Eugenol microemulsion, preparation method thereof and application of eugenol microemulsion in prevention and control of aquatic snails

Abstract

The application discloses a eugenol microemulsion for preventing and controlling snail-borne parasitic diseases, and discloses a preparation method and application thereof, and belongs to the technical field of biological pesticides. The eugenol microemulsion comprises the following components: 1-10% of eugenol, 10-40% of a solvent, 15-40% of an emulsifier, and the balance of water. The eugenol microemulsion with different dosages has a killing effect on aquatic snails. The eugenol microemulsion is compared with a commonly used aquatic snail-killing drug, i.e., niclosamide, and it is proved that the eugenol microemulsion has a significant aquatic snail-killing effect and has an activity equivalent to that of niclosamide. Moreover, the eugenol used in the application has a lower fish toxicity than niclosamide, and thus can be widely applied to the field of parasitic disease prevention and control.

Description

Technical Field

[0001] This invention relates to the fields of biological pesticides and snail-borne parasitic disease control technology, specifically to an eugenol microemulsion, its preparation method, and its application in the control of aquatic snails. Background Technology

[0002] Snail-borne parasitic diseases are those transmitted by snails or caused by consuming undercooked snails. In my country, there are many types of snail-borne parasitic diseases, which are widely distributed and cause serious harm. The most common snail-borne parasitic diseases include schistosomiasis, paragonimiasis, clonorchiasis, and Angiostrongylus cantonensis. Their intermediate hosts are aquatic and freshwater snails such as Oncomelania hupensis, Spirogyra spp., Diplodocus spp., Spirogyra spp., Spirogyra spp., Spirogyra spp., Spirogyra spp., Spirogyra spp. (also known as the water snail), and Pomacea canaliculata.

[0003] Currently, snail control mainly relies on chemical molluscicides (such as niclosamide), environmental modification (ditch hardening, soil burial, etc.), and biological control (duck farming with snails, etc.). Among these, chemical control is the most widely used due to its rapid effectiveness and ease of use. However, the long-term and large-scale use of chemical molluscicides has also brought many problems: on the one hand, snails easily develop drug resistance, leading to reduced efficacy; on the other hand, most chemical molluscicides are highly toxic to aquatic organisms such as fish and shrimp, disrupting the ecological balance of aquatic areas and posing a risk of water residue, which threatens non-target organisms and environmental safety. Environmental modification is costly and time-consuming, making large-scale implementation difficult; biological control has unstable effects and poor controllability. Therefore, the development of new, highly efficient, low-toxicity, and environmentally friendly molluscicides has become an urgent need in the field of snail-borne parasitic disease control.

[0004] In recent years, plant-derived pesticides have received widespread attention due to their natural origin, easy degradation, and environmental friendliness. Studies have shown that various plant extracts and their active ingredients have good molluscicidal effects. Among them, eugenol, a natural phenolic compound derived from plants such as clove and cinnamon, has antibacterial, antiviral, and fish-anesthetizing activities and has been widely used in agricultural crop disease control and aquaculture. Eugenol has been used as a fish anesthetic in international fisheries transportation for over 50 years and is relatively safe for non-target organisms such as fish, showing potential for development as a green molluscicide. However, eugenol itself has extremely low water solubility and is often considered insoluble in water in practical applications, making it difficult to directly formulate water-based preparations that are easy to dilute. Furthermore, its chemical properties are unstable; the phenolic hydroxyl and allyl groups in its molecular structure are easily affected by light, heat, and oxygen, leading to oxidation, polymerization, or isomerization, resulting in degradation of the active ingredient and limiting its practical application. Currently, research on eugenol is limited to specific laboratory environments, and its application in actual external ponds often fails to yield results.

[0005] Microemulsions, as a thermodynamically stable water-based formulation, possess advantages such as small particle size (10-100 nm), transparency and uniformity, good stability, and simple preparation. Preparing eugenol into microemulsions can significantly improve its dispersibility and solubility in water, enhancing its delivery efficiency to target organisms. Simultaneously, the microemulsion system effectively encapsulates the active ingredient, isolating it from adverse external factors such as light, heat, and oxygen, significantly improving the chemical stability of eugenol. Furthermore, microemulsions use water as the continuous phase, greatly reducing the use of organic solvents, aligning with the development direction of green pesticides.

