A method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish

Abstract

The invention discloses a method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish, comprising the following steps performed in sequence: S1. preparing a gradient concentration of deltamethrin standard sample exposure solution; S2. exposing zebrafish embryos to deltamethrin standard sample exposure solution for culture; S3. observing zebrafish development, fitting deltamethrin concentration zebrafish development curve, and determining the detection limit; S4. exposing zebrafish embryos to the exposure solution of the dairy product to be tested for culture, observing the zebrafish development, substituting the data into the deltamethrin concentration zebrafish development curve, the toxicity level of deltamethrin in the dairy product to be tested can be detected, and a positive detection is reported when the value is greater than the detection limit. The present invention provides a method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish, which can intuitively display the influence of deltamethrin on an organism, and scientifically and rationally determine the deltamethrin content in dairy products by combining multiple observation indicators.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety evaluation and relates to the toxicity detection of deltamethrin in dairy products, in particular to a method for evaluating the toxicity level of deltamethrin in dairy products by using zebrafish. Background Art

[0002] Deltamethrin is a second-generation pyrethroid insecticide and one of the most widely used insecticides in the world. It is widely used in agriculture and public health due to its low toxicity, high efficiency and low residue. However, most insecticides have an impact on the ecosystem, and their widespread use can lead to serious environmental problems: due to the lipophilicity of deltamethrin, biofilms and tissues can easily absorb them, and while accumulating in organisms for a long time, they may cause damage to the aquatic environment. Deltamethrin metabolites have been found in the urine of pregnant women. Exposure to deltamethrin may cause hypophosphatemia, which may be related to electrolyte redistribution and impaired renal function between intracellular or extracellular compartments. Deltamethrin also generally affects the nervous system and causes apoptosis. Studies have shown that deltamethrin can induce developmental toxicity, neurotoxicity, and cardiovascular toxicity.

[0003] At present, the detection of deltamethrin is mainly focused on instrumental methods, which can only mechanically detect the content of deltamethrin and cannot intuitively represent its physiological toxicity phenomenon. However, since the organism itself has a certain detoxification ability; and some toxic substances, such as deltamethrin, will be enriched or metabolized in the organism, which will affect the content of the substance in the organism, the instrumental detection method alone cannot reasonably characterize the impact of deltamethrin content on the organism. In the prior art, the research on deltamethrin is only carried out between some plants and mice, and only the toxicity of deltamethrin is studied on a certain organ, and there is a lack of evaluation methods for the overall toxicity level of deltamethrin to the organism. Summary of the invention

[0004] The purpose of the present invention is to provide a method for evaluating the toxicity level of cypermethrin in dairy products using zebrafish, so as to solve the problem in the prior art that there is a lack of a method that can reasonably characterize the overall toxic effect of cypermethrin content on an organism.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish comprises the following steps performed in sequence:

[0007] S1. Prepare exposure solutions of standard samples of cypermethrin with gradient concentrations;

[0008] S2. Expose zebrafish embryos to the standard sample exposure solution of deltamethrin;

[0009] S3. Observe the development of zebrafish, fit the deltamethrin concentration-zebrafish development curve, and determine the detection limit;

[0010] S4. Expose zebrafish embryos to the exposure solution of the dairy product to be tested, observe the development of the zebrafish, and substitute the data into the cypermethrin concentration-zebrafish development curve to detect the toxicity level of cypermethrin in the dairy product to be tested. When the value is greater than the detection limit, a positive detection is reported.

[0011] As a limitation, the observation of zebrafish development includes hatching rate, body length, head area, eye area, intersegmental vessel length, dorsal longitudinal anastomotic vessel length, pericardial edema ratio, pericardial area and SV-BA distance.

[0012] As another limitation, the step S1 specifically includes:

[0013] The deltamethrin standard sample concentrated solution is prepared by using the processed dairy product and dimethyl sulfoxide as solvent to form different concentrations of deltamethrin, and then diluted with culture water to obtain the deltamethrin standard sample exposure solution.

