Low-toxicity method for measuring soil nitrate nitrogen 15N based on cavity ring-down technology

Abstract

The invention discloses a low-toxicity method for measuring soil nitrate nitrogen 15N based on an optical cavity ring-down technology, which comprises the following steps: extracting 1 g of fresh soil with 10 ml of 2M KCl, determining the content of nitrate nitrogen in the soil through a flow analyzer, and diluting to 0.14-0.28 mgN / ml to obtain a soil extract for later use; the method comprises the following steps: adding 2ml of a soil leaching solution into a headspace bottle for later use, and then sequentially adding 0.2 ml of a 0.01 M VCl3 solution and 0.5 ml of 0.02 M hydroxylamine hydrochloride; transferring to a constant-temperature shaking table at the temperature of 37 DEG C, reacting for 16 hours at the speed of 125 rpm / min, extracting a gas sample by using a gas-tight injector, and injecting into an isotope measuring instrument based on an optical cavity ring-down technology to measure 15N of N2O as 15N of nitrate nitrogen. According to the method, the analysis efficiency of the whole experiment can be effectively improved, environmental hazards caused by use of high-toxicity chemical reagents are reduced, and meanwhile, the analysis cost is effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of agricultural soil testing methods, specifically relating to a method for determining soil nitrate nitrogen based on cavity ring-down technology. 15 A low-toxicity method for N. Background Technology

[0002] Nitrate nitrogen (NO3) - Nitrate nitrogen (N-N) is a typical agricultural non-point source pollutant. Due to its high solubility and fluidity, nitrate nitrogen is easily leached from the soil. The negative impacts of nitrate leaching on the environment and ecology are multifaceted, including water pollution, soil degradation and ecosystem destruction, greenhouse gas emissions, and risks to human health. Specifically, nitrate nitrogen is readily soluble in water and seeps into groundwater, leading to increased nitrate nitrogen concentrations. Groundwater is an important source of drinking water, and excessive nitrate nitrogen levels (above 10 mg / L) can harm human health, such as causing methemoglobinemia. Nitrate nitrogen also enters rivers, lakes, and other water bodies through runoff, becoming one of the main causes of eutrophication. Nitrate leaching also leads to the loss of soil nutrients, resulting in decreased soil fertility or soil acidification, which in turn affects soil quality and agricultural production. Nitrate leaching has negative impacts on farmland and surrounding ecosystems, such as reduced biodiversity (damaging aquatic ecosystems, causing the disappearance of sensitive species, and altering plant community structure) and non-target organism hazards (nitrate nitrogen entering water bodies may be converted into nitrites, which are toxic to aquatic organisms).

[0003] Nitrate nitrogen is a crucial component of the nitrogen cycle in natural ecosystems, and its sources and migration / transformation within environmental systems are a hot topic in environmental science and ecology. Accurately identifying the sources and migration / transformation of nitrate nitrogen contributes to a deeper understanding of regional nitrogen cycle characteristics and dynamics, such as the nitrogen turnover rate in soil. Nitrate nitrogen isotopes (…) 15 Nitrate nitrogen (N) is a powerful indicator of the migration and transformation of nitrate nitrogen in the environment. Analyzing nitrate nitrogen... 15 The isotopic abundance and differences of nitrogen (N) can effectively track its fate in the ecological environment. Currently, nitrate nitrogen is the most prevalent form. 15 The main methods for determining nitrogen (N) include chemical transformation and microdiffusion coupled with isotope mass spectrometry. However, current methods suffer from two major problems: first, the use of highly toxic chemical reagents, such as cadmium particles and sodium azide, causes excessive environmental pollution; second, microdiffusion methods struggle to completely remove residual ammonia nitrogen from the solution, affecting the analytical results, and are also costly and time-consuming; third, isotope mass spectrometry is expensive and requires a long analysis time. These issues hinder the determination of soil nitrate nitrogen. 15 N faces challenges such as poor safety and environmental friendliness, and high cost, making it impossible to promote its application on a large scale.

