Application method of two pesticides in rice-fish integrated culture mode

Abstract

The application provides a method for applying two pesticides in a rice-fish integrated breeding mode, carries out evaluation on death toxicity of flutriafol and flusilazole combined exposure to zebra fish, and through research on an application interval, it is proposed that an interval should exist between the application of the two pesticides, the application dose of flutriafol is 5.1-8.5 g (effective component) / mu, the application dose of flusilazole is 4-5 g (effective component) / mu, and the interval is more than 2 days. Preferably, when flusilazole is applied after flutriafol, the interval is 6 days; when flutriafol is applied after flusilazole, the interval is 4 days. The application suggests that the flutriafol and flusilazole mixture should be used carefully in agricultural production, and the interval must be strictly implemented when applied, so as to reduce the adverse effects on the ecological environment, and provide an important scientific basis for monitoring and early warning of farmland environmental pollution and remediation.

Description

Technical Field

[0001] This invention relates to the field of toxic pollution control, and more specifically, to the application methods of two pesticides in rice-fish integrated farming, particularly to the application methods of flupyrrolidone and flusilazole. Background Technology

[0002] Rice-fish integrated farming is a typical three-dimensional ecological complex planting system that organically combines rice cultivation with fish farming. Specifically, a certain number of fish are introduced into the rice paddies during the rice planting process. The shallow water environment of the paddy fields provides habitat and food sources for the fish. The fish's constant movement and foraging in the paddy fields helps to control weeds, diseases, and pests, while their excrement promotes rice growth and reduces the need for chemical fertilizers. This production method promotes the recycling of materials and multi-level flow of energy in the paddy field ecosystem, contributing to its stability. It also achieves multiple goals, including "dual use of water, multiple harvests from one field, win-win situation for rice and fish, stable grain production and increased income, green efficiency, improved quality, food safety, and ecological protection," thereby improving the utilization efficiency of paddy field resources and ultimately improving the paddy field environment. Therefore, rice-fish integrated farming has become a new technological model for the development of modern ecological circular agriculture. However, in rice-fish integrated farming systems, rice inevitably suffers from various pests and diseases, requiring the use of various pesticides to control harmful organisms.

[0003] Flupyradifurone is a neonicotinoid insecticide developed by Bayer AG. It is also a novel butenoic acid lactone compound with a novel chemical structure, making it suitable for many crops. Flupyradifurone has contact, stomach poison, and penetrating action, and can be used as a foliar spray, soil treatment, or seed treatment. Flupyradifurone is highly effective in controlling piercing-sucking pests resistant to other insecticides, including neonicotinoids, making it an effective tool for pest resistance management. Flusilazole is a fungicide used in agriculture to control fungal diseases in various crops, such as cereals, fruits, vegetables, and ornamental plants. It is effective against powdery mildew, rust, and leaf spot. Flusilazole can be used as a seed treatment or foliar spray. It works by inhibiting the biosynthesis of ergosterol, an important component of fungal cell membranes. Flupyradifurone and flusilazole are widely and frequently used in combination in agricultural production, constituting compound pollution.

[0004] Zebrafish, as a novel model organism, is frequently used in toxicology studies due to its high homology with the human genome. Zebrafish embryos are optically transparent, allowing clear observation of each developmental stage, which is helpful in observing pathological processes and toxic effects during somnolence. Furthermore, zebrafish offer advantages such as low rearing costs and high reproductive rates. Given the widespread phenomenon of pesticide contamination in agricultural production, studying its impact on zebrafish mortality toxicity is of significant guiding importance for evaluating farmland environments.

