Prochloraz pesticide ecotoxicity detection method based on pomacea canaliculata and application of prochloraz pesticide ecotoxicity detection method
By using ecotoxicological testing methods for golden apple snails, the toxic effects of imazalil on golden apple snails were comprehensively assessed, solving the problem of incomplete detection in existing technologies and realizing multi-dimensional risk assessment of aquatic ecosystems.
Patent Information
- Application Number
- CN202511872470.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-12
- Publication Date
- 2026-03-17
AI Technical Summary
Existing technologies lack systematic ecotoxicity testing methods for golden apple snails in aquatic ecosystems, especially for the ecotoxicity testing of imazalil. This results in inaccurate risk assessments and makes it difficult to fully reflect the sublethal effects, behavioral toxicity, and long-term chronic harm of pollutants.
An ecotoxicological assay for prochloraz based on golden apple snails was employed, including experimental biological preparation, acute toxicity testing, chronic toxicity testing, and histopathological examination. The toxic effects of prochloraz were assessed by observing the survival rate, escape behavior, shell height and weight changes, and histopathological changes of golden apple snails.
It provides a multi-dimensional and systematic ecotoxicological detection method that can accurately reflect the acute and chronic toxic effects of imazalil on golden apple snails, reveal the sensitivity differences and organ damage at different developmental stages, and improve the accuracy of environmental risk assessment.
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Figure CN121679006A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of environmental toxicology and ecological risk assessment, and particularly relates to a prochloraz pesticide ecological toxicity detection method based on golden apple snails and application thereof. BACKGROUND
[0002] Prochloraz (PCZ) is a broad-spectrum imidazole fungicide, which is widely used in agricultural production to prevent and control various crop diseases by inhibiting the activity of fungal cytochrome P450 14alpha-demethylase. However, it can enter surface water bodies through farmland runoff, drift and other ways, and form residues in aquatic ecosystems. Existing toxicology research mainly focuses on vertebrates and a few invertebrate models, and it has been confirmed that PCZ has multiple toxic effects such as endocrine disruption, oxidative stress induction, metabolic disorder and tissue damage. However, systematic toxicological research on aquatic mollusks, especially freshwater snails, which play an important role in water body ecosystems and are sensitive to environmental changes, is still quite lacking.
[0003] Mollusks are often used as indicator organisms for water environment monitoring. The golden apple snail is a global invasive species widely distributed in water bodies such as rice fields in southern China. It has strong reproductive capacity, clear life history, is easy to cultivate in the laboratory, and is sensitive to exogenous pollutants. However, there is currently no mature method to develop the golden apple snail as a systematic ecological toxicity detection model for imidazole fungicides (especially prochloraz). Traditional ecological risk assessment relies mainly on a single acute toxicity endpoint, which cannot fully reflect the sublethal effects, behavioral toxicity and long-term chronic hazards of pollutants, and it is also difficult to reveal the sensitivity differences of different developmental stages of organisms, resulting in inaccurate risk assessment.
[0004] Therefore, it is necessary to establish a pesticide ecological toxicity detection method that can systematically integrate multiple developmental stages and multiple biological endpoints using easily available and sensitive indicator organisms. SUMMARY
[0005] To solve the above technical problems, the first object of the present application is to overcome the shortcomings of the prior art and provide a prochloraz pesticide ecological toxicity detection method based on golden apple snails. The second object of the present application is to provide the application of golden apple snails in assessing the ecological toxicity of prochloraz pesticides.
[0006] To achieve the first object of the present application, the following technical solutions are adopted: A prochloraz pesticide ecological toxicity detection method based on golden apple snails, comprising the following steps: S1, experimental organism preparation: collecting or domesticating golden apple snails and distinguishing newly hatched individuals and adult individuals as test organisms; S2, acute toxicity test: newly hatched and adult individuals of P. canaliculata were exposed to a series of different concentrations of propanil in water, with a control group, under standard static or semi-static conditions for 96 hours of exposure, during which the number of surviving individuals, inhibited individuals and individuals exhibiting escape behavior in each group were observed and recorded regularly; S3, chronic toxicity test: adult individuals of P. canaliculata were exposed to a series of sublethal concentrations of propanil in water, with a control group, for 21 days of semi-static exposure, during which the shell height and body weight of the snails were measured and recorded regularly; S4, histopathological test: after the end of the chronic toxicity test, the liver, kidney and stomach tissues of the exposed P. canaliculata in S3 were taken, fixed, dehydrated, embedded, sectioned, HE stained, and then observed and analyzed histopathologically; S5, data processing and ecological toxicity evaluation: based on the data of S2, the 96-hour semi-lethal concentration and semi-inhibitory concentration of propanil on newly hatched and adult P. canaliculata were calculated, based on the data of S3, the effect of propanil on the growth rate of P. canaliculata was analyzed, and based on the results of S4, the degree of tissue damage of propanil to the main organs was evaluated.
