Method for evaluating toxicity of spiroxamine by using zebra fish and application of method

Abstract

The invention relates to a method for evaluating toxicity of spiroxamine by using zebra fish and application of the method, and belongs to the technical field of toxicity detection. According to the method, spiroxamine culture solutions with different concentrations are prepared for culturing zebrafish embryos, embryonic development detection and juvenile fish behavior testing are carried out, embryonic development detection includes hatching rate, heart rate and body length, and juvenile fish behavior testing includes spontaneous movement testing, vortex escape testing, swimming behavior testing, reflex behavior testing and color stimulation testing. According to the method provided by the invention, the toxic effect of spiroxamine on zebra fish embryos and juvenile fishes is systematically and comprehensively evaluated, the intuition and accuracy of spiroxamine toxicity detection are improved, and the method can be applied to evaluating the toxicity of environmental samples containing spiroxamine.

Description

Technical Field

[0001] The present invention relates to the technical field of toxicity detection, and in particular to a method for evaluating the toxicity of spiroxamine using zebrafish and its application. Background Art

[0002] Due to the threat of pathogens or pests, the global annual grain yield is reduced by 20%-40%, making it unable to meet the food demand brought about by future population growth. Therefore, pesticides / fungicides have become an indispensable part of contemporary agricultural systems to protect crops from the threat of pests and diseases. However, the excessive use of pesticides, their poor degradability, and high persistence in the ecological environment have transcended geographical boundaries and become one of the global problems threatening human health and safety.

[0003] Spiroxamine (SPX), also known as (8-tert-butyl-1,4-dioxaspiro[4,5]decane-2-ylmethyl)ethylpropylamine, is a spiroamide compound belonging to the sterol biosynthesis inhibitor and can inhibit the synthesis of C-14 demethylase. As a new type of systemic foliar fungicide, spiroxamine is a specific medicine for controlling powdery mildew, leaf rust, and Rhynchosporium secalis, and can be used alone or in combination with other bactericidal active ingredients, and is widely used in the cultivation of grains (such as wheat, barley, etc.) and fruits (such as grapes, strawberries, etc.). The market distribution of spiroxamine is extensive, and the total global annual sales volume is 3,500-4,000 tons (in terms of 100% pure substance), and the core sales regions are Eastern Europe and EU countries. Residual spiroxamine in the environment can come into contact with the human body through various channels or be ingested and absorbed by the human body through the food chain and food web. Therefore, spiroxamine exposure poses a greater risk to human health and can cause severe skin irritation.

[0004] Currently, for the toxicity research of toxic substances, researchers mostly use rats, hamsters, mice, etc. as experimental objects. The experiments have high research costs, long animal reproduction cycles, low litter sizes per birth, and it is not easy to obtain homogeneous experimental individuals. Therefore, it is not conducive to the application of the toxicity evaluation of spiroxamine. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for evaluating the toxicity of spiroxamine using zebrafish. Zebrafish has a high degree of gene homology (87%) with humans and has many advantages such as in vitro fertilization, transparent early embryos, rapid development, easy large-scale breeding, large number of eggs laid, and low culture costs. In order to evaluate the ecological environment health risk of the new fungicide spiroxamine, the present invention uses zebrafish as a model organism, exposes zebrafish embryos to a certain concentration of spiroxamine solution, and comprehensively evaluates the toxicity of spiroxamine and its effects on the embryonic development and larval behavior of zebrafish through multiple indicators.

[0006] A method for evaluating the toxicity of spiroxamine using zebrafish, which involves exposing zebrafish embryos to a spiroxamine solution and conducting embryo development detection and larval behavior tests on the zebrafish to evaluate the toxicity of spiroxamine.

[0007] Further, the embryo development detection includes hatching rate, heart rate, and body length.

[0008] Further, the larval behavior tests include spontaneous movement test, vortex escape test, swimming behavior test, reflex behavior test, and color stimulation test.