[0005] Currently, flupyrrolidone and flusilazole are still widely used as high-quality insecticides and fungicides on the market. However, there are no reports on the combined mortality toxicity effects of flupyrrolidone and flusilazole contamination on zebrafish, nor have there been in-depth research on how to properly apply these two pesticides in the same rice-fish integrated farming site from the perspective of protecting the ecosystem and reducing pollution. There is also no corresponding guidance on such application methods. Summary of the Invention

[0006] To address the aforementioned issues, this invention conducts research on the mortality toxicity of flupyrrolidone and flusilazole on zebrafish, laying a theoretical foundation for the scientific and rational use of pesticides and evaluating the ecological risks of pesticide mixed pollution, and providing guidance on application methods when flupyrrolidone and flusilazole are applied at the same location.

[0007] This invention provides two methods for applying pesticides, wherein the two pesticides are flupyrrolidone and flusilazole, and flupyrrolidone and flusilazole are applied according to the industry-recommended dosage. When flupyrrolidone and flusilazole are applied at the same location, there must be an application interval.

[0008] The industry-recommended dosages here refer to the dosages recorded in the pesticide registration certificates for flupyrrolidone and flusilazole. The application dosage for flupyrrolidone is 5.1-8.5 g (active ingredient) / mu (approximately 0.067 hectares), and for flusilazole, it is 4-5 g (active ingredient) / mu (approximately 0.067 hectares). Based on the application characteristics of flupyrrolidone and flusilazole, when the pesticides are evenly mixed with water at a depth of 10 cm in rice-fish integrated farming areas (with a water density of 1 g / mL), approximately 66,700 L of water per mu (approximately 0.067 hectares) will be evenly mixed with the pesticides. Therefore, it can be calculated that when applying according to the industry-recommended dosages, the application dosage for flupyrrolidone is 0.075–0.125 mg / L, and for flusilazole, it is 0.059–0.073 mg / L.

[0009] The application interval here refers to the time interval that must exist between the application of flupyrrolidone and flusilazole separately at the same location.

[0010] Furthermore, the application interval between flupyrrolidone and flusilazole when applied at the same location should be more than 2 days.

[0011] Preferably, when flupyrrolidone is applied first and then flusilazole is applied at the same location, the application interval is 6 days; when flusilazole is applied first and then flupyrrolidone is applied, the application interval is 4 days.

[0012] On the other hand, the present invention also provides a method for determining how to obtain the application methods of the two pesticides, mainly for determining the toxicity of the two pesticides to zebrafish.

[0013] Furthermore, the determination of the toxicity of the two pesticides to zebrafish mainly includes: toxicity testing of a single pesticide on zebrafish; toxicity testing of combined exposure to the two pesticides on zebrafish; and toxicity testing of the two pesticides after an application interval. The toxicity testing of a single pesticide on zebrafish refers to testing the toxicity of flupyrrolidone or flusilazole applied alone. The toxicity testing of combined exposure to the two pesticides on zebrafish refers to testing the toxicity of flupyrrolidone and flusilazole when they are exposed together, testing whether there is a synergistic effect in their toxicity. The toxicity testing of the two pesticides after an application interval means that there must be a certain application interval between the application of flupyrrolidone and flusilazole alone. It tests whether a short application interval still results in a synergistic effect in the toxicity of flupyrrolidone and flusilazole alone on zebrafish, thus selecting the most suitable application interval.

[0014] Furthermore, the toxicity test on zebrafish after the application interval of the two pesticides refers to first applying flupyrrolidone alone, then applying flusilazole alone after the application interval and conducting a toxicity test on zebrafish, or first applying flusilazole alone, then applying flupyrrolidone alone after the application interval and conducting a toxicity test on zebrafish.

[0015] Furthermore, the toxicity test on zebrafish during the application interval of the two pesticides refers to testing the combined toxicity of the two pesticides on zebrafish within an application interval ranging from 2 days to 10 days or more, analyzing and judging based on the test results, and selecting the application interval for flupyrrolidone and flusilazole.

[0016] Furthermore, the basis for analyzing and judging the test results is the synergistic effect of flupyrrolidone and flusilazole on zebrafish toxicity.

[0017] Furthermore, the toxicity test on zebrafish refers to the evaluation of mortality toxicity in zebrafish.

