Method for determining toxicity of pesticides to neoseiulus californicus and method for evaluating primary risk of population
By combining spraying and artificial climate chambers with *Oleander beckoniensis* as prey, the problem of pesticide toxicity determination and population impact assessment of *Neoseiuthis californica* was solved, enabling simple and accurate toxicity and population risk assessment, and improving the safety and guidance of pesticide use.
Patent Information
- Application Number
- CN202610743298.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-27
- Publication Date
- 2026-08-25
AI Technical Summary
Existing technologies are insufficient to effectively evaluate the acute toxicity and medium- to long-term population impact of pesticides on Neoseis californica, which affects the guiding significance of using mites to control mites or insects.
Using a spraying method and an artificial climate chamber, different concentrations of pesticides were sprayed in petri dishes, and the mortality and reproduction of *Neoseiuthischianus* were observed. *Oleander* was used as prey to conduct pesticide toxicity tests and preliminary population risk assessments.
This provides a simple and easy-to-use method to accurately evaluate the acute toxicity and population impact of pesticides on Neoseis californica, which is consistent with practical application scenarios and improves the safety and guidance of pesticide use.
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Abstract
Description
Technical Field
[0001] This application relates to the field of pesticide ecotoxicology technology, and in particular to a method for determining the toxicity of pesticides to Neoseis californica and a method for primary risk assessment of populations. Background Technology
[0002] Neoseuiulus californicus, also known as the California amblyseius mite, belongs to the genus Neoseuiulus in the family Phytoseiidae of the subclass Acari. It effectively controls various spider mites and is one of the most widely used predatory mites globally. In China, Neoseuiulus californicus has been promoted and applied as an important measure in building a green pest control system.
[0003] *Neoseiuyes californicus* has five distinct developmental stages: egg, larva, first nymph, second nymph, and adult. At 25°C, *Neoseiuyes californicus* completes its development in just 4-5 days. Adult females can survive for 20-40 days, with an oviposition period exceeding 14 days, laying 3-4 eggs daily (Xie, et al. 2026). In practice, using mites to control other mites often requires release approximately 40 days in advance at a predatory-to-harm ratio of 1:5 to 1:20. During this period, the predatory mite population will reproduce and expand, achieving sustained pest control. Therefore, evaluating the impact of field pesticide application on *Neoseiuyes californicus* in mite control or insect control requires not only clarifying its acute toxicity but also assessing its medium- and long-term population effects for better guidance. Summary of the Invention
[0004] In view of this, this application provides a method for determining the toxicity of pesticides to *Neoseiu Seymite californicus* and a method for primary risk assessment of the population, thus constructing a simple and easy-to-use experimental system that can evaluate both the acute toxicity of pesticides to *Neoseiu Seymite californicus* and the impact of pesticides on the *Neoseiu Seymite californicus* population and conduct primary risk assessment.
[0005] To achieve the above objectives, this application employs the following technical solution:
[0006] Option 1
[0007] A method for determining the toxicity of a pesticide to *Neoseiuthischianus*, comprising the following steps:
[0008] Prepare multiple petri dishes, place a water-soaked sponge in each petri dish, place washed and dried mulberry leaves on top of the sponge, and use resin to form a circle near the edge of the mulberry leaves to prevent the test mites from escaping.
[0009] The same number of adult female Neoseui caesarea mites were introduced into the resin ring of each petri dish as test mites, and Orinus babesi was introduced as prey.
[0010] The petri dishes were divided into multiple groups, with each group containing 3-4 petri dishes. Different concentrations of the test drug solution were sprayed into each group of petri dishes using a sprayer. The concentrations were the same for petri dishes within the same group, and different for petri dishes in different groups. One group of petri dishes was treated with water instead of the drug solution and sprayed as a control group.
[0011] Each group of culture dishes was kept in an artificial climate chamber at 25°C and 85% RH. Oleanders betani was added to each culture dish daily to ensure that adult female Neoseui caesarea had sufficient food.
[0012] After 72 hours of treatment, the number of dead and live female adult Neoseii Californians in each group of culture dishes was observed. The average mortality rate and corrected mortality rate of each group of culture dishes were calculated based on the observation data.
[0013] Specifically, the culture dish has a diameter of 12cm, the sponge has a diameter of 10cm, and a ring of resin is formed about 1cm from the edge of the mulberry leaf; the number of adult female Neoseui mites introduced into each culture dish is 30.
[0014] Specifically, if the mulberry leaves are large, they should be cut into round leaf discs with a diameter of 10cm.