[0014] As a further limitation, the step S2 specifically includes:

[0015] Transgenic zebrafish were selected for side cross breeding with wild AB zebrafish to obtain fertilized eggs. Normally developed embryos were selected and placed in solvents diluted with culture water and exposure solutions of different concentrations of deltamethrin standard samples. Parallel samples were set up for each group and cultured at a constant temperature of 27-28°C for 24-72 hours.

[0016] As a further limitation, step S3 specifically includes:

[0017] S31. Place the zebrafish fry in a lateral position with its eyes overlapped for observation. If the fry has not hatched, pierce the membrane, and then place the zebrafish fry in a lateral position with its eyes overlapped for observation, and collect images.

[0018] S32. The collected images were analyzed using Image J software, the data were standardized, and the principal component was used for dimensionality reduction, and then the least square method was used for fitting to obtain the deltamethrin concentration-zebrafish development curve;

[0019] S33. Select the concentration of the cypermethrin standard sample exposure solution that is significantly different from that of zebrafish larvae in the solvent diluted with culture water as the detection limit.

[0020] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0021] ① The method provided by the present invention for evaluating the toxicity level of deltamethrin in dairy products by using zebrafish can more intuitively demonstrate the influence of the toxicity of deltamethrin on organisms, and comprehensively observe multiple indicators to determine the detection limit, thereby more scientifically and reasonably expressing the physiological response of organisms to different contents of deltamethrin, compared with the method of detecting the content of deltamethrin by instruments in the prior art.

[0022] ② The present invention provides a method for evaluating the toxicity level of cypermethrin in dairy products using zebrafish. The method uses treated dairy products and dimethyl sulfoxide as solvents, which can eliminate the effects of other components in dairy products on organisms, making the detection of the cypermethrin content in dairy products more reasonable.

[0023] The present invention provides a method for evaluating the toxicity level of deltamethrin in dairy products by using zebrafish, which can intuitively demonstrate the influence of deltamethrin on organisms, and scientifically and rationally determine the deltamethrin content in dairy products by integrating multiple observation indicators. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The curve of deltamethrin concentration-zebrafish development in the embodiment;

[0025] Figure 2 is the body length of zebrafish cultured and exposed to dairy products in the Examples;

[0026] Figure 3 The head area of ​​zebrafish cultured in the embodiment exposed to dairy products;

[0027] Figure 4 is the eye area of ​​zebrafish cultured and exposed to dairy products in the Example;

[0028] Figure 5 The length of intersegmental vessels of zebrafish cultured in the embodiment exposed to dairy products;

[0029] Figure 6 The length of the longitudinal anastomotic vessels on the dorsal side of zebrafish cultured in the embodiment exposed to dairy products;

[0030] Figure 7 The pericardial area of ​​zebrafish cultured in the embodiment exposed to dairy products;

[0031] Figure 8 SV-BA distance of zebrafish cultured and exposed to dairy products in the examples. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0033] Example A method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish

[0034] This embodiment specifically includes the following steps performed in sequence:

[0035] S1. Preparation of exposure solutions of standard samples of cypermethrin with gradient concentrations

[0036] Dairy products were tested using gas chromatography-mass spectrometry to find out dairy products that did not contain deltamethrin as the background. 2.00 g of dairy products (accurate to 0.01 g) were accurately weighed, 0.5 g of sodium chloride, 5.0 mL of water, and 10.0 mL of acetonitrile were added, and vortexed for 60 seconds, ultrasonically extracted for 10 minutes, and then centrifuged at 4000 r / min for 5 minutes. The solution was separated into water and acetonitrile layers. 5.0 mL of acetonitrile was accurately transferred to the acetonitrile layer and blown to about 1 mL with nitrogen at 40°C.

[0037] The mixture was poured into a C18 solid phase extraction column pre-eluted with 5 mL of acetonitrile, eluted with 4 mL of acetonitrile, the eluate was collected, and nitrogen was blown to near dryness at 40°C. After vortex mixing and dissolving the residue with 0.5 mL of n-hexane, the mixture was poured into a fluorosilicone solid phase extraction column pre-eluted with 5 mL of n-hexane-ethyl acetate, eluted with 5.0 mL of n-hexane-ethyl acetate, the eluate was collected, and nitrogen was blown to near dryness at 40°C. 1.0 mL of dimethyl sulfoxide was used for vortexing and redissolving to obtain a matrix concentrate;