[0007] In the present application, the collection or domestication of P. canaliculata is carried out under standard laboratory conditions, so that the physiological state of P. canaliculata can be kept stable.
[0008] Preferably, in S1, the newly hatched individuals are 5-7 days old after hatching, with a shell length of 2-3 mm; the adult individuals are 20±5 mm or 30±5 mm in shell height. In practice, the division of newly hatched individuals and adult individuals according to the above standards is based on morphological differences and the differences in physiological and metabolic levels of each P. canaliculata individual. The newly hatched individuals have high metabolism, immature detoxification system, large surface area / volume ratio and high sensitivity to exogenous pollutants, and are the key stage for evaluating the effects of pollutants on the early survival and replenishment capacity of the population. The adult individuals have stable physiology and relatively strong tolerance, and are suitable for chronic effect and mechanism research. Before actual testing, the newly hatched individuals and adult individuals need to be fasted for 24 hours, so as to eliminate the interference of feeding activity on metabolism and pollutant absorption, ensure that all individuals are in a standardized initial state, and improve the comparability, repeatability and accuracy of test data.
[0009] Preferably, in S2, for acute toxicity test of newly hatched individuals of golden apple snails, the exposure concentration gradient of propanil includes 0.25 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L and 3.0 mg / L, because newly hatched individuals have the characteristics of high sensitivity, therefore, the exposure concentration of propanil with the above lower concentration gradient can accurately capture the low-dose response of newly hatched individuals; for acute toxicity test of adult individuals of golden apple snails, the exposure concentration gradient of propanil includes 1.6 mg / L, 3.125 mg / L, 6.25 mg / L, 12.5 mg / L and 25.0 mg / L, because adult individuals have the characteristics of relatively high tolerance, therefore, the exposure concentration of propanil with the above higher concentration gradient can cover the toxicity effect range of adult individuals.
[0010] In S2 of the present application, the semi-lethal concentration LC 50 is calculated according to the number of surviving individuals, and the semi-inhibitory concentration IC 50 is calculated according to the number of inhibited individuals, and the semi-inhibitory concentration can reflect the potential impact of pollutants on population health more than the single mortality index.
[0011] Preferably, in S2, the judgment standard of the inhibited individuals is that the shell retraction reaction is slow, does not adhere to the wall, activity is significantly weakened or is stationary for a long time after mechanical stimulation; and the judgment standard of the escape behavior is that the individual actively climbs to the container wall above the liquid surface and lasts for more than 3 seconds.
[0012] Preferably, in S2, the escape behavior is dynamically recorded at 0.5 hours, 1 hour, 2 hours, 3 hours and 4 hours in the early exposure period, and a concentration-time escape rate heat map is drawn. High-frequency observation of the escape behavior of the above test golden apple snail individuals during 0.5-4h in the early exposure period can sensitively reflect the stress and avoidance reaction caused by pollutants, which is an early warning signal of neurotoxicity or environmental stress, and through the concentration-time escape rate heat map, the behavior effect threshold and dynamic change law of the toxicant can be directly revealed.
[0013] In S3 of the present application, sub-lethal effects and long-term effects are analyzed.
[0014] Preferably, in S3, the sub-lethal concentration gradient of propanil includes 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L and 1.6 mg / L, and specifically, adult golden apple snails are selected for semi-static exposure for 21 days, and the exposure concentration is set to be sub-lethal level far below the acute lethal concentration, which can analyze the growth rate of shell height and body weight of adult golden apple snails after exposure.