[0018] This invention processes zebrafish mortality toxicity test data using DPS statistical analysis software (version V14.10), and uses probability value analysis to statistically analyze the zebrafish mortality toxicity data to determine the LC. 50 The 95% confidence limit is also specified. This invention uses the Marking additive index method to evaluate the combined toxicity of flupyrfuranone and flusilazole.

[0019] This invention conducts a study on the toxicity of combined exposure to flupyrrolidone and flusilazole on zebrafish, obtaining basic data on the combined toxicity of mixed pollution by flupyrrolidone and flusilazole. This provides basic data for correctly evaluating the biological effects of the two pesticides as pollutants on zebrafish, and provides a scientific basis for resource protection and environmental risk assessment in farmland environments.

[0020] This invention reveals a significant synergistic effect between combined exposure to flupyrrolidone and flusilazole on zebrafish mortality toxicity, indicating that their coexistence can have serious adverse effects on the ecological environment. Although flupyrrolidone and flusilazole mixtures are widely used in agricultural production to improve pest control, their coexistence can also produce toxic side effects on the ecological environment. Therefore, flupyrrolidone and flusilazole mixtures should be used cautiously in agricultural production to minimize adverse impacts on the ecological environment.

[0021] Meanwhile, through research on the application isolation period, this invention has determined that when flupyrrolidone or flusilazole is applied alone, a synergistic effect on zebrafish toxicity still exists when the application interval is less than 4 days. Therefore, it is recommended that when flupyrrolidone and flusilazole are applied at the same location, the application interval should not be less than 2 days. Preferably, when flupyrrolidone is applied first and then flusilazole is used, the application interval is 6 days; when flusilazole is applied first and then flupyrrolidone is used, the application interval is 4 days.

[0022] This study evaluated the toxicity of combined exposure to flupyrrolidone and flusilazole on zebrafish and proposed the concept of an application interval for these two pesticides. This provides a reference for the study of toxicity testing and application intervals of combined exposure to other types of pollutants on zebrafish, and will also provide important scientific basis for the monitoring, early warning and remediation of farmland environmental pollution. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0024] 1. Test materials

[0025] (1) Test organisms

[0026] The wild-type AB strain of zebrafish was used in the experiment. After purchase, it was domesticated in the laboratory. The adult fish used for collecting fish eggs had been raised in this laboratory for more than 1 month, fed with Artemia twice a day. After 30 minutes of feeding, the residual bait and feces were fished out. The ratio of light time to dark time was 14h:10h. On the eve of reproduction, healthy and sexually mature parent fish were put into the mating and spawning tank at a ratio of 1 male to 2 females. At 8 am the next day, light was given to allow them to lay eggs and be fertilized. The normal fertilized eggs that had been cleaned and disinfected were placed in an incubator at 27±1°C with light for incubation. The larvae 2 days after hatching were used for the exposure experiment;

[0027] (2) Experimental water and experimental equipment: The preparation method of the test water followed the OECD guidelines. After sufficient aeration, it was待用. Its main indicators were: the water temperature for zebrafish was 27±1°C, the pH value was 7.8±0.2, the dissolved oxygen was ≥7.8mg·L -1 , and the hardness was 230±2mg·L -1 counting, a 24-well cell culture plate was used as the apparatus for exposing larvae to the poison;

[0028] (3) Experimental chemicals: The tested pesticides flupyradifurone and flusilazole were both technical materials. Analytical pure N,N-dimethylformamide and Tween-80 were used to dissolve the pesticide technical materials and make a stock solution of a certain concentration by constant volume. The dosage of its adjuvant was not more than 0.1% by volume ratio for determination;

[0029] (4) Test quality control

[0030] When the mortality rate of zebrafish in the control group was <10%, the test was valid.

