Method for evaluating developmental toxicity of zebra fish by single or combined use of fluopyram and tebuconazole

Abstract

The invention belongs to the technical field of toxicity tests, and discloses a method for evaluating zebra fish developmental toxicity by single or combined use of fluopyram and tebuconazole. The method is simple, efficient and accurate, through a combined toxicity test and a single pesticide test, reference data is conveniently increased according to the influence of different environments and different medicaments on animals, the influence of pesticides on zebra fish development is calculated according to the death rate and the median lethal concentration of zebra fish embryos, the data is reasonably analyzed and processed, and the method is suitable for popularization and application. The problems that related evaluation standards formulated by depending on a single effect are not accurate and cannot truly reflect the change of the real environment quality are avoided, the environmental risk of effectively evaluating the pollutants in the environmental water body is improved, and an environmental threshold value safer for aquatic organisms is formulated conveniently.

Description

Technical Field

[0001] This invention belongs to the field of toxicity testing technology, specifically, it relates to a method for evaluating the developmental toxicity of fluopyram, tebuconazole alone and in combination to zebrafish. Background Technology

[0002] Regarding the toxicity of fluopyram (FLU), studies have shown that it can cause liver cell carcinogenesis in female mice by interfering with the expression of estrane and androstened receptor genes; and in male mice, it can cause excessive thyroid hormone production and thyroid tumors. However, there is currently limited research on the toxicity of fluopyram to aquatic organisms.

[0003] Due to its high usage and persistent nature, tebuconazole (TBU) is widely present in the environment. As aquatic organisms, TBU is considered moderately toxic, and its toxic effects on aquatic life have received considerable attention.

[0004] Currently, many environmental toxicological effects cannot be explained by the mechanism of action of a single pollutant. Several studies have shown that even when multiple pollutants are at the "safe" level of relevant water quality benchmarks, they may still have significant combined toxic effects on aquatic organisms. Evaluation standards based on single effects cannot truly reflect changes in actual environmental quality. Therefore, in order to accurately and effectively assess the environmental risks of pollutants in environmental water bodies and to establish safer environmental thresholds for aquatic organisms, it is necessary to consider the combined toxic effects of pollutants.

[0005] Zebrafish embryos are highly sensitive to toxic substances in the early stages of development. There is a good correlation between embryonic toxicity data and traditional acute toxicity data of fish. Therefore, zebrafish embryos are increasingly used as animal models for toxicology. Summary of the Invention

[0006] The purpose of this invention is to provide a method for evaluating the developmental toxicity of fluopyram, tebuconazole alone and in combination to zebrafish.

[0007] To achieve the objectives of this invention, this invention provides a method for evaluating the developmental toxicity of fluopyram, tebuconazole, and their combined use to zebrafish, comprising the following steps: (1) Wild-type AB zebrafish were used in the experiment. Male and female zebrafish were kept in circulating water at a temperature of 28 ± 1 ℃ with a light-dark cycle of 14 h:10 h. On the night before breeding, male and female zebrafish were placed on both sides of the spawning box in a 1:1 ratio. The next day, after the photoperiod started, the male and female zebrafish on both sides of the spawning box were allowed to mate and the fertilized eggs produced by the female fish were collected. (2) In order to evaluate the effects of single pesticides fluopyram and tebuconazole on the early embryonic development of zebrafish, fluopyram and tebuconazole powders were dissolved in DMSO (dimethyl sulfoxide) to prepare stock solutions of a certain concentration and stored in the dark. (3) Based on the preliminary experimental results, a concentration gradient was set: the mother liquor was diluted with circulating water to the following exposure concentrations: fluopyram at 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L, and tebuconazole at 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, and 12 mg / L, respectively, with DMSO concentration less than 0.05%; then, according to the OECD guidelines (OECD Code of Responsible Business Conduct for Multinational Corporations), after 2 hpf (2 hours after fertilization), the single toxicity of fluopyram and tebuconazole to zebrafish at different exposure concentrations was investigated; specifically, normally developing fertilized eggs were selected and placed in a six-well plate, with 20 embryos randomly placed in each well, and 3 replicates were set for each exposure concentration, with 0.05% DMSO added to the blank control group; the exposure solution was changed every 24 h during the entire exposure period; finally, the LC at 96 hpf was calculated using GraphPad Prism software to generate a fitted curve. 50 value; (4) Combined toxicity AI index: The LC of single exposure to fluopyram and tebuconazole was determined using the Marking additive index method. 50 Values ​​and LC of combined exposure of the two 50 Substituting the concentrations of fluopyram and tebuconazole into equation (I): M = CA / LC 50-A +CB / LC 50-B Formula (I) Among them, LC 50-A LC-100 of fluopyram 50 Value, LC 50-B LC50 of tebuconazole 50 Value, CA is the LC of exposure to both. 50 The value represents the concentration of fluopyram, and CB represents the LC50 of exposure to the combination of the two. 50 The concentration of tebuconazole in the value; Then convert M into an additive exponent AI. When M = 1, AI = 0; when M > 1, AI = 1 - M; when M < 1, AI = 1 / M - 1. Determine the mode of action based on the AI ​​value. If AI = 0, it is a simple additive action; if AI > 0, it is a synergistic action; if AI < 0, it is an antagonistic action.

