Method for measuring influence of microplastics on nitrogen utilization efficiency of plants

Abstract

The invention provides a method for determining the influence of micro-plastics on the nitrogen utilization efficiency of plants. The method comprises the following steps: 1) adding micro-plastics: mixing the micro-plastics with a preset concentration with soil without micro-plastic pollution; 2) plant planting and marking: planting a target plant in the soil added with the micro-plastic, and irrigating with a nitrogen fertilizer marked by a 15N stable isotope in the early growth stage; 3) sample collection: collecting the sample after the target plant grows; and 4) sample treatment and analysis: drying and grinding the collected sample, and carrying out determination, calculation and statistical analysis on the treated sample. According to the method, the influence of the micro-plastic on the nitrogen utilization efficiency of the plant is determined by utilizing a 15N stable isotope nitrogen labeling technology, the specific influence of the micro-plastic on the nitrogen absorption and utilization of the plant can be effectively revealed, and a scientific basis is provided for reasonably controlling micro-plastic pollution and protecting agricultural production and nutrient circulation of an ecological system.

Description

Technical Field

[0001] The invention relates to the fields of ecology, environmental science and agricultural technology, and in particular to a method for determining the influence of microplastics on plant nitrogen utilization efficiency. Background Art

[0002] Microplastic pollution has become a serious threat to ecosystem security. The abundance of microplastics in the soil environment is high, which has a significant impact on plant growth and nitrogen cycle. In the soil environment, microplastics not only occupy soil pores due to their physical presence, affecting soil structure and water infiltration, but also pose a potential threat to soil biological communities and ecosystem functions due to the organic pollutants, heavy metals and other harmful substances adsorbed on their surfaces. More importantly, the interaction between microplastics and soil nitrogen cycle has become a research hotspot. Nitrogen is an indispensable nutrient for plant growth, and its cycle process involves multiple complex biogeochemical processes such as nitrogen fixation, nitrification and denitrification. The presence of microplastics may indirectly or directly affect plant nitrogen utilization efficiency through multiple mechanisms such as affecting the structure of soil microbial communities, changing soil enzyme activity, and interfering with the absorption and utilization of nitrogen by plant roots.

[0003] Although studies have shown that microplastics can interact with nitrogen in the soil, scientific knowledge about how microplastics specifically affect plant nitrogen absorption, transport and utilization efficiency is still limited. This knowledge gap limits the comprehensive understanding of the ecological effects of microplastic pollution and hinders the formulation of effective management strategies and mitigation measures. Therefore, there is an urgent need to develop an accurate and sensitive method to quantitatively assess the specific impact of microplastics on plant nitrogen utilization efficiency, so as to provide solid scientific support for the formulation of targeted microplastic pollution prevention and control strategies, ensure the sustainable development of agriculture, and maintain the balance of nutrient cycles in ecosystems. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for determining the effect of microplastics on plant nitrogen utilization efficiency.

[0005] Specifically, the method for determining the effect of microplastics on plant nitrogen utilization efficiency provided by the present invention comprises the following steps: 1) Microplastic addition: Mixing different concentrations of microplastics with soil without microplastic contamination.

[0006] 2) Plant planting and labeling: Plant the target plants in the soil with microplastics added and use them in the early growth stage. 15 N stable isotope labeled nitrogen fertilizer was used for irrigation.

[0007] 3) Sample collection: Samples are collected after the target plants have grown.

[0008] 4) Sample processing and analysis: The collected samples are dried and ground, and the processed samples are measured, calculated and statistically analyzed. The present invention proposes an accurate and reliable method to measure the effect of microplastics on plant nitrogen utilization efficiency using 15N stable isotope nitrogen labeling technology to reveal the specific effect of microplastics on plant nitrogen absorption and utilization, and provide a scientific basis for the rational control of microplastic pollution, protection of agricultural production and ecosystem nutrient cycle.

[0009] Preferably, step 1) further comprises disinfecting the soil and measuring basic physical and chemical properties, wherein the basic physical and chemical properties include one or more of soil organic carbon, available phosphorus, available potassium, ammonium nitrogen, nitrate nitrogen and pH value.

[0010] Preferably, in step 1), a bottom-sealed plastic barrel is used, with an upper diameter of 10±2 cm, a bottom diameter of 12±2 cm, and a height of 10±2 cm, and 1±0.2 kg of soil is contained in the plastic barrel.

[0011] Preferably, in step 1), the added concentration of the microplastics is 0-3% based on the total weight of the soil.