[0031] 3. Evaluation method for the combined toxicity of pesticides

[0032] According to the Marking additive index method, the combined toxicity of flupyradifurone and flusilazole was evaluated. The sum of the biological toxicity effects S was calculated using the following formula: S = Am / Ai + Bm / Bi, where Am and Bm were the toxicities (LC 50 ) of each poison in the mixture, and Ai and Bi were the toxicities (LC 50 ) of poisons A and B when acting alone; S was converted into the additive index AI (Additive Index). When S≤1, AI = (1 / S) - 1.0; when S>1, AI = 1.0 - S. Finally, AI was used to evaluate the mixed effect of pesticides. When -0.2 < AI < 0.25, it was an additive effect (Addition); when AI≥0.25, it was a synergistic effect (Synergism); when AI≤-0.2, it was an antagonistic effect (Antagonism). The toxicity increase multiple = AI + 1.

[0033] Example 1 Toxicity test of insecticides on zebrafish

[0034] This embodiment selected 10 insecticides commonly used in rice-fish integrated farming models. A certain amount of each insecticide technical grade was weighed and completely dissolved in acetone. Then, 10% Tween 80 (w / v) was added, the mixture was shaken well, and finally, acetone was used to make up to a final volume, resulting in a high-concentration mother liquor. Based on preliminary experiments that clarified the effective concentration range of the pesticides, the above-mentioned insecticide mother liquor was diluted with standard dilution water at seven different concentration levels. The concentration range completely covered the industry-recommended dosage of the insecticides. A standard dilution water solution was also set up as a blank control group. A 24-well cell culture dish was used as the exposure apparatus, with each well containing 3 mL. During the experiment, 2 mL of the test drug solution and one normally developed 2-day-old hatched fish were added to each well. The fish were not fed during the experiment. Standard dilution water served as the control group. Each 24-well plate represented one replicate, and each concentration and control had three replicates. The experimental temperature was 27±1℃, and the photoperiod was 14h:10h. The experimental solution was changed every 24 hours. The LC50 after 120 hours of exposure was calculated using probability analysis. 50 Values ​​and their 95% confidence limits, LC50 values ​​of 10 pesticides on zebrafish 50 The values ​​are shown in Table 1.

[0035] Table 1: Toxicity of Insecticides to Zebrafish

[0036] Insecticide <![CDATA[LC 50 (mg / L)]]> Triazophos 1.05 Chlorpyrifos 7.06 High-efficiency cyhalothrin 0.14 Flupyradifurone 45.2 Thiazide 157 Profenofos 1.54 Miedowei 2.81 Phosphine 4.13 Phosphine 0.43 Flupyrrolidone 140

[0037] The results showed that, based on the industry-recommended dosage and median lethal concentration (LD50) of the pesticides, only flupyradifurone, thiamethoxam, and flupyradifurone were considered to have low toxicity to zebrafish among the 10 pesticides mentioned above.

[0038] Example 2: Toxicity test of bactericide on zebrafish

[0039] This embodiment selected 10 fungicides commonly used in rice-fish integrated farming and conducted toxicity tests on zebrafish for each of the 10 fungicides. The mortality and toxicity test methods for the fungicides on zebrafish were the same as in Example 1. Seven concentrations of each of the 10 fungicides were set at certain intervals, completely covering the industry-recommended dosage for the corresponding fungicide. Three replicates were set for each concentration (one replicate per 24-well plate), and three blank control groups without the fungicide were also included. DPS statistical analysis software (version V14.10) was used to statistically analyze the zebrafish mortality toxicity data using probability value analysis to determine the LC50. 50 and its 95% confidence limit, LC50 of 10 bactericides against zebrafish 50 The values ​​are shown in Table 2.

[0040] Table 2: Toxicity of bactericides to zebrafish

[0041] bactericide <![CDATA[LC 50 (mg / L)]]> Pyraclostrobin 0.035 Oxadiazon 0.32 Fludioxonil 0.081 Azoxystrobin 0.64 Oximetrimer 0.085 Carbendazim 0.65 Imazalil 0.52 pyrimethanil 9.17 Ethylcarb 5.41 Fluorosilazole 8.50

[0042] The results showed that, based on the industry-recommended dosage and median lethal concentration (LD50) of the fungicides, among the 10 fungicides mentioned above, only pyrimethanil, etoxazole, and flusilazole had relatively low toxicity to zebrafish.