[0008] Furthermore, in step (4), the concentrations of fluopyram and tebuconazole under combined exposure were 0.5 mg / L and 0.125 mg / L, 5 mg / L and 1.25 mg / L, and 2.5 mg / L and 10 mg / L, respectively.

[0009] Furthermore, hatching rate and mortality rate were recorded every 24 hours until 96 hpf; in accordance with OECD guidelines, the probability of pericardial edema in zebrafish juveniles was manually assessed at 96 hpf.

[0010] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: The method of this invention is simple, efficient, and accurate. By combining toxicity tests and single pesticide tests, it is easy to consider the effects of different environments and different pesticides on animals, and it increases the reference data. The impact of pesticides on zebrafish development is calculated based on the mortality rate and median lethal concentration of zebrafish embryos. The data is analyzed and processed reasonably, avoiding the inaccuracy of evaluation standards based on single effects, which cannot truly reflect changes in real environmental quality. This improves the effectiveness of evaluating the environmental risks of pollutants in water bodies and facilitates the establishment of safer environmental thresholds for aquatic organisms. Attached Figure Description

[0011] Figure 1 The FLU dose-response curve (A) and TBU dose-response curve (B) are shown in the preferred embodiment of the present invention.

[0012] Figure 2 The hatching rates of zebrafish embryos under single and combined exposure to FLU and TBU are shown in the preferred embodiments of the present invention.

[0013] Figure 3 The images shown are (A) a 2.5x magnified whole photograph of zebrafish under a microscope and (B) the body length of zebrafish embryos under single and combined exposure to FLU and TBU, respectively, in a preferred embodiment of the present invention.

[0014] Figure 4 The heart rate of zebrafish embryos under single and combined exposure to FLU and TBU in a preferred embodiment of the present invention.

[0015] Figure 5 This refers to the probability of cardiac malformations in zebrafish embryos under single and combined exposure to FLU and TBU in a preferred embodiment of the present invention.

[0016] Figure 6 The pericardial area of ​​zebrafish embryos under single and combined exposure to FLU and TBU in a preferred embodiment of the present invention.

[0017] Figure 7 In a preferred embodiment of the present invention, the zebrafish embryo exhibits swim bladder closure and pericardial edema.

[0018] Figure 8A This invention relates to the change in the expression level of the zebrafish embryo and the heart development-related gene tbx5a in a preferred embodiment of the present invention.

[0019] Figure 8B This invention relates to a preferred embodiment of the expression level of gata4, a gene related to heart development in zebrafish embryos.

[0020] Figure 8C This invention relates to the changes in the expression level of the nppa gene, which is related to heart development in zebrafish embryos, in a preferred embodiment of the present invention.

[0021] Figure 8D This invention relates to the expression level changes of the zebrafish embryo and the gene nkx2.5, which is related to heart development, in a preferred embodiment of the present invention.

[0022] Figure 8E This invention relates to a preferred embodiment of the expression level of myh6, a gene related to heart development in zebrafish embryos.

[0023] Figure 8F This invention relates to the changes in the expression level of the gene vmhc, which is related to heart development in zebrafish embryos, in a preferred embodiment of the present invention.

[0024] Figure 9 In a preferred embodiment of the present invention, the distance (A) and speed (B) of the zebrafish embryo’s autonomous movement within 40 minutes are shown.