[0009] Further, the reflex behavior test includes visual startle reflex behavior test and auditory startle reflex behavior test.

[0010] Further, it includes the following steps:

[0011] Step S1: Prepare spiroxamine culture solutions with different concentrations;

[0012] Step S2: Expose zebrafish embryos to spiroxamine culture solutions with different concentrations and culture for 48 - 96 hours for zebrafish embryo development detection;

[0013] Step S3: Expose zebrafish embryos to spiroxamine culture solutions with different concentrations and culture for 96 - 120 hours for larval behavior tests.

[0014] Further, in step S1, the concentration of the spiroxamine culture solution is 0.03 - 0.6 mg / L.

[0015] Further, in step S1, the pH value of the spiroxamine culture solution is 6.5 - 8.5.

[0016] Further, in step S2, the zebrafish embryos are from wild AB - type zebrafish broodstock.

[0017] Further, in step S2, the culture temperature of the zebrafish embryos is 26 - 30 °C.

[0018] The second object of the present invention is to provide the application of the above - mentioned method in evaluating the toxicity of environmental samples.

[0019] Advantages of the present invention:

[0020] The method described in the present invention systematically and comprehensively evaluates the toxic effects of spiroxamine on zebrafish embryos and larvae, realizes the rapid screening of the toxicity of spiroxamine, improves the intuitiveness and accuracy of spiroxamine toxicity detection, and can be applied to evaluate the toxicity of environmental samples containing spiroxamine. With the strong reproductive ability of zebrafish, high - throughput screening can be carried out, greatly improving the evaluation efficiency and enabling the detection of more samples in a shorter time. Brief Description of the Drawings

[0021] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to specific embodiments of the present invention in conjunction with the drawings, where

[0022] Figure 1 shows the effect of spiroxamine on the hatching rate of zebrafish embryos in Example 2 of the present invention, where CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L respectively;

[0023] Figure 2 shows the effect of spiroxamine on the body length of zebrafish in Example 3 of the present invention, where CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L respectively;

[0024] Figure 3 shows the effect of spiroxamine on the heart rate of zebrafish in Example 4 of the present invention, where CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L respectively;

[0025] Figure 4 shows the spontaneous movement frequency of zebrafish embryos after 24 h of spiroxamine exposure in Example 5 of the present invention, where CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L respectively;

[0026] Figure 5 shows the vortex escape path of zebrafish after 96 h of spiroxamine exposure in Example 6 of the present invention, where CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L respectively;

[0027] Figure 6 shows the effect of spiroxamine exposure for 120 h on the swimming behavior of zebrafish in Example 7 of the present invention, where A is the movement time, B is the movement distance, C is the average movement speed, CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L respectively;

[0028] Figure 7It is the visual startle reflex behavior of zebrafish after exposure to spiroxamine for 96 h in Example 8 of the present invention. Among them, A is the difference in movement distance within 1 min before and after transient light stimulation, B is the difference in average movement speed within 1 min before and after transient light stimulation, CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L, respectively;

[0029] Figure 8 It is the movement trajectory map and heat map of zebrafish in different color regions after exposure to spiroxamine for 120 h in Example 9 of the present invention. Among them, A is the movement trajectory map, with blue above, red on the left, green on the right, yellow below, and blank in the middle. B is the heat map, CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L, respectively;

[0030] Figure 9 It is the residence time of zebrafish in different color regions after exposure to spiroxamine for 120 h in Example 9 of the present invention. Among them, CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L, respectively;

[0031] Figure 10 It is the influence on the auditory startle reflex behavior of zebrafish after exposure to spiroxamine for 120 h in Example 10 of the present invention. Among them, A is the difference in movement distance within 1 min before and after vibration stimulation, B is the difference in average movement speed within 1 min before and after vibration stimulation, CK is the blank control, SC is the solvent blank, and C1, C2, and C3 represent spiroxamine concentrations of 0.03 mg / L, 0.3 mg / L, and 0.6 mg / L, respectively. Detailed implementation manners

