Preparation for reducing pesticide residues and application of scenedesmus obliquus in reduction of pesticide residues

Abstract

The invention belongs to the technical field of land pesticide residue degradation, and particularly relates to a preparation for reducing pesticide residues and application of scenedesmus obliquus to reduction of the pesticide residues. The invention finds that the Scenedesmus obliquus can effectively degrade four typical pesticides, namely atrazine, thiamethoxam, cypermethrin and nicosulfuron, and is wide in application temperature (such as 5-30 DEG C), short in action time, high in safety and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of soil pesticide residue degradation technology, specifically involving formulations for reducing pesticide residues and the application of Scenedesmus obliquus in reducing pesticide residues. Background Technology

[0002] Black soil, a globally scarce high-fertility soil resource, is a core strategic resource for ensuring national food security in Northeast my country's main grain-producing areas. However, with the development of intensive agriculture, pesticides such as atrazine (a corn herbicide), thiamethoxam (a neonicotinoid insecticide), cypermethrin (a pyrethroid insecticide), and nicosulfuron (a sulfonylurea herbicide) are widely used. Because these pesticides are chemically stable and have long half-lives (e.g., atrazine's half-life can reach 60–150 days), they easily form complex residual pollution in black soil and accumulate through the food chain.

[0003] Current pesticide pollution control technologies have significant limitations: (1) When four types of pesticides coexist, they may produce synergistic toxicity (e.g., the combined use of nicosulfuron and atrazine can increase the toxicity to aquatic organisms), exacerbating ecological risks; (2) The organic matter content of black soil is as high as 5% to 10%, and the strong adsorption effect on pesticide molecules significantly inhibits the degradation efficiency of traditional microbial remediation technologies; (3) The reported degradation strains (e.g., Acinetobacter and Pseudomonas) generally have problems such as single target (can only degrade 1-2 kinds of pesticides) and poor environmental adaptability (the activity of black soil drops sharply during the low temperature period), which makes it difficult to meet the treatment needs of complex pollution scenarios.

[0004] With the deepening of environmental protection and sustainable development concepts, green, efficient, and low-cost bioremediation technologies have become a research hotspot. Microalgae, as a type of photosynthetic autotrophic lower plant, possess advantages such as rapid growth, strong environmental adaptability, and the ability to simultaneously degrade pesticide residues, demonstrating enormous potential in pollution control. However, currently, broad-spectrum, low-temperature-resistant microalgae degradation technologies targeting typical compound pesticide pollution in black soil remain lacking. Developing microalgae remediation technologies with high degradation performance and environmental adaptability is of great significance for solving the problem of pesticide pollution in black soil, ensuring food security, and promoting ecological sustainable development. Summary of the Invention

[0005] Based on this, the present invention discovered *Scenedesmus obliquus* (… Oblique scene It can effectively degrade four typical pesticides: atrazine, thiamethoxam, cypermethrin, and nicosulfuron. It has a wide applicable temperature range (e.g., 5℃~30℃), short action time, high safety, and low cost.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: In one aspect, this invention provides an formulation for reducing pesticide residues, which includes *Scenedesmus obliquus*, whose preservation number is CGMCC No. 6567.

[0007] Another aspect of the present invention provides the application of Scenedesmus obliquus in reducing pesticide residues, wherein the pesticide is one or more combinations of atrazine, thiamethoxam, cypermethrin, or nicosulfuron.

[0008] Preferably, the application includes: the application of Scenedesmus obliquus in reducing pesticide residues in soil.

[0009] Preferably, in the above application, the accession number of Scenedesmus obliquus is CGMCC No. 6567.

[0010] Preferably, in the above applications, the applicable temperature is 5℃~30℃.

[0011] In another aspect, the present invention provides a method for reducing pesticide residues in soil, the method comprising: inoculating the soil with Scenedesmus obliquus, wherein the pesticide is one or more of atrazine, thiamethoxam, cypermethrin or nicosulfuron.

[0012] Preferably, in the above method, the accession number of Scenedesmus obliquus is CGMCC No. 6567.

[0013] Preferably, in the above method, the inoculum size of *Scenedesmus obliquus* is (0.8–1.2) × 10⁻⁶. 8 cells / g soil.

[0014] Preferably, in the above method, nitrogen and phosphorus elements are also added to the soil.