[0015] Preferably, in the S3, the shell height is measured by using an electronic vernier caliper to measure the maximum shell diameter, the body weight is measured by using an analytical balance to weigh the live body weight, and the shell height growth rate and the body weight growth rate are calculated, wherein the shell height is measured by using an electronic vernier caliper with an accuracy of 0.02 mm to measure the maximum shell diameter; the body weight is measured by using an analytical balance with an accuracy of 0.1 mg to weigh the live body weight; and the shell height growth rate and the body weight growth rate are calculated according to the measurement results of the shell height and the body weight.
[0016] In the S4 of the present application, the target points and mechanisms of toxic effects are revealed from the microstructure and cell level, and in the present application, the hepatopancreas, the kidney and the stomach organs are selected for in-depth analysis, wherein the hepatopancreas is the main metabolic, detoxification and energy storage organ of mollusks, and is the primary target of environmental toxicants; the kidney is responsible for excretion and osmoregulation; and the stomach is responsible for feeding and primary digestion.
[0017] Preferably, in the S4, the histopathological observation and analysis specifically include: Hepatopancreas tissue: whether the acinus structure is loose, whether the epithelial cells are vacuolar degeneration or exfoliation, whether the lacuna is dilated, whether there are basophilic cell aggregation, lipofuscin-like granule deposition and necrotic gland tube; Kidney tissue: whether the kidney crypt epithelium is swollen, vacuolated or exfoliated, whether the kidney crypt lumen is dilated, and whether the number and lumen diameter of the intertubular sinus are increased; Stomach tissue: whether the mucosa layer is thin, swollen or exfoliated, whether the submucosa layer is edematous or has a fissure, and whether the muscle layer is disorganized or broken.
[0018] In the S5 of the present application, comprehensive analysis is performed on all the aforementioned hierarchical data, and by calculating the ratio of the LC 50 / IC 50 of the newly hatched period, the qualitative understanding of the more sensitive juvenile is converted into a quantifiable risk coefficient, and at the same time, the acute behavioral response mode, the chronic growth inhibition curve and the histopathological damage score are comprehensively analyzed, so as to establish a diversified and stereoscopic ecological toxicity "portrait" of acute toxicity intensity, behavioral interference potential, chronic hazard threshold and organ damage characteristics, which can comprehensively reflect the potential risk of profoxydim to the aquatic ecological system.
[0019] To achieve the second object of the present application, the following technical scheme is adopted: An application of an apple snail in evaluating the ecological toxicity of propanil pesticide, the application comprising: using the propanil pesticide ecological toxicity detection method based on the apple snail as described above, exposing the newly hatched stage and adult stage apple snails to the water containing the propanil respectively, evaluating the acute toxicity of the propanil by determining at least one of the acute lethal effect, the acute physiological inhibition effect or the acute escape behavior response of the propanil on the newly hatched stage and adult stage apple snails; and / or, evaluating the chronic toxicity of the propanil by determining at least one of the chronic growth inhibition effect or the histopathological damage effect on the liver and pancreas, kidney, stomach of the adult stage apple snails.
[0020] Preferably, the application further comprises: based on the acute toxicity determination results, obtaining the 96-hour half lethal concentration and half inhibition concentration of the propanil on the newly hatched stage apple snails and the 96-hour half lethal concentration and half inhibition concentration of the propanil on the adult stage apple snails, and calculating the half lethal concentration ratio and the half inhibition concentration ratio of the newly hatched stage and the adult stage, for quantitatively evaluating the development stage-specific toxicity risk of the propanil.
[0021] According to the above technical solution, the application of the present application can be indicated, which is not only to use the apple snail as a test organism, but also to use it as a reproducible and reproducible toxicity test organism system, and the application value can be embodied in the following aspects: Firstly, the application of the apple snail, especially the newly hatched stage individual, can establish a high-sensitivity acute toxicity benchmark line for propanil, and the data can be used for cross comparison with the toxicity data of other pesticides or other test organisms to evaluate and analyze the relative toxicity of propanil.
[0022] Secondly, the application of the adult apple snail in chronic exposure can effectively reveal the harm that cannot be directly exposed, such as growth retardation, which can be caused by propanil at environmentally relevant concentrations, and this growth inhibition effect can predict the decline of population biomass and the weakening of competitiveness.