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0033] Example 1: Cultivation of zebrafish and exposure to spiroxamine

[0034] Cultivation conditions: temperature (27.0 - 28.5 °C), humidity (40 - 60%), pH (6.8 - 8.0), dissolved oxygen (6 - 10 mg / L), conductivity (480 - 600 μS / cm), carbonate hardness (50 - 100 mg / L), ammonia nitrogen content (<0.02 mg / L), nitrite (<0.2 mg / L), nitrate (5 - 10 mg / L), and residual chlorine (<0.2 mg / L).

[0035] Determine the number of breedings according to the required amount of fish eggs. In the evening of the day before the experiment, start breeding the zebra fish broodstock (wild AB-type zebra fish). Insert the inner cylinder of the spawning box, which has been washed and dried, into the outer cylinder, insert the partition board, and add about 2 / 3 of the standard dilution water in the cylinder. Select male and female broodstock with similar body lengths, fish ages, obvious sexual development characteristics, and being healthy and active. Add 2 female fish and 1 male fish to the left and right compartments respectively, cover the cover board, and keep it in the constant temperature room overnight in the dark.

[0036] After turning on the light the next day, open the cover board of the spawning box, pull out the partition board, and let the male and female broodstock mate and lay eggs. After 1.5 h, check the egg-laying situation of the broodstock in each cylinder, collect the corresponding fish eggs in each cylinder respectively. After washing, disinfection (soaking in 0.003% hypochlorous acid), and rinsing with standard dilution water, put them into the biochemical incubator for cultivation (set the temperature at 28±2 °C, the light intensity at 750 lx, and the light-dark ratio at 14 h:10 h). Statistically calculate the fertilization rate of the fish eggs produced by each group of broodstock, and select 3 groups of fish eggs with a fertilization rate ≥70% for mixing and standby.

[0037] Randomly pick round, plump, and intact fish eggs and add them to the 24-well plate. Remove the fish eggs with obvious abnormalities during cell division (such as asymmetry, having vesicles or no egg membrane, etc.) or damaged egg membranes. Put one fertilized egg in each well and cover the cover board. Place the above 24-well cell culture plate in the biochemical incubator for cultivation (set the temperature at 28±2 °C, the light intensity at 750 lx, and the light-dark ratio at 14 h:10 h). Set 7 concentration gradients for the acute toxicity test: CK, SC, 0.625 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L. Set 3 replicates for each group. Add the prepared spiroxamine solutions with different concentrations to the 24-well cell culture plate, 2 mL per well. Calculate the LC 50 value of 3 mg / L, which characterizes the acute toxicity of spiroxamine, and set the concentrations for the subsequent zebrafish embryo development and behavioral response tests accordingly. After the zebrafish embryos are exposed to the toxicant, measure the corresponding indicators according to the experimental requirements.

[0038] Example 2: Determination of zebrafish embryo hatching rate

[0039] The period from 48 h to 72 h after fertilization of zebrafish is called the hatching period. During this stage, the tissues and organs of the embryos develop rapidly, forming early fry and gradually breaking through the egg membrane. After exposing to low concentrations of spiroxamine (1%, 10%, and 1 / 5 of the median lethal concentration) for 48 h, 72 h, and 96 h respectively, accurately observe and record the embryo development in each well of the 24-well cell culture plate. The results are as Figure 1 shown, where CK is the blank control, SC is the solvent blank, C1 is the spiroxamine concentration of 0.03 mg / L, C2 is the spiroxamine concentration of 0.3 mg / L, and C3 is the spiroxamine concentration of 0.6 mg / L.

