A test method for assessing the activity inhibition of microbial pesticides on daphnia magna

Abstract

This invention discloses a test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa, relating to the field of pesticide detection. The method includes an outer casing, a layered test cylinder, an air supply assembly, a water quality monitoring probe, and a light source. The outer casing has a hinged lid and a transparent observation window on its front. The layered test cylinder is housed within the outer casing. This method innovatively uses a layered test cylinder composed of a test cylinder, an isolation cylinder, and a pesticide loading cylinder. The Daphnia macrocarpa in the test cylinder is isolated from the microbial pesticide in the pesticide loading cylinder, but the water in the test cylinder and the pesticide loading cylinder exchange water through an exchange port. This method realistically simulates the natural exposure state of Daphnia macrocarpa in water containing microorganisms and effectively avoids the problem of microbial adhesion interfering with Daphnia macrocarpa activity, thus significantly improving the accuracy of the test results.

Description

Technical Field

[0001] This invention relates to pesticide detection technology, specifically to a test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa. Background Technology

[0002] Microbial pesticides, as an important support for green agriculture, can significantly reduce environmental pollution and pesticide residue risks; however, their environmental safety remains a concern. Because they may enter water bodies through direct application or runoff and may be filter-fed by aquatic organisms, their aquatic ecotoxicity needs to be scientifically evaluated.

[0003] Among numerous aquatic test organisms, Daphnia magna is an internationally recognized standard test species. It is a key zooplankton in freshwater ecosystems, sensitive to pollutants, and has a short lifespan, making it easy to observe in the laboratory. Its filter-feeding habits mean it can be directly exposed to microbial pesticides through water and may also be indirectly affected by consuming contaminated microorganisms, making it an ideal model for assessing the potential impact of such pesticides on aquatic invertebrates.

[0004] However, the inherent characteristics of microbial pesticides can interfere with the observation and interpretation of traditional toxicity tests. For example, they can deepen water color and consume oxygen, potentially making it difficult to accurately distinguish between the actual toxic effects and environmental interference. Furthermore, traditional methods typically place Daphnia macrocarpa directly in the pesticide-containing solution, making it difficult to differentiate whether the activity inhibition experienced by Daphnia macrocarpa is due to toxic effects or physical adhesion / encapsulation caused by the mycelial microbial pesticide. Frequent movement of the experimental container during observation can also interfere with the distribution of Daphnia macrocarpa and the microorganisms. Therefore, a testing method is needed to overcome these interferences and objectively and specifically assess the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa. Summary of the Invention

[0005] The purpose of this invention is to provide a test method for evaluating the inhibition of Daphnia macrocarpa activity by microbial pesticides, in order to solve the problem that existing test methods cannot effectively eliminate the physical interference caused by test equipment and test reagents on Daphnia macrocarpa activity.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa, comprising a test device for the inhibition of Daphnia macrocarpa activity by microbial pesticides, the test device comprising:

[0007] The outer box has a hinged lid and a transparent observation window on the front.

[0008] A layered test cylinder is set in the outer casing and includes a test cylinder, an isolation cylinder and a drug loading cylinder arranged sequentially from the inside out. The test cylinder has an exchange hole, and an exchange pipe is connected between adjacent drug loading cylinders, and a valve is connected to the exchange pipe.

[0009] An air supply assembly, installed on the outer casing, is used to supply air into the test cylinder;

[0010] Two sets of water quality monitoring probes are installed, one in each cavity between the test tube, the isolation tube, and the drug loading tube.

[0011] The light source consists of supplementary lights mounted on the box cover and is directed toward the layered test tube.

[0012] Preferably, a lower clamping ring is fixedly connected to the bottom of the inner wall of the outer casing. The lower clamping ring is composed of two concentrically fitted rubber rings. The lower end of the test cylinder is engaged in the inner rubber ring, the lower end of the isolation cylinder is engaged between the two rubber rings, and the lower end of the drug loading cylinder is engaged outside the outer rubber ring.