[0025] Figure 10 The vertical length of the swim bladder of zebrafish embryos under single and combined exposure to FLU and TBU in a preferred embodiment of the present invention.

[0026] Figure 11A This invention relates to the changes in the expression level of the gene foxa3, which is related to swim bladder inflation and development, in a preferred embodiment of the present invention.

[0027] Figure 11B This invention relates to the changes in the expression level of pbx1a, a gene related to swim bladder inflation and development, in a preferred embodiment of the present invention.

[0028] Figure 11C This invention relates to the changes in the expression level of hprt1l, a gene related to swim bladder inflation and development, in zebrafish embryos in a preferred embodiment of the present invention.

[0029] In the picture, express P <0.05, express P <0.01, expressP <0.001, express P <0.0001. Detailed Implementation

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] In this invention, hpf refers to hours post-fertilization.

[0032] Example 1 Experimental Materials and Methods: Fluopyram (purity 99%) and Tebuconazole (purity 98.3%) were purchased from Beijing Solarbio Science & Technology Co., Ltd. FLU and TBU were dissolved in analytical grade dimethyl sulfoxide (DMSO) to prepare drug exposure stock solution (10000 mg / L), which was stored at 4 ℃ until dilution.

[0033] 1.2 Zebrafish rearing Wild-type AB zebrafish were obtained from the Wuhan Zebrafish Resource Center in China. Male and female zebrafish were housed separately in circulating water at 28 ± 1 ℃, with a light-dark cycle of 14 h:10 h. Males and females were placed on opposite sides of the spawning tank at a 1:1 ratio. On the second day, the partition of the spawning tank was removed to stimulate mating behavior, resulting in successful spawning and collection of fertilized eggs. The animal experimental procedures were approved by the Laboratory Animal Protection and Utilization Committee of the Chinese Academy of Agricultural Sciences and complied with relevant national regulations.

[0034] 1.3 Acute toxicity exposure test To evaluate the effects of fluopyram and tebuconazole on early embryonic development in zebrafish, fluopyram and tebuconazole powders were dissolved in DMSO to prepare a 10,000 mg / L stock solution, which was stored in the dark. Concentration gradients (diluted with circulating water) were established based on preliminary experimental results: fluopyram at 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L; and tebuconazole at 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, and 12 mg / L. Following OECD guidelines, after 2 hpf, normally developing fertilized eggs were carefully selected using an OLYMPUSS ZX2-1LLT stereomicroscope (Tokyo, Japan). Twenty embryos were randomly placed in each well of a six-well plate. Three replicates were set up for each exposure concentration, with a blank control group containing 0.05% DMSO. The exposure solution was changed every 24 h throughout the exposure period.

[0035] 1.4 Combined Toxicity AI Index The Marking additive index method was used to determine the median lethal concentration (LC50) of FLU and TBU for single exposure. 50-A and LC 50-B Substituting the concentrations of component A and component B (CA and CB) in the median lethal concentration of the mixture of the two components into equation (I): M = CA / LC 50-A +CB / LC 50-B Formula (I) Among them, LC 50-A LC for FLU 50 Value, LC 50-B LC for TBU 50 Value, CA is the LC of exposure to both. 50 The value represents the concentration of FLU, and CB represents the LC50 of exposure to both. 50 The concentration of TBU in the value.

[0036] Then convert M into an additive exponent AI. When M = 1, AI = 0; when M > 1, AI = 1 - M; when M < 1, AI = 1 / M - 1. Determine the mode of action based on the AI ​​value: if AI = 0, it is a simple additive effect; if AI > 0, it is a synergistic effect; if AI < 0, it is an antagonistic effect.

[0037] 1.5 Phenotypic Analysis Hatching and mortality rates were recorded every 24 hours until 96 hpf. The probability of pericardial edema in zebrafish juveniles was manually assessed at 96 hpf according to OECD basic guidelines. To assess the developmental toxicity of fluopyram and tebuconazole at 96 hpf, zebrafish juveniles were anesthetized with tricaine mesylate, fixed with methylcellulose, and mounted on glass slides. Bright-field images of the entire body (n = 12) were captured using an OLYMPUS SZX2-1LLT stereomicroscope (Tokyo, Japan). Observation and analysis were performed using Fiji software (National Institutes of Health, NIH, Bethesda, MD, USA).

