A rotifer killing composition and preparation method thereof

Abstract

The invention belongs to the technical field of rotifer killing, and particularly relates to a rotifer killing composition and a preparation method thereof. The composition is prepared by mixing a base liquid A, an emulsifier B and water. The base liquid A is prepared by mixing eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol and thujone in a mass ratio of 1-10:1-9:1-10:1-10:1-9:1-9:1-9; the B liquid is prepared by mixing the A liquid, tristyrylphenol polyoxyethylene ether, n-butanol and benzyl alcohol in a volume ratio of 1:8:2:0-4 in a reaction container at room temperature; the composition is prepared by adding deionized water to the B liquid and setting the volume to 100 ml. The composition uses herbaceous plants as preparation raw materials, can be directly sprinkled in waters contaminated by rotifers, can remove most harmful rotifers, and reduces pollution to the environment, and is environmentally friendly and practical.

Description

Technical Field

[0001] The invention belongs to the technical field of rotifer killing, and in particular relates to a rotifer killing composition and a preparation method thereof. Background Art

[0002] my country is a large country of aquaculture. Rotifers are often rampant in aquaculture, causing great economic losses. In particular, the intensive ponds in the Yellow River Basin of my country are seriously damaged, and some fish ponds have been abandoned and abandoned. Rotifers are aquatic multicellular protozoa and are natural bait for fish, shrimp and crab seedlings. In particular, rotifers are rich in amino acids and unsaturated fatty acids necessary for seedlings, so they are highly valued by the aquatic seed industry. However, it is undeniable that excessive reproduction of rotifers in water bodies will not only increase the carrying capacity of water bodies, but also increase oxygen consumption due to untimely consumption by aquatic animals, which is very likely to cause pond overturning events, especially in the adult fish stage. The risk is particularly great. At the same time, due to the large amount of rotifers eating algae, the water body cannot meet the aquaculture standards of "fat, alive, tender and refreshing", which in turn causes the relevant physical and chemical indicators of the water body (dissolved oxygen, pH, ammonia nitrogen, nitrite, etc.) to change, affecting the normal growth of aquatic animals and seriously restricting the healthy development of aquaculture. Therefore, it is particularly important to efficiently control the changes in the number of zooplankton in water bodies. At present, people mainly use chemical drugs for prevention and control in aquaculture, but chemical drugs are often highly toxic, easy to pollute water bodies, and insects are prone to drug resistance, which limits their large-scale application. Therefore, this application takes Brachionus calyciflorus as the research object and studies the killing effects of twenty-two kinds of plant essential oils on it, in order to screen out a safe, efficient, and purely natural rotifer prevention and control plan, in order to provide a reference for the prevention and control of rotifers in production. Summary of the invention

[0003] The purpose of the present invention is to provide a rotifer killing composition and a preparation method thereof, in order to address the shortcomings of the prior art that chemical drugs are used to kill rotifers, which pollute water bodies and harm beneficial algae and fish and shrimps.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A rotifer killing composition is prepared by mixing a base liquid A, an emulsifier B and water. The base liquid A is prepared by mixing eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol and thujone in a mass ratio of 1-10:1-9:1-10:1-10:1-9:1-9:1-9; the liquid B is prepared by mixing the liquid A, tristyrylphenol polyoxyethylene ether, n-butanol and benzyl alcohol in a volume ratio of 1:8:2:0-4 in a reaction container at room temperature; and the composition is prepared by adding deionized water to the liquid B and settling the volume to 100 ml.

[0006] Furthermore, the base liquid A is prepared by mixing eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a mass ratio of 9:6:9:1:2:1:7.

[0007] A method for preparing a rotifer killing composition comprises the following steps:

[0008] S1. Stir eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone essential oils in a mass ratio of 1-10:1-9:1-10:1-10:1-9:1-9:1-9 in a reaction vessel at room temperature at a speed of 100 rpm for 15-20 min, and let stand for 5-10 min until the bubbles on the surface of the solution are dissolved to obtain a base liquid A. After stirring, the A liquid is used as the base liquid to make the various essential oils preliminarily mixed, which is convenient for the next step of preparation. The weight ratio is used for preparation, which is convenient for large-scale configuration and production;

[0009] S2. Liquid A, tristyrylphenol polyoxyethylene ether, and n-butanol are mixed in a volume ratio of 1:8:2 in a reaction vessel at room temperature to obtain liquid B. After mixing the emulsifier with liquid A, the degree of mixing of various essential oils is better, which is convenient for subsequent processing and improves the mixing effect;

[0010] S3. Add deionized water to the solution B obtained in step S2 to make up to 100 ml, stir during the addition, and continue stirring for 15 to 20 minutes after the solution is completely added, and let it stand to obtain a rotifer killing composition.