[0040] After 48 h, 72 h, and 96 h of exposure to the drug, as the concentration of spiroxamine increased, there was no significant overall difference in its effect on the hatching rate of zebrafish embryos. This may be because before hatching, the chorion of zebrafish embryos has a certain obstructive effect on exogenous pollutants, forming a barrier to reduce the impact of spiroxamine on zebrafish embryos. However, after 72 h and 96 h of exposure to the drug, as the concentration of spiroxamine increased, the hatching rate showed a certain downward trend overall. In addition, compared with 72 h after exposure, the hatching rate of the spiroxamine-exposed group increased at 96 h, that is, some zebrafish just completed hatching at 96 h, which was delayed compared with the normal hatching period (the normal hatching period of zebrafish is 48 h to 72 h after fertilization), indicating that spiroxamine may delay the hatching cycle of zebrafish embryos.

[0041] Example 3: Measurement of the body length of early-stage zebrafish larvae

[0042] (1) Pipette a small amount of methylcellulose working solution into a culture dish;

[0043] (2) Dilution of the anesthetic: Usually, take 24 mL of standard dilution water and add 1 mL of MESAB stock solution (25×, 0.4%) to prepare MESAB working solution (1×, 0.016%);

[0044] (3) Place the larvae to be measured in the above MESAB working solution for 1 - 2 min. When the larvae stop swimming or do not respond to touch, the test operation can be carried out;

[0045] (4) Gently place the anesthetized larvae into the culture dish containing the methylcellulose working solution, and gently swing the position of the larvae with a dissecting needle to an appropriate shooting angle;

[0046] (5) Body length measurement: Use the microscope software ImageView to measure the length data from the tip of the zebrafish nose to the tail of the zebrafish to obtain the body length data of the larvae. Randomly select 8 larvae from each treatment group for measurement.

[0047] The results are as Figure 2As shown in the figure, where CK is the blank control, SC is the solvent blank, C1 is the spiroxamine concentration of 0.03 mg / L, C2 is the spiroxamine concentration of 0.3 mg / L, and C3 is the spiroxamine concentration of 0.6 mg / L. After 72 h of exposure to the toxicant, the body length of early-stage zebrafish larvae gradually decreased with the increase in spiroxamine concentration. Compared with the control group, there was no significant difference in the body length of zebrafish in the spiroxamine treatment groups of 0.03 mg / L and 0.3 mg / L. When the concentration of the exposure group reached 0.6 mg / L, the body length showed a significant difference compared with the control group. After 96 h of exposure to spiroxamine, the change in the body length of early-stage zebrafish larvae was similar to that after 72 h of exposure, that is, the body length gradually decreased with the increase in spiroxamine concentration. Among them, the highest concentration treatment group (0.6 mg / L) showed a significant difference compared with the control group.

[0048] Example 4: Measurement of the heart rate of early-stage zebrafish larvae

[0049] (1) Pipette a small amount of methylcellulose working solution into a petri dish.

[0050] (2) Gently place the larvae to be observed into the petri dish containing the methylcellulose working solution, and use a dissecting needle to gently swing the position of the larvae to a suitable shooting angle to ensure that the larvae are on their sides and the heartbeats can be clearly seen for counting the number of heartbeats.

[0051] (3) Heart rate measurement: Observe the heartbeat of zebrafish larvae using microscope software and record the number of heartbeats of zebrafish within 20 seconds to obtain the corresponding heart rate data. Randomly select 8 larvae from each treatment group for measurement.

[0052] The results are as Figure 3 shown in the figure, where CK is the blank control, SC is the solvent blank, C1 is the spiroxamine concentration of 0.03 mg / L, C2 is the spiroxamine concentration of 0.3 mg / L, and C3 is the spiroxamine concentration of 0.6 mg / L. After 72 h of exposure to the toxicant, the heart rate of early-stage zebrafish larvae gradually decreased with the increase in spiroxamine concentration. Among them, there was no significant difference in the heart rate of zebrafish in the low-concentration spiroxamine treatment group (0.03 mg / L) compared with the control group. When the concentration of the exposure group reached 0.3 mg / L, the heart rate showed a significant difference compared with the control group, and an obvious dose-effect relationship was presented. After 96 h of exposure to spiroxamine, the heart rate showed an obvious decreasing trend, and all three spiroxamine treatment groups showed significant differences compared with the control group.

