Method for relieving toxicity of micro-plastic to soil-plant system based on combination of nitrification inhibitor and carbon nanomaterial

Abstract

The invention belongs to the field of soil pollution treatment, and relates to a method for jointly relieving toxicity of microplastics to a soil-plant system based on a nitrification inhibitor-carbon nanomaterial. The method comprises the following steps: applying a nitrification inhibitor and a carbon nanomaterial to plant planting soil; the nitrification inhibitor is DMPP, and the carbon nanomaterial is one or more of a multi-walled carbon nanotube and a nanodiamond. The method can technically slow down microplastic pollution, improve soil health and guarantee quality safety of agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of soil pollution remediation and relates to a method for mitigating the toxicity of microplastics to soil-plant systems based on the combination of nitrification inhibitors and carbon nanomaterials. Background Technology

[0002] Microplastics in farmland soil mainly originate from agricultural mulch film residues, primarily composed of polyvinyl chloride (PVC) and polystyrene. In my country, phthalate concentrations in soils covered with plastic mulch film are 74%–208% higher than in uncovered soils. Plastic mulch film releases harmful pollutants such as phthalates, damaging the soil environment. Microplastics may affect soil quality, including soil pH and the carbon, nitrogen, and phosphorus cycles. For example, higher concentrations of microplastics inhibit the decomposition of dissolved organic matter, leading to eutrophication. Furthermore, microplastics may react with pesticides in the soil, accelerating the decomposition of soluble nitrogen and phosphorus nutrients, thus reducing soil nutrient content. In addition, microplastics reduce soil adsorption capacity, promoting the movement of other pollutants in the soil. Microplastics interfere with the activity of enzymes involved in the nitrogen cycle (such as chitinase and leucine aminopeptidase), thereby affecting soil nitrogen cycling. The direct effects of microplastics on plants include blocking root nutrient input and affecting photosynthesis. Microplastics indirectly affect plant growth through their own toxicity by disrupting the plant's antioxidant enzyme system. Changes in soil productivity caused by microplastics can also indirectly regulate plant growth. These multifaceted impacts pose significant challenges to sustainable agriculture and the protection of ecosystem functions. However, current technologies for the remediation of microplastic pollution still have many limitations. Physical removal is costly and difficult to achieve completely; chemical degradation easily produces secondary pollution; and bioremediation is time-consuming and inefficient. Especially in agricultural soils, there is a lack of green regulatory technologies that can effectively mitigate microplastic toxicity without affecting crop quality and soil health. Existing studies have shown that while the use of carbon nanomaterials alone can improve soil properties, it may lead to problems such as a decline in endophytic bacterial diversity. To date, there is still no research on nitrification inhibitors, alone or in combination with carbon nanomaterials, addressing plant growth under microplastic stress. Therefore, there is an urgent need to develop a synergistic, safe, and applicable composite regulatory strategy for farmland systems. Thus, there is an urgent need to provide a low-cost, non-toxic method for jointly mitigating the toxicity of microplastics to soil-plant systems. Summary of the Invention

[0003] This invention aims to explore the biological and environmental mechanisms by which nitrification inhibitors and carbon nanomaterials, alone and in combination, act in soil. This is crucial for improving plant biomass and quality, enhancing the microbial environment, and achieving sustainable remediation of soil pollution. This method can mitigate microplastic pollution, improve soil health, and ensure the quality and safety of agricultural products, providing a novel, efficient, and sustainable soil remediation strategy.

[0004] The technical solution provided by this invention is as follows:

[0005] A method for mitigating the toxicity of microplastics to soil-plant systems based on a combination of nitrification inhibitor and carbon nanomaterials, the method comprising: applying a nitrification inhibitor and carbon nanomaterials to the soil in which the plants are grown; wherein the nitrification inhibitor is DMPP, and the carbon nanomaterials are one or more of multi-walled carbon nanotubes and nanodiamonds.

[0006] Furthermore, the microplastic is polyvinyl chloride.

[0007] Furthermore, fertilizers are applied to the soil, including urea, trisodium phosphate, and potassium chloride.

[0008] Furthermore, the application ratio of urea, trisodium phosphate, and potassium chloride is 2:1:1.

[0009] Furthermore, the dosage of urea is 200 mg N kg. -1 Dry soil.

[0010] The present invention also provides the application of the above-mentioned method for mitigating the toxicity of microplastics to the soil-plant system based on nitrification inhibitor-carbon nanomaterials in promoting plant growth.

[0011] Furthermore, the plant in question is bok choy.