[0038] 1.6 Zebrafish Behavioral Trajectory Zebrafish embryos (96 hpf) from each treatment group were transferred to 48-well plates, one embryo per well, with 2 mL of circulating water. One embryo per concentration was used in triplicate. Swimming behavior of the zebrafish larvae was recorded using the Noldus DanioVision system for 40 minutes in a dark room at room temperature, 5 minutes after acclimatization to the 48-well plates. Recorded data included total distance (mm) and average speed (mm / s).

[0039] 1.7 RNA extraction and quantitative analysis of mRNA levels Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used to evaluate gene expression changes induced by fluopyram and tebuconazole. Exposure to fluopyram, tebuconazole, and the combined use of both pesticides was performed for 96 h at three different concentrations: fluopyram (1, 10, 20 mg / L), tebuconazole (0.25, 2.5, 5 mg / L), and combined use (FLU + TBU = 0.5 + 0.125, 5 + 1.25, 10 + 2.5 mg / L). RNA was extracted from whole zebrafish juveniles (n = 30) using TransZol Up solution (TransGen Biotech, Beijing, China). RNA purity was assessed by measuring the OD260 / OD280 ratio using a Unano-1000 micro spectrophotometer (Hangzhou UMI Instruments, China). cDNA was synthesized using AccuPower RT premix (TransGen Biotech, Beijing, China). Q-RT-PCR analysis was performed on a CFX96™ Real-Time PCR system (C1000 Touch™, Bio-Rad, USA) using the TransStart® TopTaq (TransGen-Biotech) method.

[0040] 1.8 Statistical Analysis Analyze data using GraphPad Prism 8.3 (GraphPad Software, San Diego, CA, USA).

[0041] 2. Experimental Results 2.1 Developmental toxicity of single and combined exposures to fluopyram and tebuconazole to zebrafish 2.1.1 Mortality rate and median lethal concentration like Figure 1 As shown, the mortality rate of zebrafish embryos gradually increased with increasing FLU and TBU dosages. At 96 hpf, the mortality rate in the highest FLU concentration treatment group was 61.7%, and the mortality rate in the highest TBU concentration treatment group was 100%. The median lethal concentration (LC50) of FLU for zebrafish embryos was calculated using a fitted curve generated by GraphPadPrism software. 50 The median lethal concentration (LC50) of TBU for zebrafish embryos was 30.78 mg / L. 50 The concentration was 7.211 mg / L. Figure 1 (A and B). The concentrations of each component in the combined exposure mixed solution were FLU: 11.352 mg / L and TBU: 2.838 mg / L.

[0042] 2.1.2 Combined Effect Index The Marking additive index method is developed based on the toxicity unit method. The formula is shown in equation (I).

[0043] The AI ​​value calculated by the formula is 0.311, so when FLU and TBU are mixed in equal toxicity ratios, they exhibit a synergistic effect.

[0044] 2.1.3 Hatching rate like Figure 2 As shown, based on the LC of the two drugs 50 The concentrations for the combined exposure experiment were reset. Zebrafish embryos began to hatch after 48 hpf, and all embryos in the control group hatched at 72 hpf. At 48 hpf, the hatching rates of embryos in the FLU-treated group were 65%, 67%, and 27%, respectively; the hatching rates of embryos in the TBU-treated group were 77%, 80%, and 57%, respectively; and the hatching rates of embryos in the combined treatment group were 52%, 40%, and 15%, respectively. This shows that the hatching rate of embryos in all treatment groups decreased with increasing drug concentration. Furthermore, the combined treatment group had a lower hatching rate and more pronounced developmental toxicity compared to the single treatment group.

[0045] 2.1.4 Body length like Figure 3 As shown in Figures A and B, the body length of zebrafish juveniles (n = 10) was measured using Fiji software. Compared with the body length of the control group (4.04 ± 0.12 mm), single exposure to FLU and TBU at low concentrations had little effect on the body length of zebrafish. The body length of zebrafish juveniles at the highest concentrations in both the single FLU exposure group and the combined exposure group was significantly shortened, with average lengths of 3.65 ± 0.16 mm and 3.54 ± 0.18 mm, respectively.

