A composition with effect of killing apple snails and application thereof

Abstract

The application provides a composition with a killing effect on golden apple snails and application thereof, and belongs to the technical field of pest control. Effective active ingredients of the composition include discarded tobacco leaf extract and orange peel essential oil. The composition is extracted from natural plants, has high efficiency and safety, can effectively achieve the prevention and control of harmful soft-bodied animals, i.e., golden apple snails, has a significant killing effect on the golden apple snails at a low concentration, and is verified by experiments to have no toxicity to 3-4 cm carp fry at an effective concentration, fully indicating that the composition is safe to non-target organisms and has low environmental risk.

Description

Technical Field

[0001] This invention belongs to the field of pest control technology, specifically relating to a composition with the effect of killing golden apple snails and its application. Background Technology

[0002] Golden apple snails, native to the Amazon River basin in South America, are a malignant aquatic organism that has invaded my country. Widely distributed, they feed on the tender leaves and stems of crops such as rice, leading to reduced crop yields. The direct damage to agriculture and the resulting chain of economic losses severely erode farmers' interests and undermine the foundation of food security. Furthermore, golden apple snails are highly competitive in aquatic environments, disrupting the original ecological balance and altering the biodiversity structure of aquatic ecosystems, thus affecting the stability of aquatic ecosystems. In addition, golden apple snails are closely related to the transmission of parasites, seriously threatening public health. However, current common methods for controlling golden apple snails mostly rely on chemical reagents, which have problems such as toxic residues, environmental pollution, and unsafety to non-target organisms. Summary of the Invention

[0003] The purpose of this invention is to address the current situation where the control of golden apple snails mainly relies on chemical agents, which poses risks of environmental pollution and ecological harm, and to provide an environmentally friendly and safe alternative control method. To this end, this invention provides a composition with golden apple snail-killing effects and its application.

[0004] This invention provides a composition with the effect of killing golden apple snails, characterized in that the effective active ingredients include waste tobacco leaf extract and orange peel essential oil.

[0005] Preferably, the mass ratio of the waste tobacco extract to the orange peel essential oil is 1:5.

[0006] Preferably, the waste tobacco extract is an organic solvent extract of waste tobacco; The waste tobacco leaves include waste tobacco leaves that have undergone a curing process.

[0007] Preferably, the preparation of the waste tobacco extract includes the following steps: Waste tobacco powder and ethanol aqueous solution were mixed and extracted, and the extract was collected. The extract was concentrated under reduced pressure to obtain a paste; Resuspend the extract to obtain a suspension; The suspension was subjected to petroleum ether extraction, ethyl acetate extraction and chloroform extraction in sequence to obtain the extract; The extract was dried to obtain waste tobacco leaf extract.

[0008] Preferably, the extraction is performed under heating conditions; The heating temperature is 50°C; The extraction time was 3 hours. The volume concentration of ethanol in the aqueous ethanol solution is 75%. The ratio of the ethanol aqueous solution to the waste tobacco powder, W:V, is 1:15. The extraction operation is repeated 3 to 5 times.

[0009] Preferably, the reagent used for resuspension includes water; the mass ratio of extract to water in the suspension is 1:2. The petroleum ether extraction includes: mixing the suspension with petroleum ether, performing petroleum ether extraction, and obtaining the petroleum ether extract residue; the volume ratio of petroleum ether to suspension is 1:1. The petroleum ether extraction operation was repeated 3 to 5 times; The ethyl acetate extraction includes: mixing the petroleum ether extract phase with ethyl acetate, performing ethyl acetate extraction, and obtaining the ethyl acetate raffinate phase; In the ethyl acetate extraction, the volume ratio of the petroleum ether extract residue to ethyl acetate is 1:1. The ethyl acetate extraction operation was repeated 6 to 8 times; Before performing the ethyl acetate extraction operation, adjust the pH of the petroleum ether extract residue to 3; The chloroform extraction comprises: mixing the ethyl acetate extract phase with chloroform and performing chloroform extraction to obtain a chloroform extract phase; Before performing the chloroform extraction operation, adjust the pH of the ethyl acetate raffinate to 11; Prior to chloroform extraction, sodium chloride saturated salting-out was performed in an alkaline aqueous phase. In the chloroform extraction, the volume ratio of ethyl acetate raffinate to chloroform is 1:3. The chloroform extraction operation was repeated 4 times.