[0015] Specifically, based on the average mortality rate and corrected mortality rate of each group of culture dishes obtained statistically, the lethal median concentration (LC50) of the test drug solution was further calculated. 50 .
[0016] Specifically, based on the average mortality rate and corrected mortality rate of each group of culture dishes obtained statistically, the sublethal concentration (LC50) of the test drug solution was further calculated. 30 .
[0017] The toxicity determination method in this application refers to the comprehensive toxicity determination method of Chen Xia et al. (2007), and adopts the spray method.
[0018] Option 2
[0019] A method for primary risk assessment of pesticide populations against *Neoseiu's californicus* includes the following steps:
[0020] Prepare multiple petri dishes, place a water-soaked sponge in each petri dish, place washed and dried mulberry leaves on top of the sponge, and use resin to form a circle near the edge of the mulberry leaves to prevent the test mites from escaping.
[0021] The petri dishes were divided into two groups, each containing 3-4 petri dishes. One group of petri dishes was sprayed with the same concentration of the test drug solution using a sprayer. The concentration of the test drug solution was the pre-determined sublethal dose (LC50) of the test drug solution against *Neoseiuthisches caecifolius*. 30 Concentration; another group of petri dishes were sprayed with distilled water instead of the drug solution as a control group;
[0022] In each of the two groups, the same number of mated adult females of *Neoseiuthischianus* were introduced into each culture dish, and *Oleum betani* was introduced as prey.
[0023] Each group of culture dishes was kept in an artificial climate chamber at 25°C and 85% RH. Oleanders betani was added to each culture dish daily to ensure that adult female Neoseui caesarea had sufficient food.
[0024] The number of surviving adult female Neoseis californica and the number of eggs laid were counted daily in the culture dishes of the treatment group sprayed with the test drug solution and the control group sprayed with distilled water. During this period, the eggs laid by adult female Neoseis californica and the dead adult females were removed daily to avoid squeezing the living space and affecting the experiment. After 14 days, the average mortality rate, corrected mortality rate and reproductive inhibition rate of adult female Neoseis californica in each group of culture dishes were calculated, and the comprehensive influence value E was calculated.
[0025] Specifically, the culture dish has a diameter of 12cm, the sponge has a diameter of 10cm, and a ring of resin is formed about 1cm from the edge of the mulberry leaf; each culture dish is introduced with 15 mated adult females of *Neoseiuthis Californiae*, which are pre-raised adult females of *Neoseiuthis Californiae* who have just entered the adult stage and have been introduced into adult males of *Neoseiuthis Californiae* to complete mating.
[0026] Specifically, the sublethal dose LC 30 The concentration is the sublethal dose (LC50) calculated according to the pesticide toxicity assay method for *Neoseiuthisches caesarea* described in Scheme 1. 30 concentration.
[0027] Specifically, if the mulberry leaves are large, cut them into round leaf dishes with a diameter of 10cm, and place the mulberry leaves with the front side facing up.
[0028] The primary risk assessment method used in this application is based on the method recommended by the International Organization of Biological Control (IOBC) (Hassan, S. 1992). It involves statistically analyzing the mortality rate and reproductive inhibition rate of female adult mites after 14 days of exposure to sublethal doses of pesticides, and calculating the comprehensive impact value E, thereby expanding the research on the impact of pesticides on natural enemy populations and primary risk assessment.
[0029] As can be seen from the above technical solution, this application has at least the following advantages and positive effects:
[0030] (1) Using mulberry leaves as the treatment surface has the following advantages: easy to obtain materials; the leaves are thick and tough enough that they are not easily damaged during the experiment; mulberry leaves have strong vitality, and the cut leaf discs can be placed on a wet sponge to keep the leaf activity for more than 14 days, which can be used to complete the 14-day population impact experiment of this invention.
[0031] (2) The female adult mites of Neoseis caesarea are highly active. Using wet cotton to form a circle could not prevent escape, which led to the failure of the experiment. Instead, a circle of resin was used. Because the resin is sticky, the test mites' forelegs retracted immediately after being lightly touched and no harm was caused. Practice has proven that it has a good escape prevention effect.
[0032] (3) Using Oleoma betani as prey can ensure that there is enough food for Neoseis ca. to reproduce and expand normally. Furthermore, Oleoma betani is readily available and easy to standardize.
[0033] (4) Compared with the slide immersion method and the primary risk assessment method of the population, the present invention is simpler, easier to implement and closer to the actual application scenario. Attached Figure Description
[0034] Figure 1 This is a schematic diagram of Method 2 of Embodiment 1 of this application and the experimental apparatus used in Embodiment 2. Detailed Implementation
[0035] The exemplary embodiments of the present invention are described in more detail below. These embodiments are intended to provide a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art. While exemplary embodiments of the present invention are shown, it should be understood that the invention should not be limited to the embodiments set forth herein.