[0013] Preferably, the gas delivery assembly includes an oxygen pump installed on the outer casing and an air stone connected to the bottom of the inner wall of the outer casing. The air stone is installed at the bottom of the test cylinder and is connected to the oxygen pump pipeline.

[0014] Preferably, the test tube is cylindrical.

[0015] Preferably, the test cylinder is arranged in the shape of a prism with a cross-shaped cross section.

[0016] Preferably, the steps include:

[0017] Prepare test solutions of various concentrations for microbial pesticide formulations;

[0018] Aquaculture water was injected into the test tubes of multiple layered test tubes. Test solutions of different concentrations were injected into the chamber between the drug loading tube and the isolation tube. Insulated water was injected between the outer box and the drug loading tube. The temperature was set at 20°C, the light cycle was 16h:8h, the light intensity was 2000 lux, and aeration was maintained through the air supply component to maintain the dissolved oxygen concentration in the water greater than 8.0 mg / L.

[0019] Multiple large Daphnia juveniles were placed in the aquaculture water of multiple nested test tubes and acclimatized for 2 hours. They were then removed from the isolation tubes and exposed for 24 hours. The activity status of the large Daphnia was observed and recorded at regular intervals through the observation window.

[0020] By analyzing the recorded data on the movement trajectory and activity intensity of Daphnia magna, the activity inhibition rate of Daphnia magna within each concentration group was calculated.

[0021] Preferably, individuals of the large daphnia that show no visible movement of appendages or antennae and do not respond to external stimuli during a 15-second observation period are classified as inhibited individuals. The concentration of EC that results in 50% inhibition of activity in large daphnia is calculated using a probability unit method or a nonlinear regression model. 50and its 95% confidence interval.

[0022] Compared with existing technologies, the present invention provides a test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa. It innovatively sets up a nested test tube consisting of a test tube, an isolation tube, and a pesticide loading tube. The Daphnia macrocarpa in the test tube is isolated from the microbial pesticide in the pesticide loading tube. The water in the test tube and the water in the pesticide loading tube exchange water through an osmosis port. This method can realistically simulate the natural exposure state of Daphnia macrocarpa in water containing microorganisms, and effectively avoid the problem of microbial adhesion interfering with the activity of Daphnia macrocarpa, thus significantly improving the accuracy of the test results. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0024] Figure 1 A schematic diagram of the overall structure provided for an embodiment of the present invention. Figure 1 ;

[0025] Figure 2 A schematic diagram of the overall structure provided for an embodiment of the present invention. Figure 2 ;

[0026] Figure 3 This is a schematic diagram of the overall cross-sectional structure provided in an embodiment of the present invention;

[0027] Figure 4 This is a schematic diagram of the cross-sectional structure of the sleeve test cylinder provided in an embodiment of the present invention;

[0028] Figure 5 This is a schematic diagram of the first type of test cylinder structure provided in an embodiment of the present invention;

[0029] Figure 6 This is a schematic diagram of a second type of test cylinder structure provided in an embodiment of the present invention.

[0030] Explanation of reference numerals in the attached figures:

[0031] 1. Outer casing; 2. Box cover; 3. Observation window; 4. Air stone; 5. Test cylinder; 6. Exchange port; 7. Isolation cylinder; 8. Lower clamping ring; 9. Drug loading cylinder; 10. Water quality monitoring probe; 11. Exchange pipe; 12. Valve; 13. Supplemental lighting; 14. Aeration pump. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0033] As attached Figure 1 To be continued Figure 6 As shown:

[0034] Example:

[0035] This invention provides a test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa, and applies a test device for the inhibition of Daphnia macrocarpa activity by microbial pesticides, the test device comprising:

[0036] The outer box 1 has a lid 2 hinged to it, and a transparent observation window 3 is provided on the front of the outer box 1, through which the test personnel can observe the test process inside the outer box 1.

