Application of nano-copper in killing fish ichthyophthirius multifilis
By using nano-silver copper as insecticides, the problems of traditional copper-based insecticides in terms of toxicity, dispersion, effectiveness, environmental risks, etc. are solved, and efficient killing of fish, melonworms and safety to fish and the environment are achieved.
Patent Information
- Application Number
- CN202510068208.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-03-25
AI Technical Summary
Existing copper-based pesticides have problems in toxicity, dispersion, effectiveness, environmental risks, etc., making it difficult to effectively prevent and control fish worm disease, and at the same time have adverse effects on fish and the environment.
Nanocopper is used as the active ingredient of insecticides, and the predator and cysts of fish are killed by soaking, and the efficient insecticidal properties of nanocopper suspension are utilized.
Nanocopper significantly improves the killing effect of predators and cysts of multiborn melonworms, has a low effective concentration, and has a high 96h-LC50 value for yellow croaker, which significantly improves safety and environmental friendliness.
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Figure CN119679822A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of nanomaterials, and particularly relates to an application of nano copper in killing Ichthyophthirius multifida of fish. Background Art
[0002] Ichthyophthirius multifiliis is a ciliated protozoan that parasitizes freshwater teleost fish. Ichthyophthirius multifiliis has a high penetrating ability, causing tissue damage and making the fish vulnerable to other pathogens, ultimately leading to fish death. Pelteobagrus fulvidraco is a specialty aquaculture species in my country, with high yields and stable growth. Ichthyophthirius multifiliis is one of the most serious parasitic diseases affecting the yellow catfish aquaculture industry. Since Ichthyophthirius multifiliis does not have strict host specificity, it can infect most freshwater fish and even amphibians in addition to yellow catfish, posing a major threat to fish farming. The life cycle of Ichthyophthirius multifiliis can be roughly divided into three stages: the predatory body (Theront) that can swim freely in the water, the cyst (Tomont), and the trophozoite (Trophont) that parasitizes the fish body. Since the trophozoites are embedded in the skin and other tissues in the body stage, general drugs cannot penetrate. The use of chemical drugs for external deworming is currently the main means of controlling Ichthyophthirius punctatus, such as malachite green, mercury preparations and formalin. However, due to problems such as food safety and environmental pollution, these chemical drugs have been gradually banned in production. Therefore, it is urgent to find efficient and green anti-insect drugs or pesticides.
[0003] Copper is a transition element existing in nature and one of the essential nutrients for aquatic animals. It is also the main component of many oxidases in the body. In aquaculture, copper sulfate has been used as a traditional insecticide, fungicide and water purifier. Studies have shown that the concentration range of copper sulfate that has a significant in vitro killing effect on Ichthyophthirius punctatus of different fish is 50-120μg / L, and the 96-hour half-lethal concentration (96h-LC50) of copper sulfate for different fish is 2.5-3.5. 50) varies greatly, with a concentration range of 0.001-730 mg / L (Tavares-Dias, M., Toxic, physiological, histomorphological, growth performance and antiparasitic effects of copper sulphate in fish aquaculture. Aquaculture, 2021. 535: 736350.). The copper ions in the copper sulfate solution can destroy the parasite membrane, damage the cell osmotic regulation and protein synthesis of the parasite, and destroy the physiological metabolism of the parasite, thereby hindering its growth and development or inhibiting its spread. However, due to the low utilization rate of copper atoms in copper sulfate and the high concentration during application, it has obvious toxicity to aquatic animals such as fish, and also has adverse effects on human health and the environment, thus limiting its application in industry. Current studies have shown that the toxicity of dissolved copper is significantly higher than that of other forms of copper, and the toxicity range is wider. Therefore, there is an urgent need for a copper preparation with good prevention and control effects and safety and low toxicity. Summary of the invention
[0004] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide an application of nano-copper in the preparation of drugs or insecticides for killing Ichthyophthirius multiflorus in fish, which solves the problems of traditional copper-based insecticides in toxicity, dispersibility, persistence, environmental risks, etc., and promotes the development of green fisheries.
[0005] The invention also provides a fish farming insecticide for preventing and controlling the predatory bodies and / or cysts of Ichthyophthirius multifida in fish.