[0012] This invention also provides the application of the above-mentioned method for mitigating the toxicity of microplastics to soil-plant systems based on nitrification inhibitors and carbon nanomaterials in soil remediation.

[0013] The present invention also provides the application of the above-mentioned method for mitigating the toxicity of microplastics to the soil-plant system based on the combination of nitrification inhibitors and carbon nanomaterials in reducing the nitrate content of plants.

[0014] The present invention also provides the application of the above-mentioned method for mitigating the toxicity of microplastics to soil-plant systems based on the combination of nitrification inhibitors and carbon nanomaterials in reducing soil nitrate nitrogen content.

[0015] Beneficial effects

[0016] Soil conditioners and growth regulators have the potential to mitigate the negative impacts of microplastics on soil-plant systems. Nanomaterials can enhance soil phytochemistry, optimize nutrient uptake, and modulate microbial communities to promote plant growth. Carbon nanotubes can penetrate seed coats, promote water absorption, and influence seed germination and growth. Furthermore, carbon nanotubes can capture or degrade pollutants, thereby mitigating their inhibitory effects on key microorganisms, and can modulate soil microbial communities and nitrogen-converting enzyme activity, accelerating nitrogen mineralization and nitrification processes.

[0017] This invention significantly alleviates the toxicity of polyvinyl chloride (PVC) microplastics to the soil-plant system by combining the nitrification inhibitor DMPP with carbon nanomaterials (MWCNTs / ND). Specific effects include: aboveground biomass of pak choi significantly increased by 69.1% and 104.7% compared to the PVC-contaminated group (PVT), exceeding the uncontaminated level (CK); nitrate content in plants decreased by 44.4% and 63.9%, while soluble sugar content increased by 241.8% and 140.7%; nitrate nitrogen content decreased by 92.5% and 82.3%; soil bacterial community stability was restored to the control level, while significantly altering the distribution of endophytic microbial communities: promoting endophytic bacterial communities and inhibiting endophytic fungal communities. This invention innovatively combines DMPP with carbon nanomaterials, achieving the goal of reducing microplastic toxicity through biological processes such as microbial community regulation, biostimulation, and nitrogen cycle regulation.

[0018] Current research on the mechanisms by which nitrification inhibitors and carbon nanomaterials jointly mitigate microplastic toxicity is limited. In agricultural soil remediation, microbial communities and biostimulation are core targets of in-situ bioremediation strategies. The combined effect of DMPP and carbon nanomaterials fully leverages their synergistic effect in mitigating microplastic toxicity. This technology also offers environmental benefits such as improved nutrient utilization, contributing to effective ecological solutions for addressing major environmental challenges such as microplastic pollution, soil remediation, and sustainable agriculture. Attached Figure Description

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0020] Figure 1 This is a technical roadmap of the present invention;

[0021] Figure 2 The effects of microplastics, carbon nanomaterials, and DMPP on pakchoi are shown in the figure. (a) represents biomass, (b) represents nitrate, and (c) represents soluble sugar content. Control (CK) is the blank treatment; PVT is the PVC exposure treatment; PCNT is the coexistence treatment of PVC and multi-walled carbon nanotubes; PCDT is the coexistence treatment of PVC and nanodiamonds; PDMT is the coexistence treatment of PVC and DMPP; PCNMT is the coexistence treatment of PVC, multi-walled carbon nanotubes, and DMPP; and PCDMT is the coexistence treatment of PVC, nanodiamonds, and DMPP. Lowercase letters in the figure indicate significant differences between treatments (P < 0.05).

[0022] Figure 3The effects of microplastics, carbon nanomaterials, and DMPP on soil are shown in the figure. (a) represents pH; (b) represents nitrate nitrogen content. Control (CK) is the blank treatment; PVT is the PVC exposure treatment; PCNT is the coexistence treatment of PVC and multi-walled carbon nanotubes; PCDT is the coexistence treatment of PVC and nanodiamonds; PDMT is the coexistence treatment of PVC and DMPP; PCNMT is the coexistence treatment of PVC, multi-walled carbon nanotubes, and DMPP; and PCDMT is the coexistence treatment of PVC, nanodiamonds, and DMPP. Lowercase letters in the figure indicate significant differences between treatments (P < 0.05).