[0012] Preferably, the microplastics are mixed with soil at preset concentrations of 0-0.01%, 0.1%-0.2%, 0.5%-0.6% and 1%-1.2%, respectively. The present invention uses microplastics of different concentrations to better understand the growth and nutrient absorption state of plants at different concentrations and determine their nitrogen utilization efficiency.

[0013] Further preferably, in step 1), the microplastic is polyethylene or polypropylene, and the particle diameter is 1 to 100 μm.

[0014] Preferably, in step 2), the 15 The nitrogen fertilizer labeled with N stable isotope is 15 N-labeled urea or ammonium nitrate.

[0015] More preferably, the 15 The nitrogen content of nitrogen fertilizer labeled with N stable isotope is 42%~50%. 15 N abundance>0.366%, preferably 9% to 11%. In the present invention, by adopting an optimized ratio, it is closer to the actual production situation, and the measured nitrogen absorption efficiency is more accurate.

[0016] Preferably, the 15 The corresponding field application rate of nitrogen fertilizer labeled with N stable isotope is 200~250 kg / hm 2 .

[0017] Preferably, in step 2), the target plant includes one or more of wheat, corn and rice.

[0018] Preferably, in step 3), the sample collection includes the seedling stage, growth stage and / or maturity stage of the plant growth, preferably the samples of the above-ground part, the samples of the underground part and the soil sample of the target plant are collected. In the present invention, the sample collection can be any period of the seedling stage, growth stage and maturity stage of the plant growth, such as collecting and measuring the nitrogen utilization efficiency of plants in the seedling stage or maturity stage under microplastic stress.

[0019] Preferably, in step 4), the determination comprises: determining the nitrogen content of the plant using an element analyzer; and / or determining the nitrogen content of the plant using a stable isotope mass spectrometer; 15 N and 14 N isotope determination.

[0020] Further preferably, in step 4), the statistical analysis includes: using statistical software to perform one-way analysis of variance or Duncan's multiple comparison test on the data to evaluate the significance of the effect of microplastics on plant nitrogen utilization efficiency.

[0021] Further preferably, in step 4), the calculation includes: Ndf 肥 % is the proportion of nitrogen in the plant that comes from fertilizer nitrogen, calculated as follows: Among them, the plant samples 15 N percentage is determined by mass spectrometry 15 N abundance minus natural 15 N abundance; 15 N labeled fertilizers 15 N percentage of ultra-labeled fertilizers determined by mass spectrometry 15 N abundance minus 15 N natural abundance.

[0022] Preferably, Ndf 土 % is the proportion of nitrogen in the plant that comes from soil nitrogen, calculated as follows: .

[0023] Preferably, the nitrogen absorption of plants is calculated as follows: Plant nitrogen absorption = plant dry matter mass × plant nitrogen content.

[0024] Preferably, the fertilizer nitrogen absorption is calculated as follows: Fertilizer nitrogen absorption = plant nitrogen absorption × Ndf 肥 %.

[0025] Preferably, nitrogen utilization efficiency is calculated as follows: The beneficial effects of the present invention are at least that: the present invention provides a15 The method of using N stable isotope nitrogen labeling technology to determine the impact of microplastics on plant nitrogen utilization efficiency is accurate and reliable. By using 15N stable isotope nitrogen labeling technology to determine the impact of microplastics on plant nitrogen utilization efficiency, it reveals the specific impact of microplastics on plant nitrogen absorption and utilization, and provides a scientific basis for the rational control of microplastic pollution, protection of agricultural production and ecosystem nutrient cycles. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0028] If no specific technology or conditions are specified in the examples of the present invention, the technology or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the devices, instruments, reagents, etc. is not specified, they are all conventional products that can be purchased through regular channels. The experimental reagents and raw materials involved are all commercially available, and the reagents are all analytically pure products.

[0029] In some embodiments of the present invention, experimental materials and preparation: Microplastic selection: Select the target microplastic type (polyethylene, polypropylene, etc.) to ensure that the particle size is uniform and the diameter is within the range of 1-100 microns. Test plant selection: Any target plant can be selected, such as wheat, corn, rice, etc., to ensure that the plants are in the same growth stage and growth conditions. 15 N stable isotope labeling: 15 N isotope nitrogen fertilizers, such as 15 N-labeled urea or ammonium nitrate, ensure 15 N abundance is higher than the natural abundance (about 0.366%). The specific determination steps include: 1) Soil preparation: Select soil without microplastic contamination, perform necessary disinfection, and measure its basic physical and chemical properties. The test uses a green plastic bucket with a sealed bottom, with an upper diameter of 10 cm, a bottom diameter of 12 cm, and a height of 10 cm. Each pot contains 1 kg of dry soil.