[0023] Furthermore, by using the propanil pesticide ecological toxicity detection method to expose the apple snail, histopathological analysis of target organs such as liver and pancreas can be performed, and some toxicities such as death, growth inhibition and cell damage can be associated with each other, for example, by observing extensive vacuolization of the liver and pancreas, it can be analyzed that propanil may interfere with lipid metabolism or cause organelle dysfunction.
[0024] The beneficial effects of the present application: The propanil pesticide ecological toxicity detection method of the present application is derived from the easily accessible apple snail, which is low in cost and easy to operate; the newly hatched stage apple snail individual is extremely sensitive to propanil, which is suitable for low concentration detection; The ecotoxicity detection method for prochloraz pesticide of the present invention is comprehensive. On the one hand, it can conduct acute toxicity tests, and on the other hand, it can also assess behavioral responses, long-term growth inhibition and tissue damage. By testing newly hatched golden apple snails and adult golden apple snails, the sensitivity differences at different life stages can be directly compared, and the risk of pesticide to individual populations can be analyzed.
[0025] The ecotoxicity detection method for prochloraz pesticide of the present invention is applied to assess the ecotoxicity of prochloraz pesticide, and can present the toxic effects of pesticide from multiple dimensions, providing practical and systematic experimental basis for scientifically assessing the environmental risks of prochloraz. Attached Figure Description
[0026] Figure 1 A series of charts showing the effect of imazalil exposure on the survival rate of newly hatched golden apple snails, among which... Figure 1 (A) is a graph showing the relationship between the average survival rate of newly hatched golden apple snails and the exposure concentration; Figure 1 (B) is a dose-response nonlinear regression plot based on 96h data; Figure 1 (C) shows the fitting results for different exposure times; Figure 1 (D) is the Kaplan–Meier survival curve; Figure 1 (E) is a graph showing the relationship between the survival rate of newly hatched golden apple snails and the exposure concentration under different exposure times; Figure 2 A series of charts showing the effect of imazalil exposure on the inhibition rate of newly hatched golden apple snails, among which, Figure 2 (A) is a graph showing the relationship between the average inhibition rate and exposure concentration of newly hatched golden apple snails; Figure 2 (B) is a dose-response nonlinear regression plot based on 96h data; Figure 2 (C) shows the fitting results for different exposure times; Figure 2 (D) is a comparison chart of the results between the control group and the treatment group; Figure 2 (E) is a heatmap of exposure concentration-time; Figure 3 A series of charts illustrating the escape behavior of newly hatched golden apple snails induced by imazalil exposure. Figure 3 (A) is a graph showing the relationship between the number of newly hatched golden apple snails escaping and the exposure concentration at different times; Figure 3 (B) is a heatmap of exposure concentration-time; Figure 3 (C) is a photograph of the six-well plates of the control and treatment groups at the 96-hour endpoint; Figure 4 A series of charts showing the effect of imazalil exposure on the survival rate of adult golden apple snails, among which... Figure 4 (A) is a graph showing the relationship between the average survival rate of adult golden apple snails and exposure concentration; Figure 4 (B) is a dose-response nonlinear regression plot based on 96h data;Figure 4 (C) shows the fitting results for different exposure times; Figure 4 (D) is the Kaplan–Meier survival curve; Figure 4 (E) is a graph showing the relationship between the survival rate of adult golden apple snails and the exposure concentration at different exposure times; Figure 5 A series of charts showing the effect of imazalil exposure on the inhibition rate of adult golden apple snails. Figure 5 (A) is a graph showing the relationship between the average inhibition rate and exposure concentration of adult golden apple snails; Figure 5 (B) shows the fitting results for different exposure times; Figure 5 (C) is a dose-response nonlinear regression plot based on 96h data; Figure 5 (D) is a comparison chart of the results between the control group and the treatment group; Figure 5 (E) is a heatmap of exposure concentration-time; Figure 6 A series of charts showing the effects of imazalil exposure on escape behavior and chronic growth in adult golden apple snails, including... Figure 6 (A) is a graph showing the relationship between the number of adult golden apple snails that escaped and the exposure concentration and time; Figure 6 (B) is a heatmap of exposure concentration-time; Figure 6 (C) is a graph showing the relationship between the shell height