A method for non-invasive detection of estrogen activity of water pollutants using zebrafish

Abstract

The invention discloses a method for non-invasively detecting the estrogen activity of water pollutants by zebrafish, which belongs to the technical field of ecotoxicology, and comprises the following steps: S1, male zebrafish is placed in a 17α-ethynyl estradiol (EE2) standard exposure solution for breeding, and then skin mucus samples are collected to determine the vitellogenin (VTG) concentration; S2, fitting the EE2 exposure concentration-VTG relative concentration standard curve; S3, male zebrafish is placed in a test water sample for breeding, and then mucus samples are collected to determine the VTG concentration; S4, the EE2 equivalent of the water body to be detected is calculated according to the standard curve. The present invention provides a new means for the detection of estrogen activity in water bodies, and the operation steps are simple, and animal dissection is not involved. It only needs to collect fish skin mucus and detect VTG to quickly warn of estrogen analog pollution in water bodies. Compared with the existing detection methods, it has the advantages of high efficiency, accuracy and low cost, and can effectively protect animal welfare.

Description

Technical Field

[0001] The invention relates to the technical field of ecotoxicology, and in particular to a method for non-invasively detecting the estrogen activity of water pollutants using zebrafish. Background Art

[0002] In order to protect the health of aquatic organisms, it is necessary to quickly and accurately evaluate the water ecological risks of EDCs and achieve early warning of pollution. Traditional estrogen activity detection methods require collecting aquatic animals (such as fish), dissecting them to obtain different tissue samples, and using them for the determination of biomarkers to reflect the estrogen interference effects of water pollutants.

[0003] However, these methods seriously damage animal welfare on the one hand, which is contrary to the 3Rs requirements of animal experiments (Replacement, Reduction, Refinement) currently advocated; on the other hand, large-scale and high-frequency sampling of wild organisms also causes significant interference to aquatic ecosystems, especially for protected and endangered species. At present, the detection methods of estrogenic activity in fish mainly involve in vitro and in vivo. In vitro detection methods mainly include receptor binding and DNA binding experiments, biosensors, recombinant gene yeast cells, etc. However, due to the great difference between the in vitro detection environment and living animals, the reliability of in vitro detection results is not high. In vivo detection uses model organisms, with the help of real-time fluorescence quantitative PCR, protein blotting, transcriptomics analysis, histological analysis, high-performance liquid chromatography and other technologies, to evaluate the estrogenic activity of water pollutants by observing changes in genes, hormones, proteins, pathology, etc. In vivo detection methods generally obtain biological samples through animal dissection, which cannot guarantee the welfare of experimental animals. For example, fish need to be anesthetized and dissected to remove brain, liver, gonads and other tissues; or blood can be collected by tail cutting.

[0004] The above existing estrogen activity detection methods have defects: 1) The experiment involves dissecting animals for sampling, which cannot guarantee animal welfare and has certain limitations in the number of biological samples, biological repetitions and experimental costs; 2) Small fish have problems such as difficulty in sampling, small sample size (blood and tissue), and complex operation; 3) A single individual cannot be sampled repeatedly, and the temporal dynamic changes of samples cannot be continuously monitored. Therefore, at this stage, there is an urgent need to innovate methods for detecting estrogen effects in water bodies. The present invention aims to establish a non-invasive method for detecting the estrogen activity of water pollutants, which is simple and rapid in sampling and effectively protects the welfare of aquatic animals; at the same time, the detection indicators are sensitive to the endocrine disrupting toxicity of pollutants, and can accurately warn and respond to the estrogen activity of water bodies. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide a method for non-invasively detecting the estrogen activity concentration of water pollutants by zebrafish. The sampling is simple and rapid, and the welfare of aquatic animals is effectively protected. At the same time, the detection indicators are sensitive to the endocrine disrupting toxicity of pollutants and can quickly and accurately reflect the estrogen activity of water.