[0004] Therefore, it is particularly important to develop an experimental method that is low in toxicity, high in throughput, low in cost, and suitable for widespread application. Summary of the Invention

[0005] To address the issues of high toxicity of traditional chemical reagents and high time and economic costs associated with micro-diffusion methods, this invention proposes the following technical solution:

[0006] This invention first discloses a method for determining soil nitrate nitrogen based on optical cavity ring-down technology. 15 The method for reducing the toxicity of N includes the following steps:

[0007] (1) Preparation of the sample to be tested:

[0008] Weigh 1g of fresh soil and extract it with 10ml of 2M KCl for 1h. Determine the nitrate nitrogen content of the soil using a flow analyzer and dilute it to 0.14-0.28mg N / ml.

[0009] (2) Preparation of reagents:

[0010] ① 0.01M Vanadium Chloride (VCl3): Prepare 10ml of solution according to the sample preparation, which is sufficient for the determination of 50 samples. Weigh 0.02g of VCl3 and dissolve it in 6M hydrochloric acid (HCl). Complete dissolution takes about 1 hour; it can be stored at 4℃ for several months.

[0011] ② 0.02M hydroxylamine hydrochloride (NH2OH·HCl): Weigh 0.024g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water. This reagent should be prepared and used immediately.

[0012] ③Reaction conditions:

[0013] In a headspace vial, add 2 ml of sample (soil extract), 0.2 ml of 0.01 M VCl3 solution, and then 0.5 ml of 0.02 M hydroxylamine hydrochloride. Transfer to a 37°C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, use a gas-sealed syringe to extract the gas sample and inject it into an isotope measuring instrument based on optical cavity ring-down technology to determine N2O. 15 N, as nitrate nitrogen 15 N.

[0014] The beneficial effects of this invention are as follows:

[0015] 1. This invention can effectively improve the analytical efficiency of the entire experiment:

[0016] The reduction of nitrate nitrogen to N2O for one batch of samples (95 samples, 60 standard samples, and 3 blank treatments) takes approximately one day (including overnight hydroxylamine reaction). Analysis of one batch of N2O isotopes using a nitrous oxide isotope analyzer takes 1.5 days. Each batch of 95 samples requires a total of 2.5 days, which is nearly 5 days less than the microdiffusion method, significantly improving analytical efficiency.

[0017] 2. This invention can reduce the environmental hazards caused by the use of highly toxic chemical reagents:

[0018] Cadmium is one of the most harmful heavy metals to the environment and human health. However, existing technologies use cadmium particles for reduction, which not only increases the risk of environmental pollution but also endangers the safety of laboratory personnel. By replacing highly toxic and explosive cadmium and sodium azide with low-toxicity, low-pollution vanadium chloride and hydroxylamine hydrochloride, respectively, environmentally friendly and green determination is achieved.

[0019] 3. This invention effectively reduces analysis costs:

[0020] In terms of analytical costs, the cost of sample introduction for isotope mass spectrometry is around 150-300 yuan per sample, while the cost can be reduced to around 20 yuan per sample using cavity ring-down technology. Secondly, in terms of analytical time, isotope mass spectrometry requires about 0.5-1 hour per sample, while the sample analysis time can be reduced to 5-10 minutes per sample using cavity ring-down technology, which greatly improves analytical efficiency and thus achieves low-cost, high-throughput analysis. Attached Figure Description

[0021] Figure 1 The calibration curve was plotted. Detailed Implementation

[0022] Example 1:

[0023] A method for determining soil nitrate nitrogen based on optical cavity ringback technology 15 The method for reducing the toxicity of N includes the following steps:

[0024] (1) Preparation of the sample to be tested:

[0025] Weigh 1g of fresh soil and extract it with 10ml of 2M KCl for 1h. Determine the nitrate nitrogen content of the soil using a flow analyzer and dilute it to 0.14-0.28mg N / ml.