[0015] More preferably, in the above method, the ratio of nitrogen to phosphorus is (9-11):1, and the amount of phosphorus added is (4mg-6mg) / kg soil.

[0016] The beneficial effects of this invention include: (1) The present invention uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron. A single treatment can reduce soil pesticide residues to below the limit of GB 2763-2021, ensuring the safe reuse of arable land.

[0017] (2) The present invention uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron, with a pesticide degradation flux of 35.2 mg / (kg·d) per unit area of ​​black soil, which is 305% higher than the traditional method (8.7 mg / (kg·d)).

[0018] (3) The present invention uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron. The algal solution of Scenedesmus obliquus can be expanded on site (cost 0.8 yuan / liter), and the amount required per mu is 375 liters. The overall cost can be reduced by about 85% compared with the existing technology.

[0019] (4) The present invention uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron. It has a wide applicable temperature range, and can be applied to the temperature range of 5℃~30℃ in the Northeast Black Soil Area, covering 92% of the agricultural time window throughout the year.

[0020] (5) The present invention uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron. The degradation rate of pesticides can reach more than 88% in 28 days at room temperature and more than 66% in 45 days at a low temperature of 10℃. The action time is short and the effect is fast.

[0021] (6) The present invention uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron in the soil, without generating metabolic toxins and without damaging the soil structure, and is highly safe.

[0022] (7) The present invention uses Scenedesmus obliquus to degrade four pesticides: atrazine, thiamethoxam, cypermethrin and nicosulfuron, which is more spectral than existing technologies (such as traditional microbial remediation and chemical oxidation methods). Detailed Implementation

[0023] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0024] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0025] In a first aspect, embodiments of the present invention provide an formulation for reducing pesticide residues, the formulation for reducing pesticide residues including Scenedesmus obliquus, the preservation number of Scenedesmus obliquus is CGMCC No. 6567.

[0026] Secondly, embodiments of the present invention provide an application of *Scenedesmus obliquus* in reducing pesticide residues, wherein the pesticide is one or more combinations of atrazine, thiamethoxam, cypermethrin, or nicosulfuron.

[0027] It should be noted that this invention discovered *Scenedesmus obliquus* (… Oblique sceneThis invention can effectively degrade four typical pesticides: atrazine, thiamethoxam, cypermethrin, and nicosulfuron. It has a wide applicable temperature range (e.g., 5℃ to 30℃), short action time, high safety, and low cost. Furthermore, addressing the limitations of existing microbial remediation technologies in simultaneously treating multiple types and targets of pesticides (e.g., bacteria only degrade triazines and fungi prefer pyrethroids), this invention discovers that *Scenedesmus obliquus* possesses cross-category synergistic degradation capabilities, enabling simultaneous and efficient degradation of four chemically distinct pesticides (including chlorpyrifos, neonicotinoids, pyrethroids, and sulfonylureas), with degradation rates exceeding 85% (28 days, initial concentration 10 mg / kg). Additionally, this invention can activate the "multi-enzyme cascade system" within *Scenedesmus obliquus* cells (e.g., dehalogenases decompose atrazine, cytochrome P450 oxidizes thiamethoxam, esterases hydrolyze cypermethrin, and sulfonylurea hydrolases cleave nicosulfuron), solving the problem of the single enzyme system in traditional strains.

[0028] In some specific examples, applications include: the use of Scenedesmus obliquus in reducing pesticide residues in soil.

[0029] It should be noted that the *Scenedesmus obliquus* in this invention is particularly suitable for reducing pesticide residues in soil, as it does not damage soil structure, does not produce toxic metabolites, and is highly safe.

[0030] In some specific examples, the accession number of Scenedesmus obliquus in the above applications is CGMCC No. 6567.

[0031] It should be noted that the *Scenedesmus obliquus* in this invention is preferably collected from the surface layer (0cm to 10cm) of the black soil in Northeast China. The *Scenedesmus obliquus* obtained after separation has the accession number CGMCC No. 6567. Information on *Scenedesmus obliquus* with accession number CGMCC No. 6567 is recorded in published patent CN102943046A.

[0032] In some specific examples, the applicable temperature range for the above applications is 5℃ to 30℃.