[0036] Figure 1 The diagram below shows the experimental apparatus used in Method 2 of Example 1 and Example 2. In the specific implementation process, the mulberry leaves can be trimmed into larger circular leaf discs and then placed tightly on a wet sponge. When conducting the 14-day test, the upper surface of the mulberry leaves should be facing up, which can prolong the active period of the mulberry leaves.
[0037] The experimental materials used in the following examples are as follows:
[0038] Tree resin: Tree Tanglefoot brand insect barrier manufactured by SCOTTS CANADA LTD. Other brands of tree resin that achieve similar effects can also be used.
[0039] Test reagent (the solution to be tested): Acetamiprid (70% acetamiprid water-dispersible granules, produced by Dongguan Ruidefeng Biotechnology Co., Ltd.);
[0040] The test insect sources: Neoseui californica and Oleander Bessler were provided by the Natural Enemy Industrialization Base of the Institute of Plant Protection, Fujian Academy of Agricultural Sciences, and experimental populations were established in the insect research laboratory.
[0041] Example 1: Test method for toxicity determination of acetamiprid against Neoseii caecifolium
[0042] 1. Test Methods
[0043] Five test solutions of the tested pesticide were prepared according to the recommended concentration range for the target pest. The toxicity of each solution was determined using the following two methods:
[0044] Method 1: Slide Immersion Method: Cut double-sided tape into 2cm pieces and attach them to one end of a glass slide. Select healthy adult female Neoseii Californians, gently lift them with a brush, and lightly stick their backs onto the tape, being careful not to stick the mite's legs, mouthparts, or palps. Adhere 30 mites to each slide, arranging them in several rows. Place the slides containing the adult female Neoseii Californians on a large porcelain dish (with a large, water-soaked sponge inside), and incubate at 25°C for 4 hours. After 4 hours, remove any dead individuals under a binocular dissecting microscope, replenishing the number of mites to 30. Immerse glass slides containing adult female *Neoseiuthischianus* mites in the corresponding concentrations of the test solution (five test concentrations (mg / L): 60.00, 102.00, 173.40, 294.78, and 501.13) for 5 seconds with gentle agitation. Remove the slides, absorb excess solution with absorbent paper, and return them to the large porcelain dish according to the different concentrations. After 24 hours at 25°C, examine the number of dead and survival rates of the adult female *Neoseiuthischianus* mites under a binocular dissecting microscope. Water treatment serves as a control. Each test concentration and water treatment are repeated four times. A mite is considered dead if it shows no reaction when gently touched with a brush.
[0045] Method 2, the spray method (comprehensive toxicity assay) of this application: Prepare multiple petri dishes, each of which is described in detail below. Figure 1 As shown, a 12cm diameter petri dish was used, with a 10cm diameter sponge filled with water inside. Washed and dried mulberry leaves (larger mulberry leaves can be cut into 10cm diameter circular leaf discs) were placed on top of the sponge. About 1cm from the edge of the mulberry leaves, a circle of resin was used to prevent the test mites from escaping.
[0046] Thirty female adult Neoseui mites of California were introduced into the resin ring of each petri dish as test mites, and Orinus babesi was introduced as prey.
[0047] The petri dishes were divided into six groups, each containing 3-4 petri dishes (for repeated testing). Five of the groups were sprayed with different concentrations of the test pesticide solution (simulating field application) using a tower sprayer (or other small sprayers) (the five test concentrations (mg / L) were 50, 80, 128, 204.8, and 327.68, respectively). The other group of petri dishes was treated with water instead of pesticide solution as a control group.
[0048] Each group of culture dishes was placed in an artificial climate chamber at 25°C and 85% RH. *Oleander betani* was added to each culture dish daily to ensure that adult female *Neoseiu* cyanobacteria had sufficient food.
[0049] After 72 hours of treatment, the number of dead and live mites of *Neoseiu's caeciliana* was observed. Based on the observation data, the average mortality rate and corrected mortality rate of each group of culture dishes were calculated, and then the lethal median concentration (LC50) of the test drug solution was further calculated. 50 and sublethal concentration LC 30 .
[0050] 2. Data Processing
[0051] SPSS data processing software was used for analysis.