[0046] 2.2 Cardiotoxicity of single and combined exposures to fluopyram and tebuconazole in zebrafish 2.2.1 Heart Rate Heart rate variability is an important indicator of cardiotoxicity in zebrafish. For example... Figure 4 As shown, the heart rate of zebrafish embryos decreased with increasing drug dosage in each treatment group. In the highest concentration treatment group, the heart rate from highest to lowest was: TBU > FLU > combined exposure, indicating that combined exposure had a more significant inhibitory effect on zebrafish heart rate. Therefore, it can be concluded that the combined exposure treatment group had a stronger toxic effect on the zebrafish heart compared to single exposure to either drug.

[0047] 2.2.2 Heart malformations A key phenotype of zebrafish cardiac poisoning is pericardial edema. For example... Figure 5As shown, under individual exposure to FLU and TBU, a small percentage of zebrafish juveniles developed pericardial edema at a FLU concentration of 20 mg / L, while TBU did not cause pericardial edema. Under combined exposure, the probability of pericardial edema increased with increasing dose, with probabilities of 3.3%, 16.7%, and 55% at the three concentration gradients, respectively. Combined exposure to FLU and TBU resulted in significant cardiac malformations in zebrafish juveniles.

[0048] 2.2.3 Pericardial area like Figure 6 As shown, compared with the control group, the pericardial area of ​​zebrafish juveniles in the single exposure treatment group did not show significant changes. However, the pericardial area of ​​the high-concentration combined exposure treatment group was significantly increased (5 + 1.25, 10 + 2.5), and the pericardial area of ​​the 5 + 1.25 mg / L treatment group was equivalent to three times that of the control group (0.07 mm). 2 The 10 + 2.5 mg / L treatment group was equivalent to four times that of the control group (0.1 mm). 2 Therefore, it can be concluded that the combined exposure treatment group was more toxic to zebrafish hearts, causing malformations, compared to single exposure to the two drugs.

[0049] 2.2.4 Expression levels of heart-related genes Zebrafish embryos were treated with fluopyram and tebuconazole for 96 h, and the embryos were collected. Total mRNA was extracted, and the expression of heart-related genes was detected using Q-RT-PCR (primer sequences are shown in Table 1). These included tbx5a (Gene ID: 30071), myh6 (Gene ID: 386711), nkx2.5 (Gene ID: 30696), vmhc (Gene ID: 30616), nppa (Gene ID: 321442), and gata4 (Gene ID: 30483). Figures 8A-8F As shown, compared with the control group, the mRNA levels of nkx2.5, vmhc, myh6, and tbx5a in the single-treatment groups generally showed a decreasing trend. Specifically, the vmhc mRNA expression level in the FLU-treated group initially increased and then decreased compared to the control group at low concentrations (1 mg / L), and the combined exposure treatment group showed a significant decrease compared to the single-treatment groups. The nppa and tbx5a mRNA expression levels in the TBU-treated group were increased compared to the control group. Overall, the combined exposure treatment groups showed a decreasing trend.

[0050] 2.3 Effects of single and combined exposure to fluopyram and tebuconazole on the locomotor behavior of zebrafish juveniles 2.3.1 Zebrafish Behavioral Trajectory The analytical parameters for zebrafish behavioral trajectories include the distance traveled, duration of movement, and speed over a 40-minute period. For example... Figure 9 As shown in Figures A and B, compared to the control group, the fluopyram-treated group showed increased movement distance, duration, and speed at low concentrations (1 mg / L), indicating that low concentrations of fluopyram enhance the neural activity of juvenile zebrafish, while high concentrations inhibit it. Unlike fluopyram, tebuconazole inhibited the neural activity of juvenile zebrafish at low concentrations (0.25 mg / L), and the inhibitory effect increased with increasing dose. The combined exposure to both drugs resulted in more severe inhibition of neural activity compared to single exposure.

[0051] 2.3.2 Vertical length of the swim bladder The zebrafish swim bladder, as an important functional organ, is highly susceptible to interference from environmental pollutants during its development and inflation process, such as... Figure 7 As shown, the zebrafish swim bladder can serve as an important marker organ for determining whether a zebrafish has been poisoned. The degree of inflation of the zebrafish swim bladder can be expressed by its vertical length. Figure 10 As shown, compared with the control group, the swim bladder length of the single FLU and TBU treatment groups was not significantly different at low doses (0.2032 mm), while the swim bladder length was significantly reduced in the FLU 20 mg / L treatment group (0.0852 mm) and the TBU 5 mg / L treatment group (0.0974 mm). Compared with the FLU 20 mg / L and TBU 5 mg / L treatment groups, the vertical length of the swim bladder in the combined exposure treatment group was shorter (0.0542 mg / L). Therefore, it can be concluded that single exposure to FLU and TBU at high concentrations (FLU 20 mg / L, TBU 5 mg / L) has a significant effect on zebrafish swim bladders, and the combined exposure treatment group has a greater effect on zebrafish swim bladders than single exposure.