[0010] Preferably, the preparation of the orange peel essential oil includes the following steps: Fresh orange peels were mixed with an aqueous sodium carbonate solution and soaked, then filtered to obtain an orange peel soaking solution. The orange peel soaking solution was distilled to obtain orange peel essential oil.

[0011] Preferably, the sodium carbonate in the sodium carbonate aqueous solution has a sodium carbonate mass concentration of 4%; the soaking material-to-liquid ratio is 1:10, and the soaking time is 1 hour. The distillation process was carried out at a temperature of 140°C for 30 minutes.

[0012] The present invention also provides a golden apple snail killer, characterized in that the active ingredient of the golden apple snail killer comprises the composition described in the above technical solution.

[0013] The present invention also provides the application of the composition described in the above-mentioned technical solution or the golden apple snail killer described in the above-mentioned technical solution in the control of golden apple snails.

[0014] Beneficial effects: This invention provides a composition with a killing effect on golden apple snails. The effective active ingredients of the composition include waste tobacco leaf extract and orange peel essential oil. The composition of this invention is extracted from natural plants, possessing both high efficiency and safety. The core component of the waste tobacco leaf extract, nicotine, can continuously activate the nerve receptors of the golden apple snail, leading to its nervous system over-excitation and subsequent collapse. The orange peel essential oil is rich in various active substances, which can not only open channels for the penetration of other drugs by destroying the protective layer on the surface of the golden apple snail, but also interfere with the physiological functions of the golden apple snail (such as respiratory metabolism and enzyme activity). Furthermore, toxicity testing has verified that the effective active ingredients of the composition of this invention have a significant synergistic effect, effectively controlling the harmful mollusc golden apple snail. It not only has a significant killing effect on golden apple snails at low concentrations, but also, experimental verification shows that the composition of this invention has no visible toxicity to 3-4 cm carp fry at effective concentrations, fully demonstrating that the composition of this invention is safe for non-target organisms and has low environmental risk.

[0015] Furthermore, this invention can also utilize agricultural waste (such as waste tobacco leaves and orange peels) to produce highly effective molluscicides, thus making the best use of natural resources. This not only significantly improves the comprehensive utilization efficiency and added value of agricultural products such as tobacco leaves and oranges, but also helps reduce environmental pollution problems caused by waste accumulation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 Flowchart of the process for preparing extracts from waste tobacco leaves; Figure 2 The image shows the gas chromatography-mass spectra of extracts from waste tobacco leaves; among which... Figure 2 In the figure, A represents the total ion chromatogram of waste tobacco extract. Figure 2 B in the figure represents the mass spectrometry analysis of the waste tobacco leaf extract; Figure 3 This is a flowchart of the overall preparation process of the composition; Figure 4 Diagram of an essential oil extraction apparatus; Figure 5 This is a graph showing the activity of the composition against acetylcholinesterase. Detailed Implementation

[0018] This invention provides a composition with the effect of killing golden apple snails, characterized in that the effective active ingredients include waste tobacco leaf extract and orange peel essential oil.

[0019] As one embodiment, the overall preparation process of the composition of the present invention is as follows: Figure 3 As shown. In one embodiment, the mass ratio of waste tobacco leaf extract to orange peel essential oil in this invention is 1:5. In another embodiment, the waste tobacco leaf extract in this invention is an organic solvent extract of waste tobacco leaves; the waste tobacco leaves include waste tobacco leaves processed by a curing process. In another embodiment, the raw materials for preparing the composition of this invention can be waste tobacco leaves and waste orange peels, possessing the characteristics of low cost and environmental friendliness. This not only significantly improves the comprehensive utilization efficiency and added value of agricultural products such as tobacco leaves and oranges, but also helps reduce environmental pollution problems caused by waste accumulation. In another embodiment, the composition of this invention achieves a mortality rate of 75.00% for *Pomacea canaliculata* at a concentration of 1.00 mg / L, and a 100.00% kill rate at a concentration of 2.00 mg / L. In yet another embodiment, the composition of this invention achieves a mortality rate of 0 for non-target organisms at a concentration of 8.00 mg / L, and a mortality rate of only 20.00% for non-target organisms at a concentration of 10.00 mg / L, meeting the safety requirements for application as a molluscicide. In one embodiment, the non-target organism described in this invention is a 3.00 cm to 4.00 cm carp fry.