[0043] Example 3: Toxicity test of combined exposure to insecticides and fungicides to zebrafish

[0044] This embodiment involves a pesticide mixture of an insecticide and a fungicide used for zebrafish mortality toxicity testing using an isotoxicity design. The insecticides are flonicamid, thiamethoxam, and flupyrflufenoxam (obtained in Example 1), and the fungicides are pyrimethanil, ethionil, and flusilazole (obtained in Example 2). The insecticides and fungicides are combined in pairs for combined exposure toxicity testing on zebrafish. The isotoxicity design uses the LC50 of the number of zebrafish deaths caused by a single pesticide. 50 The value is one toxicity unit. Six different concentrations of the mixed pesticide were set at logarithmic intervals based on a 1:1 mixing ratio of the two individual pesticides. The exposure test method was the same as in Example 1. The concentration range of the isotoxicity setting completely covered the industry-recommended dosages of the corresponding insecticides and fungicides. After 120 hours of exposure, the number of deaths was counted, and the LC50 values ​​of the corresponding insecticides and fungicides in the mixed system were calculated. 50 The values ​​are shown in Table 3.

[0045] Table 3: Toxicity of Mixed Pesticides to Zebrafish

[0046]

[0047]

[0048] The results showed that the combination of flupyrrolidone and flusilazole in mixed pesticides had a significant synergistic effect on zebrafish toxicity, meaning that the toxicity of the two pesticides used together on zebrafish was multiples of the sum of the toxicities of the two pesticides used alone. Other combinations had antagonistic and additive effects on zebrafish toxicity.

[0049] Example 4: Toxicity test of zebrafish treated with flupyrrolidone and flusilazole at 2-day intervals.

[0050] This embodiment involves a toxicity test of flupyrrolidone and flusilazole administered at a 2-day interval on the mortality of zebrafish, using an isotoxicity design. The isotoxicity design uses the LC-weighted average (LC-weighted average) of a single pesticide on the number of zebrafish deaths. 50The value is one toxicity unit. Nine different concentrations were set at logarithmic intervals, using two single pesticides with a 1:1 toxicity unit ratio (flupyrrolidone and flusilazole at 0.05 mg / L + 0.003 mg / L, 0.15 mg / L + 0.009 mg / L, 0.45 mg / L + 0.027 mg / L, 1.35 mg / L + 0.081 mg / L, 4.05 mg / L + 0.24 mg / L, 12.2 mg / L, etc.). The concentrations of flupyrrolidone, flusilazole, and 0.72 mg / L, 36.5 mg / L + 2.18 mg / L, 109 mg / L + 6.56 mg / L, and 328 mg / L + 19.7 mg / L were mixed. The experiment was conducted in two batches. One batch was treated with flupyrrolidone first, left at room temperature for 2 days, and then treated with flusilazole. The other batch was treated with flusilazole first, left at room temperature for 2 days, and then treated with flupyrrolidone. After both pesticides were added, zebrafish toxicity testing was conducted using the same method as in Example 1. The concentration ranges set for isotoxicity completely covered the industry-recommended dosages of flupyrrolidone and flusilazole (0.075-0.125 mg / L and 0.059-0.073 mg / L, respectively). After 120 hours of exposure, the number of deaths was counted, and the LC50 of each pesticide was calculated. 50 value.

[0051] Example 5: Toxicity test of zebrafish treated with flupyrrolidone and flusilazole at 4-day intervals.