[0052] 2.3.3 Swim bladder-related genes To investigate the effects of fluopyram and tebuconazole on zebrafish swim bladder development and aeration, zebrafish embryos were treated with fluopyram and tebuconazole for 96 h, and then collected. Total mRNA was extracted, and the expression of swim bladder development-related genes was detected using Q-RT-PCR (primer sequences are shown in Table 1). These included pbx1a (Gene ID: 58138), foxa3 (Gene ID: 30559), and hprt11 (Gene ID: 406259). Figures 11A-11CAs shown, the expression levels of all three genes decreased with increasing exposure concentration in the treatment groups. The highest gene expression levels in the combined treatment group were all lower than those in the single treatment group, indicating that the combined exposure to FLU and TBU had a greater toxic effect on zebrafish swim bladder development and aeration than the single exposure alone.

[0053] Table 1 Primer sequences involved in this invention (SEQ ID NO:1-16)

[0054] 3. Discussion This invention is the first to discover that, according to OECD standards, the median lethal concentration (LD50) of fluopyram for zebrafish embryos is 30.78 mg / L, classifying it as low toxicity. The LD50 of tebuconazole is 7.211 mg / L, classifying it as moderately toxic. In terms of mortality, FLU had mortality rates of 18% and 57% at exposure concentrations of 20 mg / L and 40 mg / L, respectively, while TBU had mortality rates of 6% and 92% at exposure concentrations of 5 mg / L and 10 mg / L, respectively. When the two pesticides were combined according to the principle of equal toxicity, the combined exposure of FLU and TBU at concentrations of 10 + 2.5 mg / L and 20 + 10 mg / L resulted in mortality rates of 20% and 100%, respectively, higher than single exposure. Therefore, it is concluded that the combined toxicity of FLU and TBU is higher than that of single exposure. This conclusion is corroborated by zebrafish developmental phenotypic data, including hatching rate, body length, and malformation rate. The additive index (AI) also indicates a synergistic effect between FLU and TBU.

[0055] The zebrafish heart is the first organ to develop and function, starting to beat at 22 hours and forming its basic cardiovascular structure at 48 hours, exhibiting a series of complex ion channels and metabolic processes. Changes in heart rate are an important indicator for evaluating cardiotoxicity. This experiment found that high-concentration exposure to fluopyram was detrimental to embryonic cardiac function, significantly inhibiting the heart rate of juvenile zebrafish. The inhibition was even more severe under combined high-concentration exposure. The probability and area of ​​pericardial edema also demonstrated that combined exposure to FLU and TBU had a more significant toxic effect on the zebrafish heart than single exposure, indicating a synergistic effect between the two pesticides in terms of toxicity.

[0056] nkx2.5 is a gene related to heart development, playing a crucial role in maintaining a normal heart rate during the early development of the conduction system in zebrafish, and is involved in heart formation, circumduction, and maturation. Gene-level data show that this gene expression is severely suppressed, resulting in a slower heart rate, consistent with experimental results. The myh6 gene is an important gene involved in zebrafish heart development. Literature indicates that the myh6 gene controls the normal contraction and relaxation of zebrafish cardiomyocytes; abnormal expression of this gene leads to abnormal development of the zebrafish heart and ventricles. This invention found that at the highest combined exposure to FLU and TBU (10⁻² 2.5 mg / L), myh6 gene expression was severely suppressed, leading to pericardial edema and increased pericardial area, consistent with phenotypic results. The vmhc, nppa, and gata4 genes are also involved in zebrafish heart growth and development. Gene expression level data show that abnormal expression of these genes leads to abnormal heart rate and pericardial edema in zebrafish.

