A detection method and application of soil contaminated by cadmium / arsenic and microplastics

Abstract

The present invention belongs to the technical field of soil pollutant detection and environmental governance, and discloses a detection method and application of cadmium / arsenic and microplastic composite contaminated soil. The detection method is first based on known detection or experimental data, and analyzes the combined toxicity effect of in-situ exposure and cultivation of earthworms in composite contaminated soil formed by different cadmium / arsenic and microplastics in different occurrence scale ranges, and the concentration of cadmium / arsenic in the soil, the main distribution characteristics of microplastics and the occurrence scale range, the main distribution characteristics and concentration of cadmium / arsenic at the level of earthworm organs, and the corresponding relationship between the six elements. Then, according to the corresponding relationship, the composite contaminated soil to be tested is tested, and a group of six elements can be directly obtained by substituting part of the detection data into the detection data, without physically separating and separately detecting the microplastics in the soil. The present invention can be widely used in technical fields such as environmental pollutant detection and ecological environment governance.

Description

Technical Field

[0001] The present invention relates to the technical field of soil pollutant detection and environmental governance, and in particular to a detection method and application of cadmium / arsenic and microplastic composite contaminated soil based on combined toxicity effect and in situ bioimaging technology. Background Art

[0002] Microplastics (MPs) refer to plastic fragments or particles with a particle size of less than 5 mm, which are mainly formed by the cleavage of plastic products through physical, chemical and biological effects in the environment. Microplastics can be further divided into micron-sized microplastics and nano-sized microplastics according to their size. Plastic fragments discarded in the environment are decomposed into microplastics (<5 mm) and even nanoplastics (<100 nm) through mechanical wear, ultraviolet radiation, weathering and biodegradation. Common microplastics are mainly composed of several common polymers, including polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyethylene terephthalate (PET), etc., and can be divided into fragments, particles, fibers and films in morphology. They all have carbon skeletons that are resistant to hydrolysis and enzymatic hydrolysis, resulting in strong biological stability, difficulty in degradation, and long-term existence in the environment. Microplastics have been frequently detected in a variety of environmental media and organisms, which can cause many toxic effects such as oxidative stress, abnormal energy metabolism and histopathological damage in animals.

[0003] Over the years, a large number of plastic mulches have been widely used in farmland. With weathering and mechanical action of the soil, the mulches will break into micron or nano-scale fragments in the soil, making agricultural plastic mulches one of the most important sources of microplastics in farmland soil. Existing studies have found that with the large-scale enrichment of microplastics in the soil, in some areas with heavy metal pollution such as cadmium / arsenic, microplastics and heavy metal pollution coexist extensively in the soil ecosystem. Microplastics can absorb pollutants such as heavy metals in the soil, thereby producing complex pollution, which has complex toxicity (combined toxicity effect) to organisms in the soil. In the article "Progress in the Study of Complex Toxicity of Microplastics" (Guangdong Chemical Industry, Issue 20, 2022), researchers found that because microplastics are easily ingested by various marine organisms and enter the food chain, and even enter the body with diet, threatening human health, and their huge specific surface area enables microplastics to absorb many pollutants, becoming a "carrier" in the process of pollutant migration in the ecological environment, forming a more complex complex pollution, affecting the environmental behavior and toxic effects of pollutants in the ecological environment. Therefore, the interaction between microplastics and pollutants such as heavy metals has become a research hotspot in environmental pollution control and ecological environmental risk assessment. This paper discusses the adsorption effect of microplastics on heavy metals and organic pollutants, the synergistic or antagonistic toxicity mechanism of microplastics on heavy metals and organic pollutants, and the transfer of microplastics in the food chain of courtyard soil-earthworms and soil-chicken, which enables microplastics to transfer through the food chain and accumulate at higher trophic levels, thereby posing potential health risks to humans. At the same time, different organisms are selective in the ingestion and accumulation of microplastics of different sizes. Therefore, detecting the occurrence size of microplastics in soil is of great significance for accurately studying their biological hazards and targeted governance. However, there is currently a lack of simple and effective means to detect the occurrence size of microplastics in soil.

[0004] In the article "Combined pollution of microplastics and heavy metals in soil: principles and processes" (Journal of Agricultural Environmental Science, 2023, 42 (8): 1675-1684), researchers found that microplastics in soil can combine with heavy metal pollutants to form combined pollution, thereby changing the environmental behavior and toxic effects of single pollutants, causing serious harm to soil ecosystems. At present, people's understanding of the combined toxic effects of microplastics and heavy metals, their formation principles, and pollution processes is still very limited. This article reviews the combined environmental behavior of microplastics and heavy metals in the soil environment, discusses the principles and processes of adsorption and desorption of heavy metals by microplastics, sorts out the migration and transformation of microplastics and heavy metals in the soil environment, and summarizes the latest research progress on the combined toxic effects of the two. Therefore, the type, concentration, and scale of microplastic polymers, the type and concentration of heavy metals, etc. will affect the effect of microplastics on the bioavailability of heavy metals. Accurately assessing the ecological risks caused by the combined pollution of microplastic scale and heavy metals in soil requires comprehensive consideration of the above factors, which inevitably leads to complex and cumbersome detection methods.

[0005] In the prior art, the detection of the occurrence scale of microplastics in the soil requires that the microplastics be physically separated and processed separately, and collected as much as possible before detection can be carried out. For example, Chinese invention application CN202410063368.6 discloses a method for separating microplastics in a soil environment and assessing pollution risks, which includes the following steps: (1) using a density separation method to separate microplastics in a soil environment; (2) using a stereoscope to count the abundance and size of microplastics; (3) calculating the geoaccumulation index to conduct an ecological risk assessment; (4) calculating the estimated daily intake of humans at different age stages to conduct a human health risk assessment. Therefore, before detection, the microplastics must be physically separated from the soil before their size can be detected, making the detection process cumbersome and costly.

[0006] Since microplastics are widely present in farmland soil, their size distribution is mostly between 0.03-5mm, and their storage shapes (film, fiber, fragments, particles) are diverse. The storage depth is between 0-30cm. The main polymer types are polypropylene (PP), polyethylene (PE), polystyrene (PS), nylon (PA), polyethylene terephthalate (PET). Microplastics affect the apparent state of plants, the health of soil animals and the diversity of soil microorganisms, thus posing a threat to the health of soil ecosystems. The toxicity of microplastics is closely related to factors such as their particle size, type, shape, exposure dose and type. The toxicity of PE microplastics to soil animals mainly depends on their particle size, shape and concentration. Among them, nano-scale microplastics are more easily internalized by cells and cause cytotoxicity. Since the diameter of muddy and sandy soil particles is less than 2 mm, and the diameter of fine sand-level soil particles is between 0.02-0.2 mm, and nano-scale (10-1000 nm) and micro-scale (1-10 μm) microplastics are much smaller than the particle size of soil particles, and have a large specific surface area and strong adhesion, it is actually difficult to completely physically separate and effectively collect nano-scale and micro-scale microplastics in the soil, and then detect them. As a result, there is still a technical gap in the effective detection of the main occurrence scales of micro-scale and nano-scale microplastics in contaminated soil by existing conventional detection technology. Due to the above-mentioned difficult-to-solve technical difficulties, the existing research results and detection technologies have not involved the problem of synchronous detection of the occurrence size of microplastics (especially micro-scale and nano-scale microplastics) in soil contaminated by cadmium / arsenic and microplastics. The existing technology is also difficult to fundamentally solve the technical difficulties of effective physical separation and collection of micro-scale and nano-scale microplastics in soil, and it is also unable to solve the technical problems of the variety and composition of microplastics. However, compared with millimeter-sized microplastics, nano- and micron-sized microplastics pose the highest risk to ecological and environmental health. If they are not tested, it will be difficult to meet the needs of ecological and environmental health risk monitoring and precise ecological and environmental governance. Summary of the invention

[0007] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to propose a new detection method and application of cadmium / arsenic and microplastic composite contaminated soil, and to propose a new technical concept, which adopts a combined toxicity effect and in situ biological imaging technology, and utilizes the synchronous change correspondence law between multiple elements to detect the main occurrence scale range of microplastics in composite contaminated soil. There is no need to carry out complex physical separation and separate detection of microplastics, which fundamentally solves the problem that microplastics cannot be effectively separated and collected from the soil, greatly simplifies the detection steps, can fill the technical gap in the detection of the occurrence scale of microplastics in composite contaminated soil, and meet the needs of ecological environmental health risk monitoring and precise ecological environmental governance.