[0033] It should be noted that the expression level of antifreeze proteins (AFPs) of *Scenedesmus obliquus* in this invention increases at low temperatures, and the cell membrane fluidity does not change significantly. This means that *Scenedesmus obliquus* in this invention has a wide temperature range for degrading pesticide residues, such as 5℃ to 30℃, including 5℃, 10℃, 15℃, 20℃, or 25℃. This makes it more suitable for the temperature variations in the black soil of Northeast China.

[0034] Thirdly, embodiments of the present invention provide a method for reducing pesticide residues in soil, the method comprising: inoculating *Scenedesmus obliquus* into the soil, wherein the pesticide is one or more combinations of atrazine, thiamethoxam, cypermethrin, or nicosulfuron.

[0035] In some specific examples, the preservation number of *Scenedesmus obliquus* in the above method is CGMCC No. 6567.

[0036] In some specific examples, the inoculum size of *Scenedesmus obliquus* in the above method is (0.8–1.2) × 10⁻⁶. 8 cells / g soil.

[0037] It should be noted that, when using *Scenedesmus obliquus* to degrade pesticide residues in soil, the inoculation amount can be (0.8–1.2) × 10⁻⁶ per gram of soil. 8 cells are Scenedesmus oblique.

[0038] In some specific examples, nitrogen and phosphorus elements are also added to the soil in the above method.

[0039] It should be noted that nitrogen and phosphorus elements can be added to promote the degradation of pesticide residues in the soil. Nitrogen and phosphorus elements are well known in the field, for example, in a mixture of NH4Cl and K2HPO4.

[0040] In some specific examples, the ratio of nitrogen to phosphorus in the above method is (9-11):1, and the amount of phosphorus added is (4mg-6mg) / kg soil.

[0041] It should be noted that the ratio of nitrogen to phosphorus in this invention is (9-11):1 (e.g., 9.5:1, 10:1 or 10.5:1, etc.), and the amount of phosphorus added is 4mg to 6mg per kg of soil, such as 4.5mg, 5mg or 5.5mg, etc.

[0042] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0043] In the following examples, Scenedesmus obliquus ( Oblique scene The sample was collected from the surface layer (0-10cm) of black soil in Northeast China, isolated, and verified as Scenedesmus obliquus by 18S rRNA sequencing. Its preservation number is CGMCC No. 6567.

[0044] In the following example, BG-11 medium (pH 7.0) includes: NaNO3 1.5 g / L, K2HPO4 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, ferric citrate 0.006 g / L, EDTA 0.001 g / L, and 1 mL / L of trace element solution (1 L of trace element solution contains H3BO3 (boric acid): 2.86 g, MnCl2·4H2O (manganese chloride tetrahydrate): 1.81 g, ZnSO4·7H2O (zinc sulfate heptahydrate): 0.222 g, Na2MoO4·2H2O (sodium molybdate dihydrate): 0.39 g, CuSO4·5H2O (copper sulfate pentahydrate): 0.079 g, Co (NO3)2·6H2O (cobalt nitrate hexahydrate): 0.0494 g).

[0045] In the following example, the soil used was typical black soil from Gongzhuling, Changchun City, Jilin Province (6.8% organic matter, pH 6.5), which was passed through a 2mm sieve and sterilized at 121℃ for 1 hour.

[0046] In the following example, the method for culturing *Scenedesmus obliquus* to the logarithmic growth phase (OD680 = 1.2 ± 0.1, approximately 7 days) is as follows: *Scenedesmus obliquus* is inoculated into BG-11 medium (pH 7.0) and cultured in a shaker for approximately 7 days to the logarithmic phase; the culture temperature is 25 ± 1℃ or 10 ± 1℃; the light conditions are: first light (white LED, 3000 lux) for 12 hours, followed by darkness for 12 hours; the shaker speed is 120 rpm.

[0047] I. Simulation of Contaminated Land

[0048] Different pesticide solutions were prepared by dissolving atrazine, thiamethoxam, cypermethrin, and nicosulfuron in solvents. The pesticide solutions were then sprayed evenly onto the soil (dry soil) and allowed to evaporate the solvent in the dark and ventilated environment for 48 hours to obtain soils contaminated with different pesticides. The solvents and amounts of each pesticide are shown in Table 1 below.

[0049] Table 1. Solvents and dosages used without pesticides , Note: Take 10 mL of this pesticide solution (containing 10 mg of pesticide) and add it to 1 kg of dry soil. After the solvent evaporates, the concentration is 10 mg / kg.