[0020] As one embodiment, the preparation of the waste tobacco extract of the present invention includes the following steps: Waste tobacco powder and ethanol aqueous solution were mixed and extracted, and the extract was collected. The extract was concentrated under reduced pressure to obtain a paste; Resuspend the extract to obtain a suspension; The suspension was subjected to petroleum ether extraction, ethyl acetate extraction and chloroform extraction in sequence to obtain the extract; The extract was dried to obtain waste tobacco leaf extract.

[0021] This invention involves extracting waste tobacco powder from an ethanol-water solution and collecting the extract. In one embodiment, the waste tobacco powder is obtained by pulverizing dried waste tobacco leaves and passing them through a 40-mesh sieve. In another embodiment, the waste tobacco leaves are dried at 70°C for 3 hours. In another embodiment, the waste tobacco leaves include those processed by a curing process. In another embodiment, the pulverizing operation can be performed using a pulverizer. In another embodiment, the extraction is carried out under heating conditions; the heating temperature is 50°C; the extraction time is 3 hours; the volume concentration of ethanol in the ethanol-water solution is 75%; the ratio of ethanol-water solution to waste tobacco powder (W:V) is 1:15; and the extraction operation is repeated 3-5 times.

[0022] After collecting the extract, the present invention concentrates the extract under reduced pressure to obtain a paste. In one embodiment, the present invention simultaneously recovers ethanol by distillation during the reduced pressure concentration of the extract. In another embodiment, the pressure during the reduced pressure concentration operation is -0.08 MPa and the temperature is 50°C.

[0023] After obtaining the extract, the present invention resuspends the extract to obtain a suspension. As one embodiment, the reagent used for resuscitation in the present invention includes water; the mass ratio of extract to water in the suspension is 1:2.

[0024] After obtaining the suspension, the present invention sequentially performs petroleum ether extraction, ethyl acetate extraction, and chloroform extraction on the suspension to obtain the extract. In one embodiment, the present invention removes substances such as solanesol from waste tobacco leaves through petroleum ether extraction; after petroleum ether extraction, the solanesol is present in the petroleum ether extract phase. In another embodiment, the present invention removes substances such as hyoscyamine from waste tobacco leaves through ethyl acetate extraction; after ethyl acetate extraction, the hyoscyamine is present in the ethyl acetate extract phase. In another embodiment, the present invention utilizes the property that phenolic acids such as rutin and chlorogenic acid are slightly soluble or sparingly soluble in chloroform, and alkaloids are readily soluble in chloroform, to obtain alkaloids from waste tobacco leaves through chloroform extraction; after chloroform extraction, the alkaloids from waste tobacco leaves are present in the chloroform extract phase, while rutin, chlorogenic acid, and other phenolic acids are present in the chloroform raffinate phase. In yet another embodiment, the extraction operation of the present invention is achieved by shaking and mixing followed by static separation. In one embodiment, the petroleum ether extraction of the present invention includes: mixing the suspension with petroleum ether, performing petroleum ether extraction, and obtaining a petroleum ether raffinate phase; the volume ratio of petroleum ether to suspension is 1:1; the petroleum ether extraction operation is repeated 3-5 times. In one embodiment, the ethyl acetate extraction of the present invention includes: mixing the petroleum ether extract phase with ethyl acetate, performing ethyl acetate extraction, and obtaining an ethyl acetate raffinate phase. In one embodiment, in the ethyl acetate extraction of the present invention, the volume ratio of the petroleum ether raffinate phase to ethyl acetate is 1:1. In one embodiment, the ethyl acetate extraction operation is repeated 6-8 times. In one embodiment, before performing the ethyl acetate extraction operation, the pH of the petroleum ether raffinate phase is adjusted to 3. In one embodiment, the pH of the petroleum ether raffinate phase is adjusted to 3 using hydrochloric acid. In one embodiment, the present invention uses ethyl acetate for small-scale, multiple extractions until colorless. In one embodiment, the chloroform extraction of the present invention includes: mixing the ethyl acetate extract phase with chloroform, performing chloroform extraction, and obtaining a chloroform extract phase. In one embodiment, before performing the chloroform extraction operation, the pH of the ethyl acetate raffinate phase is adjusted to 11. In one embodiment, the pH of the ethyl acetate raffinate is adjusted to 11 using sodium hydroxide. In another embodiment, sodium chloride saturation salting-out is performed in an alkaline aqueous phase before chloroform extraction. In another embodiment, the volume ratio of the ethyl acetate raffinate to chloroform in the chloroform extraction is 1:3. In yet another embodiment, the chloroform extraction operation is repeated four times.