growth rate of adult golden apple snails and exposure concentration and time; Figure 6 (D) is a graph showing the relationship between the weight gain rate of adult golden apple snails and exposure concentration and time; Figure 7 This image shows the macroscopic morphological changes in the soft tissues of adult golden apple snails after 21 days of chronic exposure to different concentrations of imazalil. Figure 7 (A) is a diagram showing the morphological changes of adult golden apple snails in the control group after 21 days; Figure 7 (B)- Figure 7 (F) shows the morphological changes of adult golden apple snails after 21 days of exposure to prochloraz at concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg / L, respectively; Figure 8 Microscopic images showing the pathological changes in the hepatopancreatic tissue of *Pomacea canaliculata* after 21 days of chronic exposure to different concentrations of imazalil. Figure 8 From top to bottom, the middle section consists of: Figure 8 (A)- Figure 8 (F), where, Figure 8 (A) is a pathological change diagram of the hepatopancreas tissue of the control group after 21 days; Figure 8 (B)- Figure 8 (F) shows the pathological changes in the hepatopancreas tissue of *Pomacea canaliculata* after 21 days of exposure to prochloraz at concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg / L, respectively; Figure 9Microscopic images of pathological changes in the kidney tissue of *Pomacea canaliculata* after 21 days of chronic exposure to different concentrations of prochloraz. Figure 9 From top to bottom, the middle section consists of: Figure 9 (A)- Figure 9 (F), where, Figure 9 (A) is a pathological change diagram of the kidney tissue of the control group of golden apple snails after 21 days; Figure 9 (B)- Figure 9 (F) shows the pathological changes in the kidney tissue of *Pomacea canaliculata* after 21 days of exposure to prochloraz at concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg / L, respectively; Figure 10 Microscopic images showing the pathological changes in the gastric tissue of *Pomacea canaliculata* after 21 days of chronic exposure to different concentrations of imazalil. Figure 10 From top to bottom, the middle section consists of: Figure 10 (A)- Figure 10 (F), where, Figure 10 (A) is a pathological change diagram of the stomach tissue of the control group of golden apple snails after 21 days; Figure 10 (B)- Figure 10 (F) shows the pathological changes in the gastric tissue of *Pomacea canaliculata* after 21 days of exposure to prochloraz at concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg / L. Detailed Implementation
[0027] The invention can be further understood through the specific embodiments given below, but they are not intended to limit the invention.
[0028] Example 1: Collection, identification, and standardized laboratory domestication of the golden apple snail. The golden apple snails in this embodiment were collected from rice paddies in Hangzhou, Zhejiang Province.
[0029] Species identification was performed based on the groove characteristics, aperture morphology, and number of spiral layers of the snail's shell. In the laboratory, *Pomacea canaliculata* snails were placed in oxygenated, dechlorinated tap water and domesticated for two weeks under conditions of 25±1℃, a photoperiod of 12L:12D, pH 7.0±0.2, and dissolved oxygen >6mg / L. They were fed washed cabbage leaves daily, and the water was changed and cleaned regularly. Egg masses were collected and incubated under controlled temperature and humidity conditions to obtain newly hatched juvenile snails with a shell length of 2-3mm, 5-7 days after hatching. Healthy individuals with a shell height of 25±5mm were selected as adult experimental snails. They were fasted for 24 hours before the experiment.
[0030] Example 2 Acute toxicity test of newly hatched golden apple snails Accurately weigh imazalil standard and prepare exposure solutions with ultrapure water at concentrations of 0 (control group), 0.25 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, and 3.0 mg / L. Add 5 ml of the corresponding concentration of exposure solution to each well of a six-well plate, and randomly place 10 newly hatched snails in each well. Each concentration has three replicate groups. Incubate at 25°C. Observe and record the number of escaped individuals at 0.5, 1, 2, 3, and 4 hours after exposure. Escape behavior: newly hatched snails climb to the well wall above the liquid surface and remain there for more than 3 seconds. Observe and record the number of dead and inhibited individuals at 24, 48, 72, and 96 hours after exposure. The criteria for death are: newly hatched snails showing no shell retraction, whitening of body color, and no heartbeat; the criteria for inhibition are: newly hatched snails showing sluggish response to stimulation, not adhering to the well wall, and weak activity.
[0031] The detection results of this embodiment 2 are as follows: Figures 1-3 As shown, the 96h-LC was calculated using statistical software. 50 and 96h-IC 50 .