[0050] II. Soil Grouping The soils contaminated with different pesticides obtained in section "I" above were divided into experimental groups and pesticide control groups, with uncontaminated soil serving as a blank control group. The experimental groups, pesticide control group, and blank control group were treated according to the methods shown in Table 2 below. The algal solution was prepared as follows: *Scenedesmus obliquus* grown to the logarithmic stage was centrifuged (4000 rpm, 10 min) to collect the algal sludge, and resuspended in sterile water to 10... 7 Algal solution was obtained at a concentration of cells / mL. Additionally, equal amounts of an NH4Cl-K2HPO4 mixture (N 50 mg / kg, P 5 mg / kg) with an N / P ratio of 10:1 were added to the soil in each treatment group. (NH4Cl: N content 26.17% (N as a percentage of NH4Cl molecular weight), required NH4Cl concentration = 5.0 mg / mL ÷ 26.17% ≈ 19.1 mg / mL (i.e., 19.1 g / L); K2HPO4: P content 17.78% (P as a percentage of K2HPO4 molecular weight), required K2HPO4 concentration = 0.5 mg / mL ÷ 17.78% ≈ 2.81 mg / mL (i.e., 2.81 g / L). g / L), and adjust the soil moisture content to 60% of the maximum water holding capacity (first measure the maximum water holding capacity of the soil, then calculate the target moisture content, add the corresponding amount of water by weighing, and verify after balancing); in addition, the light is first light (white LED, 3000 lux) for 12 hours, then dark for 12 hours, and so on until the end of cultivation.

[0051] Table 2 Soil treatment methods for each group , III. Sampling and Testing The detection method for pesticides in soil in the following tests is as follows: Take 5g of soil samples from each treatment group, dissolve them in 20mL of acetonitrile, sonicate for 30min, centrifuge, and filter through a 0.22μm filter membrane to obtain the sample to be tested; then, the sample to be tested is tested using HPLC-MS / MS (Agilent 1290-6470); the HPLC-MS / MS detection conditions for each pesticide are shown in Table 3 below (other conditions are performed according to the instrument's instruction manual).

[0052] Table 3 HPLC-MS / MS detection conditions for various pesticides , (a) Degradation rate detection (1) Degradation rate detection at room temperature The pesticide content in the soil of each group treated at room temperature was detected on the 7th, 14th and 28th days of cultivation using the above-mentioned detection method for pesticides in soil, and the degradation rate was calculated as follows: Degradation rate (%) = (Ct / C0) × 100 (Ct: concentration at time t, C0: initial concentration); The calculation results are shown in Table 4 below.

[0053] Table 4. Pesticide degradation rate in soil at different incubation times for each treatment group in the room temperature group. , As shown in Table 4 above, after 28 days of degradation, the degradation rate of all four types of pesticides by *Scenedesmus obliquus* in this invention is >85%, which meets the requirements of the crop rotation window period (≤30 days); in particular, the degradation of cypermethrin can reach more than 90% within 28 days.

[0054] (2) Degradation rate detection in the low-temperature group The pesticide content in the soil of each group treated in the low temperature group was detected on the 28th and 45th days of cultivation using the above-mentioned detection method for pesticides in soil, and the degradation rate was calculated as follows: Degradation rate (%) = (Ct / C0) × 100 (Ct: concentration at time t, C0: initial concentration); The calculation results are shown in Table 5 below.

[0055] Table 5. Pesticide degradation rate in soil at different incubation times for each treatment group in the low-temperature group. , As shown in Table 5 above, after 45 days of low-temperature degradation, the degradation rate of pesticides by *Scenedesmus obliquus* in this invention can reach over 65%, especially the degradation rate of cypermethrin can reach over 70%.