[0008] The technical solution provided by the present invention to solve the above problems is:

[0009] A method for detecting soil contaminated by cadmium / arsenic and microplastics, which first analyzes the combined toxicity effects of in-situ exposure to cultured earthworms in the composite contaminated soil formed by different cadmium / arsenic and microplastics in different occurrence scale ranges based on known detection or experimental data, and the corresponding relationship between the synchronous changes of the six elements, namely, the concentration of cadmium / arsenic in the soil, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, the main distribution characteristics of microplastics at the earthworm organ level and the main occurrence scale range (also called the main occurrence size range or the main occurrence particle size range), and then analyzes the corresponding relationship based on the synchronous changes. The composite polluted soil to be tested is tested according to the law; the concentration of cadmium / arsenic in the composite polluted soil of cadmium / arsenic and microplastics, the organ level distribution position of cadmium / arsenic on the earthworm cross-sectional slice, the cadmium / arsenic concentration data at different organ positions, and the main distribution characteristic data of microplastics at the earthworm organ level obtained by actual detection are substituted into the synchronous change correspondence law, and after consistency analysis, the main occurrence scale range data of microplastics in the composite polluted soil to be tested are obtained, and a complete set of six interrelated detection data are obtained, without the need to physically separate and separately detect the microplastics in the soil.

[0010] The corresponding relationship law of the synchronous changes is: there is a significant joint toxic effect between the combined pollution of cadmium / arsenic in the soil and microplastics with a main occurrence size of no more than 10 μm, and under the same cadmium / arsenic concentration, compared with the single pollution of cadmium / arsenic, as the main occurrence size range of microplastics decreases, the joint toxic effect of microplastics and cadmium / arsenic is enhanced along the path of in vitro-in vivo-body cavity-tissue and organ-circulatory system, and finally enters the tissue or organ and is enriched.

[0011] The different occurrence scale ranges of the microplastics are divided into two qualitative levels: nanoscale and microscale, where the main occurrence scale range of the nanoscale is 10-1000nm, and the main occurrence scale range of the microscale is 1-10μm; the main occurrence scale range refers to microplastics within this scale range, accounting for more than 70% of the mass ratio of all microplastics in the soil;

[0012] The specific laws of the synchronous change correspondence between the six elements are as follows:

[0013] When microplastics with cadmium complex contamination exist in the soil, compared with single cadmium pollution, the smaller the main occurrence scale of microplastics, the stronger the combined toxic effect, and the faster the growth inhibition rate increases; microplastics with a main occurrence scale range of 1-10μm, through the combined toxic effect, make the microplastics mainly concentrated in the body cavity and organ walls of earthworms, and at the same time transfer most of the cadmium from the digestive organs to the earthworm circulatory system, mainly distributed in the dorsal blood vessels, ventral blood vessels and subcutaneous tissue of earthworm slice samples; while microplastics with a main occurrence scale range of 10-1000nm further allow more microplastics to enter and concentrate in the subcutaneous tissue of earthworms, and also cause a large amount of cadmium in the earthworm body to enter and be mainly enriched in the subcutaneous tissue of earthworms, and no longer continue to diffuse to the digestive organs and circulatory system;

[0014] When microplastics contaminated with arsenic exist in the soil, compared with arsenic single pollution, the smaller the main occurrence scale range of microplastics, the stronger the combined toxicity effect, and the faster the growth inhibition rate increases; under the conditions of microplastic and arsenic combined pollution with the main occurrence scale range of 1-10μm, arsenic is mainly concentrated in the intestinal cavity and the folds of the longitudinal muscle of the earthworm, and microplastics are mainly concentrated in the earthworm epidermis and intestinal cavity; under the conditions of 10-1000nm microplastic and arsenic combined pollution, microplastics are mainly concentrated in the subcutaneous tissue of the earthworm, and arsenic is mainly concentrated in the yellow tissue and dorsal blood vessels of the earthworm.

[0015] The method for detecting soil contaminated by cadmium / arsenic and microplastics comprises the following steps:

[0016] S1: Obtain the corresponding relationship between the synchronous changes of the combined toxic effects at the sample organ level

[0017] Obtain test data of multiple earthworm slice samples exposed to soils with different concentrations of cadmium / arsenic and different occurrence scales of microplastics, and compare and analyze the test data of earthworm slice samples exposed to soils with the same concentration of single cadmium / arsenic pollution and no microplastics. Obtain the concentrations of cadmium / arsenic in soil, the occurrence scale range of microplastics, the combined toxic effects, the main distribution characteristics and concentrations of cadmium / arsenic at the earthworm organ level, the main distribution characteristics of microplastics at the earthworm organ level, and the corresponding relationship between the synchronous changes of the six factors (synchronous change trends) in multiple different composite polluted soils and single polluted soils.

[0018] S2: Detection of composite contaminated soil samples

[0019] Collect soil contaminated with cadmium / arsenic and microplastics that has at least millimeter-sized microplastics and prepare it as a soil sample to be tested. First, detect the concentration of cadmium / arsenic in the soil sample to be tested. Then, in situ expose and cultivate living earthworms in the soil to determine the growth inhibition rate. Then, prepare the earthworms into multiple cross-sectional slice samples, obtain image data of the distribution positions of cadmium / arsenic and microplastics on each earthworm cross-sectional slice sample, and detect the cadmium / arsenic concentrations at different organ positions of the multiple slice samples to obtain the distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level.

[0020] S3: Comparative analysis to obtain a set of correlation detection data

[0021] The five element data of cadmium / arsenic concentration in the soil, combined toxicity effect, main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, and main distribution characteristics of microplastics at the earthworm organ level detected in step S2 are substituted into the synchronous change correspondence law in step S1, and a comparative analysis is performed based on the changing trend of the law to obtain a group of associated data with the highest consistency with the five element associated data in the soil sample to be tested, including the main occurrence scale range data of microplastics, to complete the detection without physically separating the microplastics in the soil and testing them separately.

[0022] A method for detecting soil contaminated by cadmium / arsenic and microplastics and its application in soil microplastic detection and ecological management.

[0023] The detection method and application of cadmium / arsenic and microplastic composite contaminated soil provided by the present invention, compared with the prior art, has at least the following beneficial effects:

[0024] 1. The detection method and application provided by the present invention, in order to solve the problem that nano- and micron-sized microplastics are difficult to separate, collect and detect, proposes a new technical concept, which adopts the combined toxicity effect and in situ bioimaging technology, firstly analyzes the combined toxicity effect of in situ exposure and cultivation of earthworms in the composite contaminated soil formed by different cadmium / arsenic and microplastics in different occurrence scale ranges, and the synchronous change correspondence relationship between the six elements of cadmium / arsenic concentration in the soil, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, the main distribution characteristics of microplastics at the earthworm organ level and the occurrence scale range, and then detects the composite contaminated soil to be tested according to the synchronous change correspondence relationship law, so as to analyze and obtain data such as the main occurrence scale range of microplastics in the soil to be tested, without the need for complex physical separation and separate detection of microplastics, which greatly simplifies the detection steps, fundamentally solves the problem that microplastics cannot be effectively separated, collected and detected from the soil, can fill the technical gap in the occurrence scale detection of microplastics in composite contaminated soil, and meet the needs of ecological environmental health risk monitoring and precise ecological environmental governance.

[0025] 2. The detection method and application provided by the present invention break through the technical bias of existing research that microplastics in soil must be separated first, accurately collected and then subjected to relevant tests. It makes full use of known data or experimental data, based on the combined toxicity effect, in situ bioimaging technology and the correlation between data changes of various factors, and analyzes the law of synchronous changes from the data obtained from multiple tests, and then uses this law to detect unknown contaminated soil, completely overcoming the technical difficulties of the existing technology that it is actually difficult to completely separate and accurately collect nano- and micron-sized microplastics. Taking the main occurrence scale of microplastics in composite contaminated soil as a breakthrough, an ingenious method is used to make the obtained detection data and analysis results fully meet the needs of ecological environmental health risk monitoring and precise ecological environmental governance, thereby greatly simplifying the detection method and detection cost of composite contaminated soil, which is conducive to large-scale promotion and application.