[0052] 3. Results and Analysis
[0053] The median lethal concentration (LC50) of acetamiprid against *Neoseiuthischiana* was determined using both the slide immersion method and the spray method (comprehensive toxicity assay) employed in this invention. 50 (Table 1). As can be seen from the results in Table 1, the median lethal concentration (LC50) obtained using the 72-hour comprehensive toxicity assay method... 50 Compared with the lethal median concentration (LC50) determined by the slide immersion method (24 h) 50 The values were even lower, similar to those measured by Chen Xia (2007) for several pesticides. The reason for this might be that the simulated field application method more accurately reflects the actual situation. Furthermore, the results are more consistent with the "worst case" principle of pesticide safety evaluation. In other words, this method better reflects the most unfavorable and extreme conditions in reality that are most likely to lead to pesticide residues or exposure, ensuring that pesticide use still meets safety standards even under the most stringent circumstances.
[0054] Table 1. Results of toxicity test of acetamiprid against Neoseii calica.
[0055]
[0056] Example 2: Experiment on the primary risk assessment method of acetamiprid on the population of Neoseii calica (population impact experiment)
[0057] 1. Test Methods
[0058] Prepare multiple petri dishes, see the attached table for each petri dish. Figure 1 As shown, a 12cm diameter petri dish was used. A 10cm diameter sponge filled with water was placed in each petri dish. A washed and dried mulberry leaf (larger mulberry leaves can be cut into 10cm diameter circular leaf discs) was placed on top of the sponge. A circle of resin was used to prevent the test mites from escaping, about 1cm from the edge of the mulberry leaf.
[0059] The petri dishes were divided into two groups, each containing three petri dishes (for three replicate treatments). One group of petri dishes was sprayed with a sublethal dose (LC50) using a tower sprayer. 30 (The sublethal dose mentioned is the test result calculated according to the comprehensive toxicity assay method of this application in Example 1 and Method 2, and the LC is calculated.) 30 =71.495 mg / L (in practice, other methods can also be used to determine the sublethal dose) of the test solution (simulating field application). Another group of petri dishes were sprayed with distilled water instead of the drug solution as a control group.
[0060] After preparing a batch of newly adult female Neoseui Caliphate mites, male Neoseui Caliphate mites were introduced to complete mating. Fifteen mated female Neoseui Caliphate mites were introduced into each culture dish in both groups, and Orinus babesi was introduced as prey.
[0061] Each group of culture dishes was placed in an artificial climate chamber at 25°C and 85% RH; Oleander betani was added to each treatment daily to ensure that adult female Neoseui caesarea had sufficient food.
[0062] The number of surviving female adult mites and the number of eggs laid were counted daily in the treatment group sprayed with the test solution and the control group sprayed with distilled water. During this period, the eggs laid and the dead female mites were removed daily to avoid squeezing the living space and affecting the experiment. After 14 days, the mortality rate, corrected mortality rate and reproductive inhibition rate of female adult mites of *Neoseiuthis Californiae* in each group's culture dishes were calculated. The mortality rate of female adult mites of *Neoseiuthis Californiae* in the control group sprayed with distilled water must be ≤20% and must show "normal" reproductive capacity; otherwise, the data are invalid.
[0063] Calculate the impact of pesticides on natural enemy populations using the following formula:
[0064] E(%)=100%−[100%−Mc(%)]×Rc / Rt
[0065] E: Total Effect
[0066] Mc: Corrected Mortality, in %
[0067] Rt: Average reproductive rate of the treatment group (e.g., number of eggs laid / females)
[0068] Rc: Average reproductive rate of the control group (e.g., number of eggs laid / females)
[0069] E < 30%, Level 1, harmless; 30% - 79%, Level 2, slightly harmful; 80% - 99%, Level 3, moderately harmful; > 99%, Level 4, harmful.
[0070] 2. Data Processing
[0071] SPSS data processing software was used for analysis.
[0072] 3. Results and Analysis
[0073] The results of the study on the effects of sublethal doses of acetamiprid on the population of *Neoseiuthischianus* californicus are shown in Table 2. As can be seen from the results in Table 2, the sublethal dose (LC50)... 30 Female adult Neoseiu spp. of California, treated with acetamiprid (71.495 mg / L), continued to die during the 14-day growth period and laid fewer eggs than the control group, showing a certain degree of reproductive inhibition. The calculated comprehensive impact value E=59.7%, which is between 30% and 79%, and belongs to the mildly harmful level.
[0074] Table 2. Effects of sublethal doses of acetamiprid on the survival and reproduction inhibition of *Neoseiuthischianus*.