[0032] Example 3 Acute toxicity test of adult golden apple snail Imazalil exposure solutions with concentrations of 0 (control group), 1.6 mg / L, 3.125 mg / L, 6.25 mg / L, 12.5 mg / L, and 25.0 mg / L were prepared. Glass containers with caps and vents were used. 600 mL of imazalil exposure solution was added to each container, and 5 adult snails were placed inside. Three parallel groups were set up for each concentration. Environmental conditions were the same as in Example 2. No feeding was performed during the exposure period, and escape behavior was observed as in Example 2. Mortality and inhibition were recorded at 24, 48, 72, and 96 hours. Mortality criteria: no response to stimulation in adult golden apple snails, pale body color, relaxed legs, and floating; inhibition criteria: sluggish response and reduced activity in adult golden apple snails.
[0033] The detection results of Example 3 are as follows: Figures 4-6 As shown in (A) and (B), calculate 96h-LC. 50 and 96h-IC 50 .
[0034] Example 4: Detection of chronic toxicity and growth inhibition in adult golden apple snails Imazalil exposure solutions with concentrations of 0 (control group), 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, and 1.6 mg / L were prepared. Semi-static exposure was used, with the imazalil exposure solution completely replaced every 3 days. Individual containers were used for each snail's exposure, with 200 mL of solution per container. Five replicates were established for each concentration. During the exposure period, snails were fed a small amount of vegetable leaves daily, and the food was promptly removed. On days 0, 3, 6, 9, 12, 15, 18, and 21, the shell height of each snail was measured using electronic calipers, and the body weight of each snail after drying was measured using an analytical balance. The growth rates of shell height and body weight at each time point were calculated.
[0035] Experimental results are as follows Figure 6 (C) Figure 6 As shown in (D), the results show that golden apple snails exhibit concentration-dependent growth inhibition in response to prochloraz-exposed solutions.
[0036] Example 5: Preparation and Observation of Histopathological Samples After 21 days of chronic exposure, the golden apple snails were anesthetized in ice water and then frozen at -20°C. The liver, pancreas, kidneys, and stomach tissues were dissected and removed. They were immediately fixed in 4% neutral buffered formaldehyde for more than 24 hours. Subsequently, they were dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. 4μm thick sections were cut using a paraffin microtome, spread, baked, dewaxed with xylene, rehydrated with graded ethanol, stained with hematoxylin and eosin (H&E), dehydrated, cleared, and mounted with neutral resin. Full-field digital images were obtained using a digital slide scanner and observed under 20× and 40× objectives. The liver and pancreas were examined primarily for acinar structure, epithelial cell vacuolation, and inflammatory infiltration. The results are as follows: Figure 8 As shown; The kidneys were closely observed for morphology of the renal tubular epithelium, luminal dilation, and interstitial changes. The results are as follows: Figure 9 As shown; The integrity of the gastric mucosa, submucosa, and muscularis propria was observed, and the results were as follows: Figure 10 As shown.
[0037] Depend on Figures 8-10 The results showed that the degree of damage to various organs increased progressively from low to high concentrations.
[0038] Example 6: Data Processing and Comprehensive Ecotoxicity Assessment Acute toxicity assessment: Based on data from Examples 2 and 3, the 96-h-LC50 of newly hatched golden apple snails was calculated. 50 =0.51mg / L, 96h-IC 50 =0.46 mg / L; Adult golden apple snail 96h-LC 50 =2.62 mg / L, 96 h-IC 50=1.84 mg / L. Calculate the toxicity susceptibility coefficient at the developmental stage (LC50 for the new hatching period). 50 / Adult LC 50 The value was approximately 0.195, indicating that the juveniles were about 5 times more sensitive to imazalil than the adults, suggesting a high risk to the juvenile stage of the population.
[0039] Behavioral toxicity assessment: Analysis of escape heatmaps: Figure 3 (B) Figure 6 (B) It was found that significant escape behavior could be induced even at low concentrations, and the response changed over time, proving that imazalil has rapid neurotoxicity or stress toxicity.
[0040] Chronic toxicity assessment: Two-way ANOVA of the growth data of Example 4 showed that concentration and time had extremely significant effects on shell height and weight gain rate (p<0.001); the high concentration group (≥0.8 mg / L) showed almost stagnant growth, indicating a significant chronic inhibitory effect.