[0053] Example 5: Spontaneous movement test of zebrafish embryos

[0054] After 24 h of spiroxamine exposure, 10 zebrafish embryos were randomly selected from each control group and treatment group, and a stereomicroscope was used to observe and measure the effect of spiroxamine on the spontaneous movement of zebrafish embryos. The selected embryos should be placed in a petri dish in advance for 4 min of light adaptation, and then, under normal light intensity, the spontaneous tail curling of zebrafish embryos in each treatment group was observed using a stereomicroscope. A counter was used to record the number of spontaneous movements of the embryos within 1 min.

[0055] By measuring the spontaneous movement frequency of zebrafish embryos after 24 h of spiroxamine exposure, it was determined whether spiroxamine induced neurodevelopmental toxicity in zebrafish embryos. The results are as Figure 4 shown, where CK is the blank control, SC is the solvent blank, C1 is the spiroxamine concentration of 0.03 mg / L, C2 is the spiroxamine concentration of 0.3 mg / L, and C3 is the spiroxamine concentration of 0.6 mg / L. Compared with the control group, there was no significant difference in the spontaneous movement frequency of embryos in the low-concentration exposure group (0.03 mg / L), and the toxic effect of spiroxamine was small. However, the spontaneous movement frequency of zebrafish embryos within 1 min was significantly increased when exposed to concentrations of 0.3 mg / L and 0.6 mg / L. High-concentration spiroxamine may interfere with the early embryonic development of zebrafish and cause abnormal neurobehavior in zebrafish embryos.

[0056] Example 6: Zebrafish vortex escape rate test

[0057] 80 zebrafish larvae after 96 h of spiroxamine exposure were transferred to a petri dish, and a pipette was used to gently blow the water flow along the edge of the petri dish to gather the larvae in the middle of the petri dish. After stopping the blowing of the water flow, the larvae would swim towards the edge of the petri dish. When the vortex water flow completely stopped, the number of larvae remaining in the middle area (with a diameter of 3 cm) of the petri dish and the number of larvae that escaped were counted, and the escape rate was calculated by dividing the number of escaped larvae by the total number (80).

[0058] The test results showed that the vortex escape rates of zebrafish larvae after exposure to different concentrations (C1 = 0.03, C2 = 0.3, and C3 = 0.6 mg / L) of spiroxamine were significantly lower than those of the control group. Moreover, with the increase in the spiroxamine concentration, the vortex escape rate of the larvae showed an obvious downward trend, and there was a certain dose-effect relationship between the spiroxamine concentration and the vortex escape rate of the larvae. There was no significant difference in the vortex escape rates of zebrafish larvae between the medium- and high-concentration treatment groups (the 0.3 and 0.6 mg / L spiroxamine exposure groups) ( Figure 5 ). Through this study, by evaluating the effect of spiroxamine on the vortex escape rate of zebrafish larvae, it was found that spiroxamine may interfere with the early neurodevelopment of zebrafish. When zebrafish receive external stimuli, it affects the signal transmission of Mauthner sensory neurons and induces abnormal neurobehavior.