[0052] This embodiment involves a toxicity test of flupyrrolidone and flusilazole administered at 4-day intervals on zebrafish mortality, using an isotoxicity design. The isotoxicity design uses the LC-weighted average (LC-weighted average) of a single pesticide on the number of zebrafish deaths. 50 The value is one toxicity unit. Nine different concentrations were set at logarithmic intervals, using two single pesticides with a 1:1 toxicity unit ratio (flupyrrolidone and flusilazole at 0.05 mg / L + 0.003 mg / L, 0.15 mg / L + 0.009 mg / L, 0.45 mg / L + 0.027 mg / L, 1.35 mg / L + 0.081 mg / L, 4.05 mg / L + 0.24 mg / L, 12.2 mg / L, etc.). The concentrations of flupyrrolidone, flusilazole, and 0.72 mg / L, 36.5 mg / L + 2.18 mg / L, 109 mg / L + 6.56 mg / L, and 328 mg / L + 19.7 mg / L were mixed. The experiment was conducted in two batches. One batch was treated with flupyrrolidone first, left at room temperature for 4 days, and then treated with flusilazole. The other batch was treated with flusilazole first, left at room temperature for 4 days, and then treated with flupyrrolidone. After both pesticides were added, zebrafish toxicity testing was conducted using the same method as in Example 1. The concentration ranges set for isotoxicity completely covered the industry-recommended dosages of flupyrrolidone and flusilazole (0.075-0.125 mg / L and 0.059-0.073 mg / L, respectively). After 120 hours of exposure, the number of deaths was counted, and the LC50 of each pesticide was calculated. 50 value.

[0053] Example 6: Toxicity test of zebrafish treated with flupyrrolidone and flusilazole at 6-day intervals.

[0054] This embodiment involves a toxicity test of flupyrrolidone and flusilazole administered at a 6-day interval on zebrafish mortality, using an isotoxicity design. The isotoxicity design uses the LC-weighted average (LC-weighted average) of a single pesticide on the number of zebrafish deaths. 50 The value is one toxicity unit. Nine different concentrations were set at logarithmic intervals, using two single pesticides with a 1:1 toxicity unit ratio (flupyrrolidone and flusilazole at 0.05 mg / L + 0.003 mg / L, 0.15 mg / L + 0.009 mg / L, 0.45 mg / L + 0.027 mg / L, 1.35 mg / L + 0.081 mg / L, 4.05 mg / L + 0.24 mg / L, 12.2 mg / L, etc.). The concentrations of flupyrrolidone, flusilazole, and 0.72 mg / L, 36.5 mg / L + 2.18 mg / L, 109 mg / L + 6.56 mg / L, and 328 mg / L + 19.7 mg / L were mixed. The experiment was conducted in two batches: one batch was treated with flupyrrolidone first, left at room temperature for 6 days, and then treated with flusilazole; the other batch was treated with flusilazole first, left at room temperature for 6 days, and then treated with flupyrrolidone. After both pesticides were added, zebrafish toxicity testing was conducted using the same method as in Example 1. The concentration ranges set for isotoxicity completely covered the industry-recommended dosages of flupyrrolidone and flusilazole (0.075-0.125 mg / L and 0.059-0.073 mg / L, respectively). After 120 hours of exposure, the number of deaths was counted, and the LC50 of each pesticide was calculated. 50 value.

[0055] Example 7: Toxicity test of zebrafish treated with flupyrrolidone and flusilazole at 10-day intervals.

[0056] This embodiment involves a toxicity test of flupyrrolidone and flusilazole administered at a 10-day interval on zebrafish mortality, using an isotoxicity design. The isotoxicity design uses the LC-weighted average (LC-weighted average) of a single pesticide on the number of zebrafish deaths. 50The value is one toxicity unit. Nine different concentrations were set at logarithmic intervals, using two single pesticides with a 1:1 toxicity unit ratio (flupyrrolidone and flusilazole at 0.05 mg / L + 0.003 mg / L, 0.15 mg / L + 0.009 mg / L, 0.45 mg / L + 0.027 mg / L, 1.35 mg / L + 0.081 mg / L, 4.05 mg / L + 0.24 mg / L, and 12.2 mg / L, respectively). (Concentrations of +0.72 mg / L, 36.5 mg / L + 2.18 mg / L, 109 mg / L + 6.56 mg / L, and 328 mg / L + 19.7 mg / L were mixed). The experiment was conducted in two batches. One batch was treated with flupyrrolidone first, left at room temperature for 10 days, and then flusilazole was added. The other batch was treated with flusilazole first, left at room temperature for 10 days, and then flupyrrolidone was added. After both pesticides were added, zebrafish toxicity testing was conducted, using the same method as in Example 1. The concentration ranges set for isotoxicity completely covered the industry-recommended dosages of flupyrrolidone and flusilazole (0.075-0.125 mg / L and 0.059-0.073 mg / L, respectively). After 120 hours of exposure, the number of deaths was counted, and the LC50 of each pesticide was calculated. 50 value.

