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HJ 1069-2019 English PDF

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HJ 1069-2019: Water quality--Determination of the acute toxicity--Zebrafish (Danio rerio) eggs method
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Basic data

Standard ID	HJ 1069-2019 (HJ1069-2019)
Description (Translated English)	Water quality--Determination of the acute toxicity--Zebrafish (Danio rerio) eggs method
Sector / Industry	Environmental Protection Industry Standard
Classification of Chinese Standard	Z16
Classification of International Standard	13.060
Word Count Estimation	23,246
Date of Issue	2019
Date of Implementation	2020-06-30
Issuing agency(ies)	Ministry of Ecology and Environment

HJ 1069-2019: Water quality--Determination of the acute toxicity--Zebrafish (Danio rerio) eggs method

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Water quality--Determination of the acute toxicity--Zebrafish (Danio rerio) eggs method National Environmental Protection Standard of the People's Republic of China Water quality-Determination of acute toxicity-Zebrafish egg method Water quality-Determination of the acute toxicity -Zebrafish (Danio rerio) eggs method 2 0 1 9-1 2-3 1 Posted 2 0 2 0-0 6-3 0 Implementation Released by the Ministry of Ecology and Environment

Foreword ... 1 Scope ... 1 2 Normative references ... 1 3 Terms and definitions ... 1 4 Methodology ... 2 5 Interference and cancellation ... 2 6 Reagents and materials ... 2 7 Instruments and equipment ... 3 8 Sample ... 4 9 Test procedure ... 5 10 Calculation and Representation of Results ... 7 11 Effectiveness, sensitivity, and precision ... 7 12 Quality Assurance and Quality Control ... 8 13 Test report ... 8 14 Waste disposal ... 8 Appendix A (informative) Zebrafish eggs related developmental stages ... 9 Appendix B (Informative) Domestication and maintenance of zebrafish fingerlings ... 10 Appendix C (Informative) Reference pictures of spawning boxes ... 11 Appendix D (Informative) Zebrafish Egg Identification ... 12 Appendix E (informational appendix) 24-well cell culture plate test layout setting scheme ... 15 Appendix F (Informative) Recommended Data Record Form ... 16 Appendix G (informative) EC50 calculation of EC50 ... 18

Foreword

In order to implement the "Environmental Protection Law of the People's Republic of China" and the "Law of the People's Republic of China on Water Pollution Control", protect the ecology The environment, safeguard human health, standardize the zebrafish egg method for the determination of acute toxicity of water quality, and formulate this standard. This standard specifies the zebrafish egg method for the determination of acute toxicity in surface water, groundwater, domestic sewage and industrial wastewater. Appendices A to G of this standard are informative appendices. This standard is issued for the first time. This standard is formulated by the Department of Eco-Environmental Monitoring, Laws and Standards Department of the Ministry of Ecology and Environment. This standard was drafted. Changzhou Environmental Monitoring Center of Jiangsu Province, Environmental Benchmark and Risk Assessment of Chinese Academy of Environmental Sciences State Key Laboratory. This standard is verified by. Environmental Analysis and Testing Technology Center of Chinese Academy of Environmental Sciences, China Environmental Monitoring Station, Health Research on Quality Standards of Agricultural Products of Nanjing Institute of Environmental Sciences, Zhejiang Province Environmental Monitoring Center, Zhejiang Academy of Agricultural Sciences Institute and Jiangsu Guochuang Environmental Technology Co., Ltd. This standard was approved by the Ministry of Ecology and Environment on December 31,.2019. This standard will be implemented from June 30, 2020. This standard is explained by the Ministry of Ecology and Environment. Water quality-Determination of acute toxicity-Zebrafish egg method

1 Scope

This standard specifies the zebrafish egg method for the determination of acute toxicity to water quality. This standard applies to the determination of acute toxicity of surface water, groundwater, domestic sewage and industrial wastewater.

2 Normative references

The content of this standard refers to the clauses in the following documents. For undated references, the valid version applies to this standard. GB/T 6920 Determination of pH value of water quality Glass electrode method GB/T 13267 Determination of acute toxicity of water quality substances to freshwater fish (zebrafish) HJ 91.1 Technical Specifications for Sewage Monitoring HJ 506 Determination of dissolved oxygen in water HJ/T 91 Technical specifications for surface water and sewage monitoring HJ/T 164 Technical Specifications for Groundwater Environmental Monitoring