[0026] 3. The present invention proposes for the first time a method for detecting the main size of microplastics in soil using cross-sections of earthworms grown in situ in contaminated soil. Focusing on the technical difficulties of existing technologies in completely separating and collecting microplastics in soil, the present invention proposes detection based on LA-ICP-MS, and based on the synchronization and correlation of changes among multiple factors that are easy to detect, the main size range of soil microplastics can be obtained after analysis. Using this method, on the one hand, detection personnel can quickly and accurately obtain the main size range of microplastics in the soil of contaminated sites, which is very important. Researchers can use this sample and detection data to further study and analyze the ecotoxicological effects and mechanisms of complex contaminated soils. On the other hand, it provides the possibility that microplastics can serve as cadmium / arsenic carriers, increase the biological effectiveness of cadmium / arsenic to soil organisms, and provide data support for the risk control research of complex contaminated soils.

[0027] 4. The detection method provided by the present invention can further use the AI ​​model to deeply learn the close correlation between the combined toxicity of microplastics and other heavy metals and their particle size, type, shape, exposure dose and species. For example, the toxicity of PE microplastics to soil animals mainly depends on their particle size, shape and concentration, so as to obtain a more powerful AI model, conduct more and more detailed detection data analysis, and obtain more research results; using the improved detection method of the present invention, the absorption, transport and accumulation process of microplastics of different sizes in earthworms can be visualized using bioimaging technology, so as to facilitate further qualitative or quantitative research on the effects of microplastics of different occurrence scales on earthworms ( Eisenia Fetida ), and can detect the toxic effects of microplastics in different occurrence scales on organisms from both the physiological and molecular levels, making the test results more credible.

[0028] 5. The detection method and application provided by the present invention can intuitively analyze in situ the impact of microplastics in the soil on the exposure concentration of heavy metals in organisms, and can be widely used in scientific research, teaching, contaminated soil remediation and other fields, broadening the scope of application and meeting the needs of ecological environmental health risk monitoring and precise ecological environmental governance; and it is low-cost, high-efficiency, and has strong practicality and economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the occurrence scale range of different microplastics and the distribution of cadmium / arsenic in the slice samples of the embodiment of the present invention;

[0030] Figure 2 This is a schematic diagram of the detection results of the main occurrence scale range of microplastics in soil obtained in an embodiment of the present invention;

[0031] Figure 3This is a schematic diagram showing the effect of soils in different treatment groups on earthworm growth inhibition rate in an embodiment of the present invention;

[0032] Figure 4 This is a schematic diagram of the effect of soil from different plots on the earthworm growth inhibition rate in an embodiment of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] The experimental supplies, instruments and techniques used in the following embodiments of the present invention are commercially available and conventional techniques unless otherwise specified.

[0035] Example 1

[0036] See also Figures 1 to 3 The present invention provides a method for detecting soil contaminated by cadmium / arsenic and microplastics. The method selects farmland soil contaminated by cadmium and microplastics or arsenic and microplastics around a chemical enterprise as the implementation scenario, and captures native earthworms for detection, which fundamentally solves the problem that microplastics cannot be effectively separated, collected and detected from the soil. The earthworm used is Eisenia fetida, which has soil environment indicators. (Eisenia fetida)。

[0037] The detection method of cadmium / arsenic and microplastic composite contaminated soil provided in this embodiment is to first analyze the combined toxicity effect of in situ exposure cultivation of earthworms in composite contaminated soil formed by different cadmium / arsenic and microplastics in different occurrence scale ranges based on existing relevant detection data or specially collected experimental data, and the synchronous change correspondence relationship law among the six elements, namely, the concentration of cadmium / arsenic in the soil, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, the main distribution characteristics and main occurrence scale range of microplastics at the earthworm organ level, and then detect the composite contaminated soil to be tested according to the synchronous change correspondence law; the concentration of cadmium / arsenic in the composite contaminated soil of cadmium / arsenic and microplastics to be tested, the organ level distribution position of cadmium / arsenic on the earthworm cross-sectional slice, the cadmium / arsenic concentration data at different organ positions, and the main distribution characteristics data of microplastics at the earthworm organ level obtained by actual detection are substituted into the synchronous change correspondence law, and the main occurrence scale range data of microplastics in the composite contaminated soil to be tested are obtained after consistency analysis, so as to obtain a complete set of detection data with six elements interrelated with each other, without the need to physically separate the microplastics in the soil and separately detect their main occurrence scales.

[0038] Through preliminary data analysis and induction, the corresponding relationship law of synchronous changes is obtained: there is a significant joint toxic effect between the combined pollution of cadmium / arsenic in the soil and microplastics with a main storage size of no more than 10μm, and under the same cadmium / arsenic concentration, compared with the single pollution of cadmium / arsenic, as the main storage size range of microplastics decreases, the joint toxic effect of microplastics and cadmium / arsenic is enhanced along the path of in vitro-in vivo-body cavity-tissue and organ-circulatory system, and finally enters the tissue or organ and is enriched.

[0039] Existing studies have shown that the combined toxic effect of cadmium / arsenic in the soil and microplastics with a main size greater than 10 μm under complex pollution conditions is significantly smaller than that of microplastics below 10 μm; since microplastics with a main size greater than 10 μm are relatively easy to separate and collect by physical methods, there is no need to use the detection method of the present invention; at the same time, microplastics larger than 10 μm are larger in size and difficult to enter the earthworm's body through ingestion and skin absorption, and are therefore not suitable for the detection method provided by the present invention.

[0040] This embodiment divides the different occurrence scale ranges of the microplastics into two qualitative levels: nanometer and micrometer, where the main occurrence scale range of the nanometer is 10-1000nm, and the main occurrence scale range of the micrometer is 1-10μm; the main occurrence scale range refers to the microplastics within this scale range, accounting for more than 70% of the mass ratio of all microplastics in the soil (or other definitions are given). Microplastics smaller than 10nm are no longer included in the detection range because they account for too low a proportion and are difficult to identify.

[0041] The method for detecting soil contaminated by cadmium / arsenic and microplastics provided in this embodiment comprises the following steps:

[0042] S1: Obtain the corresponding relationship between the synchronous changes of the combined toxic effects at the sample organ level

[0043] The test data of multiple earthworm slice samples exposed and cultured in soils with different concentrations of cadmium / arsenic and microplastics with different occurrence scales were obtained respectively, and compared with the test data of earthworm slice samples exposed and cultured in soils with single cadmium / arsenic pollution and no microplastics with the same concentration, as well as other existing relevant test data collected, and compared and analyzed to obtain the concentration of cadmium / arsenic in soil, the occurrence scale range of microplastics, the combined toxicity effect, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, the main distribution characteristics of microplastics at the earthworm organ level, and the synchronous change correspondence relationship law (synchronous change trend) among the six elements in multiple different composite polluted soils and single polluted soils respectively. The synchronous change correspondence relationship law among the six elements obtained in this embodiment is specifically as follows:

[0044] When microplastics with cadmium complex contamination exist in the soil, compared with single cadmium pollution, the smaller the main occurrence scale of microplastics, the stronger the combined toxic effect, and the faster the growth inhibition rate increases; microplastics with a main occurrence scale range of 1-10μm, through the combined toxic effect, make the microplastics mainly concentrated in the body cavity and organ walls of earthworms, and at the same time transfer most of the cadmium from the digestive organs to the earthworm circulatory system, mainly distributed in the dorsal blood vessels, ventral blood vessels and subcutaneous tissue of earthworm slice samples; while microplastics with a main occurrence scale range of 10-1000nm further allow more microplastics to enter and concentrate in the subcutaneous tissue of earthworms, and also cause a large amount of cadmium in the earthworm body to enter and be mainly enriched in the subcutaneous tissue of earthworms, and no longer continue to diffuse to the digestive organs and circulatory system;

[0045] When microplastics contaminated with arsenic exist in the soil, compared with arsenic single pollution, the smaller the main occurrence scale range of microplastics, the stronger the combined toxicity effect, and the faster the growth inhibition rate increases; under the conditions of microplastic and arsenic combined pollution with the main occurrence scale range of 1-10μm, arsenic is mainly concentrated in the intestinal cavity and the folds of the longitudinal muscle of the earthworm, and microplastics are mainly concentrated in the earthworm epidermis and intestinal cavity; under the conditions of 10-1000nm microplastic and arsenic combined pollution, microplastics are mainly concentrated in the subcutaneous tissue of the earthworm, and arsenic is mainly concentrated in the yellow tissue and dorsal blood vessels of the earthworm.