[0059] Example 7: Zebrafish Swimming Behavior Test

[0060] The test and analysis were carried out using an automated zebrafish behavior analysis system, which can automatically track and analyze the movement, activity, and behavior of zebrafish, and can conduct precise research on the behavioral trajectories of zebrafish. The test environment is controllable and the test results are reliable. The specific test operation method is as follows:

[0061] (1) Transfer the zebrafish larvae after 120 h of spiroxamine exposure to a 48-well cell culture plate, with one concentration per row (8 fish per concentration);

[0062] (2) Place the 48-well cell culture plate in the automated zebrafish behavior analysis system;

[0063] (3) Open the real-time behavior trajectory analysis software;

[0064] (4) Set the movement area and adjust relevant parameters: Set the movement area (circle the movement area of each zebrafish according to the page prompt), scale (facilitate the software to identify and record the movement speed and distance of zebrafish), sensitivity (facilitate the software to identify and capture the movement of zebrafish), discrimination criteria for low-speed and high-speed movement, total test duration, data recording method, etc.;

[0065] (5) Measure the free movement of zebrafish in a non-stimulated state: Dark-adapt the zebrafish for 10 min, and then give light for 10 min, and record and analyze the movement data of zebrafish under light conditions.

[0066] By testing the movement behavior of zebrafish in each treatment group after 120 h of spiroxamine exposure under non-stimulated conditions ( Figure 6 A), it can be seen that spiroxamine exposure at different concentrations has no significant effect on the total movement time of zebrafish larvae. Compared with the control group, the total movement distance of zebrafish larvae in the spiroxamine treatment group increased significantly, and with the increase of spiroxamine concentration, the total movement distance of zebrafish larvae showed a trend of first increasing and then decreasing ( Figure 6 B). Compared with the control group, the average movement speed of zebrafish larvae in the spiroxamine treatment group also increased significantly, but there was no significant difference among the spiroxamine treatment groups ( Figure 6C). Exposure to 0.3 mg / L of spiroxamine (C2) significantly increased the swimming distance and average swimming speed of zebrafish larvae. Exposure to spiroxamine at this concentration may cause anxiety-like behavior in zebrafish larvae. When the concentration of spiroxamine reached 0.6 mg / L (C3), the swimming distance of zebrafish larvae decreased, and the average swimming speed also showed a downward trend. In summary, exposure to spiroxamine can interfere with the neurodevelopment of zebrafish larvae, thereby affecting their locomotor behavior and inducing abnormal neurobehavior. Among them, low-concentration spiroxamine may cause anxiety-like behavior in zebrafish larvae, while high-concentration spiroxamine may inhibit their locomotor activity to a certain extent.

[0067] Example 8: Visual startle reflex behavior test of zebrafish

[0068] The test analysis software is an automated zebrafish behavior analysis system, which can automatically track and analyze the movement, activity and behavior of zebrafish, and can conduct precise behavioral trajectory research on zebrafish. The test environment is controllable and the test results are reliable. The specific test operation method is as follows:

[0069] (1) Transfer the zebrafish larvae exposed to spiroxamine for 96 h to a 48-well cell culture plate, with one concentration per row (8 fish per concentration);

[0070] (2) Place the 48-well cell culture plate in the automated zebrafish behavior analysis system;

[0071] (3) Turn on the real-time behavior trajectory analysis software;

[0072] (4) Set the movement area and adjust relevant parameters: Set the movement area (circle the movement area of each zebrafish according to the page prompt), scale (facilitate the software to identify and record the swimming speed and distance of zebrafish), sensitivity (facilitate the software to identify and capture the movement of zebrafish), discrimination criteria for low-speed and high-speed movement, total test duration, data recording method, etc.;

[0073] (5) Set the transient light stimulation mode: Set relevant data such as the dark and light cycles and light intensity;

[0074] (6) Measure the changes in the locomotor behavior of zebrafish larvae before and after transient light stimulation after dark adaptation: Allow zebrafish to undergo dark adaptation for 10 min, and then give transient light stimulation, and record and analyze the changes in the locomotor behavior of zebrafish within 1 min before and after transient light stimulation.