[0006] Therefore, providing an eugenol microemulsion that can effectively control aquatic snails and its preparation method is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a microemulsion with natural eugenol as the active ingredient, its preparation method, and its application in the control of aquatic snails. The present invention can solve the problems of existing molluscicides, such as environmental pollution, high toxicity to fish, low application efficiency, and short duration of effect, providing a green and efficient new option for the control of snail-borne parasitic diseases.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] An eugenol microemulsion comprises the following raw materials by mass percentage: 1%-10% eugenol, 10%-40% solvent, 15%-40% emulsifier, and the balance being water.

[0010] The eugenol microemulsion of this invention, as a plant-derived molluscicide, effectively controls aquatic snails by adding eugenol, thereby breaking the transmission chain of snail-borne parasitic diseases. It boasts advantages such as being green and pollution-free, having good molluscicidal effect, and low usage cost. This microemulsion combines the triple advantages of high-efficiency molluscicide, environmental and biosafety, and stable and controllable formulation, providing a sustainable solution for the green control of aquatic snails that can replace chemical molluscicides.

[0011] Preferably, the eugenol microemulsion comprises the following raw materials by mass percentage: 5% eugenol, 20% emulsifier, 15% solvent, and the balance being water.

[0012] Furthermore, the solvent is any one or a mixture of anhydrous ethanol, methanol, isopropanol, cyclohexanone, and xylene; preferably anhydrous ethanol.

[0013] Furthermore, the emulsifier is any one or a mixture of calcium dodecylbenzenesulfonate, castor oil polyoxyethylene ether, and fatty alcohol polyoxyethylene ether; preferably calcium dodecylbenzenesulfonate.

[0014] The present invention also provides a method for preparing the above-mentioned eugenol microemulsion, comprising the following steps: (1) At room temperature, the eugenol technical material is mixed with the solvent and stirred on a magnetic stirrer until completely dissolved to form a homogeneous oil phase; (2) While maintaining continuous stirring, add the emulsifier directly to the oil phase and stir to mix it evenly, so as to promote the full interpenetration and pre-emulsification of each component and form a mixed system; (3) Add water droplets to the mixture under magnetic stirring and continue stirring until the system spontaneously forms a clear and transparent eugenol microemulsion with uniform particle size distribution.

[0015] The microemulsion prepared by this invention should have a transparent or translucent pale blue opalescent appearance. If the microemulsion product appears cloudy, milky white, and opaque, it indicates that the oil and water phases have not achieved effective nanoscale solubilization, resulting in a common emulsion with coarse particle size and thermodynamic instability. After standing or slight centrifugation, it will exhibit stratification and oil-water separation, failing to meet the quality standards for microemulsions.

[0016] This substandard microemulsion will affect the efficacy of the molluscicide and should not be used.

[0017] Furthermore, the stirring rate in step (1) is 300-500 rpm.

[0018] Furthermore, the magnetic stirring speed in step (3) is 500-700 rpm; The water droplet acceleration rate is 80-100 mL / min.

[0019] This invention also provides the application of the above-mentioned eugenol microemulsion in the control of aquatic snails.

[0020] Furthermore, the aquatic snail is any one or a mixture of Oncomelania, Bubble Snail, and Two-umbilical Snail.