[0025] After obtaining the extract, the present invention dries the extract to obtain waste tobacco leaf extract. As one embodiment, the drying operation of the present invention can be rotary drying. As one embodiment, the main component of the waste tobacco leaf extract of the present invention is nicotine, with a purity of up to 99.75%.

[0026] As one embodiment, the preparation of the orange peel essential oil of the present invention includes the following steps: Fresh orange peels were mixed with an aqueous sodium carbonate solution and soaked, then filtered to obtain an orange peel soaking solution. The orange peel soaking solution was distilled to obtain orange peel essential oil.

[0027] This invention involves soaking fresh orange peel in a sodium carbonate aqueous solution, followed by filtration to obtain an orange peel soaking solution. As one embodiment, the sodium carbonate concentration in the aqueous solution is 4% by mass; the soaking material-to-liquid ratio is 1:10, and the soaking time is 1 hour. As another embodiment, the orange peel used in this invention can be waste orange peel from agricultural processing. As yet another embodiment, the orange peel used in this invention is granular orange peel with a size of 1.00 cm to 2.00 cm.

[0028] After obtaining the orange peel infusion, the present invention performs distillation on the orange peel infusion to obtain orange peel essential oil. As one embodiment, the distillation temperature is 140°C and the distillation time is 30 minutes.

[0029] This invention also provides a golden apple snail killer, characterized in that the active ingredient of the golden apple snail killer comprises the composition described in the above-mentioned technical solution. As one embodiment, the golden apple snail killer of this invention further comprises pharmaceutically acceptable excipients. As one embodiment, the excipients of this invention are DMSO, Tween 20, and water. As one embodiment, the concentration of the composition in the golden apple snail killer of this invention is ≥1.00 mg / L. As another embodiment, the concentration of the composition in the golden apple snail killer of this invention is 1.00 mg / L to 10.00 mg / L.

[0030] This invention also provides the application of the composition or the golden apple snail killer described in the above-mentioned technical solutions in the control of golden apple snails. As one embodiment, the composition or golden apple snail killer of this invention has a significant killing effect on golden apple snails even at low concentrations. At a concentration of 1.00 mg / L, the killing rate of golden apple snails after 72 hours is 75.00%, and at a concentration of 2.00 mg / L, the killing rate after 72 hours is 100.00%. As another embodiment, the composition or golden apple snail killer of this invention has no obvious toxic effects on non-target organisms. At a concentration of 8.00 mg / L and below, the mortality rate of non-target organisms is 0, and at a concentration of 10.00 mg / L, the mortality rate of non-target organisms is only 20.00%, meeting the safety requirements for use as a molluscicide.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a composition with the effect of killing golden apple snails and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 Preparation of components of a composition with the effect of killing golden apple snails 1. Preparation of extracts from waste tobacco leaves According to such Figure 1 The process flow chart shown illustrates the preparation of waste tobacco leaf extract. Specifically, waste tobacco leaves are dried in a 70℃ oven for 3 hours, then pulverized and passed through a 40-mesh sieve to obtain fine waste tobacco leaf powder. 100.00 g of the fine waste tobacco leaf powder is taken, and 1500.00 mL of 75% ethanol is added. The mixture is heated at 50℃ for 3 hours and extracted three times. The filtrates from the three extractions are combined, and the ethanol is recovered by vacuum distillation at a pressure of -0.08 MPa and a temperature of 50℃, yielding 68.00 g of a paste-like extract. The paste is dissolved in 140.00 mL of water to prepare a suspension, and extracted four times with 140.00 mL of petroleum ether. The raffinate is adjusted to pH=3 with hydrochloric acid and extracted repeatedly with ethyl acetate in small amounts until colorless. The raffinate is adjusted to pH=11 with sodium hydroxide, saturated with sufficient sodium chloride, and extracted four times with 420.00 mL of chloroform. The chloroform is recovered by vacuum distillation, yielding 2.03 g of waste tobacco leaf extract. The gas chromatography-mass spectra of the obtained waste tobacco leaf extract are shown below. Figure 2 As shown in the spectrum, the component with a retention time of 14.51 min is the main component of the waste tobacco extract. According to the mass spectrum analysis, it is nicotine: Cas# 54-11-5, with a content of 99.75%. Therefore, the main component of the waste tobacco extract of this invention is nicotine, with a purity of up to 99.75%.