[0037] The test tube is set in the outer box 1 and includes a test tube 5, an isolation tube 7 and a drug loading tube 9 arranged sequentially from the inside out. The test tube 5 is provided with an exchange hole 6 with a diameter of 80-120μm. This diameter allows the test solution containing microbial pesticides to flow freely, while confining the large Daphnia inside to prevent it from escaping but ensuring that it is fully exposed to the pesticide environment. An exchange pipe 11 is connected between adjacent drug loading tubes 9, and a valve 12 is connected to the exchange pipe 11.

[0038] An air supply assembly, installed on the outer casing 1, is used to supply air into the test cylinder 5 to maintain the dissolved oxygen concentration in the water inside each layer of the test cylinder at a constant saturation level.

[0039] Two sets of water quality monitoring probes 10 are set up and are respectively installed in the cavity between the test tube 5, the isolation tube 7 and the drug loading tube 9. They monitor the water temperature, pH value, dissolved oxygen and conductivity in the test tube in real time or at intervals to ensure water quality stability during the test.

[0040] The light source consists of a supplementary light 13 mounted on the cover 2 and is directed toward the layer test tube.

[0041] As can be seen from the above, this method innovatively sets up a nested test cylinder consisting of a test cylinder 5, an isolation cylinder 7, and a pesticide loading cylinder 9. The Daphnia magna in the test cylinder 5 is isolated from the microbial pesticide in the pesticide loading cylinder 9. However, the water in the test cylinder 5 and the water in the pesticide loading cylinder 9 exchange water through the communication port 6. This can not only realistically simulate the natural exposure state of Daphnia magna in water containing microorganisms, but also effectively avoid the problem of microbial adhesion interfering with the activity of Daphnia magna, thus effectively improving the accuracy of the test results.

[0042] A lower clamping ring 8 is fixedly connected to the bottom of the inner wall of the outer casing 1. The lower clamping ring 8 is composed of two concentrically fitted rubber rings. The lower end of the test cylinder 5 is clamped in the inner rubber ring, the lower end of the isolation cylinder 7 is clamped between the two rubber rings, and the lower end of the drug-filling cylinder 9 is clamped outside the outer rubber ring. The lower clamping ring 8, composed of rubber rings, can tightly fit the lower end interface of the test cylinder 5, the isolation cylinder 7, and the drug-filling cylinder 9 to seal, thereby preventing the liquid in the isolation cylinder 7 from exchanging with the liquid in the drug-filling cylinder 9.

[0043] The gas delivery assembly includes an aeration pump 14 installed on the outer casing 1 and an air stone 4 connected to the bottom of the inner wall of the outer casing 1. The air stone 4 is installed at the bottom of the test cylinder 5 and is connected to the aeration pump 14 through a pipeline. The aeration pump 14 pumps air through the pipeline to the air stone 4. After the air stone 4 disperses and forms small bubbles, the small bubbles come into contact with the water in the test cylinder 5 to exchange oxygen, thereby maintaining the dissolved oxygen concentration in the water in the test cylinder 5 at a relatively saturated state, thus avoiding the adverse effects of oxygen deficiency on the test results.

[0044] In one embodiment, the test cylinder 5 is cylindrical, which facilitates the user's observation of the activity of the large daphnia inside the test cylinder 5.

[0045] In one embodiment, the test tube 5 is arranged in the shape of a cross-section prism to increase the contact area between the water in the test tube 5 and the water in the isolation tube 7. After the test personnel withdraw from the isolation tube 7, the surface area of ​​the water containing microbial pesticides inside the drug-filled tube 9 surrounding the test tube 5 is larger, which can improve the exchange efficiency between the microbial pesticide water and the water inside the test tube 5, reduce the contact distance between the microorganisms and the large Daphnia magna, and make the environment of the large Daphnia magna in the test tube 5 more closely match the water environment containing microbial pesticides, further improving the test effect.