[0046] Step S1 further includes the following specific steps:

[0047] S1-1: Prepare a single contaminated soil of cadmium / arsenic with a set concentration, then in situ expose and cultivate earthworms in the soil, determine the growth inhibition rate, and then prepare and test earthworm slice samples to obtain the main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level, and obtain a set of interrelated test data of four elements (the concentration of cadmium / arsenic in the soil, the main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level) under the condition of the single contaminated soil;

[0048] S1-2: Add microplastics of known main occurrence scale range to the single cadmium / arsenic contaminated soil with the concentration set in S1-1 to prepare composite contaminated soil, then expose and cultivate earthworms in situ in the soil, determine the growth inhibition rate, and then prepare and test earthworm slice samples to obtain the concentration of cadmium / arsenic in the soil, the occurrence scale range of microplastics, the main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level, and obtain a set of six interrelated test data (cadmium / arsenic concentration in the soil, growth inhibition rate, microplastic occurrence scale range, main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level, and main distribution characteristics of microplastics at the earthworm organ level) under the conditions of composite contaminated soil;

[0049] S1-3: Changing the soil pollution conditions in steps S1-1 and S1-2, setting different cadmium / arsenic concentrations, adding different known main occurrence scale ranges of microplastics, repeating steps S1-1 to S1-3 multiple times, and obtaining multiple sets of four-element or six-element interrelated detection data respectively;

[0050] S1-4: Obtain the synchronous changes of the combined toxic effects at the sample organ level

[0051] Based on the combined toxicity effect, a comparative analysis was conducted on multiple groups of data obtained from steps S1-1 to S1-3 to obtain the concentration of cadmium / arsenic in the soil, the growth inhibition rate, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, and the corresponding change relationship between the main distribution characteristics of microplastics at the earthworm organ level and the main occurrence scale range data, that is, the synchronous change correspondence law between the six element data at the sample organ level.

[0052] S2: Detection of composite contaminated soil samples

[0053] In the area of ​​the plot that needs to be tested and analyzed, collect soil contaminated with cadmium / arsenic and microplastics that is visually detected to have at least millimeter-level microplastics, prepare it as a soil sample to be tested, first detect the concentration of cadmium / arsenic in the soil sample to be tested, then expose and cultivate living earthworms in situ in the soil, determine the growth inhibition rate, and then prepare the earthworms into multiple cross-sectional slice samples, obtain the distribution position image data of cadmium / arsenic and microplastics on each earthworm cross-sectional slice sample, and detect the cadmium / arsenic concentration at different organ positions of the multiple slice samples to obtain the distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level; specifically comprising the following steps:

[0054] S2-1: Collect sufficient amount of soil contaminated by cadmium / arsenic and microplastics with visual detection of millimeter-sized microplastics to prepare the soil sample to be tested;

[0055] S2-2: Perform sample pretreatment by first using conventional methods to detect the concentrations of cadmium and arsenic in the soil samples to be tested;

[0056] S2-3: Expose the cultivated earthworms in situ in the soil sample to be tested and measure the growth inhibition rate;

[0057] S2-4: Prepare the earthworms cultivated in situ in the soil into multiple cross-sectional slice samples, obtain the distribution position image data of cadmium / arsenic and microplastics on each earthworm cross-sectional slice sample, and detect the cadmium / arsenic concentrations at different organ positions of the multiple slice samples to obtain the distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level.

[0058] S3: Comparative analysis to obtain a set of correlation detection data

[0059] The five element data of cadmium / arsenic concentration in the soil, combined toxicity effect, main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, and main distribution characteristics of microplastics at the earthworm organ level detected in step S2 are substituted into the synchronous change correspondence law in step S1, and a comparative analysis is performed based on the changing trend of the law to obtain a group of associated data with the highest consistency with the five element associated data in the soil sample to be tested, including the main occurrence scale range data of microplastics, to complete the detection without physically separating the microplastics in the soil and testing them separately.

[0060] The in-situ exposure cultivation of earthworms in the soil is to cultivate living earthworms in the soil, allow them to grow naturally until they become adult earthworms with a complete reproductive ring structure, and then directly capture or trap them;

[0061] The preparation of earthworm slice samples comprises the following steps:

[0062] A1: Sample processing of fresh adult earthworms: The captured fresh earthworms were cleaned of their intestines, anesthetized with alcohol, and then fixed with 4% paraformaldehyde;

[0063] A2: Sectioning: After the head and reproductive ring of the adult earthworm sample are embedded in paraffin, multiple slices are cut continuously along the cross section according to the set thickness to obtain multiple tissue sections;

[0064] A3: Tissue section pretreatment

[0065] The tissue sections were dewaxed in a gradient manner using a transparent agent (TO), anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, and distilled water for 10 minutes in each step to obtain the dewaxed sections. A number of complete sections were then screened out using an ordinary optical microscope as the section samples to be tested in preparation for the machine.

[0066] The slice sample detection comprises the following steps:

[0067] B1: Slice sample cross-section imaging measurement

[0068] First, collect the image data of the cross section of the sample to be tested (save and apply to image feature extraction and analysis later), then use LA-ICP-MS surface scanning to determine the cumulative distribution of cadmium or arsenic and obtain the surface scanning results; LA-ICP-MS setting parameters are: Energy: 3.5jcm -2 , beam spot: 20um, frequency: 20Hz, speed: 30ums -1 ;

[0069] B2: Interpretation of cross-sectional measurement data of sliced ​​samples

[0070] The surface scan results of the sliced ​​samples to be tested were processed. The surface scan results were calibrated for cadmium / arsenic content using the "TraceElements" module of the iolite software and SRM610 as an external standard;

[0071] Use iolite's Imaging module to map the corrected matrix data, including adjusting the image resolution, contrast, image color, and scale;

[0072] Then, the surface scan images and matrix data of each element were output respectively to obtain the distribution data of cadmium / arsenic at the organ level on the cross section of earthworm slices.

[0073] For the image data of the two cross sections (two sides) of the collected slice samples, appropriate conventional graphic image analysis tools can be used to extract the characteristic information and perform characteristic analysis to obtain the required data; especially during the experiment, the distribution characteristics of microplastics in different main occurrence scale ranges in the cross-sectional slices of earthworms can be obtained through graphic image analysis tools, and their change patterns can be further analyzed. In the subsequent detection of new soil samples and slice samples to be tested, a variety of distribution characteristic data can be obtained through graphic image analysis technology, and substituted into the synchronous change correspondence relationship pattern. Through calculation, the results can be directly obtained to simplify the detection steps.

[0074] The application of the above-mentioned detection method for cadmium / arsenic and microplastic composite contaminated soil in soil microplastic detection and ecological management can provide corresponding data support for qualitative detection of the main occurrence scale range of microplastics in soil, joint toxicity analysis and ecological management.

[0075] The embodiment of the present invention first determines the corresponding relationship between the synchronous changes of multiple factors, and then determines the heavy metal distribution characteristics of the earthworm cross section in the composite polluted soil. The method mainly adopts a combination of in vitro detection and biological exposure experiments, and synchronous in situ imaging (laser confocal microscopy + laser etching) in vivo. The earthworms naturally growing in situ in the contaminated soil are captured, and the cross-sectional slice samples of their heads are selected. The distribution data of cadmium / arsenic and microplastics in the cross-sectional slices of the earthworms are obtained by using graphic image analysis and LA-ICP-MS. The cadmium / arsenic concentrations in different organ positions in different slices are analyzed to obtain the main distribution characteristics of cadmium / arsenic and microplastics at the organ level of the earthworms. Based on the combined toxicity effect, the distribution characteristics of cadmium / arsenic and microplastics of earthworms exposed to single cadmium / arsenic pollution are compared and analyzed (consistency analysis), so as to obtain the main existence size of microplastics in the composite polluted soil, without the need for the complicated operation steps of first separating and then detecting the microplastics in the soil, which fundamentally solves the problem of separation and collection of nano- and micro-scale microplastics.

[0076] The embodiment of the present invention uses bio-imaging technology to visualize the absorption, transport, and accumulation process of microplastics of different sizes in earthworms. It can also be further applied to study the combined toxic effects of microplastics of different sizes and heavy metals on earthworms under complex pollution conditions.

[0077] This example analyzes the cumulative distribution and toxic effects of nano-MPs and micro-MPs in the soil environmental indicator organism Eisenia fetida, and reveals in situ the absorption, transport, distribution characteristics, combined toxic effects and toxicity mechanisms of different-sized MPs and Cd or As complex pollutants in the earthworm. This law is further used to detect the main occurrence scale range of nano-MPs and micro-MPs in the soil to be tested, eliminating the steps of physical separation and collection of microplastics.