[0033] 2. Preparation of Orange Peel Essential Oil use Figure 4 The essential oil extraction apparatus shown is used to prepare orange peel essential oil. Specifically, fresh orange peel is chopped into granules of 1.00 cm to 2.00 cm; 1000.00 g of orange peel granules are soaked in 10.00 L of 4% sodium carbonate aqueous solution for 1 h; the contents are distilled at 140°C for 30 min to obtain 9.80 g of orange peel essential oil.

[0034] Example 2 Preparation of Pomacea canaliculata exterminant According to the mass ratio of waste tobacco leaf extract to orange peel essential oil = 1:5, the two plant extracts prepared in Example 1 were accurately weighed using an analytical balance, and DMSO (2.00 mL) was added to dissolve them completely. Then, Tween 20 (1.00 mL) and water (6.00 L) were added and mixed evenly to prepare the golden apple snail killer.

[0035] Example 3 Golden apple snail killer application test and fish toxicity test 1. Mollusc extermination experiment Experimental subjects: Adult golden apple snails with good activity and shell lengths of 25.00 mm to 40.00 mm were collected from Yangzonghai Wetland in Yunnan Province. After being rinsed clean with tap water, the snails were raised in dechlorinated tap water. Snails of uniform size, with no obvious damage to their shells and strong vitality were selected for the experiment.

[0036] Experimental method: The experiment was conducted by immersion method, with DMSO + Tween 20 as the control.

[0037] Using the golden apple snail exterminant described in Example 2, solutions with concentrations of 1.00 mg / L, 2.00 mg / L, 5.00 mg / L, and 10.00 mg / L were prepared as concentration groups for the immersion test. Eight snails were placed in the center of the bottom of each concentration group, and one experiment was set up for each concentration group.

[0038] Control group experimental method: Add DMSO (2.00 mL), then add Tween 20 (1.00 mL) and water (6.00 L), mix well, and place 8 snails in the center of the bottom to set up 1 experiment.

[0039] The snails were covered with plastic mesh to prevent them from escaping. The number of dead snails was recorded after soaking for 24 h, 48 h, and 72 h, and the mortality rate after 72 h was calculated. The specific data are shown in Table 1.

[0040] Table 1 Results of the snail eradication test

[0041] 2. Fish venom test Experimental subjects: Healthy carp were selected as experimental subjects, with a weight ranging from 1.34 g ± 0.37 g to 1.42 g ± 0.21 g and an average length ranging from 3.60 cm ± 0.25 cm to 4.36 cm ± 0.25 cm. Before conducting the fish toxicity experiment, a prepared 6.00 L aquarium was filled with water and exposed to sunlight for two days to remove chlorine. Newly purchased fish were placed in the prepared dechlorinated water for at least one week of acclimatization training. The water was changed every two days to keep it clean, and appropriate food (special goldfish food) was provided to ensure the accuracy and reliability of the experimental data.

[0042] Experimental method: The experiment was conducted by immersion method, with DMSO and Tween 20 as controls.

[0043] Using the golden apple snail killer described in Example 2, solutions with concentrations of 2.00 mg / L, 5.00 mg / L, 8.00 mg / L, and 10.00 mg / L were prepared as concentration groups for the immersion test. Ten fish were placed in each concentration group, and one test was set up for each concentration group.

[0044] Control group experimental method: Add DMSO (2.00 mL), then add Tween 20 (1.00 mL) and water (6.00 L), mix well, and release 10 fish to set up 1 experiment.

