish species

Deviation index of fish dominance family= (fish dominance family species ratio-fish dominance family species ratio reference value)/fish dominance family species ratio reference value

c. Conservation index of important protective species

In the water body, the biological species detected by the environmental DNA sample are evaluated, and the biological species are subject to national class 1 and class 2 protection aquatic organisms, local protection species and aquatic germplasm resource protection zone protection species, and reflect the protection species conditions, and the index calculation method comprises the following steps:

important protective species conservation index=important protective species number present value/important protective species number reference value

d. Foreign invasive species index

Investigation of the condition of wading foreign invasive species through historical data collection, field investigation and environmental DNA sample detection results;

e. benthonic animal BMWP index

The BMWP index was calculated from the zoobenthos species detected from the environmental DNA samples according to the following formula

BMWP＝∑Fi

Wherein F is _i For the sensitivity value of Kei

f. Benthonic Hilsenhoff index (BI biological index)

The Hi lsenhoff index (BI biological index) was calculated from the phylum of the zoobenthos species detected from the environmental DNA sample according to the following formula

Wherein: pi-relative abundance of species i

Contamination resistance value of Bi-species i corresponding family

S-number of species

Shannon-Wiener diversity index (H')

Based on the species composition and abundance ratio detected by the environmental DNA sample, the shannon-wiener diversity index was calculated in combination with the following formula:

H'＝-∑( ⁵ pilnpi)

wherein: h' -shannon-wiener diversity index;

pi-the ratio of the number of i-th fish individuals or biomass to the total fish individuals or total biomass (biomass ratio is in positive correlation with the ratio of ambient DNA abundance).

h. Uniformity index (Evenness)

J'＝H'/ln S

Wherein J' is the uniformity index

H' is shannon-wiener diversity index

S is the number of species.

7. The method for water ecology monitoring and comprehensive evaluation based on the environmental DNA technology of claim 1, wherein the method comprises the following steps: in the fourth step, the method for evaluating and assigning the index items is as follows,

a. ecological flow/water level satisfaction

Calculating the percentage of the minimum daily average flow of 4-9 months and 10-3 months to the average flow of the same period for years respectively, calculating the scoring values according to scoring criteria respectively, and taking the lowest scoring of the two to be scoring for the satisfaction degree of the river ecological water;

b. quality of water

Assigning points according to the proportion of the water quality class river length of the investigation and monitoring to the total length of the target river;

c. habitat condition index

Obtaining habitat monitoring data according to a habitat investigation method, respectively scoring 10 parameters of the habitat according to the habitat scoring data, wherein the score range of each parameter is 0-20, dividing the habitat into four evaluation grades, accumulating and calculating the total score (H) of the habitat of each monitoring point by 10 parameter scores, and assigning scores according to grading evaluation standards;

d. index of fish preservation

The index monitors the current value of the fish species number through the environmental DNA, calculates the fish preservation index, and carries out scoring by adopting a linear interpolation method according to a fish preservation index scoring table;

e. index of degree of deviation of dominant fish

The index monitors the deviation degree of the current value and the reference value of the dominant fish through the environmental DNA and assigns according to the deviation degree index of the dominant fish;

f. conservation index of important protective species

The index is classified by monitoring the ratio of the current value of the number of species of the important protection fish to the standard value through the environmental DNA.

g. Foreign invasive species index

The index is assigned by collecting or investigating a historical and current wading foreign invasive species situation assignment table;

h. benthonic animal BMWP index

The index is calculated according to the environmental DNA monitoring result and then assigned according to the benthonic animal BMWP index assignment table.

i. Benthonic Hilsenhoff index (BI biological index)

The index is assigned according to a BI biological index assignment table after being calculated according to an environmental DNA monitoring result;

j. Shannon-Wiener (Shannon-Wiener) diversity index

The index is assigned according to a shannon-wiener diversity index assignment table after being calculated according to an environmental DNA monitoring result;

k. uniformity index (Evenness)

The index is assigned according to a uniformity index assignment table after being calculated according to the environmental DNA monitoring result.

8. The method for water ecology monitoring and comprehensive evaluation based on the environmental DNA technology of claim 1, wherein the method comprises the following steps: in the fifth step, the method for calculating the final score and grade of the target river water ecology evaluation comprises the following steps:

The comprehensive index method is adopted to carry out comprehensive evaluation on the quality of the water ecology environment, the water resource, the water environment, the water habitat and the water biology indexes are weighted and summed to construct a comprehensive evaluation index WEQI of the quality of the water ecology environment, the index is used for representing the overall quality condition of each evaluation unit and the water environment, and the calculation formula of the comprehensive evaluation index WEQI of the quality of the water ecology environment is as follows:

wherein: WEQI-comprehensive evaluation index of water ecological environment quality;

xi-an evaluation index score;

wi-evaluation index weight

The water ecological environment quality grade is classified into five grades according to the value of the water ecological environment quality comprehensive evaluation index (WEQI).