[0057] Example 8: Analysis of Zebrafish Toxicity Test Results

[0058] After the experiment, the mortality rate of zebrafish in the blank control group after 5 days of exposure was less than 10%. Therefore, this experiment met the quality control requirements and was valid.

[0059] Analysis of the experimental results in Examples 1, 2, and 3 revealed that both flupyrrolidone and flusilazole significantly affected zebrafish mortality at the tested concentrations. Furthermore, for the same pesticide, mortality increased with increasing exposure concentration. When flupyrrolidone and flusilazole were used in an equal toxicity ratio, they exhibited a significant synergistic effect on zebrafish mortality, with an AI value of 1.92, indicating a toxicity increase factor of 2.92, as shown in Table 4. This demonstrates that the coexistence of these two pesticides can have a significant synergistic adverse effect on the zebrafish population.

[0060] Table 4. Mortality Toxicity Effects of Combined Exposure to Flupyrrolidone and Flusilazole on Zebrafish

[0061]

[0062] LC-1 inhibitory effect of exposure to α-fluopyranozone and flusilazole alone on zebrafish mortality 50 (95% confidence limit).

[0063] LC-1 inhibitory effect of combined exposure to b-flupyrrolidone and flusilazole on zebrafish mortality 50 (95% confidence limit).

[0064] c-addition exponent value

[0065] The experimental results of Examples 4, 5, 6, and 7 are analyzed below, and the results are shown in Table 5:

[0066] Table 5. Mortality Toxicity Effects of Alternating Flupyrrolidone and Flusilazole on Zebrafish

[0067]

[0068] Note: Order of administration: A means flupyrrolidone is added first, and flusilazole is added after the interval period; B means flusilazole is added first, and flupyrrolidone is added after the interval period.

[0069] The test results show that:

[0070] In Example 4, when the interval between flupyrrolidone and flusilazole treatment was 2 days, a significant synergistic effect was observed on zebrafish mortality: when flupyrrolidone was added first, followed by flusilazole 2 days later, the AI ​​value was 0.98, meaning the toxicity increased by a factor of 1.98, indicating a significant adverse effect on the zebrafish population; when flusilazole was added first, followed by flupyrrolidone 2 days later, the AI ​​value was 0.81, slightly lower than the former, but still had a significant adverse effect on zebrafish mortality, with a toxicity increase factor of 1.84.

[0071] In Example 5, when the interval between flupyrrolidone and flusilazole treatment was 4 days, two scenarios emerged regarding zebrafish mortality: When flupyrrolidone was administered first, followed by flusilazole 4 days later, the AI ​​value was 0.28, indicating a 1.28-fold increase in toxicity, demonstrating a significant adverse effect on the zebrafish population; when flusilazole was administered first, followed by flupyrrolidone 4 days later, the AI ​​value was 0.13, indicating an additive effect rather than a synergistic one on zebrafish mortality. Therefore, a 4-day isolation period is appropriate when flusilazole is administered first, followed by flupyrrolidone.