[0075]
[0076] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for determining the toxicity of a pesticide to *Neoseiuthischianus*, characterized in that: Includes the following steps: Prepare multiple petri dishes, place a water-soaked sponge in each petri dish, place washed and dried mulberry leaves on top of the sponge, and use resin to form a circle near the edge of the mulberry leaves to prevent the test mites from escaping. The same number of adult female Neoseui caesarea mites were introduced into the resin ring of each petri dish as test mites, and Orinus babesi was introduced as prey. The petri dishes were divided into multiple groups, with each group containing 3-4 petri dishes. Different concentrations of the test drug solution were sprayed into each group of petri dishes using a sprayer. The concentrations were the same for petri dishes within the same group, and different for petri dishes in different groups. One group of petri dishes was treated with water instead of the drug solution and sprayed as a control group. Each group of culture dishes was kept in an artificial climate chamber at 25°C and 85% RH. Oleanders betani was added to each culture dish daily to ensure that adult female Neoseui caesarea had sufficient food. After 72 hours of treatment, the number of dead and live female adult Neoseii Californians in each group of culture dishes was observed. The average mortality rate and corrected mortality rate of each group of culture dishes were calculated based on the observation data.
2. The method for determining the toxicity of pesticides to *Neoseiuthisches caesarea* according to claim 1, characterized in that: The culture dish was 12cm in diameter, and the sponge was 10cm in diameter. A ring of resin was formed 1cm from the edge of the mulberry leaf. The number of adult female Neoseui mites introduced into each culture dish was 30.
3. The method for determining the toxicity of pesticides to *Neoseiuthisches caesarea* according to claim 1, characterized in that: If the mulberry leaves are large, cut them into round leaf discs with a diameter of 10cm.
4. The method for determining the toxicity of pesticides to *Neoseiuthisches caesarea* according to any one of claims 1-3, characterized in that: Based on the average mortality rate and corrected mortality rate of each group of culture dishes obtained from statistics, the lethal median concentration (LC50) of the test drug solution was further calculated. 50 .
5. The method for determining the toxicity of pesticides to *Neoseiuthisches caesarea* according to any one of claims 1-3, characterized in that: Based on the average mortality rate and corrected mortality rate of each group of culture dishes obtained from statistics, the sublethal concentration (LC50) of the test drug solution was further calculated. 30 .
6. A method for primary risk assessment of pesticide populations against *Neoseiu's californicus*, characterized in that: Includes the following steps: Prepare multiple petri dishes, place a water-soaked sponge in each petri dish, place washed and dried mulberry leaves on top of the sponge, and use resin to form a circle near the edge of the mulberry leaves to prevent the test mites from escaping. The petri dishes were divided into two groups, each containing 3-4 petri dishes. One group of petri dishes was sprayed with the same concentration of the test drug solution using a sprayer. The concentration of the test drug solution was the pre-determined sublethal dose (LC50) of the test drug solution against *Neoseiuthisches caecifolius*. 30 Concentration; another group of petri dishes were sprayed with distilled water instead of the drug solution as a control group; In each of the two groups, the same number of mated adult females of *Neoseiuthischianus* were introduced into each culture dish, and *Oleum betani* was introduced as prey. Each group of culture dishes was kept in an artificial climate chamber at 25°C and 85% RH. Oleanders betani was added to each culture dish daily to ensure that adult female Neoseui caesarea had sufficient food. The number of surviving adult female mites of Neoseis caecifolius and the number of eggs laid were counted daily in the culture dishes of the treatment group sprayed with the test solution and the control group sprayed with distilled water. During this period, the eggs laid by adult female mites of Neoseis caecifolius and the dead adult female mites were removed daily to avoid squeezing the living space and affecting the experiment. Fourteen days later, the average mortality rate, corrected mortality rate, and reproductive inhibition rate of adult female Neoseii Californians in each culture dish were calculated, and the overall impact value E was calculated.
7. The method for primary risk assessment of pesticide populations against *Neoseiuthischianus* according to claim 6, characterized in that: The culture dish has a diameter of 12cm and the sponge has a diameter of 10cm. A ring of resin is formed 1cm from the edge of the mulberry leaf. Each culture dish contains 15 mated adult females of *Neoseiuthis Californiae*. These mated adult females are pre-bred and introduced into the males of *Neoseiuthis Californiae* after mating.
8. The method for primary risk assessment of pesticide populations against *Neoseiu's californicus* according to claim 6, characterized in that: The sublethal dose LC 30 The concentration is the sublethal dose (LC50) calculated using the pesticide toxicity determination method for *Neoseiuthisches caesarea* according to claim 5. 30 concentration.
9. The method for primary risk assessment of pesticide populations against *Neoseiu's californicus* according to claim 6, characterized in that: If the mulberry leaves are large, cut them into round leaf dishes with a diameter of 10cm, and place the mulberry leaves with the front side facing up.