[0041] Histopathological damage assessment: Based on the observations in Example 5, the damage to the liver, pancreas, kidneys, and stomach was analyzed and assessed. It was found that the degree of damage to the liver, pancreas, kidneys, and stomach was significantly positively correlated with the exposure concentration, confirming concentration-dependent toxicity at the microscopic level.
[0042] Comprehensive Risk Assessment: By analyzing and integrating the results from the above four levels, the following conclusions are drawn: Imazalil exhibits clear developmental stage-specific toxicity to golden apple snails (extremely sensitive during the newly hatched stage), and also has multiple toxic effects, including acute lethality, sublethal physiological inhibition, rapid behavioral disturbance, chronic growth inhibition, and damage to multiple organs and tissues. Even long-term exposure at concentrations below the acute lethal level may have a profound impact on the health of golden apple snail populations by inhibiting growth and damaging key organs.
[0043] Example 7: Specific application of Pomacea canaliculata in assessing the ecotoxicity of prochloraz. Application scenario: Water near the drainage outlet of a paddy field in a certain area is suspected of being contaminated by pesticides, and it is necessary to assess the potential ecological risks of imazalil.
[0044] Application steps: Y1. Golden apple snails collected from the area or domesticated using laboratory standards.
[0045] Y2. Acute Toxicity Screening: Using newly hatched juvenile snails, a 96-hour exposure test was conducted on the suspected water body (or after concentration / dilution), following the method in Example 2. If a significant increase in mortality, inhibition rate, or enhanced escape behavior was observed, and this was correlated with the concentration gradient, the pollutant (including but not limited to imazalil) in the water body was preliminarily determined to have acute ecotoxicity. To further confirm whether it is imazalil, a standard sample can be used for comparative verification.
[0046] Y3. Toxicity Characterization and Risk Quantification: If a detailed risk assessment of pure imazalil is required, the methods described in Examples 1-6 should be fully implemented. The LC values of newly hatched and adult snails should be obtained through parallel testing. 50 / IC 50 .
[0047] The specific application is as follows: Based on the results of acute toxicity tests, the 96-hour median lethal concentration (LD50) and median inhibitory concentration (LC50) of imazalil for newly hatched golden apple snails, as well as for adult golden apple snails, are obtained. The ratio of L50 to L50 and the ratio of L50 to L50 for newly hatched snails to adults are calculated to quantitatively assess the developmental stage-specific toxicity risk of imazalil.
[0048] Calculations show that the LC of both is... 50 The ratio is 0.51 / 2.62≈0.19, which is much less than 1. This ratio can quantitatively indicate that imazalil poses a significantly greater threat to early life stages than to adults. This data can be used as a key coefficient to assess its risk to the reproduction of aquatic invertebrate populations.
[0049] Chronic and mechanistic level assessment: Referring to Example 4, a 21-day chronic exposure experiment was conducted to assess the long-term effects on adult growth at sublethal concentrations; referring to Example 5, histopathological examination was used to identify target organs of toxicity, such as the liver, pancreas, and kidneys, to provide a basis for understanding the toxicological mechanism and developing targeted protective measures.
[0050] As can be seen from the above embodiments, the detection method provided by the present invention is highly systematic, has a clear endpoint, and provides abundant data. The application of golden apple snails in this method system can be used efficiently and in multiple dimensions for the ecological risk assessment of imazalil.
[0051] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.
Claims
1. A method for detecting the ecological toxicity of the pesticide pyrithiobac-acid based on the apple snail, characterized in that, The method comprises the following steps: S1, experimental biological preparation: collecting or domesticating apple snails and distinguishing newly hatched individuals and adult individuals as test organisms; S2, acute toxicity detection: exposing newly hatched apple snails and adult apple snails to a series of different concentrations of prohexadione calcium aqueous solution respectively, setting a control group, and performing 96-hour exposure under standard static or semi-static conditions, and observing and recording the number of surviving individuals, the number of inhibited individuals and the number of individuals exhibiting escape behavior in each group during the period; S3, chronic toxicity detection: exposing adult apple snails to a series of sublethal concentrations of prohexadione calcium aqueous solution, setting a control group, and performing semi-static exposure for 21 days, and measuring and recording the shell height and body weight of the snails regularly during the period; S4, histopathological detection: after the end of the chronic toxicity detection, the liver pancreas, kidney and stomach tissues of the exposed apple snails in S3 are taken, fixed, dehydrated, embedded, sliced, HE stained, and then histopathological observation and analysis are performed; S5, data processing and ecological toxicity evaluation: based on the data of S2, the 96-hour semi-lethal concentration and semi-inhibitory concentration of prohexadione calcium on newly hatched and adult apple snails are calculated, based on the data of S3, the influence of prohexadione calcium on the growth rate of apple snails is analyzed, and based on the results of S4, the degree of tissue damage of prohexadione calcium to the main organs is evaluated.