[0011] The advantages of the present invention are as follows: the present invention selects eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone essential oils that have a killing effect on rotifers (wherein eugenol and limonene have high toxicity to rotifers, carvacrol, methyl salicylate, and thujone can effectively inhibit rotifer egg laying, and eugenol, allicin, and patchouli alcohol have a strong inhibitory effect on the hatching of rotifer eggs. Compared with the essential oil used alone, the compounded examples 2 and 4 have better effects in killing rotifers, egg laying, and egg hatching rate, indicating that the combination and compounding of the essential oils can not only play a multi-faceted role in the killing of rotifers, but also play a role in the killing of rotifers. The preparation comprises the following components: tristyrylphenol polyoxyethylene ether and n-butanol, which are effective ingredients and can evenly disperse the essential oil components in water to form a transparent microemulsion, thereby avoiding the potential safety hazard caused by the use of flammable liquids such as alcohol. Benzyl alcohol added to the preparation as a preservative can prolong the effective time of the plant components and prevent the preparation from becoming ineffective due to corruption. The composition has the characteristics of strong rotifer killing ability, high safety to non-target organisms, low cost and easy use. When used, 100 mL of the composition can effectively kill rotifers in waters within 1000 L. DETAILED DESCRIPTION Example

[0012] Although the preparation of the composition described in the present invention is described by taking the preparation of 100 ml as an example, the protection scope of the invention is not limited to this protection scope. In real life, the compositions prepared in proportion to the proportions prepared in the embodiments of the present invention all fall within the protection scope of the present invention.

[0013] A rotifer killing composition is prepared by mixing a base liquid A, an emulsifier B and water. The base liquid A is prepared by mixing eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol and thujone in a mass ratio of 1-10:1-9:1-10:1-10:1-9:1-9:1-9; the liquid B is prepared by mixing the liquid A, tristyrylphenol polyoxyethylene ether, n-butanol and benzyl alcohol in a volume ratio of 1:8:2:0-4 in a reaction container at room temperature; and the composition is prepared by adding deionized water to the liquid B and settling the volume to 100 ml.

[0014] Furthermore, the base liquid A is prepared by mixing eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a mass ratio of 9:6:9:1:2:1:7.

[0015] A method for preparing a rotifer killing composition comprises the following steps:

[0016] S1. Stir eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone essential oils in a mass ratio of 1-10:1-9:1-10:1-10:1-9:1-9:1-9 in a reaction vessel at room temperature at a speed of 100 rpm for 15-20 min, and let stand for 5-10 min until the bubbles on the surface of the solution are dissolved to obtain a base liquid A. After stirring, the A liquid is used as the base liquid to make the various essential oils preliminarily mixed, which is convenient for the next step of preparation. The weight ratio is used for preparation, which is convenient for large-scale configuration and production;

[0017] S2. Liquid A, tristyrylphenol polyoxyethylene ether, and n-butanol are mixed in a volume ratio of 1:8:2 in a reaction vessel at room temperature to obtain liquid B. After mixing the emulsifier with liquid A, the degree of mixing of various essential oils is better, which is convenient for subsequent processing and improves the mixing effect;

[0018] S3. Add deionized water to the solution B obtained in step S2 to make up to 100 ml, stir during the addition, and continue stirring for 15 to 20 minutes after the solution is completely added, and let it stand to obtain a rotifer killing composition.

[0019] Application Example 1

[0020] Every 100 mL of the composite microemulsion contains 4 mL of tristyrylphenol polyoxyethylene ether, 1 mL of n-butanol, and 94.5 mL of deionized water, and the rest is a mixture of eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a weight ratio of 1:1:1:1:1:1:1:1:1.

[0021] Application Example 2

[0022] Every 100 mL of the composite microemulsion contains 4 mL of tristyrylphenol polyoxyethylene ether, 1 mL of n-butanol, and 94.5 mL of deionized water, and the rest is a mixture of eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a weight ratio of 10:1:10:5:1:4:4.

[0023] Application Example 3

[0024] Every 100 mL of the composite microemulsion contains 4 mL of tristyrylphenol polyoxyethylene ether, 1 mL of n-butanol, and 94.5 mL of deionized water, and the rest is a mixture of eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a weight ratio of 3:2:4:6:8:6:6.

[0025] Application Example 4

[0026] Every 100 mL of the composite microemulsion contains 4 mL of tristyrylphenol polyoxyethylene ether, 1 mL of n-butanol, and 94.5 mL of deionized water, and the rest is a mixture of eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a weight ratio of 9:6:9:1:2:1:7.