[0023] Figure 4 The effects of microplastics, carbon nanomaterials, and DMPP on the activities of different types of enzymes in soil are shown. (a) represents chitinase, (b) represents urease, and (c) represents acid phosphatase. Control (CK) is the blank treatment; PVT is the PVC exposure treatment; PCNT is the co-existence treatment of PVC and multi-walled carbon nanotubes; PCDT is the co-existence treatment of PVC and nanodiamonds; PDMT is the co-existence treatment of PVC and DMPP; PCNMT is the co-existence treatment of PVC, multi-walled carbon nanotubes, and DMPP; and PCDMT is the co-existence treatment of PVC, nanodiamonds, and DMPP. Lowercase letters in the figure indicate significant differences between treatments (P < 0.05).

[0024] Figure 5 The effects of microplastics, carbon nanomaterials, and DMPP on soil bacteria, endophytic bacteria, soil fungi, and α-diversity of endophytic fungal communities are shown in the figure. The treatments are: CK (control), blank control; PVT (PVC exposure); PCNT (PVC and multi-walled carbon nanotubes coexisting); PCDT (PVC and nanodiamonds coexisting); PDMT (PVC and DMPP coexisting); PCNMT (PVC, multi-walled carbon nanotubes and DMPP coexisting); and PCDMT (PVC, nanodiamonds and DMPP coexisting). Lowercase letters in the figure indicate significant differences between treatments (P < 0.05).

[0025] Figure 6 The effects of microplastics, carbon nanomaterials, and DMPP on the topological parameters of collinear networks in soil communities are shown in the figure. (a) and (b) represent soil bacteria, (c) and (d) represent soil fungi, (e) and (f) represent endophytic bacteria, and (g) and (h) represent endophytic fungi. CK represents the control group; PVT represents PVC exposure; PCNT represents the coexistence of PVC and multi-walled carbon nanotubes; PCDT represents the coexistence of PVC and nanodiamonds; PDMT represents the coexistence of PVC and DMPP; PCNMT represents the coexistence of PVC, multi-walled carbon nanotubes, and DMPP; and PCDMT represents the coexistence of PVC, nanodiamonds, and DMPP. Lowercase letters in the figure indicate significant differences between treatments (P < 0.05). Detailed Implementation

[0026] Example 1

[0027] 1. Test soil:

[0028] From the Chang'an Research Base of Hunan Agricultural University (N28°10′, E113°4′). After removing residual leaves and debris, the experimental soil was air-dried, ground, and sieved through a 2mm sieve for later use. The soil pH was 5.34.

[0029] 2. Chemical reagents and materials:

[0030] The nitration inhibitor DMPP was produced by Shanghai Titan Technology Co., Ltd., with a purity >99.0%. Urea was supplied by Sinopharm Chemical Reagent Co., Ltd. and Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Multi-walled carbon nanotubes were purchased from Shenzhen Suiheng Technology Co., Ltd. (Shenzhen, China), with a purity ≥95%. Other properties included: average diameter (≤7 μm), specific surface area (≥250 m² / m³). 2 / g), bulk density (0.08 g / cm³) 3 The nanodiamonds were purchased from Shenzhen Suiheng Technology Co., Ltd. (Shenzhen, China), with a particle size of 30 nm and a purity of ≥95%.

[0031] 3. Test instruments:

[0032] Ultraviolet spectrophotometer; high-speed and low-speed centrifuges, drying ovens, etc.

[0033] 4. The specific technical solution of this invention is as follows:

[0034] 4.1 Experimental Design:

[0035] This experiment included seven treatments, each with four replicates: blank control (CK), polyvinyl chloride exposure (PVT), polyvinyl chloride coexisting with multi-walled carbon nanotubes (PCNT), polyvinyl chloride coexisting with nanodiamonds (PCDT), polyvinyl chloride coexisting with DMPP (PDMT), polyvinyl chloride, multi-walled carbon nanotubes and DMPP coexisting (PCNMT), and polyvinyl chloride, nanodiamonds and DMPP coexisting (PCDMT).

[0036] After surface sterilization of the plant seeds, they were germinated in the dark. Each pot was filled with 2.5 kg of soil, with polyvinyl chloride (PVC) added according to the previous treatment protocol. Microplastics and carbon nanomaterials were thoroughly mixed with the soil at doses of 5.0 g / kg and 1.0 mg / kg, respectively. Fifteen selected germinated seeds were then added to each pot and evenly distributed. All pots were placed in an environment with natural light and room temperature. Day 0 was the day of sowing. Thinning was performed on day 14, ensuring four seedlings remained in each pot. Fertilizer and nitrification inhibitor were added on the third and fourth days after thinning, respectively. The fertilizer and DMPP were dissolved in distilled water and applied on days 16 and 17, respectively. Urea was applied at a dosage of 200 mg N / kg. -1 The application rate of dry soil, trisodium phosphate, and potassium chloride is 100 mg N / kg. -1 Dry soil. Watering was conducted regularly during the experiment. Plant and soil samples were collected on day 42 to determine the biomass, quality index, soil chemical properties, enzyme activity, electron transport system activity, and endophytic and soil microbial communities of the Chinese cabbage.