[0057] The swim bladder is an air-filled sac-like structure crucial for the survival and growth of fish. The development and inflation of the swim bladder are highly sensitive to environmental pollutants, easily leading to developmental abnormalities. Genes pbx1a, hprt1l, and foxa3 are essential for the development of the mesocortical layer of the swim bladder. pbx1 is expressed in the central nervous system, pharyngeal arch, and during individual development of the swim bladder, affecting its normal expansion. Experimental data on gene expression levels show that the expression of these genes is significantly suppressed, impacting the development of the swim bladder structure. The early swim bladder development in zebrafish mainly consists of three stages: the budding stage (36–65 hpf), the elongation stage (65–96 hpf), and the inflation stage (96–120 hpf). Normal expression of the genes pbx11a, hprt1l, and foxa3 is essential for swim bladder development, regulating the development of the mesocortex. Studies have shown that swim bladder development affects zebrafish swimming behavior and neural control; swim bladder inflation is closely related to buoyancy control and swimming depth in zebrafish. This invention found that zebrafish with abnormal swim bladder development swam significantly shorter and slower distances and speeds within a specified time compared to zebrafish with normal swim bladder development. Therefore, FLU and TBU affect normal zebrafish swim bladder development, thereby influencing zebrafish locomotion, and the combined use of the two pesticides produces a more severe toxic effect on the swim bladder than either pesticide alone.

[0058] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for evaluating the developmental toxicity of fluopyram, tebuconazole, and their combined use to zebrafish, characterized in that... Includes the following steps: (1) Wild-type AB zebrafish were used in the experiment. Male and female zebrafish were kept in circulating water at a temperature of 28 ± 1 ℃ with a light-dark cycle of 14 h:10 h. On the night before breeding, male and female zebrafish were placed on both sides of the spawning box in a 1:1 ratio. The next day, after the photoperiod started, the male and female zebrafish on both sides of the spawning box were allowed to mate and the fertilized eggs produced by the female fish were collected. (2) In order to evaluate the effects of single pesticides fluopyram and tebuconazole on the early embryonic development of zebrafish, fluopyram and tebuconazole powders were dissolved in DMSO to prepare stock solutions of a certain concentration and stored in the dark. (3) Based on the preliminary experimental results, the concentration gradient was set up as follows: the mother liquor was diluted with circulating water to the following exposure concentrations: fluopyram at 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L, and tebuconazole at 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, and 12 mg / L, respectively, with DMSO concentration less than 0.05%; then, according to the OECD guidelines, after 2 hpf, the single toxicity of fluopyram and tebuconazole to zebrafish at different exposure concentrations was investigated; specifically, normally developing fertilized eggs were selected and placed in a six-well plate, with 20 embryos randomly placed in each well, and 3 replicates were set for each exposure concentration. 0.05% DMSO was added to the blank control group; the exposure solution was changed every 24 h during the entire exposure period; finally, the LC at 96 hpf was calculated using GraphPad Prism software to generate a fitting curve. 50 value; (4) Combined toxicity AI index: The LC of single exposure to fluopyram and tebuconazole was determined using the Marking additive index method. 50 Values ​​and LC of combined exposure of the two 50 Substituting the concentrations of fluopyram and tebuconazole into equation (I): M = CA / LC 50-A +CB / LC 50-B Formula (I) Among them, LC 50-A LC-100 of fluopyram 50 Value, LC 50-B LC50 of tebuconazole 50 Value, CA is the LC of exposure to both. 50 The value represents the concentration of fluopyram, and CB represents the LC50 of exposure to the combination of the two. 50 The concentration of tebuconazole in the value; Then convert M into an additive exponent AI. When M = 1, AI = 0; when M > 1, AI = 1 - M; when M < 1, AI = 1 / M - 1. Determine the mode of action based on the AI ​​value. If AI = 0, it is a simple additive action; if AI > 0, it is a synergistic action; if AI < 0, it is an antagonistic action.

2. The method according to claim 1, characterized in that, In step (4), the concentrations of fluopyram and tebuconazole under combined exposure were 0.5 mg / L and 0.125 mg / L, 5 mg / L and 1.25 mg / L, and 2.5 mg / L and 10 mg / L, respectively.

3. The method according to claim 1 or 2, characterized in that, Hatching rate and mortality rate were recorded every 24 hours until 96 hpf; in accordance with OECD guidelines, the probability of pericardial edema in zebrafish juveniles was manually assessed at 96 hpf.

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