[0006] Technical solution: In order to achieve the above purpose, the nano copper of the present invention is used in the preparation of drugs or insecticides for killing Ichthyophthirius multiflorus in fish.
[0007] Furthermore, the nano copper can effectively kill the predators of Ichthyophthirius multiflorus in fish and can be used in the preparation of drugs or pesticides for killing Ichthyophthirius multiflorus in fish.
[0008] Furthermore, the effective concentration of the nano-copper for killing fish predators of Ichthyophthirius multifida is 10-100 μg / L.
[0009] Furthermore, the nano-copper has a killing effect of more than 90% on the predators of Ichthyophthirius multifida, and the effective concentration of the nano-copper is not less than 80 μg / L.
[0010] Furthermore, the nano-copper has an effective concentration of 80-100 μg / L, and the killing effect of the nano-copper on killing fish predators of Ichthyophthirius multiflorus reaches more than 90%.
[0011] Furthermore, the nano copper can effectively kill the cysts of Ichthyophthirius multifida in fish and can be used in the preparation of a drug or insecticide for killing Ichthyophthirius multifida in fish.
[0012] Furthermore, the effective concentration of the nano-copper for killing Ichthyophthirius multifida cysts in fish is 60-120 μg / L.
[0013] Furthermore, the nano-copper has a therapeutic effect of more than 90% on killing Ichthyophthirius multifida cysts in fish, and the effective concentration of the nano-copper is not less than 100 μg / L.
[0014] Furthermore, the nano-copper has a therapeutic effect of more than 90% on killing Ichthyophthirius multifida cysts in fish, and the effective concentration of the nano-copper is 100-120 μg / L.
[0015] Furthermore, the nano copper is used to kill the predatory bodies or cysts of Ichthyophthirius multifleus in fishes by an immersion method.
[0016] Furthermore, the fish is Pelteobagrus fulvidraco.
[0017] The invention discloses a fishery insecticide for preventing and controlling the predatory bodies and / or cysts of Ichthyophthirius multifida of fishes. The fishery insecticide contains nano copper as an effective ingredient and uses distilled water as a solvent.
[0018] Furthermore, nano copper powder is weighed, added with distilled water and mixed thoroughly to prepare an insecticide for fish farming.
[0019] The nano-copper is a nano-copper suspension, the average size of the nano-copper monomer is 128±28nm, and the average size of the nano-copper aggregate is 485±16nm.
[0020] Effect of Nano-Copper on 4h-EC of Predators 50 The 22h-EC of cysts was 29.7μg / L. 50 It is 64.5μg / L.
[0021] 96h-LC of nano-copper on yellow catfish 50 The value is 82.4 mg / L, and the safe concentration is 34.8 mg / L.
[0022] The therapeutic index (TI) of nano-copper against predators and cysts was 2774 and 1278, respectively.
[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:
[0024] The nano copper prepared by the present invention has a good killing effect on Ichthyophthirius multifida. The 4h half effective insecticidal concentration (4h-EC 50 ) was 29.7 μg / L, and the 22h-EC 50The 96h-LC50 of nano-copper on yellow catfish 50 ) is 82.4 mg / L, and the safe concentration is 34.8 mg / L. The 4h half effective insecticidal concentration (4h-EC 50 ) was 75.2 μg / L, and the 22h-EC 50 The 96h-LC of copper sulfate on yellow catfish 50 The safe concentration is 2.3 mg / L, and the safe concentration is 0.7 mg / L. This shows that the effective insecticidal concentration of nano-copper on predators and the toxicity to yellow catfish are significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is an electron microscope photo of 50μg / L nano copper of the present invention;
[0026] Figure 2 The 4h-EC of nano-copper and copper sulfate on predators 50 and R 2 value;
[0027] Figure 3 The morphology of predators after nano-copper treatment, where (A) is the predator of the control group, (B-D) are the predators after 4 hours of treatment with 40, 60 and 100 μg / L nano-copper, respectively, and the arrows indicate dead predators;
[0028] Figure 4 is the 22h EC of nano-copper and copper sulfate on the encapsulated 50 and R 2 value;
[0029] Figure 5 These are the hatched and unhatched cysts after nanocopper treatment, where (A) is the cysts in the control group that hatched normally into predators, and (B) is the unhatched cysts in the treatment group. DETAILED DESCRIPTION
[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.