2. The method according to claim 1, wherein the method is characterized by, In S1, the newly hatched individuals are individuals hatched for 5-7 days and with a shell length of 2-3 mm; the adult individuals are adult snails with a shell height of 20±5 mm or 30±5 mm.
3. The method according to claim 1, wherein the method is characterized by, In S2, for the acute toxicity detection of newly hatched apple snails, the exposure concentration gradient of prohexadione calcium includes 0.25 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L and 3.0 mg / L; for the acute toxicity detection of adult apple snails, the exposure concentration gradient of prohexadione calcium includes 1.6 mg / L, 3.125 mg / L, 6.25 mg / L, 12.5 mg / L and 25.0 mg / L.
4. The method according to claim 1, wherein the method is characterized by, In S2, the judgment standard of the inhibited individuals is that the shell retraction reaction is slow, not wall-adhering, significantly weakened activity or long-term stationary after mechanical stimulation; the judgment standard of the escape behavior is that the individual actively climbs to the container wall above the liquid surface and lasts for more than 3 seconds.
5. The method according to claim 1, wherein the method is characterized by, In S2, the observation of escape behavior is dynamically recorded at 0.5 hours, 1 hour, 2 hours, 3 hours and 4 hours in the early exposure, and a concentration-time escape rate heat map is drawn.
6. The method according to claim 1, wherein the method is characterized by, In S3, the sublethal concentration gradient of prohexadione calcium includes 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L and 1.6 mg / L.
7. The method according to claim 1, wherein the method is characterized by, In S3, the shell height is measured by using an electronic vernier caliper to measure the maximum shell diameter, and the body weight is weighed by using an analytical balance to weigh the live body weight, and the shell height growth rate and the body weight growth rate are calculated.
8. The method according to claim 1, wherein the method is characterized by, In S4, the histopathological observation and analysis specifically includes: Liver pancreas tissue: observing whether the acinus structure is loose, whether the epithelial cells are vacuolar degeneration or shedding, whether the lacuna is dilated, whether there are alkaline cell aggregation, lipofuscin-like granule deposition and necrotic gland tube; Kidney tissue: whether the epithelium of renal crypt is swollen, vacuolated or exfoliated, whether the lumen of renal crypt is dilated, whether the number and lumen diameter of intertubular sinus are increased; Stomach tissue: whether the mucosa layer is thinned, swollen or exfoliated, whether the submucosa layer is edematous or has fissures, whether the muscle layer is disaggregated or ruptured.
9. The use of an apple snail in the evaluation of the ecological toxicity of propanil pesticide, characterized by, The application comprises: using the ecological toxicity detection method of propanil pesticide based on the golden apple snail according to any one of claims 1 to 8, exposing the newly hatched and adult golden apple snails to the water containing the propanil respectively, evaluating the acute toxicity of the propanil by determining at least one of the acute lethal effect, the acute physiological inhibition effect or the acute escape behavior response of the propanil on the newly hatched and adult golden apple snails; and / or, evaluating the chronic toxicity of the propanil by determining at least one of the chronic growth inhibition effect or the histopathological damage effect of the propanil on the liver and pancreas, kidney and stomach of the adult golden apple snails.
10. The use of a golden apple snail in the evaluation of the ecological toxicity of propanil according to claim 9, characterized in that, The application further comprises: based on the acute toxicity determination results, obtaining the 96-hour semi-lethal concentration and semi-inhibition concentration of the propanil on the newly hatched golden apple snails and the 96-hour semi-lethal concentration and semi-inhibition concentration of the propanil on the adult golden apple snails, and calculating the semi-lethal concentration ratio and semi-inhibition concentration ratio of the newly hatched and adult stages, which are used for quantitatively evaluating the development stage-specific toxicity risk of the propanil.