[0045] The mortality rate of fish after soaking for 24 h, 48 h and 72 h was recorded respectively, and the mortality rate after 72 h was calculated. The specific data are shown in Table 2.

[0046] Table 2 Results of fish toxicity test

[0047] Experimental Results Analysis and Discussion: As shown in Tables 1 and 2, the composition of this invention exhibits a 75.00% kill rate against golden apple snails at a concentration of 1.00 mg / L, a 100.00% kill rate at a concentration of 2.00 mg / L, no kill effect on fish fry at a concentration of 8.00 mg / L, and a fish fry mortality rate of only about 20.00% at a concentration of 10.00 mg / L. This indicates that the composition of this invention has a significant lethal effect on golden apple snails and good biosafety for non-target fish species.

[0048] 3. Toxicity Assessment Referring to NYT1154.7-2006, the co-toxicity coefficient (CTC) was calculated using the Sun Yunpei method, and the CTC value was used to evaluate the combined toxicity of the agents. A CTC value less than 80.00 indicates an antagonistic effect, a CTC value greater than 120.00 indicates a synergistic effect, and a CTC value between 80.00 and 120.00 indicates an additive effect. The specific CTC calculation method is as follows: ① Calculate the median lethal concentration (LC50) for each single agent and mixture. 50 Calculate the toxicity index (TI) of each single dose relative to the standard dose according to Formula I.

[0049] TI = (LC of standard reagent) 50 LC-10 of the test reagent 50 ) × 100, formula I.

[0050] ②Calculate the theoretical toxicity index (TTI) of the mixture according to Formula II based on the toxicity index of each single agent and its proportion in the mixture.

[0051] TTI = (TI of waste tobacco extract × percentage of waste tobacco extract in the mixture) + (TI of orange peel essential oil × percentage of orange peel essential oil in the mixture), Formula II.

[0052] ③Based on the measured LC of the mixture 50 Calculate its actual toxicity index (ATI) according to Formula III.

[0053] ATI = (LC50 of standard reagent) 50 LC of the mixture 50 ) × 100, Formula III.

[0054] ④ Calculate the co-toxicity coefficient (CTC) according to formula IV based on the actual and theoretical toxicity indices of the mixture.

[0055] CTC = (ATI / TTI) × 100, Formula IV.

[0056] The regression equation for the toxicity of waste tobacco leaf extract to *Pomacea canaliculata* after 72 hours was y = 5.40x - 2.68, and the LC50 / mg·L⁻¹ (95% confidence interval) was 3.13 (2.61-3.64), where x is the logarithm of the drug concentration. The regression equation for the toxicity of orange peel essential oil to *Pomacea canaliculata* after 72 hours was y = 6.82x + 0.32, and the LC50 / mg·L⁻¹ (95% confidence interval) was 0.90 (0.32-1.06).

[0057] Calculate LC using SPSS Statistics 27 Win64 software 50 Using waste tobacco extract as the standard reagent, the actual toxicity index (ATI), theoretical toxicity index (TTI), and co-toxicity coefficient (CTC) against *Pomacea canaliculata* were calculated. The results are shown in Table 3.

[0058] Table 3. Toxicity Index and Coefficients

[0059] Table 3 shows that, based on the implementation results of the embodiments, the LC50 of the combined golden apple snail killer of the present invention against golden apple snails was calculated. 50At a concentration of 0.76 mg / L, it can achieve a median lethality at a relatively low concentration with a small confidence interval, indicating reliable data. This demonstrates that the combined insecticide for golden apple snails described in this invention has a significant lethal effect on golden apple snails. Toxicity assessment generally uses the CTC value to evaluate the combined toxicity of the agents. The CTC value is the ratio of the actual toxicity index (ATI) of the mixed agents to the theoretical toxicity index (TTI) summed proportionally. A CTC value greater than 120.00 indicates a synergistic effect, and the higher the value, the greater the synergistic effect. The co-toxicity coefficient of the combined insecticide used in the examples against golden apple snails is 134.38, exceeding 120.00, indicating that the combined insecticide for golden apple snails described in this invention has a synergistic effect.