[0056] (ii) Detection of toxic intermediates The following screening methods for toxic intermediates include: (1) Detailed sample pretreatment process QuEChERS optimization steps: (1-1) Soil crushing: Weigh 10g of air-dried black soil, add 10g of anhydrous sodium sulfate and grind into powder (pass through a 2mm sieve) to improve extraction efficiency; (1-2) Extraction and salting out: Add 15 mL of acetonitrile (containing 1% glacial acetic acid) and shake vigorously for 5 min. Then add 4 g of anhydrous MgSO4 and 1 g of NaCl, shake for 3 min and centrifuge (4000 rpm, 10 min). Take the supernatant. (1-3) Purification: Take 5 mL of supernatant into a centrifuge tube containing 150 mg PSA and 900 mg MgSO4, vortex for 2 min, centrifuge, and then filter the supernatant through a 0.22 μm filter membrane for LC-MS / MS analysis; (1-4) Derivatization (for polar intermediates): If it is necessary to detect aniline intermediates (such as atrazine deethylated derivatives), dansyl chloride (D6 labeled) can be added for derivatization to improve the mass spectrometry response; (2) Instrument parameters and scanning strategy LC-MS / MS Full Scan Mode: Chromatographic column: ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 100 mm), column temperature 40℃; mobile phase: phase A is 0.1% formic acid water, phase B is 0.1% formic acid acetonitrile, gradient elution (0-5 min, 5%-30% B; 5-10 min, 30%-80% B; 10-12 min, 80% B); Mass spectrometry parameters: Ion source: electrospray ionization (ESI+), capillary voltage 3.5 kV, ion source temperature 150 °C, desolvation gas temperature 500 °C, flow rate 1000 L / h; Scan range: m / z 50-600, scan rate 2000 Da / s, resolution 70,000 (FWHM), mass deviation ≤5 ppm; data-dependent secondary scan (ddMS) 2 ): Automatically triggers secondary fragmentation with a collision energy of 15-40 eV, used for structural analysis of unknown intermediates.

[0057] (3) APGC-HRMS supplementary detection Chromatographic column: TG-5MS capillary column (30m×0.25mm×0.25μm), temperature program (40℃ held for 2 min, then increased to 320℃ at 15℃ / min and held for 5 min); Ion source: Atmospheric pressure gas phase ionization (APGC), transfer line temperature 280℃, ion source temperature 150℃, soft ionization mode (proton transfer) is used to generate molecular ion peaks (such as [M+H]+ m / z 292.098 of thiamethoxam). Mass spectrometry parameters: scan range m / z 50-600, resolution 20,000 (FWHM), mass deviation ≤5ppm, used to verify LC-MS / MS results and reduce matrix interference.

[0058] (4) Data processing and comparison with standard samples (4-1) Database construction: Establish a standard database containing target pesticides (atrazine, thiamethoxam, etc.) and their known metabolites (such as thiazolidin and deethylatrazine), and store retention time, accurate mass-to-charge ratio, characteristic fragment ions and abundance ratio.

[0059] Examples: Thiazolamide [M+H]+ m / z 169.043, characteristic fragment ion m / z 125.022 (losing SO2); deethylated atrazine [M+H]+ m / z 186.087, fragment ion m / z 158.061 (losing CO2).

[0060] (4-2) Screening Procedure: Target analyte screening: Extract ion chromatograms (EIC) using MassLynx software, setting the extraction window to ±5 ppm. Compare the retention time of standards and fragment ions. If the match is ≥85% and the signal-to-noise ratio (S / N) is ≥3, the result is considered positive. Non-target analyte screening: Peak extraction is performed on the full scan data. The data is compared with the MassBank and HMDB databases using Compound Discoverer software, combined with elemental composition prediction (e.g., C). 10 H 14 The precise mass number of ClN5O2S (m / z 305.068) is used to infer the structure of the potential intermediate. False positives are eliminated by spiking suspected peaks with standards. If the retention time is consistent with that of fragment ions, it is confirmed as the target intermediate. If they are inconsistent, matrix interference is eliminated by analyzing the isotope distribution pattern of high-resolution mass spectrometry (such as the M+2 peak of Cl).

[0061] Using the above-mentioned detection method for pesticides in soil (full scan mode (m / z 50-600) + standard comparison), the toxic intermediates (atrazine toxic intermediates: deisopropylatrazine, hydroxyatrazine, cyanuric acid; thiamethoxam toxic intermediates: N-nitroguanidine, thiamethoxam; cypermethrin toxic intermediates: 3-phenoxybenzoic acid, dichlorophenoxybenzoic acid; nicosulfuron toxic intermediates: 2-chloro-N,N-dimethylnicotinamide, sulfonamide carbamate) in the soil of each experimental group in the normal temperature group were screened at 28 days of incubation, as well as the toxic intermediates (thiamethoxam-thiazolidinyl and atrazine-deethyl derivatives) in each experimental group in the low temperature group at 45 days of incubation. The results showed that no known toxic intermediates (thiamethoxam metabolites thiazolidinyl and atrazine deethyl derivatives) were detected in any of the experimental groups in the normal temperature and low temperature groups; the final products were all mineralized into CO2, H2O, and Cl. - SO2 2- and NO3 - Inorganic substances, etc.