[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.
[0032] Preparation method of nano copper suspension: weigh 0.5 g of nano copper using an electronic balance, take 499.5 mL of distilled water in a measuring bucket, mix the nano copper and distilled water thoroughly with an ultrasonic magnetic stirrer, and then dilute them to 200 μg / L using a gradient as the mother liquor.
[0033] The mother solution was diluted to 50 μg / L, and the aggregation state and size of nano-copper in water were observed by transmission electron microscopy imaging ( Figure 1 ), and calculated the average size of monomers and aggregates. The average size of nano-copper monomers was 128±28nm, and the average size of nano-copper aggregates was 485±16nm.
[0034] Collection and counting method of Ichthyophthirius cysts: Place the yellow catfish infected with Ichthyophthirius cysts in a beaker filled with 100 mL of distilled water, stimulate it slightly with a glass rod and let it stand for 0.5 h. The mature Ichthyophthirius cysts will fall off and settle at the bottom of the beaker. Suck out the Ichthyophthirius cysts with a rubber-tipped pipette, place them in a culture dish, and wash them repeatedly with distilled water for 3 times. Count the cysts under a microscope and collect the cysts for subsequent experiments.
[0035] Collection and counting method of Ichthyophthirius cysts: Place cleaned Ichthyophthirius cysts in a 96-well plate, with 10 cysts and 200 μL of distilled water in each well. Then place the 96-well plate in a constant temperature water bath at 22°C for incubation. After 22 hours of incubation, stir the distilled water containing the predators evenly, pipette 100 μL and fix it with formaldehyde solution, and count them through a plankton counting plate and microscope after gradient dilution. The number of predators in the mother solution is calculated based on the average number of predators and the dilution multiple. The calculation formula is: average number of Ichthyophthirius cysts × dilution multiple.
[0036] Example 1
[0037] Application of Nano-Copper and Copper Sulfate in Killing Ichthyophthirius multifida Predators
[0038] The prepared Ichthyophthirius punctatus predators were placed in a 96-well plate, with 100 predators in each well. Different concentrations of nanocopper and copper sulfate solution were added to the 96-well plate in sequence. The final concentrations of nanocopper in each well were 10, 20, 40, 60, 80, and 100 μg / L, and the concentrations of copper sulfate were 50, 60, 70, and 80 μg / L, respectively. Only distilled water was added to the control group. The 96-well plate was placed in a constant temperature water bath at 22°C, and the experiment was repeated 3 times. After 4 hours, the morphology of the Ichthyophthirius punctatus predators was observed under a microscope, and the survival of the predators was recorded. The predators were considered dead when the following conditions occurred: the insect body became round, the structure was blurred, the original transparency was lost, the cilia stopped moving, or the insect body was broken.
[0039] The results of nano-copper insecticide are shown in Table 1. After 4 hours, the average mortality rate of the Ichthyophthirius predators in the control group was 2.3%. As the concentration of nano-copper solution increased, the mortality rate of the Ichthyophthirius predators showed a clear upward trend. When the nano-copper concentration was 10 μg / L, the mortality rate of the predators was 25.7%, significantly higher than that of the control group. When the nano-copper concentration was 40 μg / L, the mortality rate of the predators was 54.3%, and when the nano-copper concentration was 80 μg / L, the mortality rate of the Ichthyophthirius predators reached 96.7%. After calculation ( Figure 2 ), 4h EC of nano copper killing Ichthyophthirius punctatus predators 50 After treatment with the nano-copper suspension, most of the predators changed their morphology, lost their ability to move, and died. Figure 3 Shown are the morphologies of predators after treatment with different concentrations of nano-copper, where Figure A is the predator in the control group, with no dead predators, and Figures BD are the predators after treatment with 40, 60, and 80 μg / L nano-copper for 4 hours, with arrows pointing to dead predators.
[0040] The results of copper sulfate killing insects are shown in Table 2. After 4 hours, the average mortality rate of the Ichthyophthirius punctatus predators in the control group was 0.7%. When the copper sulfate concentration was 80 μg / L, the mortality rate of the predators was 42.3%. Figure 2 ), 4h EC of copper sulfate against Ichthyophthirius punctatus predators 50 The mortality rate of Ichthyophthirius punctatus predators after 4 hours of treatment with nano-copper at a concentration of 80 μg / L was 96.7%, while the mortality rate of Ichthyophthirius punctatus predators after 4 hours of treatment with copper sulfate at a concentration of 80 μg / L was only 42.3%. It can be seen that the effective insecticidal concentration of nano-copper on predators is significantly lower than that of copper sulfate.