[0060] Example 4 On-site testing of the snail-killing properties of golden apple snail killer. In Nanjie Community, Nanjian Town, Nanjian County, a water pit approximately 1.00 m long, 1.00 m wide, and 0.10 m deep was selected. Adult golden apple snails with good activity and shell lengths of 25.00 mm to 40.00 mm were collected. Before administering the pesticide, plastic sheeting was tied to wooden sticks inserted into silt to form a 1.00 m long, 1.00 m wide, and 0.20 m high enclosure. The bottom of the plastic sheeting was buried in silt, and soil was piled on the outside to prevent water overflow. The water depth was controlled to an average of approximately 0.10 m. The mortality of the golden apple snails was observed 1, 2, and 3 days after pesticide administration. Fine netting was placed around each plot to prevent the snails from migrating. Water was introduced and maintained at a depth of approximately 10.00 cm and allowed to stand for one day before the experiment. All naturally occurring golden apple snails in each plot were manually removed before the experiment.

[0061] This trial was divided into a water group (negative control group) and a target drug group.

[0062] Using the golden apple snail exterminant described in Example 2, solutions with concentrations of 2.00 mg / L, 5.00 mg / L, and 10.00 mg / L (concentrations of the solution in the pool) were prepared as the target drug concentration groups. Twenty golden apple snails were placed in the center of each concentration group in the experimental pool. One experiment was conducted for each concentration group, along with a corresponding negative control group. The experimental data are summarized in Table 4.

[0063] Table 4. Results of on-site snail eradication tests

[0064] As shown in Table 4, the golden apple snail killer prepared from waste tobacco leaf extract and orange peel essential oil described in Example 1 still exhibited good snail-killing activity in the field test.

[0065] Comparative Example 1 The only difference between this snail killer and the one described in Example 2 is that orange peel essential oil was not added.

[0066] Example 5 The only difference between this and the golden apple snail killer described in Example 2 is that the mass ratio of waste tobacco extract and orange peel essential oil is 1:1.

[0067] Example 6 The only difference between this and the golden apple snail killer described in Example 2 is that the mass ratio of waste tobacco extract and orange peel essential oil is 1:2.

[0068] Example 7 The only difference between this and the golden apple snail killer described in Example 2 is that the mass ratio of waste tobacco extract and orange peel essential oil is 2:1.

[0069] Example 8 Molluscicide and fish toxicity tests of waste tobacco leaf extract and orange peel essential oil in other mass ratios. The golden apple snail killers described in Comparative Example 1 and Examples 5-7 were prepared into solutions with concentrations of 1.00 mg / L, 2.00 mg / L, 5.00 mg / L, and 10.00 mg / L, respectively, and snail eradication tests were conducted using the immersion method. The snail mortality rates after immersion for 24 h, 48 h, and 72 h were recorded, and the specific data are summarized in Table 5.

[0070] Table 5 Results of Molluscicide Test

[0071] The golden apple snail killers described in Comparative Example 1 and Examples 5-7 were prepared into solutions with concentrations of 2.00 mg / L, 5.00 mg / L, 10.00 mg / L, and 16.00 mg / L, respectively, and fish toxicity tests were conducted using the immersion method. The mortality rates of the fish after immersion for 24 h, 48 h, and 72 h were recorded, and the specific data are summarized in Table 6.

[0072] Table 6 Results of fish toxicity tests

[0073] As shown in Tables 5 and 6, Comparative Example 1 demonstrates that the combined waste tobacco extract and orange peel essential oil in this invention significantly enhances the snail-killing effect compared to a single-component waste tobacco extract, and also exhibits higher biocompatibility. Examples 5-7 show that a 1:5 ratio of waste tobacco extract and orange peel essential oil in this invention represents the lowest effective snail-killing concentration and offers the highest biocompatibility.

[0074] Example 9 Assay of acetylcholinesterase activity in golden apple snails using a combination of waste tobacco leaf extract and orange peel essential oil The test used an "Acetylcholinesterase ELISA Detection Kit," which employs a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double antibodies. The sample, standard, and horseradish peroxidase-labeled detection antibody were added sequentially to the pre-coated microwells containing acetylcholinesterase antibody. After incubation and thorough washing, the sample was developed using the substrate 3,3',5,5'-tetramethylbenzidine (TMB). TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the acetylcholinesterase content in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample activity.