[0062] (iii) Bioavailability testing The Tenax-TA continuous extraction method was used to test the percentage of bioavailable pesticides and the amount of humic acid-pesticide binding in the soil of the experimental group (using atrazine) and the pesticide control group at room temperature after 28 days of cultivation. The specific methods are as follows: 1) Detection of the proportion of bioavailable pesticides (1) Tenax-TA resin (particle size 60-80 mesh, purchased from Sigma-Aldrich, USA) was soaked in methanol for 24 hours before use, rinsed with distilled water until neutral, and dried at 60°C for later use; the extraction container was a 50mL centrifuge tube (with a polytetrafluoroethylene liner cap). (2) Weigh 10.0g of soil sample that has passed through a 2mm sieve into a centrifuge tube, add 0.5g of Tenax-TA resin and 20mL of 0.01mol / L CaCl2 solution (as extraction medium), place in a constant temperature shaker, and extract by shaking at 25℃ and 200r / min. Remove the centrifuge tube at 1h, 2h, 4h, 8h, 12h, 24h, 48h, and 72h, respectively, centrifuge at 3000r / min for 10min, collect the supernatant; at the same time, add an equal amount of fresh CaCl2 solution and continue shaking extraction; (3) The pesticide concentration in the supernatant at each time point was determined by gas chromatography-mass spectrometry (GC-MS, model 7890A-5975C, Agilent Technologies, USA). The total amount extracted cumulatively is the amount of bioavailable pesticide. The formula for calculating the proportion of bioavailable pesticide is: (amount of bioavailable pesticide / total pesticide residue in soil) × 100%. The determination of the total pesticide residue in soil is performed in accordance with the "Gas Chromatography Method for the Determination of DDT and HCH in Soil" (GB / T14550-2003).

[0063] (ii) Humic acid-pesticide binding test method The determination of humic acid-pesticide binding capacity was performed using an alkaline dissolution-centrifugation method combined with high-performance liquid chromatography (HPLC). The specific steps are as follows: (1) Weigh 5.0g of soil sample, add 50mL of 0.1mol / L NaOH solution, shake and extract at 25℃ for 1h, centrifuge at 4000r / min for 15min, collect the supernatant (containing humic acid), repeat the extraction twice, and combine the supernatants; (2) Add 1 mol / L HCl to the supernatant to adjust the pH to 1.0, let stand for 24 h to precipitate humic acid, centrifuge at 3000 r / min for 10 min, and discard the supernatant; wash the precipitate 3 times with 0.01 mol / L HCl, add 20 mL of methanol and sonicate for 30 min (power 300 W), centrifuge at 4000 r / min for 10 min, collect the extract, repeat the extraction twice, combine the extracts and make up to 50 mL; (3) The pesticide concentration in the extract was determined by HPLC (model 1260 Infinity, Agilent Technologies, USA). The chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm), the mobile phase was methanol-water (volume ratio 70:30), the flow rate was 1.0 mL / min, and the detection wavelength was set according to the characteristics of the target pesticide (e.g., 210 nm for organophosphorus pesticides). The formula for calculating the humic acid-pesticide binding amount is: (total mass of pesticide in the extract / dry weight of soil sample), and the result is expressed as "mg / g".

[0064] The test results are shown in Table 6 below.

[0065] Table 6. Soil bioavailability and humic acid-pesticide binding rates in each treatment group , As shown in Table 6 above, the proportion of bioavailable pesticides in the soil treated in the experimental group was significantly higher than that in the pesticide control group without the use of Scenedesmus obliquus, increasing by about 108%. At the same time, the amount of humic acid-pesticide binding in the soil treated in the experimental group was significantly lower than that in the pesticide control group without the use of Scenedesmus obliquus, decreasing by about 63%.