[0038] Accurately weigh deltamethrin and add it to the matrix concentrate to prepare deltamethrin standard sample concentrates with deltamethrin concentrations of 0.1 mg / L, 1 mg / L, 10 mg / L, 25 mg / L, and 50 mg / L respectively;

[0039] When exposed to zebrafish, the deltamethrin standard sample concentrate was diluted 1000 times with culture water to obtain 0.1 μg / L, 1 μg / L, 10 μg / L, 25 μg / L, and 50 μg / L deltamethrin standard sample exposure solutions;

[0040] S2. Expose zebrafish embryos to standard cypermethrin sample exposure solution

[0041] Two transgenic zebrafish, TG (flk:GFP) and TG (cmlc2:GFP), were selected for cross-breeding with wild AB zebrafish. The fertilized eggs were transferred into culture dishes, and the fertilized eggs with abnormal development (including dead eggs) and impurities were removed. They were rinsed with culture water for 2-3 times, and then the embryos with normal development were selected under a stereomicroscope for exposure experiments.

[0042] Take a 96-well imaging plate, add 200 μL of matrix concentrate diluted 1000 times with culture water, different concentrations of deltamethrin standard sample exposure solution and one selected embryo to each well, set up a normal control group (diluted matrix concentrate) and a matrix spike group (different concentrations of deltamethrin standard sample exposure solution) in the experiment, set up 20 parallel samples for each treatment group, put the 96-well imaging plate into a constant temperature incubator for 72 hours, and the constant temperature culture temperature was 27°C;

[0043] S3. Observe the development of zebrafish, fit the deltamethrin concentration-zebrafish development curve, and determine the detection limit

[0044] The zebrafish larvae were placed in a lateral position with their eyes overlapped for observation. For those that had not hatched, the membrane was gently pricked with dissecting forceps, and then the larvae were placed in a lateral position with their eyes overlapped for observation with an egg-pulling needle. Images were collected using an inverted fluorescence microscope. The observation indicators were: hatching rate, body length, head area, eye area, intersegmental vessel length, dorsal longitudinal anastomotic vessel length, pericardial edema ratio, pericardial area, and SV-BA distance.

[0045] Add a scale bar to the acquired image and export it to a picture format (PNG, JPEG, etc.), and use Image J software to circle and analyze it;

[0046] The data were standardized using SPSS software for 9 variables, and then the principal component was used for dimensionality reduction and then fitted using the partial least squares regression method to obtain the deltamethrin concentration-zebrafish development curve, as shown in the figure. Figure 1 As shown;

[0047] When the concentration of deltamethrin standard sample was 50 ppb, there was a significant difference between the matrix spiked group and the control group, so 50 ppb was used as the detection limit;

[0048] S4. Select two transgenic zebrafish, TG (flk:GFP) and TG (cmlc2:GFP), and cross them with wild AB zebrafish. Transfer the fertilized eggs into a culture dish, remove the abnormally developed fertilized eggs (including dead eggs) and impurities, rinse them with culture water 2-3 times, and then select the embryos with normal development under a stereo microscope for exposure experiments;

[0049] Accurately weigh 2.00 g (accurate to 0.01 g) of the dairy product to be tested, add 0.5 g of sodium chloride, 5.0 mL of water, and 10.0 mL of acetonitrile, vortex extract for 60 seconds, ultrasonic extract for 10 minutes, and then centrifuge at 4000 r / min for 5 minutes. The solution is separated into water and acetonitrile layers. Accurately transfer 5.0 mL of acetonitrile to the acetonitrile layer and blow it to about 1 mL at 40°C with nitrogen;

[0050] Pour it into a C18 solid phase extraction column pre-eluted with 5 mL of acetonitrile, elute with 4 mL of acetonitrile, collect the eluate, blow it to nearly dryness with nitrogen at 40°C, vortex and dissolve the residue with 0.5 mL of n-hexane, pour it into a fluorosilicone solid phase extraction column pre-eluted with 5 mL of n-hexane-ethyl acetate, elute with 5.0 mL of n-hexane-ethyl acetate, collect the eluate, blow it to nearly dryness with nitrogen at 40°C, vortex and redissolve with 1.0 mL of dimethyl sulfoxide to obtain a dairy sample concentrate;