[0046] The testing method steps include:

[0047] Prepare test solutions of various concentrations for microbial pesticide formulations;

[0048] Aquaculture water is injected into the test tube 5 of the multi-layered test tubes. Test solutions of different concentrations are injected into the chamber between the medicine loading tube 9 and the isolation tube 7. Insulated water is injected between the outer box 1 and the medicine loading tube 9. The temperature is set at 20°C, the light cycle is 16h:8h, the light intensity is 2000 lux, and aeration is maintained through the air supply component to maintain the dissolved oxygen concentration in the water greater than 8.0 mg / L.

[0049] Multiple large Daphnia juveniles were placed in the aquaculture water of multiple test tubes and acclimatized for 2 hours. They were then removed from the isolation tube 7 and exposed for 24 hours. The activity status of the large Daphnia was observed and recorded at regular intervals through the observation window 3. External camera equipment could be used for continuous or timed shooting, and video analysis software could be used to help determine the movement trajectory and activity intensity.

[0050] By analyzing the recorded data on the movement trajectory and activity intensity of Daphnia magna, the activity inhibition rate of Daphnia magna within each concentration group was calculated.

[0051] To facilitate researchers' understanding of the activity of Daphnia macrocarpa in water environments containing different concentrations of microbial pesticides, and to allow researchers to understand the Daphnia macrocarpa's response to changes in microbial pesticide concentrations, researchers can, after recording the activity of Daphnia macrocarpa in each test chamber, rotate valve 12 to connect exchange pipe 11. Through exchange pipe 11, the researchers can connect to the pesticide loading cylinder 9 of adjacent test chambers, thereby changing the concentration of microbial pesticides in the water of adjacent test chambers. Researchers can obtain activity data of Daphnia macrocarpa when microbial pesticides change in the environment, increasing the amount of experimental data. By continuously opening the exchange pipe 11 between different adjacent loading cylinders 9, more experimental data can be obtained, which helps to improve the authenticity of the experiment.

[0052] Individuals of the large daphnia that show no visible movement of appendages or antennae and do not respond to external stimuli during a 15-second observation period are considered suppressed. The concentration of EC that causes 50% of the large daphnia's activity to be suppressed is calculated using a probability unit method or a nonlinear regression model. 50 and its 95% confidence interval.

[0053] EC 50 It is one of the most critical input parameters for conducting a preliminary environmental risk assessment. The typical process includes:

[0054] Predicted Environmental Concentration (PEC): Based on pesticide application rates, runoff models, etc., estimate the possible concentration of pesticides in water bodies;

[0055] Predicted ineffective concentration (PNEC): The EC obtained from the experiment... 50 (or more sensitive endpoints such as EC) 10 Divide the concentration threshold that is considered safe for the ecosystem by an assessment factor (e.g., 100, 50 or 10, depending on data quality, number of species, etc.).

[0056] Risk Quotient (RQ) calculation: RQ = PEC / PNEC; if RQ < 1, the risk is generally considered acceptable; if RQ > 1, it indicates that there is unacceptable risk, which may require stricter control, restrictions on use, or further research.

[0057] The 95% confidence interval, as a statistical result, is direct proof of the superiority of the method of this invention.

[0058] Reflects the accuracy and variability of the data: narrow confidence intervals (e.g., EC50). 50 =10 mg / L, 95% CI: 9.5-10.5 mg / L), indicating excellent repeatability, concentrated data points, and highly accurate estimation. Wide interval (e.g., EC50). 50 If the concentration is 10 mg / L and the concentration is 95% CI (5-20 mg / L), it indicates that the experimental data has high dispersion and large uncertainty.