[0021] The beneficial effects of this invention are as follows: Eugenol microemulsions of different dosages exhibit a killing effect on aquatic snails. A comparison with niclosamide, a commonly used aquatic snail-killing drug, confirmed that the eugenol microemulsion has a significant snail-killing effect, comparable to that of niclosamide. Furthermore, the eugenol used in this invention has lower toxicity to fish than niclosamide, thus allowing for widespread application in the prevention and control of schistosomiasis. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: 1% Eugenol Microemulsion (1) Weigh the following by mass fraction: 1% eugenol, 15% calcium dodecylbenzenesulfonate emulsifier, 10% anhydrous ethanol solvent, and 74% deionized water. At room temperature, mix the eugenol technical material with the solvent and stir on a magnetic stirrer at 400 rpm until completely dissolved to form a homogeneous oil phase; (2) While maintaining continuous stirring, add the emulsifier directly to the oil phase and stir to mix it evenly to form a mixed system; (3) Add water dropwise to the mixture at a rate of 90 mL / min under magnetic stirring at 600 rpm, and continue stirring until the system spontaneously forms a 1% eugenol microemulsion with a clear and transparent appearance and uniform particle size distribution.

[0024] Example 2: 5% Eugenol Microemulsion (1) Weigh out the following by mass fraction: 5% eugenol, 20% calcium dodecylbenzenesulfonate emulsifier, 15% anhydrous ethanol solvent, and 60% deionized water. At room temperature, mix the eugenol technical material with the solvent and stir on a magnetic stirrer at 400 rpm until completely dissolved to form a homogeneous oil phase; (2) While maintaining continuous stirring, add the emulsifier directly to the oil phase and stir to mix it evenly to form a mixed system; (3) Add water dropwise to the mixture at a rate of 90 mL / min under magnetic stirring at 600 rpm, and continue stirring until the system spontaneously forms a 5% eugenol microemulsion with a clear and transparent appearance and uniform particle size distribution.

[0025] Example 3: 10% Eugenol Microemulsion (1) Weigh out the following by mass fraction: 10% eugenol, 40% calcium dodecylbenzenesulfonate emulsifier, 40% anhydrous ethanol solvent, and 10% deionized water. At room temperature, mix the eugenol technical material with the solvent and stir on a magnetic stirrer at 400 rpm until completely dissolved to form a homogeneous oil phase; (2) While maintaining continuous stirring, add the emulsifier directly to the oil phase and stir to mix it evenly to form a mixed system; (3) Add water dropwise to the mixture at a rate of 90 mL / min under magnetic stirring at 600 rpm, and continue stirring until the system spontaneously forms a 10% eugenol microemulsion with a clear and transparent appearance and uniform particle size distribution.

[0026] Experimental Example 1: Indoor Immersion Test Eugenol used in Experiment 1 was purchased from Jiangxi Hairui Natural Plant Co., Ltd., with an eugenol content >99%. Indoor immersion tests were conducted on aquatic snails using the eugenol microemulsions prepared in Examples 1-3 above. The Oncomelania snail, Diplodocus snail, and Microbubella snail used in the experiments were provided by the Jiangsu Provincial Institute of Schistosomiasis Control.

[0027] Eugenol microemulsions were prepared at active ingredient dosages of 0.125, 0.25, and 0.5 mg / L. The positive controls used in the experiment were eugenol technical and 50% molluscicide ethanolamine salt wettable powder (50% WP), which was purchased from Jiangsu Aijin Crop Science and Technology Group Co., Ltd.

[0028] The experimental steps are as follows: a. Eugenol microemulsion was prepared into three equal concentrations (0.125, 0.25, and 0.5 mg / L) using dechlorinated water. 1000 mL of each concentration solution was poured into beakers, and immersion tests were conducted on *Oncomelania hupensis*, *Synapticus pulmonata*, and *Dystomum chinense*. Ten test snails were placed in each beaker and immersed in the solution to prevent them from crawling out. A control solution and a blank control were also included. The laboratory temperature was 26±1℃. b. Parallel soaking schemes of 24h and 48h were set up. After soaking, the medicine was poured out, the test snails were rinsed 3 times with dechlorinated water, and then fed for 48h. The life or death of the snails was then determined by the water culture method or the knocking method. c. Record the total number of snails and the number of dead snails in each treatment, and calculate the mortality rate for each type of snail. If the snail mortality rate in the blank control group is greater than 10%, the experiment should be repeated. Each experiment should be repeated 3 times.

[0029] Wherein, the snail mortality rate = number of dead snails / total number of snails treated × 100%.