[0051] The concentrated solution of the dairy product sample was diluted 1000 times with culture water to obtain the exposure solution of the dairy product sample;

[0052] Take a 96-well imaging plate, add 200 μL of the dairy sample exposure solution to be tested and one selected embryo to each well, set up 20 parallel samples, and place the 96-well imaging plate in a constant temperature incubator for 72 hours at a constant temperature of 27°C;

[0053] The zebrafish larvae were placed in a lateral position with their eyes overlapped for observation. For those that had not hatched, the membrane was gently pricked with dissecting forceps, and then the larvae were placed in a lateral position with their eyes overlapped for observation with an egg-pulling needle. Images were collected using an inverted fluorescence microscope. The observation indicators were: hatching rate, body length, head area, eye area, intersegmental vessel length, dorsal longitudinal anastomotic vessel length, pericardial edema ratio, pericardial area, and SV-BA distance.

[0054] Add a scale to the acquired image and export it to a picture format (PNG, JPEG, etc.), and use Image J software to circle and analyze it, such as Figure 2~Figure 8 As shown;

[0055] The hatching rate was 100%, the body length was 3.409mm, and the head area was 0.155mm 2 , the eye area is 0.05mm 2 The length of intersegmental vessels was 0.156 mm, the length of dorsal longitudinal anastomotic vessels was 2.234 mm, the proportion of pericardial edema was 0%, and the pericardial area was 0.024 mm 2 , SV-BA distance is 0.27mm;

[0056] The data was standardized using SPSS software and substituted into the cypermethrin concentration-zebrafish development curve, and the concentration of cypermethrin in dairy products was detected to be 0.002 ppb, which was less than 50 ppb, and the detection result was negative.

Claims

1. A method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish, characterized in that: The process includes the following steps: S1. Prepare exposure solutions of standard samples of cypermethrin with gradient concentrations; S2. Expose zebrafish embryos to the standard sample exposure solution of deltamethrin; S3. Observe the development of zebrafish, fit the deltamethrin concentration-zebrafish development curve, and determine the detection limit; S4. Expose zebrafish embryos to the exposure solution of the dairy product to be tested, observe the development of the zebrafish, and substitute the data into the cypermethrin concentration-zebrafish development curve to detect the toxicity level of cypermethrin in the dairy product to be tested. When the value is greater than the detection limit, a positive detection is reported.

2. The method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish according to claim 1, characterized in that: The zebrafish development is observed, and the observation indicators include hatching rate, body length, head area, eye area, intersegmental blood vessel length, dorsal longitudinal anastomotic blood vessel length, pericardial edema ratio, pericardial area and SV-BA distance.

3. A method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish according to claim 1 or 2, characterized in that: The step S1 specifically includes: The deltamethrin standard sample concentrated solution is prepared by using the processed dairy product and dimethyl sulfoxide as solvent to form different concentrations of deltamethrin, and then diluted with culture water to obtain the deltamethrin standard sample exposure solution.

4. The method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish according to claim 3, characterized in that: The step S2 specifically includes: Transgenic zebrafish were selected for side cross breeding with wild AB zebrafish to obtain fertilized eggs. Normally developed embryos were selected and placed in solvents diluted with culture water and exposure solutions of different concentrations of deltamethrin standard samples. Parallel samples were set up for each group and cultured at a constant temperature of 27-28°C for 24-72 hours.

5. The method for evaluating the toxicity level of deltamethrin in dairy products using zebrafish according to claim 3, characterized in that: The step S3 specifically includes: S31. Place the zebrafish fry in a lateral position with its eyes overlapped for observation. If the fry has not hatched, pierce the membrane, and then place the zebrafish fry in a lateral position with its eyes overlapped for observation, and collect images. S32. The collected images were analyzed using Image J software, the data were standardized, and the principal component was used for dimensionality reduction, and then the least square method was used for fitting to obtain the deltamethrin concentration-zebrafish development curve; S33. Select the concentration of the cypermethrin standard sample exposure solution that is significantly different from that of zebrafish larvae in the solvent diluted with culture water as the detection limit.

Images