[0078] In this embodiment, when collecting detection data through a specially designed experiment, soil with known pollutant parameters is first prepared, earthworms are cultivated in situ in the composite contaminated soil, and the growth inhibition rate is determined; then adult earthworms growing naturally in situ in the contaminated soil are captured, and their head cross-sections are selected after paraffin embedding, and LA-ICP-MS is used to obtain the distribution data of cadmium / arsenic in the cross-sectional slices of earthworms, and then the cadmium / arsenic concentrations in different organ positions in different slices are analyzed to obtain the main distribution characteristics and concentration data of cadmium / arsenic and microplastics at the organ level of earthworms, and compared and analyzed with the cadmium / arsenic distribution characteristics and concentration data of earthworms exposed to single cadmium / arsenic pollution, the corresponding change relationship between multiple elements (parameters) can be obtained. According to this law, and some elements (parameters) of the soil to be tested, the main occurrence scale range of microplastics in the soil to be tested can be obtained. In other embodiments, relevant existing detection data can also be collected and utilized in various ways.

[0079] In this embodiment, the specific process of preparing the test sample is as follows:

[0080] For every 5 adult earthworms with reproductive rings (Eisenia fetida Eisenia Fetida ) were placed in containers containing 250g of different treated paddy soil and exposed for 14 days, and the growth inhibition rate of earthworms in each treatment was measured. The concentrations of 70nm, 1μm, and 10μm PS-MPs were all set to 0.9% (w / w), and the Cd concentration was set to 10mg / kg -1 , the As concentration was set to 5 mg / kg -1 The specific processing settings are shown in Table 1.

[0081] Table 1 Treatment group settings for the combined toxicity exposure experiment

[0082]

[0083] After 12 hours of bowel cleaning of adult earthworms exposed and cultured for 14 days, two fresh samples were taken from each treatment. After anesthesia with alcohol, they were fixed with 4% paraformaldehyde. The head and reproductive ring of the earthworm were embedded in paraffin, and multiple tissue slices with a thickness of 10 μm were cut continuously along the cross section. The cross section of the 40th ring of the head was taken, and 5 slices were cut continuously. The slices with complete shapes were selected with an ordinary optical microscope for pretreatment and testing.

[0084] The steps of slice preprocessing are:

[0085] The slices were washed with TO (transparent agent), anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, and distilled water in sequence, with each step washing for 10 minutes. After gradient dewaxing, they can be used as slice samples for observation on the machine.

[0086] The steps for imaging and measuring the cross section of a sliced ​​sample are:

[0087] LA-ICP-MS was used to measure the cumulative distribution of cadmium or arsenic in each section of the sample, and the graphic image data of the corresponding section was obtained and then interpreted; the LA-ICP-MS setting parameters were: Energy: 3.5 J cm -2 , beam spot: 20um, frequency: 20Hz, speed: 30ums -1 ;

[0088] The steps for interpreting the cross-sectional measurement data of a sliced ​​sample are:

[0089] The surface scan results of the sliced ​​samples were analyzed using the "Trace Elements" module of the iolite (Paton et al., 2011) software, with SRM610 as the external standard, to calibrate the cadmium / arsenic content (including signal background selection, sample effective range selection, instrument sensitivity calibration, and element content calculation); the calibrated matrix data were mapped using the iolite Imaging module, including manual adjustments to the image resolution, contrast, image color, and scale; finally, the surface scan images and matrix data of each element were output separately to obtain the distribution status of cadmium / arsenic in all treatments on the earthworm cross section; through the analysis of a large number of earthworm cross-section slice data, it was found that there was a strong correlation between the changes in the cumulative distribution of cadmium / arsenic in earthworm organs and the main size range of microplastics. The experimental results are as follows: Figure 1 As shown, the qualitative detection results of whether the main occurrence scale of microplastics in the soil is nanometer or micrometer can be accurately obtained.

[0090] After multiple experiments and analyses, this example found that under the conditions of 10μm microplastics and cadmium composite pollution, the cadmium in the earthworm body is mainly distributed in the dorsal blood vessels, ventral blood vessels, gizzard and metanephric duct, with a small amount attached to the epidermis, and the microplastics are mainly concentrated in the earthworm body cavity and organ walls; under the conditions of 70nm microplastics and cadmium composite pollution, the cadmium is mainly distributed in the dorsal blood vessels, ventral blood vessels and subcutaneous tissue of the earthworm, and the microplastics are mainly concentrated in the subcutaneous tissue of the earthworm. Therefore, 10μm microplastics can cause more cadmium to transfer from the digestive organs to the earthworm circulatory system (metanephric duct); 70nm microplastics cause a large amount of cadmium in the earthworm body to be enriched in the subcutaneous tissue, less diffused to the digestive organs and circulatory system, and the toxic effect is faster and stronger. Under the conditions of 10μm microplastics and arsenic combined pollution, arsenic was mainly concentrated in the intestinal cavity and the wrinkled area of ​​the longitudinal muscle of the earthworm, and microplastics were mainly concentrated in the earthworm epidermis and intestinal cavity; under the conditions of 70nm microplastics and arsenic combined pollution, arsenic was mainly concentrated in the yellow tissue and dorsal blood vessels of the earthworm, and microplastics were mainly concentrated in the subcutaneous tissue of the earthworm. Only a small amount of microplastic accumulation was observed in the earthworm body cavity. By observing and comparing the distribution of cadmium / arsenic at the organ level of earthworms, the main occurrence scale range of microplastics in soils contaminated with microplastics and cadmium / arsenic combined pollution can be determined.

[0091] In this embodiment, the cross-sectional measurement data of the earthworm slice sample obtained from the soil to be tested is interpreted, and finally the surface scan map and matrix data of each element are output respectively. The distribution of cadmium / arsenic in the earthworm cross section is obtained as follows: Figure 2 Observation Figure 2 It can be seen that in the soil samples of the cadmium and microplastics composite pollution site, the cadmium in the earthworm is mainly distributed in the dorsal blood vessels, ventral blood vessels, gizzard and metanephric duct, with a small amount attached to the epidermis, and the microplastics are mainly concentrated in the earthworm body cavity and organ walls, indicating that the main occurrence scale of microplastics in this site is 1-10μm; cadmium is mainly distributed in the dorsal blood vessels, ventral blood vessels and subcutaneous tissue of earthworms, and microplastics are mainly concentrated in the subcutaneous tissue of earthworms, indicating that the main occurrence scale of microplastics in this site is 10-1000nm;

[0092] In soil samples from sites contaminated with microplastics and arsenic, arsenic is mainly concentrated in the intestinal cavity and the wrinkled areas of the longitudinal muscles of earthworms, and microplastics are mainly concentrated in the epidermis and intestinal cavity of earthworms, indicating that the scale range of microplastics at this site is 1-10μm; arsenic is mainly concentrated in the yellow tissue and dorsal blood vessels of earthworms, and microplastics are mainly concentrated in the subcutaneous tissue of earthworms. Only a small amount of microplastic accumulation can be observed in the earthworm body cavity, indicating that the scale range of microplastics at this site is 10-1000nm.

[0093] In this example, the growth inhibition rate of earthworms in different treatment groups was tested. Figure 3 Observation Figure 3It can be seen that the toxic effect of soil contaminated with microplastics and cadmium / arsenic is significantly greater than that of soil contaminated with single cadmium / arsenic, and the toxic effect of 70nm microplastics and cadmium / arsenic combined pollution is significantly greater than the toxic effect of 10μm microplastics and cadmium / arsenic combined pollution.

[0094] The technical solution provided by the embodiments of the present invention overcomes the technical difficulties of the prior art that requires complex operations and a variety of precision detection instruments to separate nano- to micron-sized microplastics from the soil and then collect them before detection. There is no need for separate physical separation, collection and detection of microplastics, thus filling the technical gap in this field. The detection samples of the present invention are easy to collect, have a wide range of applications, a simple processing method, and low testing costs. The present invention can meet the needs of ecological environmental health risk monitoring, ecotoxicology research, and precise ecological environmental governance by significantly reducing the detection cost.

[0095] The present embodiment provides an application of the detection method, which is based on the in situ bio-imaging technology, and is used for diversified detection of soil contaminated with cadmium / arsenic and microplastics in different occurrence scale ranges. A combination of in vivo and in vitro methods is used to determine the data of multiple pollutants in the contaminated soil and their combined toxic effects. By obtaining image data of cross-sectional slices of earthworms cultured in situ in the soil and obtaining data such as the distribution characteristics of heavy metals and microplastics in different organs, the data on the main occurrence particle size range of microplastics in soil samples contaminated with cadmium / arsenic and microplastics can be obtained without the need to separate, collect and detect the microplastics separately, which greatly simplifies the detection steps and detection instruments, and improves the accuracy of the detection results.