[0030] The results of the indoor tests of the eugenol microemulsions of Examples 1-3 on Oncomelania hupensis, Spirometra pulmonata and Spirometra bivalves are shown in Table 1.

[0031] Table 1 Mortality rate of Oncomelania snails in indoor experiments

[0032] As shown in Table 1, the indoor test results indicate that, using the same effective dose of eugenol, the eugenol technical grade group showed no killing effect on Oncomelania hupensis, Bufo davidii, and Diplodocus aquaticus 1 and 2 days after application. Compared with the eugenol technical grade group, the eugenol microemulsion described in this invention showed better killing effects on Oncomelania hupensis, Bufo davidii, and Diplodocus aquaticus 1 and 2 days after indoor immersion application, with the 5% eugenol microemulsion showing the best snail-killing effect. In particular, the mortality rate of Oncomelania hupensis, Bufo davidii, and Diplodocus aquaticus 100% was achieved on the second day for all concentrations of eugenol microemulsion groups, and the eugenol microemulsion still showed a certain control effect on aquatic snails at lower doses. However, eugenol technical grade is essentially insoluble in water and is considered an insoluble substance in practical applications. Furthermore, its chemical properties are unstable and easily affected by light, heat, and oxygen, leading to oxidation, polymerization, or isomerization, resulting in degradation of the active ingredients. Therefore, direct application cannot effectively kill aquatic snails. Compared with chemical molluscicides, the eugenol microemulsions in Examples 1-3 have relatively lower immediate effects on day 1, but their plant-derived characteristics give them better environmental compatibility.

[0033] Experimental Example 2: Outdoor Field Immersion Test A small ditch on Jiangxinzhou Island in Dantu District, Zhenjiang City, Jiangsu Province, was selected for field immersion tests on Oncomelania snails using the eugenol microemulsions prepared in Examples 1-3. The eugenol microemulsions were prepared according to the effective ingredient dosages of 0.125, 0.25, and 0.5 mg / L described in Experimental Example 1. Positive controls included eugenol technical grade and 50% WP, while a water control group was also included.

[0034] The experimental steps are as follows: a. The ditch is divided into 5 equal sections, each with a water volume of 2 m³. 3 ~5 m 3 Three sections were designated as the experimental group, one as the control group, and one as the blank control. Three snail-inoculation points were set up at equal intervals in each section, and three snail bags containing 30 snails were placed at each point. Three equal concentrations of eugenol microemulsion were prepared in the experimental group's ditches, and the snail bags were immersed in the solution to prevent the snails from crawling out. A control group and a blank control were also included. The field experiment was conducted at temperatures ranging from 15℃ to 35℃. b. Take one snail bag at each point on days 1, 2 and 3 after drug application, rinse three times with dechlorinated water, and allow to recover for 48 hours. Then, use the water culture method or the tapping method to determine whether the snails are alive or dead. c. Record the total number of snails and the number of dead snails in each treatment to calculate the snail mortality rate. If the snail mortality rate in the blank control group is greater than 10%, the experiment should be repeated.

[0035] The mortality rate of Oncomelania snails = (number of dead snails / total number of snails treated) × 100%.

[0036] The results of outdoor field tests on Oncomelania hupensis using the eugenol microemulsions prepared in Examples 1-3 are shown in Table 2.

[0037] Table 2 Mortality rate of Oncomelania snails in outdoor field tests

[0038] As shown in Table 2, the field test results indicate that, using the same effective dose of eugenol, the eugenol technical grade group showed no killing effect on Oncomelania snails 1, 2, and 3 days after application. Compared with the eugenol technical grade group, the eugenol microemulsion prepared in this invention showed better molluscicidal effects on Oncomelania snails 1, 2, and 3 days after outdoor field application. The 5% eugenol microemulsion showed the best molluscicidal effect. In particular, the mortality rate of Oncomelania snails in all concentrations of eugenol microemulsion groups reached 100% on the 3rd day, and the eugenol microemulsion still had a certain control effect on aquatic snails at lower doses. Compared with chemical molluscicides, the eugenol microemulsion had a relatively lower rapid effect on the 1st day, but the mortality rate reached 100% on the 3rd day, and its plant-derived characteristics gave it better environmental compatibility.