[0030] 2) Addition of microplastics: Mix microplastics evenly into the soil at a preset concentration (such as 0%, 0.1%, 0.5%, 1%, etc.; soil weight ratio) to ensure that the microplastics are evenly distributed in the soil.

[0031] 3) Plant planting and labeling: Plant the target plants in soil containing or not containing microplastics, and irrigate and fertilize according to conventional management measures. Add 0.1g of pure nitrogen, corresponding to 15 N-labeled urea 0.21 g (containing 46% N, 15 N abundance is 10.14%, provided by Shanghai Research Institute of Chemical Industry (in plants) corresponding to a field dosage of 225 kg / hm 2 In the early growth stage, use 15 Water the soil with nitrogen fertilizer labeled with N stable isotope to ensure that the plants can fully absorb and utilize the labeled nitrogen. Mix the labeled urea and dissolve it in 30 mL of distilled water and apply it evenly to the soil.

[0032] 4) Sample collection: Collect samples of the above-ground and underground parts of plants, as well as corresponding soil samples, at different stages of plant growth (such as seedling stage, growth stage, and maturity stage).

[0033] 5) Sample processing and analysis: The collected samples were dried and ground, and the stable isotope mass spectrometer (Delta V Advantage, Germany) was used to measure the 15 N and 14 N isotope measurements were performed to calculate 15 The abundance of N isotopes.

[0034] Ndf 肥 % indicates the proportion of nitrogen in the plant that comes from fertilizer nitrogen, and its size can reflect the plant's 15 N fertilizer absorption competition, Ndf 土 % indicates the proportion of nitrogen in each organ of the plant that comes from soil nitrogen. 15 N percentage is determined by mass spectrometry 15 N abundance minus natural 15 N abundance (0.366%), 15 N labeled fertilizers 15 N percentage of ultra-labeled fertilizers determined by mass spectrometry 15 N abundance minus 15 Natural abundance of N (0.366%). Total nitrogen content of plant samples was determined using an elemental analyzer.

[0035] In the following embodiments of the present invention, nitrogen comes from fertilizer nitrogen (Ndf 肥 %): Crop nitrogen from soil (Ndf 土 %): Calculation of nitrogen absorption by plants: Plant nitrogen absorption (g / plant) = plant dry matter weight × plant nitrogen content.

[0036] Calculation of fertilizer nitrogen (15N) absorption: Fertilizer nitrogen (15N) absorption (mg / plant) = plant nitrogen absorption × Ndf 肥 %.

[0037] Calculation of nitrogen use efficiency: Statistical analysis: Statistical software (such as SPSS, R, etc.) was used to perform one-way analysis of variance (ANOVA), Duncan's multiple range test, etc. to assess whether the effect of microplastics on plant nitrogen utilization efficiency is significant.

[0038] Example 1 Polyethylene microplastics were selected, and the particle size was uniform, with a diameter of 100 microns. Oats were selected as the test plants.

[0039] Soil without microplastic pollution was selected, disinfected as necessary, and its basic physical and chemical properties were measured. The experiment used a green plastic bucket with a sealed bottom, with an upper diameter of 10 cm, a bottom diameter of 12 cm, and a height of 10 cm, and each pot was filled with 1 kg of dry soil.

[0040] Microplastics are evenly mixed into the soil at a preset concentration (e.g. 0%, 0.5%, 2%; soil weight ratio) in a repeatable manner to ensure uniform distribution of microplastics in the soil.

[0041] Plant appropriate amount of oat seeds in soil with different concentrations of microplastics, and thin out to 5 oats of uniform size per pot. Irrigate and fertilize according to conventional management measures. Add 0.1g of pure nitrogen, corresponding to 15 N-labeled urea 0.21 g (containing N 46%, 15 N abundance is 10.14%, provided by Shanghai Research Institute of Chemical Industry (in plants) corresponding to a field dosage of 225 kg / hm 2 .

[0042] In the early growth stage, use 15 Water the soil with nitrogen fertilizer labeled with N stable isotope to ensure that the plants can fully absorb and utilize the labeled nitrogen. Mix the labeled urea and dissolve it in 30 mL of distilled water and apply it evenly to the soil.