[0096] Example 2

[0097] The detection method and application of cadmium / arsenic and microplastic composite contaminated soil provided in this embodiment is a further improvement and perfection based on Example 1, which is basically the same as Example 1, except that:

[0098] This embodiment selects the combined pollution of cadmium and microplastics and the combined pollution of farmland soil by arsenic and microplastics downstream of a mining area as implementation scenarios, and captures native earthworms for testing.

[0099] Soil samples with single cadmium / arsenic contamination and soil samples with combined cadmium / arsenic and microplastic contamination were prepared and divided into two groups, and experiments and data collection were carried out separately to improve the accuracy of the obtained synchronous change correspondence law and detection results.

[0100] The steps for preparing the test samples are:

[0101] The contaminated land is divided into several equal-sized areas. The soil samples must be collected along a certain route and according to the principles of "random" and "multi-point mixing". "Random" means that each sampling point is arbitrarily determined, so that all points in the sampling unit have an equal chance of being sampled.

[0102] Distribution form: Sampling points are arranged according to the terrain, area size and fertility uniformity, and five soil samples are collected from each plot. For plots with small area, flat terrain and relatively uniform fertility, the diagonal sampling method, also known as the five-point method, can be used; for plots with medium area and square shape and uneven fertility, the chessboard sampling method, also known as the plum blossom distribution method, should be used; for plots with large area, long or complex shape and uneven fertility, the snake sampling method, also known as the "S" shape distribution method, is used. Generally, the "S" shape distribution sampling is used, which can better overcome the errors caused by agronomic measures such as tillage and fertilization.

[0103] The collected soil samples were mixed and air-dried, and then passed through a 5 mm sieve to ensure that the soil in each plot was no less than 500 g. Water was added to the soil's maximum field water holding capacity and then cultured for 3 days. Ten laboratory-cultured earthworms ( E.fetida ), after a 14-day exposure experiment, the earthworms were washed and cleaned for 12 hours, and the growth inhibition rate of earthworms in the soil of each plot was measured. Two fresh earthworm samples were taken from each contaminated site, anesthetized with alcohol, fixed with 4% paraformaldehyde, and the head and reproductive ring of the earthworm were embedded in paraffin and cut into 10μm tissue slices; the cross section of the 40th ring of the head was taken, and 5 slices were cut continuously, and the most complete slice samples were selected for testing using an ordinary optical microscope.

[0104] When preparing soil and earthworm slice samples, the following steps are further included:

[0105] Two groups of soil samples were prepared, one group was cadmium / arsenic contaminated soil alone (Group A), and the other group was cadmium / arsenic and microplastics composite contaminated soil (Group B). Earthworms were exposed and cultivated in situ and then collected separately. The earthworm samples of Group A and Group B were exposed to the soil of cadmium / arsenic contaminated sites and cadmium / arsenic and microplastics composite contaminated sites for 14 days before being collected and tested.

[0106] Prepare two sets of slice specimens:

[0107] Collect live samples of earthworms from Group A captured in soil contaminated with cadmium or arsenic alone, and from Group B captured in soil contaminated with cadmium, arsenic and microplastics;

[0108] After the captured earthworms were cleaned for 12 h, two live samples were taken from each treatment. After alcohol anesthesia, they were fixed with 4% paraformaldehyde, and the head and reproductive ring of the earthworms were embedded in paraffin and cut into 10 μm tissue slices.

[0109] Take the cross section of the 40th ring of the head, and cut 5 slices in succession with a thickness of 10 μm per slice. Clean the slices in turn with TO (transparent agent), anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, and distilled water, and then dewax them in a gradient manner to obtain the processed slice samples. Finally, use an ordinary optical microscope to screen the most complete slice samples for testing in preparation for observation on the machine.

[0110] The imaging measurement of the cross section of the sliced ​​sample includes the following steps:

[0111] Set the LA-ICP-MS parameters as follows: Energy: 3.5 J cm -2 , beam spot: 20um, frequency: 20Hz, speed: 30ums -1 The processed slice samples were loaded onto the machine, and then the cumulative distribution data of cadmium or arsenic of the two groups of slice samples were processed respectively. After the cumulative distribution of cadmium or arsenic was determined by LA-ICP-MS surface scanning, two groups of cumulative distribution data of cadmium or arsenic were obtained respectively.

[0112] The surface scan results were calibrated for cadmium / arsenic content using the "Trace Elements" module of the iolite (Paton et al., 2011) software, with SRM610 as an external standard (including signal background selection, sample effective interval selection, instrument sensitivity calibration, and element content calculation). The calibrated matrix data were mapped using the Imaging module of iolite, including image resolution, contrast, image color, scale, etc.

[0113] Output two groups of data, A and B, including the surface scan map and matrix data of each element, to obtain the distribution data of cadmium / arsenic and microplastics in the cross section of earthworms respectively; compare and analyze the cadmium / arsenic distribution of group B data with that of group A data to determine the main occurrence scale range data of microplastics in microplastics and cadmium / arsenic composite contaminated soil. The distribution of cadmium / arsenic in the cross section of earthworms obtained in this example is different from Figure 2 The content shown is essentially the same.

[0114] This example shows that the toxic effect of 10-1000nm microplastics and cadmium / arsenic combined pollution is significantly greater than that of 1-10μm microplastics and cadmium / arsenic combined pollution, which is consistent with the results of the determination of the size range of microplastics in the contaminated site through earthworm cross-section image analysis data. Figure 4 shown.

[0115] This example verifies the feasibility and accuracy of the technical solution of the present invention by comparing two groups of samples. At the same time, since composite contaminated sites may have different degrees of contamination due to spatial heterogeneity, this example emphasizes that when evaluating composite contaminated sites, attention should be paid to the location and randomness of earthworm capture, otherwise the test results can only represent the results of certain points, but cannot represent the microplastics occurrence status of the entire contaminated site.

[0116] Example 3

[0117] The detection method and application of cadmium / arsenic and microplastic composite contaminated soil provided in this embodiment are further improved and perfected on the basis of embodiment 1 and embodiment 2, which are basically the same as embodiment 1 and embodiment 2, except that:

[0118] This embodiment uses AI models and transfer learning and other technologies to conduct deep machine learning and deployment of the corresponding relationship of synchronous changes based on joint toxic effects, so as to improve the automation and intelligence of detection and expand the scope of application, and refine the granularity of the interval division of the main occurrence scale range of plastics (the main occurrence scale range can be divided into more levels). The AI ​​model is an AI algorithm set built with deep learning algorithms as the basic architecture; the optional AI algorithm set includes: machine learning algorithms, deep learning algorithms, spatiotemporal analysis algorithms, convolutional neural networks, random forests, logistic regression, linear regression, integrated learning methods (such as Bagging, Boosting, etc.), generative adversarial networks, etc.

[0119] The method for detecting soil contaminated by cadmium / arsenic and microplastics, step S3 also includes the step of deep learning and automatic analysis of the corresponding relationship law of synchronous changes based on the combined toxic effect through AI model training, specifically: constructing a transfer learning AI model based on deep learning, using multiple experimental detections obtained or existing relevant image data and text data (derived from relevant research literature, databases, etc.), and pre-processing them as training data sets, pre-training the AI ​​model, and through training, enabling it to have the ability to infer and predict the corresponding relationship law of synchronous changes of multiple elements based on the combined toxic effect, and use it as an intelligent model for detecting soil contaminated by cadmium / arsenic and microplastics, to automatically detect the soil samples and earthworm slice samples to be tested and output the results.

[0120] In this embodiment, a generative adversarial network (GAN) deep learning network is first constructed, and then the image data and text data to be tested are analyzed and processed through a pre-trained AI model, and the results are output.

[0121] This embodiment uses cGAN (conditional generative adversarial network) to introduce conditional variables on the basis of GAN, so that the generator can generate image samples that meet specific conditions. Such conditional variables can be the category, attribute or any other information that helps guide the generation process. This embodiment specifies more conditional image generation tasks based on limited experimental data and other existing data, as well as the relationship between the six elements through transfer learning; in these tasks, cGAN generates corresponding image samples according to given conditions (adjusting some elements and parameters), such as generating new images based on the original image and adjusted parameters, etc. These generated image samples can be used to enhance training data sets, assist image analysis and other tasks. The GAN network can also be combined with a variety of AI models to enhance the ability to analyze large amounts of image data and extract feature data.