[0026] (2) Preparation of reagents:

[0027] ① 0.01M Vanadium Chloride (VCl3): Prepare 10ml of solution according to the sample preparation, which is sufficient for the determination of 50 samples. Weigh 0.02g of VCl3 and dissolve it in 6M hydrochloric acid (HCl). Complete dissolution takes about 1 hour; it can be stored at 4℃ for several months.

[0028] ② 0.02M hydroxylamine hydrochloride (NH2OH·HCl): Weigh 0.024g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water. This reagent should be prepared and used immediately.

[0029] ③Reaction conditions:

[0030] In a headspace vial, add 2 ml of sample (soil extract), 0.2 ml of 0.01 M VCl3 solution, and then 0.5 ml of 0.02 M hydroxylamine hydrochloride. Transfer to a 37°C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, use a gas-sealed syringe to extract the gas sample and inject it into an isotope measuring instrument based on optical cavity ring-down technology to determine N2O. 15 N, as nitrate nitrogen 15 N.

[0031] Comparative Example 1:

[0032] 1) 0.1M Vanadium Chloride (VCl3): Prepare 10ml of solution according to the sample preparation, which can be used to determine 50 samples. Weigh 0.2g of VCl3 and dissolve it in 10ml of 6M hydrochloric acid (HCl). Complete dissolution takes about 1 hour; it can be stored at 4℃ for several months.

[0033] 2) 0.24mM hydroxylamine hydrochloride (NH2OH·HCl): Weigh 2.24mg of hydroxylamine hydrochloride and dissolve it in 100ml of deionized water. This reagent should be prepared and used immediately.

[0034] 3) Reaction conditions:

[0035] In a 50 ml headspace vial, add 2 ml of sample (soil extract), 0.2 ml of 0.1 M VCl3 solution, and then 0.25 ml of 0.42 mM hydroxylamine hydrochloride. After reacting in a 55 °C oven for 50 minutes, transfer to a 37 °C constant-temperature shaker and react at 125 rpm / min for 16 hours. Then, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0036] Comparative Example 2:

[0037] 1) 0.05M Vanadium Chloride (VCl3): Prepare 10ml of solution according to the sample preparation, which can be used to determine 50 samples. Weigh 0.1g of VCl3 and dissolve it in 10ml of 6M hydrochloric acid (HCl). Complete dissolution takes about 1 hour; it can be stored at 4℃ for several months.

[0038] 2) 0.24mM hydroxylamine hydrochloride (NH2OH·HCl): Weigh 2.24mg of hydroxylamine hydrochloride and dissolve it in 100ml of deionized water. This reagent should be prepared and used immediately.

[0039] 3) Reaction conditions:

[0040] In a 50 ml headspace vial, add 2 ml of sample (soil extract), followed by 0.2 ml of 0.1 M VCl3 solution and then 0.25 ml of 0.42 mM hydroxylamine hydrochloride. Transfer to a 37 °C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0041] Comparative Example 3:

[0042] 1) 0.02M Vanadium Chloride (VCl3): Prepare according to sample requirements, generally 10ml is sufficient for 50 sample determinations. Weigh 0.031g VCl3 and dissolve it in 10ml of 6M hydrochloric acid (HCl). Complete dissolution takes approximately 1 hour; it can be stored at 4℃ for several months.

[0043] 2) 0.24mM hydroxylamine hydrochloride (NH2OH·HCl): Weigh 2.24mg of hydroxylamine hydrochloride and dissolve it in 100ml of deionized water. This reagent should be prepared and used immediately.