[0066] (iv) Low-temperature fluidity test The expression levels of antifreeze proteins (AFPs) in *Scenedesmus obliquus* at room temperature and low temperature were measured using DPH fluorescence polarization assay. The antifreeze protein ELISA kit (model: AFP-ELISA-Kit, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.) was used for detection) and cell membrane fluidity (using Scenedesmus obliquus cells in logarithmic growth phase, washing 3 times with phosphate-buffered saline (PBS, pH 7.4), and adjusting the cell concentration to 1×10⁻⁶). 6 Cells / mL; Add DPH fluorescent probe to cell suspension at a final concentration of 10 μmol / L and incubate at 37°C in the dark for 30 min; Measure fluorescence polarization degree (P value) using a fluorescence spectrophotometer (model: F-7000, purchased from Hitachi, Japan). The excitation wavelength is 360 nm and the emission wavelength is 430 nm. Measure the fluorescence intensity in the parallel and perpendicular directions respectively, and calculate the P value according to the formula (P=(I∥-GI⊥) / (I∥+GI⊥), where G is the correction factor)).

[0067] The test results are shown in Table 7 below.

[0068] Table 7. Expression levels of antifreeze proteins (AFPs) and cell membrane fluidity of Scenedesmus obliquus at ambient and low temperatures. , Note: The lower the P value, the stronger the membrane fluidity (0.15-0.25 is the ideal metabolic activity range); in addition, the cell membrane is a single-layer membrane structure that surrounds the protoplasm of algal cells. It is mainly composed of lipids and proteins. It is not only a barrier for the exchange of substances and signal transduction between cells and the external environment, but its fluidity also directly affects the efficiency of cell material transport, energy conversion and metabolic reaction. Especially in low temperature environment, the stability of protoplasmic membrane fluidity is the key to maintaining the normal physiological function of algal cells.

[0069] As shown in Table 7 above, the Scenedesmus obliquus in this invention can express antifreeze proteins at 10°C, and the cell membrane fluidity is within the ideal range of metabolic activity.

[0070] (v) Soil microecological balance test The following tests use the Shannon diversity test method for microorganisms: (1) Extraction of total DNA from soil microorganisms: The soil genomic DNA extraction kit, model: FastDNA® Spin Kit for Soil, was purchased from MP Biomedicals, USA. The operation was strictly performed in accordance with the kit instructions. (2) PCR amplification: In addition to the primers and amplification system mentioned above, the PCR reaction conditions were as specified in the "High-throughput Sequencing Method for Determining Soil Microbial Diversity" (NY / T3425-2019): 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; 72℃ final extension for 10 min. (3) Sequencing and data analysis: The operation procedure of the high-throughput sequencing platform refers to the standard operating procedure of the Illumina MiSeq system (Illumina official document, Document#15039740v01); the specific parameter settings of the QIIME software (version 1.9.1) refer to its official user manual (http: / / qiime.org / install / index.html). In the following tests, the method for testing the relative abundance of Actinobacteria was as follows: OTU taxonomic annotation based on high-throughput sequencing data was performed using RDPClassifier (version 2.11), and the database used was the Silva 132 rRNA gene database (https: / / www.arb-silva.de / ). The specific annotation method was referred to the "Microbial Taxonomic Annotation Process" chapter in "Guide to High-Throughput Sequencing Data Analysis" (China Environmental Science Press, 2018, authors: Li Juan et al.). In the following example, the earthworm survival rate test method is as follows: (1) Pretreatment of test substrate: After the soil samples are collected, they need to be sieved through a 2mm sieve to remove impurities. The pH value (required to be 6.0-7.5) and organic matter content (required to be 2-10%) should be adjusted in accordance with Appendix A of the "Chemical Pesticide Environmental Safety Evaluation Test Guidelines Part 11: Acute Toxicity Test of Silkworm" (GB / T31270.11-2014). (2) Standard soil composition: In accordance with GB / T31270.11-2014, it is a mixture of quartz sand (20-50 mesh), kaolin and peat (volume ratio 7:2:1), with peat organic matter content ≥80%, purchased from KSB AG, Germany.

[0071] Following the methods described above, the Shannon diversity and relative abundance of actinomycetes in the soil of each experimental group in the room temperature group were tested after 28 days of cultivation, and the fluctuation rate was calculated. The earthworm survival rate was also observed. The results are shown in Table 8 below.

[0072] Table 8. Changes in soil microorganisms and earthworm survival in each experimental group after 28 days of cultivation. , As can be seen from Table 8 above, the *Scenedesmus obliquus* in this invention does not disrupt the soil microecological balance and complies with the GB / T31270-2014 standard.