[0122] In the above embodiments of the present invention, it is found that the bioaccumulation of microplastics in earthworms depends on the main particle size of microplastics and the exposure time, and there is a synchronous change correspondence relationship with the same trend between the various factors. Among them, PS-MPs with a main particle size of 70nm are mainly absorbed through the skin and diffuse through epidermal cells to subcutaneous tissue; PS-MPs with main particle sizes of 1μm and 10μm are mainly absorbed through feeding and transferred along the intestine to the tail; 70nm PS-NPs are mainly accumulated in earthworms. E. fetida head and subcutaneous tissues, and aggregated at the accumulation site; 1μm and 10μm PS-MPs adhered to E.fetida The epidermis of the middle and tail segments accumulates in large quantities in the intestine, where 10 μm PS-MPs are E.fetida The accumulation in the body is the largest, and 1μm PS-MPs are more likely to be excreted in the feces. Exposure to 70nm PS-MPs at a 10% (w / w) food concentration can induce E.fetida The above studies show that nano-MPs can cause oxidative stress damage, disrupt the energy balance of earthworms, and cause significant tissue damage to earthworm muscle fibers and epidermis. E.fetida The accumulation amount in the body is the lowest, while the induced toxic effect is the strongest, indicating that the main particle size is an important factor affecting the biological toxicity of microplastics.

[0123] In the law of synchronous change correspondence, it is found that different particle sizes of MPs have E.fetida The accumulation of cadmium and arsenic transport in organs has the following effects: nano- and micro-sized MPs can significantly increase E.fetida 10μm PS-MPs reduce the concentration of As in the body and reduce the concentration of Cd; 10μm PS-MPs adsorb Cd in the soil solution, reduce the concentration of Cd available to earthworms, and promote the transfer of Cd from the gizzard to the digestive tract. 70nm PS-MPs carry Cd into E.fetida The subcutaneous tissue accumulates and aggregates, which prevents Cd from enteringE.fetida As enters the circulation system along with 10μm PS-MPs. E.fetida It is released in the intestine, migrates and accumulates in the intestine following the material circulation of the intestinal cavity. 70nm PS-MPs+As promotes the conversion of As(V) into As(III) and accumulates in the yellow cells of earthworms. This result shows that In E.fetida In vivo, PS-MPs smaller than 10 μm have obvious carrier effects on As, and PS-MPs of 70 nm have obvious carrier effects on Cd.

[0124] In the law of synchronous change correspondence, the effects of mixed exposure of MPs with different particle sizes on cadmium and arsenic toxicity and their mechanism of action are as follows: PS-MPs with different particle sizes (10μm, 1μm and 70nm) have no significant difference in their adsorption capacity for Cd or As; PS-MPs particle size does not affect the desorption degree of Cd in simulated intestinal solution, and the 70nm PS-MPs have the highest desorption concentration of As and promote the accumulation of As in earthworms. In terms of toxicity, Cd treatment alone increased E.fetida The sucrose and starch metabolism of earthworms was enhanced by As treatment alone, which upregulated the citric acid cycle and promoted the energy consumption of earthworms without causing obvious cytotoxicity. The 10μm and 1μm PS-MPs did not cause E.fetida Both 70nm PS-MPs+Cd and 70nm PS-MPs+As caused lysosomal membrane damage, while 70nm PS-MPs+Cd treatment promoted E.fetida The metabolism of amino sugars and nucleotide sugars was affected, and purine metabolism was disturbed, but no obvious growth inhibition and death were induced; 70nm PS-MPs+As E.fetida The toxicity was strong at the individual, cellular and molecular levels, inhibiting the citric acid cycle of earthworms and failing to provide sufficient energy for the urea cycle. 4 +Cannot be converted into urea and excreted from the body, resulting in cell ammonia poisoning, which is the cause E.fetida Key molecular mechanisms of cytotoxicity.

[0125] In the law of synchronous change correspondence, the effect of MPs on the dynamic bioaccumulation of cadmium and arsenic in earthworms is as follows: at a higher concentration (60 mg / kg -1 )Cd and (40mgkg -1 ) In soils contaminated with As, the addition of MPs can significantly reduce soil pH, increase DOC content and increase DGT-Cd / As concentration. The accumulation of Cd and As in earthworms is affected by time and exposure concentration. The presence of MPs significantly increases the absorption rate constant of earthworms ( ku), indicating that MPs have a significant promoting effect on the bioaccumulation of Cd / As in earthworms. MPs can improve the bioavailability of Cd or As in soil and increase the exposure risk of soil animals.

[0126] The above-mentioned embodiments of the present invention focus on analyzing and comparing the changing trends such as the main distribution characteristics of cadmium / arsenic and microplastics at the earthworm organ level based on experimental data or known related detection data obtained by conventional detection combined with in situ biological imaging, and analyzing the law of the synchronous change relationship of multiple factors related to the combined toxic effect, and then using this law to detect unknown contaminated soil samples, key data such as the main occurrence scale range of microplastics can be obtained to support corresponding evaluations and decisions without the need to physically separate and collect microplastics, filling the technical gap in the field of synchronous detection of microplastic-related factors in complex contaminated soils, and can be widely used in technical fields such as environmental pollutant detection and ecological environment governance.

[0127] It should be noted that in other embodiments of the present invention, other different schemes obtained by making specific selections within the scope of the steps, instruments, algorithms, models, process parameters and conditions recorded in the present invention can all achieve the technical effects recorded in the present invention, so the present invention will no longer list them one by one.

[0128] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. All equivalent changes made according to the components, proportions and processes of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting soil contaminated by cadmium / arsenic and microplastics, characterized in that: First, based on known detection or experimental data, the combined toxicity effect of in-situ exposure to cultivated earthworms in the composite contaminated soil formed by different cadmium / arsenic and microplastics in different occurrence scale ranges is analyzed, and the synchronous change correspondence between the six elements, namely, the concentration of cadmium / arsenic in the soil, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, the main distribution characteristics and main occurrence scale range of microplastics at the earthworm organ level, is obtained. Then, the composite contaminated soil to be tested is tested according to the synchronous change correspondence law; the combined toxicity effect of in-situ exposure to cultivated earthworms in the soil to be tested, the concentration of cadmium / arsenic in the soil contaminated by cadmium / arsenic and microplastics, the distribution position of cadmium / arsenic at the earthworm organ level, the cadmium / arsenic concentration data at different organ positions, and the main distribution characteristics of microplastics at the earthworm organ level obtained by actual detection are substituted into the synchronous change correspondence law, and a comparative analysis is performed based on the change trend of the law to obtain a set of correlation data with the highest consistency with the correlation data of the five elements in the soil sample to be tested, that is, the main occurrence scale range data of microplastics in the composite contaminated soil to be tested are obtained, and a complete set of detection data related to the six elements are obtained; The microplastics in different occurrence scale ranges are divided into two qualitative levels: nanoscale and micronscale, where the main occurrence scale range of the nanoscale is 10-1000nm, and the main occurrence scale range of the microscale is 1-10μm; the main occurrence scale range refers to microplastics within this scale range, accounting for more than 70% of the mass ratio of all microplastics in the soil; The combined toxicity effect of the in situ exposure cultured earthworms is characterized by the growth inhibition rate.

2. The method for detecting soil contaminated by cadmium / arsenic and microplastics according to claim 1, characterized in that: The specific laws of the synchronous change correspondence between the six elements are as follows: When microplastics with cadmium complex contamination exist in the soil, compared with single cadmium pollution, the smaller the main occurrence scale of microplastics, the stronger the combined toxic effect, and the faster the growth inhibition rate increases; microplastics with a main occurrence scale range of 1-10μm, through the combined toxic effect, make the microplastics mainly concentrated in the body cavity and organ walls of earthworms, and at the same time transfer most of the cadmium from the digestive organs to the earthworm circulatory system, mainly distributed in the dorsal blood vessels, ventral blood vessels and subcutaneous tissue of earthworm slice samples; while microplastics with a main occurrence scale range of 10-1000nm further allow more microplastics to enter and concentrate in the subcutaneous tissue of earthworms, and also cause a large amount of cadmium in the earthworm body to enter and be mainly enriched in the subcutaneous tissue of earthworms, and no longer continue to diffuse to the digestive organs and circulatory system; When microplastics contaminated with arsenic exist in the soil, compared with arsenic single pollution, the smaller the main occurrence scale range of microplastics, the stronger the combined toxicity effect, and the faster the growth inhibition rate increases; under the conditions of microplastic and arsenic combined pollution with the main occurrence scale range of 1-10μm, arsenic is mainly concentrated in the intestinal cavity and the folds of the longitudinal muscle of the earthworm, and microplastics are mainly concentrated in the earthworm epidermis and intestinal cavity; under the conditions of 10-1000nm microplastic and arsenic combined pollution, microplastics are mainly concentrated in the subcutaneous tissue of the earthworm, and arsenic is mainly concentrated in the yellow tissue and dorsal blood vessels of the earthworm.