[0072] In Example 6, when the interval between flupyrrolidone and flusilazole treatments was 6 days, the effects on zebrafish mortality were additive: when flupyrrolidone was added first, followed by flusilazole 6 days later, the AI ​​value was 0.023, indicating an additive effect rather than a synergistic one on zebrafish mortality; when flusilazole was added first, followed by flupyrrolidone 6 days later, the AI ​​value was 0.072, slightly higher than the former, also indicating an additive effect rather than a synergistic one on zebrafish mortality. Therefore, a 6-day isolation period is appropriate when flupyrrolidone is added first, followed by flusilazole.

[0073] In Example 7, when the interval between flupyrrolidone and flusilazole treatments was 10 days, the effects on zebrafish mortality were additive: when flupyrrolidone was added first, followed by flusilazole 10 days later, the AI ​​value was -0.023, indicating no synergistic effect on zebrafish mortality, but rather an additive effect. Therefore, when flupyrrolidone was added first and then flusilazole, the synergistic effect on zebrafish toxicity disappeared after a 10-day isolation period; when flusilazole was added first, followed by flupyrrolidone 10 days later, the AI ​​value was -0.089, slightly lower than the former, indicating no synergistic effect on zebrafish mortality, but rather an additive effect. Therefore, when flusilazole was added first and then flupyrrolidone, the synergistic effect on zebrafish toxicity disappeared after a 10-day isolation period. This demonstrates that after a 10-day interval, the synergistic effect of alternating flupyrrolidone and flusilazole treatments on zebrafish toxicity completely disappeared.

[0074] Therefore, when applying flupyrrolidone and flusilazole at the same rice-fish integrated farming site, the flupyrrolidone and flusilazole are applied according to industry-recommended dosages. The application dosage of flupyrrolidone is 5.1–8.5 g (active ingredient) / mu, and the application dosage of flusilazole is 4–5 g (active ingredient) / mu. That is, when the application dosage of flupyrrolidone is 0.075–0.125 mg / L and the application dosage of flusilazole is 0.059–0.073 mg / L, the application interval shall not be less than 2 days. Preferably, when flupyrrolidone is applied first and then flusilazole, a 6-day application interval must be implemented; when flusilazole is applied first and then flupyrrolidone, a 4-day application interval must be implemented.

[0075] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for reducing the toxicity of two pesticides to zebrafish under the rice-fish integrated farming model, characterized in that, The two pesticides are an insecticide and a fungicide, respectively. The insecticide is flupyrrolidone, and the fungicide is flusilazole. The fish introduced in the rice-fish integrated farming model are zebrafish. When flupyrrolidone and flusilazole are applied at the same location, there must be an application interval. The application dosage of flupyrrolidone is 5.1-8.5 g / mu, and the application dosage of flusilazole is 4-5 g / mu. When flupyrrolidone is applied first and then flusilazole, the application interval is 6 days. When flusilazole is applied first and then flupyrrolidone, the application interval is 4 days.

2. The method for determining the application method as described in claim 1, characterized in that, The toxicity of two pesticides to zebrafish was determined.

3. The determination method as described in claim 2, characterized in that, The determination of the toxicity of the two pesticides to zebrafish mainly includes: toxicity test of a single pesticide to zebrafish; toxicity test of combined exposure of the two pesticides to zebrafish; and toxicity test of the two pesticides to zebrafish after the application interval.

4. The determination method as described in claim 3, characterized in that, The toxicity test on zebrafish after the application interval of the two pesticides refers to first applying flupyrrolidone alone, then applying flusilazole alone after the application interval, and conducting a toxicity test on zebrafish; or first applying flusilazole alone, then applying flupyrrolidone alone after the application interval, and conducting a toxicity test on zebrafish.

5. The determination method as described in claim 4, characterized in that, The toxicity test of the two pesticides on zebrafish during the application interval refers to testing the combined toxicity of the two pesticides on zebrafish by applying them at intervals ranging from 2 days to 10 days, analyzing the test results, and selecting the appropriate application interval for the two pesticides.

6. The determination method as described in claim 5, characterized in that, The toxicity test on zebrafish refers to the evaluation of mortality toxicity in zebrafish.

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