[0027] Application Example 5

[0028] Every 100 mL of the composite microemulsion contains 4 mL of tristyrylphenol polyoxyethylene ether, 1 mL of n-butanol, and 94.5 mL of deionized water, and the rest is a mixture of eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a weight ratio of 1:9:1:2:4:9:9.

[0029] Application Example 6

[0030] Every 100 mL of the composite microemulsion contains 4 mL of tristyrylphenol polyoxyethylene ether, 1 mL of n-butanol, and 94.5 mL of deionized water, and the rest is a mixture of eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a weight ratio of 4:2:4:10:9:4:2.

[0031] Effect test

[0032] The specific tests are as follows:

[0033] Test 1 Acute toxicity test of plant extracts on rotifer larvae

[0034] Eugenol (98%, 20220603), thujone (92%, 20220603), limonene (98%, E809010), carvacrol (99%, C234255), methyl salicylate (99%, D998355), patchouli alcohol (90%, Z228482), and allicin (82%, B225112) were purchased from Guangzhou Anai Biotechnology Co., Ltd.; Chulailing (containing 10% phoxim) was purchased from Henan Anjin Biotechnology Co., Ltd.; tristyrylphenol polyoxyethylene ether (analytical grade) and n-butanol (analytical grade) were purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; stereo microscope, ZX500TR, was purchased from Shenzhen Zhongxun Optical Instrument Co., Ltd.; AuW120D analytical balance, Japan SHIMADZU INSTRUMENTS LIMITED.

[0035] Accurately weigh 500 mg of the corresponding plant extract in a volumetric flask, then add 4 mL of tristyrylphenol polyoxyethylene ether (emulsifier) ​​and 1 mL of n-butanol (auxiliary agent), and add deionized water to 100 mL to obtain a 5 mg / mL concentration of the corresponding essential oil microemulsion mother solution (set a solvent control group without adding essential oil components). Take the mother solution and dilute it to 7 gradients of 0.1 mg / L, 0.3 mg / L, 0.6 mg / L, 1 mg / L, 2 mg / L, 6 mg / L, and 10 mg / L, respectively. Pipette 1 mL of the corresponding concentration gradient of essential oil test solution into a cell culture plate (12-well plate), and put 30 rotifer larvae within 4 hours of age in each well. Each group and each concentration are set up 3 times, and deionized water is set as a blank control. The experiment was carried out in a constant temperature incubator at (25±1)℃ and without light. After 24 hours, the number of rotifer deaths in each well was observed and recorded under a stereomicroscope. The Probit method in SPSS 17.0 software was used to calculate the 24-hour half-lethal concentration (LC) of each essential oil on rotifers. 50 .

[0036] As shown in Table 1, by comparing the lethal median concentrations of rotifers in 7 plant essential oil microemulsions and 6 application examples at 24 h, it was found that the LC values ​​of the compound essential oil microemulsions in Example 1, Example 2, and Example 4 were 50The concentrations of essential oils in the formula were 0.326, 0.278 and 0.290 mg / L respectively, and their toxicity was 1.51, 1.77 and 1.69 times that of phoxim respectively. Compared with the single plant extracts, the toxicity of Examples 1, 2 and 4 to rotifers increased after compounding, indicating that the essential oils have a synergistic effect after being combined in appropriate proportions.

[0037]

[0038] Experiment 2 Effects of plant extracts on the production and hatching rate of dormant rotifer eggs

[0039] Accurately weigh 500 mg of the corresponding plant extract in a volumetric flask, then add 4 mL of tristyrylphenol polyoxyethylene ether (emulsifier) ​​and 1 mL of n-butanol (auxiliary agent), and add deionized water to make the volume to 100 mL to obtain a 5 mg / mL concentration of the corresponding essential oil microemulsion mother solution. The mother solution was diluted to 0.01 mg / L (1 / 10 of the minimum killing concentration gradient) to obtain the test solution of the corresponding treatment group. Take 6 newly hatched rotifer larvae and inoculate them into a test tube containing more than 12 mL of the test solution (containing 3.0×106ells / mL Chlorella). Suspend the algae solution at the bottom of the test tube every 6 hours. After 7 days, collect and count the dormant rotifer eggs. The number of dormant eggs includes the dormant eggs carried by sexual females. The experiment set up an acetone control group and a blank control group, with 3 replicates for each group. The experiment was carried out at a temperature of (25±1)℃ in the dark. As shown in Table 2 below, compared with the control group, the egg-laying amount of other treatment groups decreased to varying degrees after medication. Among the single essential oils, thujone had the lowest egg-laying amount, averaging 7.3 eggs, followed by carvacrol (7.7) and methyl salicylate (10.3). Among the compound essential oils, Examples 3 and 5 had the lowest egg-laying amount, averaging 6.7 and 5.3 eggs, which were also lower than thujone in the single plant extract.