[0075] This embodiment utilizes the golden apple snail killer described in Example 2, prepared at a concentration of 0.76 mg / L (LC). 50 The *Pomacea canaliculata* was treated with a solution of [drug name missing] using an immersion method. Three experiments were conducted for each concentration group, with corresponding negative control groups. After 24 and 72 hours of treatment, the snails were dissected, and their foot segments were collected and crushed with an appropriate amount of physiological saline. The mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was used for acetylcholinesterase activity testing. The test results are shown below. Figure 5 As shown. Based on the test results, it is inferred that the molluscicidal mechanism of the composition described in this invention is related to the inhibition of acetylcholinesterase activity.

[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composition with the effect of killing golden apple snails, characterized in that, The active ingredients include waste tobacco leaf extract and orange peel essential oil.

2. The composition according to claim 1, characterized in that, The mass ratio of the waste tobacco extract to the orange peel essential oil is 1:

5.

3. The composition according to claim 1, characterized in that, The waste tobacco leaf extract is an organic solvent extract of waste tobacco leaves; The waste tobacco leaves include waste tobacco leaves that have undergone a curing process.

4. The composition according to claim 1, characterized in that, The preparation of the waste tobacco extract includes the following steps: Waste tobacco powder and ethanol aqueous solution were mixed and extracted, and the extract was collected. The extract was concentrated under reduced pressure to obtain a paste; Resuspend the extract to obtain a suspension; The suspension was subjected to petroleum ether extraction, ethyl acetate extraction and chloroform extraction in sequence to obtain the extract; The extract was dried to obtain waste tobacco leaf extract.

5. The composition according to claim 4, characterized in that, The extraction is performed under heating conditions; The heating temperature is 50°C; The extraction time was 3 hours. The volume concentration of ethanol in the aqueous ethanol solution is 75%. The ratio of the ethanol aqueous solution to the waste tobacco powder, W:V, is 1:

15. The extraction operation is repeated 3 times.

6. The composition according to claim 4, characterized in that, The reagent used for resuspension includes water; the mass ratio of extract to water in the suspension is 1:

2. The petroleum ether extraction includes: mixing the suspension with petroleum ether, performing petroleum ether extraction, and obtaining the petroleum ether extract residue; the volume ratio of petroleum ether to suspension is 1:

1. The petroleum ether extraction operation was repeated 3 to 5 times; The ethyl acetate extraction includes: mixing the petroleum ether raffinate with ethyl acetate and performing ethyl acetate extraction to obtain the ethyl acetate raffinate. In the ethyl acetate extraction, the volume ratio of the petroleum ether extract residue to ethyl acetate is 1:

1. The ethyl acetate extraction operation was repeated 6 to 8 times; Before performing the ethyl acetate extraction operation, adjust the pH of the petroleum ether extract residue to 3; The chloroform extraction comprises: mixing the ethyl acetate raffinate with chloroform and performing chloroform extraction to obtain a chloroform extract phase; Before performing the chloroform extraction operation, adjust the pH of the ethyl acetate raffinate to 11; Prior to chloroform extraction, sodium chloride saturated salting-out was performed in an alkaline aqueous phase. In the chloroform extraction, the volume ratio of ethyl acetate raffinate to chloroform is 1:

3. The chloroform extraction operation was repeated 4 times.

7. The composition according to claim 1, characterized in that, The preparation of the orange peel essential oil includes the following steps: Fresh orange peels were mixed with an aqueous sodium carbonate solution and soaked, then filtered to obtain an orange peel soaking solution. The orange peel soaking solution was distilled to obtain orange peel essential oil.

8. The composition according to claim 6, characterized in that, The sodium carbonate aqueous solution has a sodium carbonate mass concentration of 4%; the soaking material-to-liquid ratio is 1:10, and the soaking time is 1 hour. The distillation process was carried out at a temperature of 140°C for 30 minutes.

9. A poisoning agent for golden apple snails, characterized in that, The active ingredient of the golden apple snail killer comprises the composition according to any one of claims 1 to 8.

10. The use of the composition according to any one of claims 1 to 8 or the golden apple snail killer according to claim 9 in the control of golden apple snails.

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