[0037] 4.2 Biomass and Quality Indicators of Chinese Cabbage

[0038] Fresh plant samples were cleaned and their total fresh weight was calculated. The plant samples were then divided into different portions for analysis of moisture content, quality indicators, and endophytic bacterial and fungal communities. Moisture content was calculated by blanching some fresh samples at 105 °C for 30 minutes, followed by drying at 65 °C to constant weight. The nitrate content of the pak choi was determined using the salicylic acid-sulfuric acid colorimetric method. The soluble sugar content of the pak choi was determined using the anthrone colorimetric method.

[0039] 4.3 Soil chemical properties and enzyme activity

[0040] Air-dried soil samples were used to determine soil pH, while fresh soil samples were used to determine nitrate nitrogen, enzyme activity, and soil microbial community. Soil pH and nitrate nitrogen were determined according to the methods described in Soil Agrochemical Analysis. Chitinase and acid phosphatase were determined using the p-nitrophenol (pNP) colorimetric method, and soil urease activity was determined using the indophenol blue method.

[0041] 4.4 Soil microbial community analysis

[0042] Freshly harvested plant samples underwent surface sterilization, followed by DNA extraction from soil and endophytic bacteria and fungi using the Fast DNA SPIN Kit. The extracted DNA was detected by 1.0% agarose gel electrophoresis. Purified PCR products were sequenced on the Illumina Miseq platform (Majorbio, China). Raw data were stored in fastq format and uploaded to NCBI database. Alpha diversity analysis was performed at the operational taxonomic unit (OTU) level. Bacterial and fungal OTUs were compared with the SILVA and PR2 databases, respectively, to present the taxonomic composition of bacterial and fungal communities. Principal coordinate analysis (PCoA) was used to analyze changes in bacterial and fungal community composition across different samples. Selected OTU data (relative abundance > 0.1%) were analyzed using the Spearman correlation matrix via the WGCNA package, and the co-occurrence network was visualized using the Fruchterman Reingold layout in Gephi software. The igraph package was used to calculate the topological features of the co-occurrence network.

[0043] 4.5 Data Analysis Methods

[0044] One-way ANOVA combined with Duncan's multiple range test was used to assess the significant effects of microplastics, DMPP, and carbon nanomaterials on the biomass, quality, soil chemistry, enzyme activity, and soil and endophytic microbial community of *Pak choi*, with a significance level of P < 0.05. Pearson correlation analysis was used to explore the linear correlation between different parameters. The raw data were standardized and dimensionality reduced, and pathway analysis was performed using maximum likelihood estimation. All the above procedures were performed using IBM SPSS Statistics 26.

[0045] This invention investigates the combined use of nitrogen management measures and carbon nanomaterials to mitigate the toxicity of microplastic polyvinyl chloride (PVC) to the soil-plant system. Microplastics and carbon nanomaterials were thoroughly mixed with soil at doses of 5.0 g / kg and 1.0 mg / kg, respectively, and then added to pak choi seeds. Nitrogen and nitrification inhibitors were added on the third and fourth days after thinning, respectively. After 42 days, samples were taken to measure pak choi biomass and quality indicators, soil physicochemical enzyme activity, and soil and endophytic bacterial and fungal communities. The relationships between nitrification inhibitors, carbon nanomaterials, and soil microbial community structure and diversity, pak choi biomass and quality, and soil abiotic factors were quantified, and the functional mechanisms by which soil microbial community structure, diversity, and soil abiotic factors drive changes in pak choi biomass and quality were elucidated. The study found that microplastic polyvinyl chloride (PVC) significantly increased soil nitrate nitrogen content, while the nitrate nitrogen content of additional carbon nanomaterials (multi-walled carbon nanotubes, nanodiamonds) and DMPP alone or in combination was reduced by 74.4%, 82.4%, 90.4%, 92.5%, and 82.3%, respectively, compared with the PVC-polluted group (PVT). Microplastic PVC significantly increased soil urease activity, by 32.2% compared with the control, while the additional carbon nanomaterials and DMPP alone or in combination significantly reduced urease activity (16.6%–39.2%). Microplastic PVC significantly reduced plant biomass, by 32.4% compared with the control, while the plant biomass of additional carbon nanomaterials and DMPP alone or in combination was increased by 33.8%, 11.4%, 66.6%, 69.1%, and 104.7%, respectively, compared with the PVC-polluted group (PVT). Microplastic PVC significantly reduced soluble sugar content, by 39.2% compared to the control group. In contrast, additional carbon nanomaterials, either alone or in combination with DMPP, significantly increased plant soluble sugar content (63.3%–241.8%) and significantly reduced plant nitrate content (24.3%–63.9%). Microplastic PVC significantly reduced soil bacterial community stability, while additional carbon nanomaterials restored soil bacterial community stability to the control level. Furthermore, additional carbon nanomaterials exhibited a double-edged sword effect on endophytic microbial communities: significantly inhibiting α-diversity and stability of endophytic bacterial communities, but promoting α-diversity and stability of endophytic fungal communities. Therefore, considering the adverse effects of nanomaterials on endophytic bacterial communities, future research should weigh the combined effects of nitrification inhibitors and carbon-based nanomaterials on the soil-plant system and explore the most beneficial applications for pollution remediation. This study provides a theoretical and practical basis for mitigating the toxic effects of microplastics and offers valuable insights for developing appropriate remediation measures.