[0044] 3) Reaction conditions:

[0045] In a 50 ml headspace vial, add 2 ml of sample (soil extract), followed by 0.2 ml of 0.02 M VCl3 solution, and then 0.25 ml of 0.42 mM hydroxylamine hydrochloride. Transfer to a 37 °C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0046] Comparative Example 4:

[0047] 1) 0.02M Vanadium Chloride (VCl3): Prepare according to sample requirements, generally 10ml is sufficient for 50 sample determinations. Weigh 0.031g VCl3 and dissolve it in 10ml of 6M hydrochloric acid (HCl). Complete dissolution takes approximately 1 hour; it can be stored at 4℃ for several months.

[0048] 2) 0.1M hydroxylamine hydrochloride (NH2OH·HCl): Weigh 0.07g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water. This reagent should be prepared and used immediately.

[0049] 3) Reaction conditions:

[0050] In a 50 ml headspace vial, add 2 ml of sample (soil extract), followed by 0.2 ml of 0.02 M VCl3 solution and then 0.50 ml of 0.01 M hydroxylamine hydrochloride. Transfer to a 37 °C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0051] Comparative Example 5:

[0052] 1) 0.02M Vanadium Chloride (VCl3): Prepare according to sample requirements, generally 10ml is sufficient for 50 sample determinations. Weigh 0.031g VCl3 and dissolve it in 10ml of 6M hydrochloric acid (HCl). Complete dissolution takes approximately 1 hour; it can be stored at 4℃ for several months.

[0053] 2) 0.05M hydroxylamine hydrochloride (NH2OH·HCl): Weigh 0.035g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water. This reagent should be prepared and used immediately.

[0054] 3) Reaction conditions:

[0055] In a 50 ml headspace vial, add 2 ml of sample (soil extract), followed by 0.2 ml of 0.02 M VCl3 solution and then 0.50 ml of 0.01 M hydroxylamine hydrochloride. Transfer to a 37 °C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0056] Comparative Example 6:

[0057] 1) 0.01M Vanadium Chloride (VCl3): Prepare according to sample requirements, generally 10ml is sufficient for 50 sample determinations. Weigh 0.031g VCl3 and dissolve it in 10ml of 6M hydrochloric acid (HCl). Complete dissolution takes approximately 1 hour; it can be stored at 4℃ for several months.

[0058] 2) 0.1M hydroxylamine hydrochloride (NH2OH·HCl): Weigh 0.07g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water. This reagent should be prepared and used immediately.

[0059] 3) Reaction conditions:

[0060] In a 50 ml headspace vial, add 2 ml of sample (soil extract), 0.2 ml of 0.02 M VCl3 solution, and then 0.50 ml of 0.1 M hydroxylamine hydrochloride. Transfer to a 37 °C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0061] Comparative Example 7:

[0062] 1) 0.01M Vanadium Chloride (VCl3): Prepare according to sample requirements, generally 10ml is sufficient for 50 sample determinations. Weigh 0.031g VCl3 and dissolve it in 10ml of 3M hydrochloric acid (HCl). Complete dissolution takes approximately 1 hour; it can be stored at 4℃ for several months.

[0063] 2) 0.02M hydroxylamine hydrochloride (NH2OH·HCl): Weigh 0.14g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water. This reagent should be prepared and used immediately.

[0064] 3) Reaction conditions:

[0065] In a 50 ml headspace vial, add 2 ml of sample (soil extract), followed by 0.2 ml of 0.02 M VCl3 solution and then 0.50 ml of 0.02 M hydroxylamine hydrochloride. Transfer to a 37°C constant-temperature shaker and react at 125 rpm / min for 16 h. Then, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0066] Comparative Example 8:

[0067] 1) 0.01M Vanadium Chloride (VCl3): Prepare according to sample requirements, generally 10ml is sufficient for 50 sample determinations. Weigh 0.031g VCl3 and dissolve it in 10ml of 6M hydrochloric acid (HCl). Complete dissolution takes approximately 1 hour; it can be stored at 4℃ for several months.

[0068] 2) 0.02M hydroxylamine hydrochloride (NH2OH·HCl): Weigh 0.14g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water. This reagent should be prepared and used immediately.