[0006] The technical solution of the present invention is: a method for non-invasively detecting the estrogen activity of water pollutants using zebrafish, comprising the following steps:

[0007] S1. Using 17α-ethynyl estradiol EE2 as a model estrogen pollutant, standard exposure solutions were prepared with EE2 starting from 0 ng / L in a stepwise concentration, and male zebrafish were placed in standard exposure solutions of various concentrations for stable culture for 7 days. During the culture process, new standard exposure solutions of the same concentration were replaced every day. After the culture was completed, skin surface mucus was non-invasively collected from male zebrafish cultured at various EE2 concentrations and prepared into mucus samples. The VTG concentration of the mucus samples was determined using an ELISA kit;

[0008] S2, taking the mucus sample with 0 ng / L EE2 concentration in the exposure solution in step S1 as the control group, converting the VTG concentration of each mucus sample in step S1 into a relative concentration by dividing it by the VTG concentration of the control group, and then fitting the EE2 exposure concentration and the VTG relative concentration to obtain the EE2 exposure concentration-VTG relative concentration standard curve I;

[0009] S3, using aerated tap water as a control water sample, placing male zebrafish in the test water sample and the control water sample respectively, and performing stable culture under the same culture conditions and culture time as in step S1, replacing new identical water samples every day during the exposure process, and collecting male zebrafish mucus non-invasively after the culture is completed and preparing mucus samples, and determining the VTG concentration of the mucus samples using an ELISA kit;

[0010] S4. Divide the VTG concentration of the mucus sample in the test water sample in step S3 by the VTG concentration of the mucus sample in the control water sample to obtain the VTG relative concentration of the test water sample, and substitute it into the EE2 exposure concentration-VTG concentration standard curve I obtained in step S2 to obtain the EE2 concentration equivalent of the water body to be tested.

[0011] Preferably, in step S1, the gradient concentration of EE2 is 0 ng / L, 1 ng / L, 2.5 ng / L, 5 ng / L, 10 ng / L, 25 ng / L, 50 ng / L, and 100 ng / L.

[0012] Preferably, the culture conditions in step S1 specifically include: culture in a stable environment of 28±0.5°C, 14h light / 10h dark photoperiod, 6.7-7.2mg / L dissolved oxygen, pH 7.0-7.4, feeding brine shrimp in the morning and feeding feed in the afternoon.

[0013] Preferably, in steps S1 and S3, non-invasively collecting mucus from the skin surface of male zebrafish and preparing a mucus sample specifically comprises: placing a plurality of male zebrafish on ice after anesthetizing them in a culture dish, into which a PBS buffer containing protease inhibitors has been pre-added, lightly scraping mucus from the body surface with a cell scraper, collecting the mucus of all zebrafish in the culture dish as a crude sample, and centrifuging the crude sample to obtain the supernatant to prepare a mucus sample.

[0014] Further, 6 to 8 zebrafish were anesthetized on ice and placed in a petri dish. Further, the volume of mucus scraped from each fish was 10 to 20 microliters.

[0015] Furthermore, 400-500 μL of PBS buffer containing protease inhibitors is pre-added to the culture dish.

[0016] Furthermore, preparing the mucus sample specifically includes: centrifuging the crude sample at 4° C. and 10,000 g for 15 minutes, transferring the supernatant and storing it in a -80° C. environment to obtain the mucus sample.

[0017] Furthermore, the PBS buffer containing protease inhibitors is prepared by adding one tablet of protease inhibitor to every 10-15 mL of PBS solution and mixing evenly. The protease inhibitors are commercially available products, and the source is protease inhibitors (Rochec Omplete TM ,Mini,EDTA-free Protease Inhibitor Cocktail; Catalog No.: 04693159001, 0.25g / piece). The preparation method of PBS buffer is as follows: first prepare 1L of 1X PBS solution (using 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4), adjust the pH to 7.4, and the rest is water.

[0018] Furthermore, at least 5 parallel mucus samples are taken at each EE2 concentration in step S1, and the VTG concentrations of the parallel mucus samples are measured and the average value is taken, which is the VTG concentration at the EE2 concentration.

[0019] Furthermore, at least 5 parallel mucus samples are taken in step S3. The VTG concentration of each parallel mucus sample of the test water sample is measured and the average value is taken, which is the VTG concentration in the test water sample environment.

[0020] In the present invention, a commercially available fish-specific ELISA test kit is used to quantitatively analyze the VTG concentration of the mucus sample. The exposure solution in step S1 is prepared by placing EE2 in aerated tap water.

[0021] The beneficial effects of the present invention are:

[0022] 1. Through exposure of standard solution under step concentration, the standard curve I of EE2 exposure concentration-VTG relative concentration is fitted; only skin mucus of fish cultured in the water environment to be tested is collected and VTG is tested to obtain EE2 equivalent, so as to realize rapid warning of estrogen analog pollution in water. Compared with previous detection methods, it has the advantages of high efficiency, accuracy and low cost, and can effectively assist large-scale ecological survey projects.