[0041] Table 1 Average mortality of Ichthyophthirius punctatus predators after 4 h of nano-copper treatment
[0042]
[0043]
[0044] Note: The differences among the groups were analyzed by t-test. There are significant differences among the groups with different letters.
[0045] Table 2 Average mortality of Ichthyophthirius punctatus predators after 4 h of copper sulfate treatment
[0046]
[0047] Note: The differences among the groups were analyzed by t-test. There are significant differences among the groups with different letters.
[0048] Example 2
[0049] Application of Nano-Copper in Killing Ichthyophthirius multifeldae Cysts
[0050] The cysts were collected manually and placed in 24-well plates with 10 cysts per well. Nanocopper suspensions with concentrations of 60, 70, 80, 100 and 120 μg / L were added to the 24-well plates. Copper sulfate treatment groups (40, 60, 80 and 100 μg / L) were set up at the same time, and only distilled water was added to the control group. The 24-well plates were placed in a constant temperature water bath, and the survival of Ichthyophthirius cysts was observed and counted under a microscope after 22 hours. The method for judging the death of cysts was: no splitting and hatching of predators or rupture of the cyst membrane occurred. The experimental temperature was 22.0±1.0℃, and the experiment was repeated 3 times.
[0051] The results of nano-copper insecticide are shown in Table 3. When the nano-copper concentration was 60 μg / L, the mortality rate of cysts was 43%, which was significantly different from the control group. As the nano-copper concentration increased, the mortality rate of cysts increased. When the nano-copper concentration was 120 μg / L, the cyst lethality was 100%, and no cysts split into predators after 22 hours. The hatching rate of cysts was also positively correlated with the nano-copper concentration (Table 3). After calculation ( Figure 4 ), 22h EC of nano copper killing cysts 50 The concentration of 1 μg / L was 64.5 μg / L (95% confidence interval was 60.7-67.6 μg / L). Figure 5 As shown, Figure A shows that the cysts in the control group hatched normally into predators, and Figure B shows the unhatched cysts in the treatment group (120 μg / L). It can be seen that the cysts in the control group hatched normally, while the cyst membranes of the cysts in the nanocopper treatment group ruptured or stopped dividing.
[0052] The results of copper sulfate killing insects are shown in Table 4. When the copper sulfate concentration is 40 μg / L, the mortality rate of cysts is 50.0%, and when the concentration is 100 μg / L, the mortality rate of cysts is 100%. Figure 4 ), 22h EC of copper sulfate killing cysts 50 The effective insecticidal concentration of nano-copper on cysts is slightly higher than that of copper sulfate. At the same time, it can be seen from the above experiments that the use of nano-copper in the present invention can significantly increase the mortality rate of Ichthyophthirius punctatus predators, but cannot significantly increase the average mortality rate of cysts, proving that replacing copper sulfate in the prior art with nano-copper to improve the killing effect of Ichthyophthirius punctatus on different fish is not obvious, and has an unexpected technical effect.
[0053] Table 3 Average mortality of cysts after 22h of nano-copper treatment
[0054]
[0055] Note: The differences among the groups were analyzed by t-test. There are significant differences among the groups with different letters.
[0056] Table 4 Average mortality of cysts after 22h treatment with copper sulfate
[0057]
[0058] Note: The differences among the groups were analyzed by t-test. There are significant differences among the groups with different letters.