[0069] 3) Reaction conditions:

[0070] In a 50 ml headspace vial, add 2 ml of sample (soil extract), followed by 0.50 ml of 0.02 M hydroxylamine hydrochloride, and immediately after, 0.2 ml of 0.02 M VCl3 solution. Heat the solution in a 55 °C oven for 50 minutes, then transfer it to a 37 °C constant-temperature shaker and continue the reaction at 125 rpm / min for 16 hours. Finally, extract the gas sample using a gas-locked syringe and measure the N2O using an isotope analyzer based on cavity ring-down technology. 15 N, as nitrate nitrogen 15 N.

[0071] The recovery rate of N2O prepared in Example 1 was determined, and a standard curve was obtained using laboratory standard samples to obtain the slope and R. 2 The calibration curve is as follows: Figure 1 As shown in Table 1, the test results are as follows.

[0072] Table 1. N2O recovery rate and calibration curve parameters of the embodiments and comparative examples of the present invention.

[0073]

[0074]

[0075] As shown in Table 1, the N2O recovery rate of the method described in this invention can reach over 50%, and the calibration curve R² reaches 0.99. Therefore, it can effectively achieve the recovery of nitrate nitrogen. 15 Determination of N.

[0076] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0077] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include such modifications and variations.

Claims

1. A method for determining soil nitrate nitrogen based on optical cavity ring-down technique 15 Methods for reducing the toxicity of N include: (1) Sample preparation: Fresh soil was extracted in KCl solution, and the nitrate nitrogen content of the soil was determined by a flow analyzer. Then it was diluted to obtain soil extract for later use. (2) Reaction conditions: Soil extract, VCl3 solution, and hydroxylamine hydrochloride solution were added sequentially to a headspace flask. The mixture was then transferred to a constant-temperature shaker for reaction. The gas sample was extracted using a gas-locked syringe and then injected into an isotope measuring instrument based on cavity ring-down technology to determine N2O. 15 N, as nitrate nitrogen 15 N.

2. The method for determining soil nitrate nitrogen based on optical cavity ringback technology according to claim 1 15 The low-toxicity method of N, wherein: The mass-to-volume ratio of the fresh soil to the KCl solution in step (1) is 1g:10ml; The concentration of the soil extract is 0.14-0.28 mg N / ml.

3. The method for determining soil nitrate nitrogen based on cavity ring-down technology according to claim 1 15 The low-toxicity method of N, wherein: The concentration of the KCl solution in step (1) is 2M; The extraction time is 1 hour.

4. The method for determining soil nitrate nitrogen based on optical cavity ringback technology according to claim 1 15 The low-toxicity method of N, wherein: The volume ratio of the soil extract, VCl3 solution, and hydroxylamine hydrochloride solution in step (2) is 20:2:

5.

5. The method for determining soil nitrate nitrogen based on optical cavity ringback technology according to claim 1 15 The low-toxicity method of N, wherein: The reaction conditions in step (2) are as follows: at 37°C, the reaction is carried out at a speed of 125 rpm / min for 16 hours.

6. The method for determining soil nitrate nitrogen based on optical cavity ringback technology according to claim 1 15 The low-toxicity method of N, wherein: The VCl3 solution in step (2) is prepared by the following method: Weigh 0.02g of VCl3 and dissolve it in 6M hydrochloric acid until completely dissolved to obtain a VCl3 solution with a concentration of 0.01M. Store the solution in a refrigerator at 4℃ for later use.

7. The method for determining soil nitrate nitrogen based on cavity ringback technology according to claim 1 15 The low-toxicity method of N, wherein: The hydroxylamine hydrochloride solution in step (2) is prepared by the following method: Weigh 0.024g of hydroxylamine hydrochloride and dissolve it in 10ml of deionized water to obtain a 0.02M hydroxylamine hydrochloride solution. This reagent should be prepared and used immediately.

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