[0059] Highlighting the innovative value of the method of this invention:

[0060] Traditional methods suffer from poor environmental control and significant observational interference, leading to large differences in responses among individual Daphnia magna and significant data fluctuations, resulting in calculated EC values. 50 Confidence intervals are often very wide;

[0061] This invention, through precise control of dissolved oxygen, light, and water quality, and employing non-destructive observation, significantly reduces individual response variability caused by non-testing factors (environmental stress, human interference). Therefore, with the same sample size, the data obtained using this method are more stable, and the calculated EC... 50 The 95% confidence interval will narrow significantly;

[0062] Narrow confidence intervals provide strong data support for the beneficial effect of this testing method in "improving the accuracy and repeatability of test results".

[0063] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa, characterized in that, A test apparatus for inhibiting the activity of Daphnia macrocarpa using a microbial pesticide, the test apparatus comprising: Outer box (1), with a box cover (2) hinged on the outer box (1), and a transparent observation window (3) provided on the front of the outer box (1); The test tube is set in the outer box (1) and includes a test tube (5), an isolation tube (7) and a drug loading tube (9) arranged sequentially from the inside to the outside. The test tube (5) is provided with an exchange hole (6). An exchange pipe (11) is connected between adjacent drug loading tubes (9), and a valve (12) is connected to the exchange pipe (11). An air supply assembly, installed on the outer casing (1), is used to supply air into the test cylinder (5); Two sets of water quality monitoring probes (10) are provided and are respectively installed in the cavity between the test tube (5), the isolation tube (7) and the drug loading tube (9); The light source consists of a supplementary lamp (13) mounted on the box cover (2) and is directed toward the layer test tube.

2. The test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa according to claim 1, characterized in that, The bottom of the inner wall of the outer box (1) is fixedly connected to a lower clamping ring (8), which is composed of two concentric rubber rings. The lower end of the test cylinder (5) is clamped in the inner rubber ring, the lower end of the isolation cylinder (7) is clamped between the two rubber rings, and the lower end of the drug loading cylinder (9) is clamped outside the outer rubber ring.

3. The test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa according to claim 1, characterized in that, The gas delivery assembly includes an oxygen pump (14) installed on the outer casing (1) and an air stone (4) connected to the bottom of the inner wall of the outer casing (1). The air stone (4) is installed at the bottom of the test cylinder (5) and is connected to the oxygen pump (14) via a pipeline.

4. The test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa according to claim 1, characterized in that, The test tube (5) is cylindrical.

5. The test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa according to claim 1, characterized in that, The test tube (5) is a prism with a cross-shaped cross section.

6. The test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa according to claim 1, characterized in that the steps are as follows: include: Prepare test solutions of various concentrations for microbial pesticide formulations; Aquaculture water was injected into the test tube (5) of multiple layered test tubes. Test solutions of different concentrations were injected into the chamber between the medicine loading tube (9) and the isolation tube (7). Insulated water was injected between the outer box (1) and the medicine loading tube (9). The temperature was set at 20°C, the light cycle was 16h:8h, the light intensity was 2000 lux, and aeration was maintained through the air supply component to maintain the dissolved oxygen concentration in the water greater than 8.0 mg / L. Multiple large Daphnia juveniles were placed in the aquaculture water of multiple layered test tubes and acclimatized for 2 hours. They were then removed from the isolation tube (7) and exposed for 24 hours. The activity status of the large Daphnia was observed and recorded at regular intervals through the observation window (3). By analyzing the recorded data on the movement trajectory and activity intensity of Daphnia magna, the activity inhibition rate of Daphnia magna within each concentration group was calculated.

7. The test method for evaluating the inhibitory effect of microbial pesticides on the activity of Daphnia macrocarpa according to claim 6, characterized in that, Individuals of the large daphnia that show no visible movement of appendages or antennae and do not respond to external stimuli during a 15-second observation period are considered suppressed. The concentration of EC that causes 50% of the large daphnia's activity to be suppressed is calculated using a probability unit method or a nonlinear regression model. 50 and its 95% confidence interval.

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