[0039] Experimental Example 3: Study on the acute toxicity of eugenol to aquatic organisms Experimental fish species: zebrafish, body length 20-25mm, weight 0.4±0.1g; small crucian carp, body length 50-70mm, weight 1.5±0.2g; eel, body length 100-150mm, weight 5±1g; red swamp crayfish, body length 30-50mm, weight 15±5g.

[0040] Experimental method: The 5% eugenol microemulsion prepared in Example 2 was diluted with dechlorinated water to concentrations of 5, 10, and 20 mg / L, respectively. Four experimental organisms were placed in each treatment, and each treatment was replicated in three times.

[0041] Using molluscicide as a positive control, a blank control group was also included. Mortality was observed and recorded 72 hours after the start of the experiment, and the mortality rate was calculated. During the experiment, the water temperature was maintained at 25±1℃, and dissolved oxygen was ≥5.0 mg / L. The experimental results are shown in Table 3.

[0042] Table 3. Safety test results of eugenol microemulsion on aquatic organisms (72-hour mortality rate)

[0043] Table 3 shows that within the concentration range of 5-20 mg / L, the toxicity of eugenol to zebrafish, eel, and crucian carp exhibits a clear concentration-dependent effect; at 20 mg / L, all fish species died. It has no significant toxic effect on *Procambarus clarkii*. At 5 mg / L, no fish species died, while the mortality rate of aquatic snails in the indoor experiment reached a maximum of 93.33% on the second day, and the mortality rate of *Oncomelania hupensis* in the outdoor experiment reached a maximum of 100% on the third day. Compared with molluscicides, eugenol is significantly safer for aquatic organisms (molluscicides cause total mortality at 0.5 mg / L). This invention's eugenol microemulsion exhibits excellent control effects against *Oncomelania hupensis*, with long-lasting efficacy and high control effect. Furthermore, it utilizes the safe biological extract eugenol, making it an environmentally friendly and highly effective novel biological molluscicide, which is conducive to widespread application and has significant implications for the control of aquatic mollusks.

[0044] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An eugenol microemulsion, characterized in that, The raw materials include the following percentages by mass: eugenol 1%-10%, solvent 10%-40%, emulsifier 15%-40%, and the balance is water.

2. The eugenol microemulsion according to claim 1, characterized in that, The solvent is any one or a mixture of anhydrous ethanol, methanol, isopropanol, cyclohexanone, and xylene.

3. The eugenol microemulsion according to claim 1, characterized in that, The emulsifier is any one or a mixture of calcium dodecylbenzenesulfonate, castor oil polyoxyethylene ether, and fatty alcohol polyoxyethylene ether.

4. A method for preparing the eugenol microemulsion according to any one of claims 1-3, characterized in that, Includes the following steps: (1) At room temperature, the eugenol technical material is mixed with the solvent and stirred on a magnetic stirrer until completely dissolved to form a homogeneous oil phase; (2) While maintaining continuous stirring, add the emulsifier directly to the oil phase and stir to mix it evenly to form a mixed system; (3) Add water droplets to the mixture under magnetic stirring and continue stirring until the system spontaneously forms a clear and transparent eugenol microemulsion with uniform particle size distribution.

5. The method for preparing an eugenol microemulsion according to claim 4, characterized in that, The stirring rate in step (1) is 300-500 rpm.

6. The method for preparing an eugenol microemulsion according to claim 4, characterized in that, The magnetic stirring speed mentioned in step (3) is 500-700 rpm; The water droplet acceleration rate is 80-100 mL / min.

7. The application of the eugenol microemulsion according to any one of claims 1-3 or the eugenol microemulsion prepared by the method according to any one of claims 4-6 in the control of aquatic snails.

8. The application of the eugenol microemulsion according to claim 7 in the control of aquatic snails, characterized in that, The aquatic snails are any one or a mixture of Oncomelania, Bubble Snail, and Diplodocus.

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