[0059] Example 3
[0060] 96h acute toxicity of nano-copper and copper sulfate to yellow catfish
[0061] Healthy yellow catfish were placed in fish tanks containing different nano-copper treatment concentrations, with 8 fish in each tank. According to the results of the previous preliminary experiments, the nano-copper treatment concentrations were set to 50, 60, 70, 80 and 90 mg / L, and the copper sulfate treatment concentrations were set to 1, 2, 3, 4 and 5 mg / L. At the same time, aerated tap water was set as the control group. During the experiment, the yellow catfish were observed every 12 hours, the number of deaths was recorded, and the mortality rate of the yellow catfish was calculated at 24, 48, 72 and 96 hours. During the experiment, a glass rod was used to touch the tail of the yellow catfish. If the fish did not react at all, it was judged to be completely dead. The yellow catfish was fished out in time after death to prevent it from affecting the water quality. The safe concentration (Sc) is calculated using the following formula: Sc = 48h-LC 50 ×0.3 / (24h-LC 50 / 48h-LC 50 )
[0062] The results of acute toxicity of nano-copper showed that after treatment with 50mg / L nano-copper, the mortality rate of yellow catfish was less than 50%. In the group treated with 90mg / L nano-copper, yellow catfish swam violently, and some fish gradually became restless, and most of the yellow catfish swam around the bottom of the water body; as time went on, the mortality rate of yellow catfish was 58.4% after 96h (Table 5). After calculation (Table 6), the 96h-LC of nano-copper on yellow catfish 50 The concentration of nano-copper is 82.4 mg / L, the 95% confidence interval is 78.5-88.0 mg / L, and the safe concentration is 34.8 mg / L, which is much higher than the effective concentration of nano-copper on the predators and cysts of Ichthyophthirius fulvidraco. Therefore, nano-copper can be used on yellow catfish within a safe range.
[0063] The results of acute toxicity of copper sulfate showed that after treatment with 2 mg / L copper sulfate, the mortality rate of yellow catfish was less than 50%. After 96 hours of treatment with 5 mg / L copper sulfate, the mortality rate of yellow catfish reached 100% (Table 7). After calculation (Table 8), the 96h-LC of copper sulfate on yellow catfish 50 The safe concentration of nano-copper is 2.3 mg / L, the 95% confidence interval is 1.0-3.5 mg / L, and the safe concentration is 0.7 mg / L. Therefore, the safe concentration of nano-copper for yellow catfish is much higher than that of copper sulfate, indicating that the safety of nano-copper for fish is relatively high.
[0064] Table 5 Mortality of yellow catfish after 96h of nano-copper treatment
[0065]
[0066] Table 6 Data of acute toxicity test of nano-copper on yellow catfish
[0067]
[0068] Table 7 Mortality of Pelteobagrus fulvidraco after 96 h of copper sulfate treatment
[0069]
[0070] Table 8 Data of acute toxicity test of copper sulfate on yellow catfish
[0071]
Claims
1. Application of nano copper in the preparation of drugs or insecticides for killing Ichthyophthirius multiflorus in fish.
2. The use according to claim 1, characterized in that: The nano copper can effectively kill the predators of Ichthyophthirius multiflorus in fishes and can be used in the preparation of medicines or pesticides for killing Ichthyophthirius multiflorus in fishes.
3. The use according to claim 2, characterized in that: The effective concentration of the nano copper for killing the fish predators of Ichthyophthirius multifida is 10-100 μg / L.
4. The use according to claim 2, characterized in that: The killing effect of the nano-copper on killing the fish predator Ichthyophthirius multifida is more than 90%, and the effective concentration of the nano-copper is not less than 80 μg / L.
5. The use according to claim 1, characterized in that: The nano copper can effectively kill the cysts of Ichthyophthirius multifida in fish and can be used in the preparation of medicine or insecticide for killing Ichthyophthirius multifida in fish.
6. The use according to claim 5, characterized in that: The effective concentration of the nano copper for killing Ichthyophthirius multifida cysts in fish is 60-120 μg / L.
7. The use according to claim 5, characterized in that: The therapeutic effect of the nano-copper on killing Ichthyophthirius multifida cysts in fish reaches more than 90%, and the effective concentration of the nano-copper is not less than 100 μg / L.
8. The use according to claim 1, characterized in that: The nano copper is used to kill the predatory bodies or cysts of Ichthyophthirius multifida of fishes by an immersion method.
9. A fishery insecticide for controlling the predatory bodies and / or cysts of Ichthyophthirius multifida, characterized in that: The fishery insecticide uses nano copper as an effective ingredient and distilled water as a solvent.
10. The fishery insecticide according to claim 9, characterized in that: Weigh nano copper particles, add distilled water and mix thoroughly to prepare an insecticide for fishery use.