[0075] By testing the visual startle reflex behavior of zebrafish larvae in each group after 96 h of exposure to spiroxamine, it can be seen that ( Figure 7) When transient light stimulation is given to dark - adapted zebrafish, the changes in the movement distance and average movement speed of zebrafish larvae in each treatment group at the dark - light alternation moment show two response effects: excitation and inhibition. Compared with the control group, after zebrafish receive sudden transient light stimulation, the change in the total movement distance of zebrafish larvae in the low - concentration spiroxamine treatment group C1 (0.03 mg / L) is similar to that of the control group, showing no significant difference, and the average movement speed increases significantly, showing a certain degree of excitatory response to sudden transient light, which may cause anxiety - like behavior in zebrafish; in the medium - concentration treatment group C2 (0.3 mg / L), the changes in the total movement distance and average movement speed of zebrafish larvae are significantly decreased compared with the control group, indicating that zebrafish still show an excitatory response to sudden transient light, but the overall excitatory response is weakened compared with the control group; in the high - concentration spiroxamine treatment group (0.6 mg / L), the total movement distance and average movement speed of zebrafish larvae are significantly lower than those of other groups, and compared with the dark condition, the total movement distance and average movement speed decrease, that is, high - concentration spiroxamine induces the excitatory response of zebrafish to turn into an inhibitory response, probably because high - concentration spiroxamine interferes with the visual motor nerves of zebrafish larvae, reducing their response to external transient light stimulation and even losing the startle reflex behavior, indicating that high - concentration spiroxamine may inhibit the motor activity of zebrafish larvae to a certain extent.

[0076] Example 9: Zebrafish Color Stimulation Behavior Test

[0077] Use an automated zebrafish behavior analysis system for test analysis. This system can automatically track and analyze the movement activities and behaviors of zebrafish, and can conduct precise behavioral trajectory research on zebrafish. The test environment is controllable and the test results are reliable. The specific test operation method is as follows:

[0078] (1) After 120 h of exposure experiment, randomly select zebrafish larvae and transfer them to the color preference device (cross - maze);

[0079] (2) Place the color preference device in the automated zebrafish behavior analysis system;

[0080] (3) Open the real - time behavior trajectory analysis software;

[0081] (4) Set the movement area and adjust relevant parameters: respectively set the movement area (including four color partitions of blue, red, green, and yellow and the middle blank area, where the upper part is blue, the left is red, the right is green, and the lower part is yellow), scale (facilitating the software to identify and record the movement speed and movement distance of zebrafish), sensitivity (facilitating the software to identify and capture the movement of zebrafish), discrimination criteria for low - speed and high - speed movement, total test duration, data recording method, etc.;

[0082] After the zebrafish larvae are dark-adapted for 10 min, normal light stimulation is given, and the changes in the locomotor behavior of zebrafish within 20 min of continuous light are recorded, and the residence time in each color area is analyzed.

[0083] From the locomotor trajectory maps and heat map residence times ([ Figure 8 ) of zebrafish larvae in each group after exposure to spiroxamine for 120 h, it can be seen that in this experiment, the control group of zebrafish showed an obvious preference for blue. However, as the concentration of spiroxamine increased, the residence time of zebrafish in the blue area showed a downward trend, and the bias towards blue weakened. This may be because exposure to high concentrations of spiroxamine affected the visual function of zebrafish larvae, thereby interfering with their color preference. In addition, compared with the control group, high-concentration spiroxamine (0.6 mg / L) also inhibited the locomotor behavior of zebrafish larvae and reduced their total locomotion time ([ Figure 9 ).

[0084] Example 10: Zebrafish Auditory Startle Reflex Behavior Test

[0085] In this experiment, an automated zebrafish behavior analysis system was used for analysis. This system can automatically track and analyze the movement, activity, and behavior of zebrafish, and can conduct precise behavioral trajectory research on zebrafish. The test environment is controllable and the test results are reliable. The specific test operation method is as follows:

[0086] (1) Transfer the zebrafish larvae after exposure to spiroxamine for 120 h to a 48-well plate, with one concentration per row (8 fish per well);

[0087] (2) Place the 48-well cell culture plate in the automated zebrafish behavior analysis system;