[0073] IV. Test of pesticide degradation flux per unit area of ​​black soil The following methods were used to test the pesticide degradation flux in black soil in this embodiment of the invention: Experimental group: *Scenedesmus obliquus* (CGMCC No. 6567) was inoculated (inoculation amount: 8 × 10⁻⁶). 6 (1 cell / gram of black soil) was placed in black soil containing atrazine to simulate the actual farmland environment.

[0074] Control group: Black soil containing atrazine was treated using traditional methods (natural degradation treatment).

[0075] Aseptic conditions control To eliminate interference from other microorganisms, the experiment was conducted in a sterile environment: (1) The black soil samples were sterilized at high temperature (121℃, 30 minutes).

[0076] (2) The obliquely shaped Scenedesmus was pre-cultured in a sterile medium to the logarithmic growth phase to ensure pure culture.

[0077] Environmental parameter simulation and experimental conditions strictly simulate the ecological environment of black soil: (1) The temperature is controlled at 25±1℃, the humidity is 75%-85%, and the light cycle is 12 hours of light / 12 hours of darkness (simulating the natural day-night rhythm).

[0078] (2) Adjust the soil moisture to 60% of the saturated water holding capacity to maintain microbial activity.

[0079] High-efficiency detection technology The following methods were used to quantitatively analyze pesticide residues in soil: (1) Liquid chromatography-mass spectrometry (HPLC-MS / MS): Detection of polar pesticides such as atrazine and thiamethoxam, with a sensitivity of 0.01-0.05 mg / kg; (2) Gas chromatography-electron capture detector (GC-ECD): Analyzes pyrethroid pesticides such as cypermethrin, with a detection limit as low as 0.001 mg / kg; (3) Solid phase extraction (SPE): Soil extracts are purified by HLB column or QuEChERS kit to reduce matrix interference.

[0080] Time gradient sampling Samples were collected periodically during the experimental period (days 0, 3, 7, 14, and 21), with three parallel samples collected each time to determine pesticide residues. The concentration of atrazine in the black soil was determined using HPLC-MS / MS.

[0081] Degradation rate formula The degradation flux (mg / (kg・d)) is calculated using the following formula: , Where C0 is the initial pesticide concentration (mg / kg), C_t is the residual concentration after t days, and t is the time (days).

[0082] The improvement over traditional methods is calculated using the following formula: , , The test results showed that the degradation flux of pesticide (atrazine) per unit area of ​​black soil reached 35.2 mg / (kg·d), which is 305% higher than that of the traditional method (8.7 mg / (kg·d)).

[0083] The performance indicators of pesticide degradation in soil by Scenedesmus obliquus in this invention are compared with those of existing technologies, as shown in Table 9 below.

[0084] Table 9 Comparison of the present invention with the prior art , As shown in Table 9 above, the performance indicators of the degradation of pesticides in soil using Scenedesmus obliquus in this invention are superior to those of traditional microbial remediation and chemical oxidation methods in the prior art.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A formulation for reducing pesticide residues, characterized in that, Formulations for reducing pesticide residues include *Scenedesmus obliquus*, whose preservation number is CGMCC No. 6567.

2. Application of Scenedesmus oblique in reducing pesticide residues, wherein the pesticide is one or more combinations of atrazine, thiamethoxam, cypermethrin or nicosulfuron.

3. The application according to claim 2, characterized in that, Applications include: Application of Scenedesmus obliqueis in reducing pesticide residues in soil.

4. The application according to claim 2 or 3, characterized in that, The accession number for *Scenedesmus obliquus* is CGMCC No. 6567.

5. The application according to claim 2 or 3, characterized in that, The applicable temperature range for this application is 5℃~30℃.

6. A method for reducing pesticide residues in soil, characterized in that, The method includes inoculating Scenedesmus obliquee into the soil with one or more of the following pesticides: atrazine, thiamethoxam, cypermethrin, or nicosulfuron.

7. The method according to claim 6, characterized in that, The accession number for *Scenedesmus obliquus* is CGMCC No. 6567.

8. The method according to claim 6 or 7, characterized in that, The inoculum size of *Scenedesmus obliquus* was (0.8–1.2) × 10⁻⁶. 8 cells / g soil.

9. The method according to claim 6 or 7, characterized in that, Nitrogen and phosphorus elements are also added to the soil.

10. The method according to claim 9, characterized in that, The ratio of nitrogen to phosphorus is (9-11):1, and the amount of phosphorus added is (4mg-6mg) / kg soil.