[0023] 2. While sensitively responding to the endocrine disrupting toxicity of estrogen pollutants, it does not involve animal dissection, effectively protects animal welfare, and reduces the use of experimental animals.

[0024] 3. Considering that male fish are more sensitive to the estrogen activity of endocrine disruptors than female fish, future sampling can only collect mucus samples from male fish surfaces, without the need to collect samples from female fish, which further reduces sample usage and interference with the aquatic ecosystem.

[0025] 4. Use fish skin mucus VTG to reflect estrogen pollution in water bodies. This non-invasive operation has no effect on fish vitality and health, so a fish can be sampled repeatedly, thereby accurately revealing the long-term changes in the ecotoxicological effects of pollutants.

[0026] 5. The response pattern of VTG in male fish skin mucus to estrogen activity is very consistent with that in liver and blood, ensuring the reliability of the test results.

[0027] 6. The pollution situation of real aquatic environment is complex, and multiple estrogen-active pollutants often coexist. VTG in fish mucus can be used as a comprehensive biomarker to integrate the combined toxic effects of pollutants and provide clues for subsequent ecological investigations.

[0028] 7. Estrogen analogs in water significantly affect the sexual differentiation and reproductive function of fish, leading to the decline of fish populations. The non-invasive detection of VTG in fish mucus provides a suitable method for studying the population changes of protected and endangered species, and can explain the ecological effects of estrogen pollutants without affecting animal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The EE2 exposure concentration-VTG relative concentration standard curve of Example 1 of the present invention is

[0030] Figure 2 is the EE2 equivalent of the typical estrogen analogue of Example 2 of the present invention

[0031] Figure 3 Figure 2 is the time-dependent changes in VTG content in liver, blood and skin mucus of male zebrafish after exposure to EE2 for different time periods.

[0032] Figure 4 Figure 2. Changes in VTG content in liver, blood and skin mucus of male zebrafish after exposure to 10 representative estrogenic pollutants.

[0033] Figure 5 For the consistency analysis of VTG changes in liver, blood and skin mucus DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The drugs used in the following examples are all commercially available products unless otherwise specified, and the methods used are all conventional methods in the art unless otherwise specified.

[0035] In the following examples, the non-invasive sampling method of skin mucus is as follows:

[0036] 1) PBS buffer preparation: First prepare 1L of 1X PBS solution (8g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4), adjust the pH to 7.4; then add a tablet of protease inhibitor (Roche Omplete TM ,Mini,EDTA-free Protease Inhibitor Cocktail; Catalog No.: 04693159001, 0.25g / piece) to prevent protein degradation.

[0037] 2) 6-8 male zebrafish were anesthetized on ice and placed in a plastic culture dish supplemented with 400 μL PBS buffer. Mucus samples were gently scraped from the body surface of each fish using a cell scraper.

[0038] 3) The mucus of 6-8 male zebrafish was combined into a crude sample in a culture dish, which was used as a biological replicate to increase the representativeness of the sample and reduce the influence of individual differences.

[0039] The method for processing the crude sample into a mucus sample is as follows:

[0040] 4) Use a 1 mL pipette to transfer all the liquid in the plastic culture dish to a 1.5 mL plastic centrifuge tube, centrifuge at 4°C and 10,000 g for 15 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0041] 5) The mucus supernatant sample was stored at -80°C to obtain a mucus sample for subsequent ELISA experiments to detect vitellogenin VTG.

[0042] 6) Determine the total protein content of the mucus sample, and the ELISA test results of VTG are corrected with the corresponding total protein concentration.

[0043] The VTG content of mucus samples was determined as follows:

[0044] The VTG concentration of mucus samples was quantified using a commercial fish-specific ELISA test kit. The procedure is as follows:

[0045] 1) Standard dilution: Dilute the VTG standard with standard diluent to prepare a series of concentration gradients (50 μg / L, 100 μg / L, 200 μg / L, 400 μg / L, 800 μg / L).

[0046] 2) Sample addition: Set up blank wells (blank wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and wells for samples to be tested. Accurately add 50μL of standard solution to the standard wells of the ELISA plate; first add 40μL of sample diluent to the wells for samples to be tested, and then add 10μL of the sample to be tested (the sample is finally diluted 5 times). During the sample addition process, the sample needs to be added to the bottom of the ELISA well, try not to touch the well wall, and use a pipette to blow and mix thoroughly.