3. The method for detecting soil contaminated by cadmium / arsenic and microplastics according to any one of claims 1 to 2, characterized in that: It includes the following steps: S1: Obtain the corresponding relationship between the synchronous changes of the combined toxic effects at the sample organ level The test data of multiple earthworm slice samples exposed to soils with different concentrations of cadmium / arsenic and different occurrence scales of microplastics were obtained, and compared with the test data of earthworm slice samples exposed to soils with single cadmium / arsenic pollution and no microplastics at the same concentration. The combined toxicity effect of in situ exposure and cultivation of earthworms in multiple different composite polluted soils and single polluted soils was obtained, and the synchronous change correspondence between the six factors of cadmium / arsenic concentration in the soil, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, the main distribution characteristics and main occurrence scale range of microplastics at the earthworm organ level was obtained. S2: Detection of composite contaminated soil samples Collect soil contaminated with cadmium / arsenic and microplastics that has at least millimeter-sized microplastics and prepare it as a soil sample to be tested. First, detect the concentration of cadmium / arsenic in the soil sample to be tested. Then, in situ expose and cultivate living earthworms in the soil to determine the growth inhibition rate. Then, prepare the earthworms into multiple cross-sectional slice samples, obtain image data of the distribution positions of cadmium / arsenic and microplastics on each earthworm cross-sectional slice sample, and detect the cadmium / arsenic concentrations at different organ positions of the multiple slice samples to obtain the distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level. S3: Comparative analysis to obtain a set of correlation detection data The five element data of the combined toxic effects of in situ exposure to cultivated earthworms in the soil detected in step S2, the concentration of cadmium / arsenic in the soil, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, and the main distribution characteristics of microplastics at the earthworm organ level are substituted into the synchronous change correspondence law in step S1, and a comparative analysis is conducted based on the changing trend of this law to obtain a set of associated data with the highest consistency with the five element associated data in the soil sample to be tested, including the main occurrence scale range data of microplastics, to complete the detection.

4. The method for detecting soil contaminated by cadmium / arsenic and microplastics according to claim 3, characterized in that: S1 includes the following steps: S1-1: Prepare a single contaminated soil with a set cadmium / arsenic concentration, then in situ expose and cultivate earthworms in the soil, determine their growth inhibition rate, and then prepare and test earthworm slice samples to obtain the main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level. Under the condition of the single contaminated soil, a set of four interrelated test data including growth inhibition rate, cadmium / arsenic concentration in the soil, and main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level are obtained; S1-2: Add microplastics of known main occurrence scale range to the single contaminated soil with the cadmium / arsenic concentration set in S1-1 to prepare composite contaminated soil, then in situ expose and cultivate earthworms in the soil, determine their growth inhibition rate, and then prepare and test earthworm slice samples to obtain the cadmium / arsenic concentration in the soil, the main occurrence scale range of microplastics, the main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level, and the main distribution characteristics of microplastics at the earthworm organ level. Under the conditions of the composite contaminated soil, a set of six interrelated detection data including the cadmium / arsenic concentration in the soil, the growth inhibition rate, the main occurrence scale range of microplastics, the main distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level, and the main distribution characteristics of microplastics at the earthworm organ level are obtained; S1-3: Changing the soil pollution conditions in steps S1-1 and S1-2, setting different cadmium / arsenic concentrations, adding microplastics of different known main occurrence scales, repeating steps S1-1 to S1-2 multiple times, and obtaining multiple sets of four-element or six-element interrelated detection data respectively; S1-4: Obtaining the corresponding relationship between the synchronous changes of the combined toxic effects at the sample organ level Based on the combined toxicity effect, a comparative analysis was performed on multiple groups of data obtained from steps S1-1 to S1-3 to obtain the concentration of cadmium / arsenic in the soil, the growth inhibition rate, the main distribution characteristics and concentration of cadmium / arsenic at the earthworm organ level, the corresponding change relationship between the main distribution characteristics of microplastics at the earthworm organ level and the main occurrence scale range data, that is, the law of synchronous change correspondence between the six element data at the sample organ level.

5. The method for detecting soil contaminated by cadmium / arsenic and microplastics according to claim 3, characterized in that: Step S2 includes the following specific steps: S2-1: Collect sufficient amount of soil contaminated with cadmium / arsenic and microplastics containing at least millimeter-sized microplastics to prepare the soil sample to be tested; S2-2: Perform sample pretreatment by first using conventional methods to detect the concentrations of cadmium and arsenic in the soil samples to be tested; S2-3: In situ exposure of living earthworms in the soil sample to be tested, and determination of growth inhibition rate; S2-4: Prepare the earthworms cultivated in situ in the soil into multiple cross-sectional slice samples, obtain the distribution position image data of cadmium / arsenic and microplastics on each earthworm cross-sectional slice sample, and detect the cadmium / arsenic concentrations at different organ positions of the multiple slice samples to obtain the distribution characteristics and concentration data of cadmium / arsenic at the earthworm organ level.

6. The method for detecting soil contaminated by cadmium / arsenic and microplastics according to claim 4 or 5, characterized in that: In situ exposure cultivation of earthworms in soil is to cultivate living earthworms in the soil, allowing them to grow naturally into adult earthworms with complete reproductive ring structures, and then directly capture or trap them; Preparation of earthworm slice samples, including the following steps: A1: Sample processing of fresh adult earthworms: The captured earthworms were cleaned of their intestines, anesthetized with alcohol, and then fixed with 4% paraformaldehyde; A2: Sectioning: After the head and reproductive ring of the adult earthworm sample are embedded in paraffin, multiple slices are cut continuously along the cross section according to the set thickness to obtain multiple tissue sections; A3: Tissue section pretreatment The tissue sections were dewaxed in a gradient manner using a transparent agent, anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, and distilled water, with each step lasting 10 minutes. Dewaxed sections were obtained, and then several complete sections were screened out using an ordinary optical microscope as section samples for machine testing.

7. The method for detecting soil contaminated by cadmium / arsenic and microplastics according to claim 4 or 5, characterized in that: The biopsy sample test includes the following steps: B1: Slice sample cross-section imaging measurement First, collect the image data of the cross section of the sample to be tested, and then use LA-ICP-MS to scan the surface to determine the cumulative distribution of cadmium or arsenic and obtain the surface scan results; the LA-ICP-MS setting parameters are: energy 3.5jcm -2 , beam spot 20um, frequency 20Hz, speed 30ums -1 ; B2: Interpretation of cross-sectional measurement data of sliced ​​samples The surface scan results of the sliced ​​samples to be tested were processed. The surface scan results were calibrated for cadmium / arsenic content using the "TraceElements" module of the iolite software and SRM610 as an external standard; Use iolite's Imaging module to map the corrected matrix data, including adjusting the image resolution, contrast, image color, and scale; Then, the surface scan images and matrix data of each element were output respectively to obtain the distribution data of cadmium / arsenic at the organ level on the cross section of earthworm slices.

8. The method for detecting soil contaminated by cadmium / arsenic and microplastics according to claim 3, characterized in that: The step S3 also includes the step of deep learning and automatic analysis of the corresponding relationship law of synchronous changes based on the combined toxic effect through AI model training, specifically: constructing a transfer learning AI model based on deep learning, using image data and text data obtained from multiple experimental tests or existing ones, and using them as training data sets after preprocessing, to pre-train the AI ​​model. Through training, the AI ​​model is enabled to have the ability to infer and predict the corresponding relationship law of synchronous changes of multiple factors based on the combined toxic effect, and serve as an intelligent model for detecting cadmium / arsenic and microplastic composite contaminated soil, and automatically detects the soil samples and earthworm slice samples to be tested and outputs the results.

9. Application of the method for detecting soil contaminated by cadmium / arsenic and microplastics as described in any one of claims 1 to 8 in soil microplastic detection and ecological management.

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