[0043] At the mature stage of plant growth, samples of above-ground and below-ground plant parts, as well as corresponding soil samples, were collected.

[0044] The collected samples were dried and ground, and the samples were analyzed by stable isotope mass spectrometry (Delta VAdvantage, Germany). 15 N and 14 N isotope measurements were performed to calculate 15 The abundance of N isotopes.

[0045] The nitrogen utilization efficiency of oats under microplastic concentrations of 0%, 0.5%, and 2% was calculated by the formula (Table 1).

[0046] Table 1 Effects of microplastics on nitrogen utilization efficiency of oats

[0047] By calculating the nitrogen source from fertilizer nitrogen (Ndf 肥 %), plant nitrogen content, and plant biomass, and the nitrogen utilization efficiency was calculated according to the formula.

[0048] Table 2 Statistical analysis of the effect of microplastics on nitrogen utilization efficiency of oats

[0049] Statistical analyses such as one-way analysis of variance (ANOVA) and Duncan's multiple range test showed that compared with the control, both 2% and 0.5% concentrations of microplastics significantly reduced nitrogen utilization efficiency.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the effect of microplastics on plant nitrogen utilization efficiency, characterized in that: The following steps are involved: 1) Microplastic addition: mixing different concentrations of microplastics with soil without microplastic contamination; 2) Plant planting and labeling: Plant the target plants in the soil with microplastics added and use them in the early growth stage. 15 N stable isotope labeled nitrogen fertilizer for irrigation; 3) Sample collection: Samples are collected after the target plants grow; 4) Sample processing and analysis: The collected samples are dried and ground, and the processed samples are measured, calculated and statistically analyzed.

2. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to claim 1, characterized in that: Step 1) also includes disinfecting the soil and measuring its basic physical and chemical properties.

3. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to claim 1 or 2, characterized in that: In step 1), the microplastics are added at a concentration of 0-3% based on the total weight of the soil; preferably, the microplastics are mixed with the soil at preset concentrations of 0-0.01%, 0.1%-0.2%, 0.5%-0.6% and 1%-1.2%, respectively.

4. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to claim 3, characterized in that: In step 1), the microplastic is polyethylene or polypropylene, and the particle diameter is 1~100μm.

5. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to any one of claims 1 to 4, characterized in that: In step 2), 15 The nitrogen fertilizer labeled with N stable isotope is 15 N-labeled urea or ammonium nitrate; preferably, the 15 The nitrogen content of nitrogen fertilizer labeled with N stable isotope is 42%~50%. 15 N abundance>0.366%, preferably 9%~11%.

6. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to any one of claims 1 to 5, characterized in that: In step 2), the target plant includes one or more of wheat, corn and rice.

7. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to any one of claims 1 to 6, characterized in that: In step 3), the sample collection includes the seedling stage, growth stage and maturity stage of plant growth, and preferably samples of the above-ground part, underground part and soil of the target plant are collected.

8. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to any one of claims 1 to 7, characterized in that: In step 4), the determination includes: using an element analyzer to determine the nitrogen content of the plant; and / or using a stable isotope mass spectrometer to determine the nitrogen content of the sample. 15 N and 14 N isotope determination.

9. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to any one of claims 1 to 8, characterized in that: In step 4), the statistical analysis includes: using statistical software to perform one-way analysis of variance or Duncan's multiple comparison test on the data to evaluate the significance of the effect of microplastics on plant nitrogen utilization efficiency.

10. The method for determining the effect of microplastics on plant nitrogen utilization efficiency according to any one of claims 1 to 9, characterized in that: In step 4), the calculation includes: Ndf 肥 % is the proportion of nitrogen in the plant that comes from fertilizer nitrogen, calculated as follows: ； Among them, the plant samples 15 N percentage is determined by mass spectrometry 15 N abundance minus natural 15 N abundance; 15 N labeled fertilizers 15 N percentage of ultra-labeled fertilizers determined by mass spectrometry 15 N abundance minus 15 N natural abundance; and / or, Ndf 土 % is the proportion of nitrogen in the plant that comes from soil nitrogen, calculated as follows: ； And / or, the nitrogen absorption of plants is calculated as follows: Plant nitrogen absorption = plant dry matter × plant nitrogen content; And / or, fertilizer nitrogen absorption is calculated as follows: Fertilizer nitrogen absorption = plant nitrogen absorption × Ndf 肥 %; And / or, nitrogen use efficiency is calculated as follows: 。

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