[0047] 3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0048] 4) Washing: Dilute the 30X concentrated washing solution to 1X with distilled water. Carefully peel off the sealing film, discard the liquid, spin dry, add 250 μL washing solution to each well, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0049] 5) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0050] 6) Incubation: the same operation as 3) above.

[0051] 7) Washing: the same operation as in 4).

[0052] 8) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, mix well, and develop color at 37°C in the dark for 10 minutes.

[0053] 9) Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color should immediately turn to yellow).

[0054] 10) Measurement: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be completed within 15 minutes after the addition of the stop solution.

[0055] 11) Result analysis: Based on the standard concentration and OD value, calculate the linear regression equation of the standard curve; substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual VTG concentration of the sample.

[0056] Example 1

[0057] The present invention provides a method for non-invasively detecting the estrogen activity of water pollutants using zebrafish, comprising the following steps:

[0058] S1. After male zebrafish were cultured in EE2 standard exposure solution, skin mucus samples were collected to determine the VTG concentration, as follows:

[0059] Using 17α-ethynyl estradiol EE2 as a model estrogen analog, EE2 was prepared into standard exposure solutions at step concentrations (0, 1, 2.5, 5, 10, 25, 50, 100 ng / L). Male zebrafish were placed in the standard exposure solutions of various concentrations for stable culture for 7 days. During the culture process, new standard exposure solutions of the same concentration were replaced every day. The culture environment was a stable environment of 28±0.5℃, 14h light / 10h dark photoperiod, 6.9 mg / L dissolved oxygen, and pH 7.2. The fish were fed with brine shrimp in the morning and feed in the afternoon.

[0060] After the culture was completed, skin surface mucus was non-invasively collected from male zebrafish cultured at each EE2 concentration and prepared into mucus samples. The VTG concentration of the mucus samples was determined using an ELISA kit. There were 5 biological replicates (n=5) at each EE2 concentration.

[0061] S2. Fitting the EE2 exposure concentration-VTG relative concentration standard curve I, as follows:

[0062] The mucus sample with EE2 concentration of 0 ng / L in the exposure solution in step S1 was used as the control group. The VTG concentration of each mucus sample in step S1 was divided by the VTG concentration of the control group to convert it into a relative concentration, and the EE2 exposure concentration was fitted with the VTG relative concentration to obtain the EE2 concentration-VTG relative concentration standard curve I (R 2 =0.993), such as Figure 1 As shown, the curve equation is y = 1.066 / [1+(x / 11.71) 1.234 ]-0.039.

[0063] S3. After male zebrafish were cultured in the test water sample, mucus samples were collected to determine the VTG concentration, as follows:

[0064] Using aerated tap water as a control water sample, male zebrafish were placed in the test water sample and the control water sample, and were cultured stably under the same culture conditions and culture time as in step S1. During the culture process, new water samples were replaced every day. After the culture was completed, the male zebrafish mucus was non-invasively collected and prepared into mucus samples. The VTG concentration of the mucus sample was determined using an ELISA kit, with 5 biological replicates (n=5);

[0065] S4. Calculate the EE2 equivalent of the water body to be tested, as follows:

[0066] The VTG concentration in the test water sample environment obtained in step S3 is specifically 466.84 μg / g protein, and the VTG concentration in the mucus sample in the control water sample is specifically 530.47 μg / g protein. Therefore, the VTG relative concentration of the water sample to be tested is converted to 0.88; the VTG relative concentration measured in this embodiment is substituted into the EE2 exposure concentration-VTG relative concentration standard curve I obtained in step S2 to obtain the EE2 concentration equivalent of the water body to be tested, which is 2.65 ng / L EE2.

[0067] Example 2

[0068] The present invention provides a method for non-invasively detecting the estrogen activity of water pollutants using zebrafish, comprising the following steps:

[0069] S1-S2: According to steps S1-S2 of Example 1, the standard curve I of EE2 exposure concentration-VTG relative concentration is obtained, and the curve equation is y=1.066 / [1+(x / 11.71) 1.234 ]-0.039(R 2 =0.993);

[0070] S3. After male zebrafish were placed in a typical estrogen analog exposure solution, mucus samples were collected to measure VTG concentration