[0046] DMPP, as a nitrification inhibitor, can influence soil abiotic properties and microbial community characteristics, while also promoting plant growth. Carbon nanomaterials can improve nitrogen availability, optimize soil microbial communities, and promote nutrient cycling processes. This invention innovatively combines DMPP with carbon nanomaterials, achieving the goal of reducing microplastic toxicity through biological processes such as microbial community regulation, biostimulation, and nitrogen cycle regulation.

[0047] Current research on the mechanisms by which nitrification inhibitors and carbon nanomaterials jointly mitigate microplastic toxicity is limited. In agricultural soil remediation, microbial communities and biostimulation are core targets of in-situ bioremediation strategies. The combined effect of DMPP and carbon nanomaterials fully leverages their synergistic effect in mitigating microplastic toxicity. This technology also offers environmental benefits such as improved nutrient utilization, contributing to effective ecological solutions for addressing major environmental challenges such as microplastic pollution, soil remediation, and sustainable agriculture.

Claims

1. A method for mitigating the toxicity of microplastics to soil-plant systems based on a combination of nitrification inhibitors and carbon nanomaterials, characterized in that, The method includes: applying a nitrification inhibitor and carbon nanomaterials to the soil in which the plants are grown; the nitrification inhibitor is DMPP, and the carbon nanomaterials are one or more of multi-walled carbon nanotubes and nanodiamonds.

2. The method for mitigating the toxicity of microplastics to soil-plant systems based on the combined use of nitrification inhibitors and carbon nanomaterials according to claim 1, characterized in that, The microplastic is polyvinyl chloride.

3. The method for mitigating the toxicity of microplastics to soil-plant systems based on the combined use of nitrification inhibitors and carbon nanomaterials according to claim 1, characterized in that, Fertilizers are applied to the soil, including urea, trisodium phosphate, and potassium chloride.

4. The method for mitigating the toxicity of microplastics to soil-plant systems based on the combined use of nitrification inhibitors and carbon nanomaterials according to claim 3, characterized in that, The application ratio of urea, trisodium phosphate, and potassium chloride is 2:1:

1.

5. The method for mitigating the toxicity of microplastics to soil-plant systems based on the combined use of nitrification inhibitors and carbon nanomaterials according to claim 4, characterized in that, The dosage of urea is 200 mg N kg. -1 Dry soil.

6. The application of the method for mitigating the toxicity of microplastics to the soil-plant system based on the combination of nitrification inhibitors and carbon nanomaterials as described in any one of claims 1 to 5 in promoting plant growth.

7. The application according to claim 6, characterized in that, The plant in question is bok choy.

8. The application of the method for mitigating the toxicity of microplastics to the soil-plant system based on the combined use of nitrification inhibitors and carbon nanomaterials as described in any one of claims 1 to 5 in soil remediation.

9. The application of the method for mitigating the toxicity of microplastics to the soil-plant system based on the combined use of nitrification inhibitors and carbon nanomaterials as described in any one of claims 1 to 5 in reducing the nitrate content in plants.

10. The application of the method for mitigating the toxicity of microplastics to soil-plant systems based on the combined use of nitrification inhibitors and carbon nanomaterials as described in any one of claims 1 to 5 in reducing soil nitrate nitrogen content.

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