[0040]

[0041] Take 6 newly hatched rotifer larvae and inoculate them into a 14 mL test tube containing EPA culture medium (containing 3.0×106 cells / mL Chlorella). Suspend the algae liquid at the bottom of the test tube every 6 hours. Collect the dormant rotifer eggs after 7 days. The number of dormant eggs includes the dormant eggs carried by sexual females. The experiment was carried out at a temperature of (25±1)℃ and in the dark. Randomly select 20 dormant eggs from each treatment group and place them in a 12-well cell culture plate containing 1 mL of the test liquid culture medium for incubation. The incubation temperature is (25±1)℃; the light condition is L:D=12h:12h. The hatched larvae are sucked out in time, and the hatching rate is calculated after continuous observation for 2 days.

[0042] As shown in Table 3 below, among all treatment groups, phoxim had the strongest inhibitory effect on the hatching of dormant eggs, with an average hatching rate of 5%, followed by Example 4, which was 6.5%. The hatching rates of dormant eggs in Example 2 and the patchouli alcohol group were both 8.5%, while the hatching rates of the other treatment groups were all above 10%.

[0043]

[0044] Experiment 3 Observation on the effect of plant extracts on the control of rotifers in water bodies

[0045] Chlorella in the logarithmic growth phase was inoculated into the sterilized culture medium, and the inoculation density was maintained at 3.0×10 6 Cells / mL. The expanded cultured rotifers were filtered through a mesh silk sieve, rinsed with filtered water and added to the algae solution. The rotifer inoculation density was 3-6 tails / mL. After that, they were cultured in a light incubator at a temperature of 25±1°C and a light condition of L:D=12h:12h. After the rotifers adapted for two days, plant extracts were added to the algae rotifer culture solution, and the algae solution without rotifers was used as a blank control group. The experimental volume was 3L, and 3 replicates were set for each treatment. 1mL of the test water was taken at 2d, 4d, and 7d, and the rotifer density was counted using a zooplankton counting plate.

[0046]

[0047] As shown in Table 4, the rotifer density was 5 / mL before medication. One day after adding the drug, the rotifer density of Examples 2, 4, limonene, and eugenol dropped to 0, while the control group was 32 / mL at this time, indicating that the drugs in different groups all played a role in inhibiting the growth and reproduction of rotifers. On the second day, the rotifer density of Examples 2 and 4 was 8 and 5 / mL, respectively, and the rotifer density of other treatment groups was above 10 / mL. On the third day, the rotifer density of Example 4 was 9 / mL, which was lower than that of other treatment groups. Compared with the control group, Example 4 has a significant effect on preventing and controlling the infestation of rotifers, and the comprehensive effect is better than that of single plant extracts.

[0048] The above-mentioned eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol and thujone essential oils are all essential oils of the brand of ANAI. The room temperature in the above steps refers to 15-30 degrees, and the above-mentioned reaction container is a reactor.

Claims

1. A rotifer killing composition, characterized in that: The composition is prepared by adding deionized water to liquid B to make the volume to 100 ml, wherein the liquid B is prepared by mixing a base liquid A liquid, tristyrylphenol polyoxyethylene ether, n-butanol, and benzyl alcohol in a volume ratio of 1:8:2:0-4 in a reaction container at room temperature, and the base liquid A liquid is prepared by mixing eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone in a mass ratio of 9:6:9:1:2:1:

7.

2. The method for preparing the rotifer killing composition according to claim 1, characterized in that: The following steps are involved: S1. Add eugenol, patchouli alcohol, limonene, allicin, methyl salicylate, carvacrol, and thujone essential oils in a mass ratio of 9:6:9:1:2:1:7 in a reaction container, stir at a speed of 100 rpm for 15 to 20 minutes at room temperature, and let it stand for 5 to 10 minutes until the bubbles on the surface of the solution are dissolved to obtain a base solution A; S2. The base liquid A, tristyrylphenol polyoxyethylene ether, n-butanol, and benzyl alcohol are mixed in a volume ratio of 1:8:2:0-4 in a reaction vessel at room temperature to obtain liquid B; S3. Add deionized water to the solution B obtained in step S2 to make up to 100 ml, stir during the addition, and continue stirring for 15 to 20 minutes after the solution is completely added, and let it stand to obtain a rotifer killing composition.

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