[0071] Using aerated tap water as the control water sample, male zebrafish were placed in 10 typical estrogen analogs exposure solutions and control water samples, including diethylstilbestrol (DES, diethylstilbestrol, 100ng / L), estradiol (E2, 17-estradiol, 200ng / L), estrone (E1, estrone, 2.7μg / L), zearalenone (ZON, Zearalenone, 3.2μg / L), 3,3'-diindolylmethane (DIM, 10μg / L), 4-nitrophenol (4-NP, 4-Nitrophenol, 14μg / L), bisphenol A (BPA, bisphenol A) and 1,4-diaminophenol (1, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 57, 58, 59, 61, 61, 62, 63, 64, 67, 68, 70, 71, 72, 73, 77, 78, 79, 80 A, 100 μg / L), 2,4-dichlorophenol (2,4-DCP, 2,4-dichlorophenol, 300 μg / L), 4-tert-octylphenol (4-T-OP, 4-tert-octylphenol, 500 μg / L), genistein (GEN, 5 mg / L). The same culture conditions and culture time as in step S1 of Example 1 were used for stable culture. During the culture process, new water samples were replaced every day. After the culture was completed, the mucus of male zebrafish was non-invasively collected and prepared into mucus samples. The VTG concentration of the mucus sample was determined using an ELISA kit, with 5 biological replicates (n=5);

[0072] S4. Calculation of EE2 equivalents of estrogen analogs

[0073] In this embodiment, the relative concentrations of 10 typical estrogen analogs VTG (the VTG concentrations of the mucus samples of the 10 typical estrogen analogs divided by the VTG concentrations of the mucus samples in the control water sample are the relative concentrations) are substituted into the EE2 exposure concentration-VTG relative concentration standard curve I obtained in step S2 to obtain the EE2 concentration equivalents (ng / L) of different estrogen analogs, as shown in FIG. Figure 2 shown.

[0074] Table 1 VTG relative concentration-EE2 concentration equivalent data of 10 typical estrogen analogs

[0075]

[0076] Verification test

[0077] 1. Verification of the sensitivity of male zebrafish skin mucus VTG to estrogen EE2

[0078] Male zebrafish were exposed to EE2 at concentrations of 0 and 100 ng / L. The exposure lasted for one week, and the exposure solution was changed every day. Zebrafish were cultured in a stable environment of 28±0.5℃, 14h light / 10h dark photoperiod, 6.7-7.2 mg / L dissolved oxygen, and pH 7.0-7.4. Brine shrimp were fed in the morning and feed in the afternoon. Mucus, blood, and liver samples of male zebrafish were collected 1 day, 2 days, 4 days, and 7 days after exposure. An ELISA kit was used to detect the changing trend of VTG content with exposure time, and the toxic response sensitivity of different tissues was compared. There were 5 biological replicates (n=5) in each group.

[0079] Figure 3 Time-dependent changes in VTG content in liver, blood and skin mucus of male zebrafish after exposure to EE2 for different time periods (*p<0.05; **p<0.01; ***p<0.001). Figure 3 It can be seen that the liver VTG content of male fish decreased significantly only on the seventh day of EE2 exposure, and the blood VTG content decreased significantly on the fourth and seventh days of exposure. However, the skin mucus VTG content increased significantly on the first day of EE2 exposure, and then decreased on the second, fourth, and seventh days of exposure. Therefore, compared with the liver and blood, skin mucus is the most sensitive to estrogen and can provide timely warning of estrogen activity in the water.

[0080] 2. Reliability verification of the estrogenic effect of water pollutants on male zebrafish skin mucus VTG

[0081] Male zebrafish were exposed to 10 representative pollutants with estrogenic activity, including diethylstilbestrol (DES, 100 ng / L), estradiol (E2, 200 ng / L), estrone (E1, 2.7 μg / L), zearalanone (ZON, 3.2 μg / L), 3,3'-diindolylmethane (DIM, 10 μg / L), 4-nitrophenol (4-NP, 14 μg / L), bisphenol A (BPA, 100 μg / L), 2,4-dichlorophenol (2,4-DCP, 300 μg / L), 4-tert-octylphenol (4-T-OP, 500 μg / L), and genistein (GEN, 5 mg / L). The exposure lasted for one week. The culture environment and sampling methods were consistent with those in Experiment 1.

[0082] Figure 4 Changes in VTG content in liver, blood and skin mucus of male zebrafish after exposure to 10 representative estrogenic pollutants (*p<0.05; **p<0.01; ***p<0.001).