[0088] (3) Turn on the real-time behavioral trajectory analysis software;

[0089] (4) Set the movement area and adjust relevant parameters: respectively set the movement area (circle the movement area of each zebrafish according to the page prompt), scale (facilitate the software to identify and record the movement speed and distance of zebrafish), sensitivity (facilitate the software to identify and capture the movement of zebrafish), discrimination criteria for low-speed and high-speed movement, total test duration, data recording method, etc.;

[0090] (5) Set the vibration stimulation mode: set relevant data such as the non-stimulus and vibration stimulation periods, vibration frequency, etc.;

[0091] (6) Measure the changes in the locomotor behavior of zebrafish larvae before and after suddenly receiving vibration stimulation, and record and analyze the locomotor behavior changes of zebrafish within 1 min before and after vibration stimulation.

[0092] The results are as Figure 10As shown, when zebrafish larvae were given vibration stimulation, the changes in the movement distance and average movement speed of zebrafish larvae in each group at the alternating moment of no stimulation - vibration stimulation showed an excitatory response effect, indicating that vibration stimulation triggered the acoustic escape response of zebrafish. When the concentration of spiroxamine reached 0.6 mg / L, the total movement distance of zebrafish larvae decreased, and the average movement speed decreased significantly, indicating that high - concentration spiroxamine may inhibit the motor activity of zebrafish larvae to a certain extent. Compared with the control group, there was no significant difference in the total movement distance and average movement speed of zebrafish larvae in the spiroxamine treatment group (C1) at 0.03 mg / L. However, in the spiroxamine treatment groups at 0.3 mg / L (C2) and 0.6 mg / L (C3), the total movement distance and average movement speed of zebrafish larvae decreased significantly, and the acoustic escape response weakened, probably because high - concentration spiroxamine interfered with the auditory nerves of zebrafish larvae, reducing their response to vibration stimulation. In summary, spiroxamine exposure can interfere with the development of the auditory nerves of zebrafish larvae, affect their auditory startle reflex behavior, and induce abnormal neurobehavioral changes.

[0093] Obviously, the above - mentioned embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of this invention.

Claims

1. A method for evaluating the toxicity of spiroxabiodinium using zebrafish, characterized in that: Zebrafish embryos were exposed to spiroxamine solution, and embryonic development and juvenile behavior tests were performed on zebrafish to evaluate the toxicity of spiroxamine.

2. The method according to claim 1, characterized in that: The embryo development tests include hatching rate, heart rate and body length.

3. The method according to claim 1, characterized in that: The juvenile fish behavior test includes a spontaneous movement test, a vortex escape test, a swimming behavior test, a reflex behavior test, and a color stimulation test.

4. The method according to claim 3, characterized in that: The reflex behavior test includes a visual startle reflex behavior test and an auditory startle reflex behavior test.

5. The method according to claim 1, characterized in that The following steps are involved: Step S1, preparing spiroxabiocin culture solutions of different concentrations; Step S2, exposing zebrafish embryos to spiroxabiodinium culture solutions of different concentrations, and culturing for 48-96 hours to perform zebrafish embryo development detection; Step S3, exposing zebrafish embryos to spiroxabiodinium culture medium of different concentrations, culturing for 96-120 hours and then conducting behavioral tests on the juveniles.

6. The method according to claim 5, characterized in that: In step S1, the concentration of the spiroxabiocin culture solution is 0.03-0.6 mg / L.

7. The method according to claim 5, characterized in that: In step S1, the pH value of the spiroxabiocin culture solution is 6.5-8.

5.

8. The method according to claim 5, characterized in that: In step S2, the zebrafish embryos are derived from wild AB type zebrafish breeders.

9. The method according to claim 5, characterized in that: In step S2, the culture temperature of the zebrafish embryos is 26-30°C.

10. Use of the method according to any one of claims 1 to 9 in evaluating the toxicity of environmental samples.

Images