[0083] Figure 4The results showed that compared with the control group, the VTG content in the liver of male zebrafish was significantly reduced in the groups exposed to 6 estrogen analogs, the VTG content in the blood was significantly reduced after exposure to 8 estrogen analogs, and the VTG content in the skin mucus was significantly reduced after exposure to 9 estrogen analogs. This proves that skin mucus VTG is more sensitive to the estrogen activity of pollutants. In addition, the correlation analysis of the VTG test results of the liver, blood, and skin mucus of all exposure groups found that the change trend of mucus VTG was very consistent with the change trend of VTG in the liver and blood (R=0.79, p=0.004; R=0.74, p=0.0098; Figure 5 ), which further proves that it is reliable to use changes in skin mucus VTG content to reflect the estrogen effects of water pollutants.

Claims

1. A method for non-invasively detecting estrogen activity of water pollutants using zebrafish, characterized in that: The following steps are involved: S1. Using 17α-ethynyl estradiol EE2 as a model estrogen pollutant, standard exposure solutions were prepared with EE2 at step concentrations of 0 ng / L, 1 ng / L, 2.5 ng / L, 5 ng / L, 10 ng / L, 25 ng / L, 50 ng / L, and 100 ng / L, and male zebrafish were placed in the standard exposure solutions of various concentrations for stable culture for 7 days. During the culture process, new standard exposure solutions of the same concentration were replaced every day. After the culture was completed, skin surface mucus was non-invasively collected from male zebrafish cultured at various EE2 concentrations and prepared into mucus samples, and the VTG concentration of the mucus samples was determined using an ELISA kit; S2, taking the mucus sample with 0 ng / L EE2 concentration in the exposure solution in step S1 as the control group, converting the VTG concentration of each mucus sample in step S1 into a relative concentration by dividing it by the VTG concentration of the control group, and then fitting the EE2 exposure concentration and the VTG relative concentration to obtain the EE2 exposure concentration-VTG relative concentration standard curve I; S3, using aerated tap water as a control water sample, placing male zebrafish in the test water sample and the control water sample respectively, and performing stable culture under the same culture conditions and culture time as in step S1, replacing new identical water samples every day during the exposure process, and collecting male zebrafish mucus non-invasively after the culture is completed and preparing mucus samples, and determining the VTG concentration of the mucus samples using an ELISA kit; S4, dividing the VTG concentration of the mucus sample in the test water sample in step S3 by the VTG concentration of the mucus sample in the control water sample to obtain the VTG relative concentration of the test water sample, and substituting it into the EE2 exposure concentration-VTG concentration standard curve I obtained in step S2 to obtain the EE2 concentration equivalent of the water body to be tested; In the above steps S1 and S3, non-invasively collecting mucus from the skin surface of male zebrafish and preparing a mucus sample specifically includes: placing 6 to 8 male zebrafish on ice after anesthetizing them in a culture dish, into which PBS buffer containing protease inhibitors has been pre-added, gently scraping mucus from the body surface with a cell scraper, collecting mucus from all zebrafish in the culture dish as a crude sample, and centrifuging the crude sample to obtain the supernatant to prepare a mucus sample.

2. The method for non-invasively detecting estrogen activity of water pollutants using zebrafish as claimed in claim 1, characterized in that: The culture conditions in step S1 specifically include: culture in a stable environment of 28±0.5°C, 14h light / 10h dark photoperiod, 6.7-7.2mg / L dissolved oxygen, pH 7.0-7.4, feeding brine shrimp in the morning and feeding feed in the afternoon.

3. The method for non-invasively detecting estrogen activity of water pollutants using zebrafish as claimed in claim 1, characterized in that: 400-500 μL of PBS buffer containing protease inhibitors was pre-added into the culture dish.

4. The method for non-invasively detecting estrogen activity of water pollutants using zebrafish as claimed in claim 1, characterized in that: The PBS buffer containing protease inhibitors is obtained by adding one tablet of protease inhibitor into every 10-15 mL of PBS solution and mixing evenly.

5. The method for non-invasively detecting estrogen activity of water pollutants using zebrafish as claimed in claim 1, characterized in that: The preparation of the mucus sample specifically includes: centrifuging the crude sample at 4° C. and 10,000 g for 15 minutes, transferring the supernatant and storing it in a -80° C. environment to obtain the mucus sample.

6. The method for non-invasively detecting estrogen activity of water pollutants using zebrafish as claimed in claim 1, characterized in that: At least five parallel mucus samples were prepared for biological replication at each EE2 concentration in step S1.

7. The method for non-invasively detecting estrogen activity of water pollutants using zebrafish as claimed in claim 1, characterized in that: In step S3, at least five parallel mucus samples were prepared for biological repetition.

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