3 terms and definitions

The following terms and definitions apply to this standard. 3.1 Fish egg Refers to egg cells that are in the entire developmental stage before rupture of the egg membrane. See Appendix A for zebrafish egg-related developmental stages. 3.2 Dilution level The original water sample accounts for the reciprocal of the total number of points of the diluted water sample, which is generally expressed by D. For example, if the water sample is not diluted, Multiple D = 1; take 250 ml water sample and dilute to 1000 ml (that is, the volume fraction is 25%), then the dilution multiple D = 4. 3.3 Lowest ineffective dilution The minimum dilution factor that does not produce a test effect during the test. This standard refers to the lowest water sample when not less than 90% of the eggs are alive. Dilution multiple, expressed as LID. 3.4 EC50 It refers to the percentage of the original water sample that caused 50% of zebrafish eggs to have an effect on the total volume of the diluted water sample after 48 hours of exposure. EC50 Means. EC50 in this standard is LC50. 3.5 Reference substance In tests, known positive control substances used to verify the sensitivity of the method, such as 3,4-dichloroaniline in this standard.

4 Method principle

Multiwell cell culture plates were used to control 4-cells under conditions controlled by microplate controls, negative controls, and positive controls. Stage to 128-cell stage zebrafish fertilized eggs are placed in water samples with different dilutions and cultured at 26 ° C ± 1 ° C for 48 hours. h. Calculate the LID or EC50 value based on the statistics of survival and death of fish eggs to characterize the acute toxicity of water samples.

5 Interference and cancellation

The turbidity or chromaticity of the water sample interferes with the end point of the test. A plastic dropper (7.11) can be used to slowly remove the porous cell culture plate (7.8) After a certain amount of water sample has been sucked out, observe it to avoid touching the eggs. After the observation, if you need to continue to expose, use plastic drops. Tube (7.11) returns the original water sample to restore the original exposed volume.

6 Reagents and materials

6.1 Test organism 6.1.1 Fish species Use robust, disease-free, deformity-free, wild-type zebrafish with high fertility and fertilization rates as breeding fish for spawning. to make The fish body is about 3.5 cm long and the fish age is between June and 24 months. The belly of healthy females is bright and plump. Healthy male body The figure is slender, the abdomen is flat, and the body has a golden yellow sheen. Breeders should not be given any medication for 6 months before spawning. Fish breeding See Appendix B for specific conditions for maintenance and maintenance. 6.1.2 Fish eggs Ensure that the fertilization rate of the test fish eggs is ≥70%, and it is in the 4-cell phase to 128-cell phase (see Appendix A). 6.2 Reagents Unless otherwise specified, analytical reagents that meet national standards are used. Test water using fresh distilled water or Ionized water meets the requirements of pH 6.5 ~ 8.5 and conductivity < 10 μS/cm. 6.2.1 Sodium hydroxide (NaOH). 6.2.2 Calcium chloride (CaCl2 · 2H2O). 6.2.3 Magnesium sulfate (MgSO4 · 7H2O). 6.2.4 Sodium bicarbonate (NaHCO3). 6.2.5 Potassium chloride (KCl). 6.2.6 3,4-dichloroaniline (C6H5Cl2N), excellent grade pure. 6.2.7 Hydrochloric acid. ρ (HCl) = 1.19 g/ml. 6.2.8 Hydrochloric acid solution. c (HCl) = 0.1 mol/L. Measure 8.3 ml of hydrochloric acid (6.2.7) and make up to 1000 ml with water. 6.2.9 Sodium hydroxide solution. c (NaOH) = 0.1 mol/L. Weigh 4 g of sodium hydroxide (6.2.1), dissolve in a small amount of water, and make up to 1000 ml with water. 6.2.10 Reference material 3,4-dichloroaniline stock solution. ρ (C6H5Cl2N) = 100 mg/L. Weigh 0.05 g of 3,4-dichloroaniline (6.2.6), dissolve it in a small amount of standard dilution water (6.2.16), and use standard dilution water (6.2.16) Make up to 500 ml, let it stand for 24 h, and adjust the pH to 7.0. Keep refrigerated in the dark, the stock solution can store 6 month. 6.2.11 Reference substance 3,4-dichloroaniline working solution. ρ (C6H5Cl2N) = 3.7 mg/L. Measure 3.7 ml of reference material 3,4-dichloroaniline stock solution (6.2.10), and use the standard of dissolved oxygen concentration to reach the air saturation value. Adjust the volume of quasi-diluted water (6.2.16) to 100 ml, and equilibrate to 26 ° C ± 1 ° C according to step 8.2.1 before use. The solution as positive In contrast, it is available immediately before use. 6.2.12 Stock solution of calcium chloride. ρ (CaCl2 · 2H2O) = 11.76 g/L. Weigh 11.76 g of calcium chloride (6.2.2), dissolve in a small amount of water, and make up to 1000 ml with water. Can be stored for 6 months. 6.2.13 Magnesium sulfate stock solution. ρ (MgSO4 · 7H2O) = 4.93 g/L. Weigh 4.93 g of magnesium sulfate (6.2.3), dissolve in a small amount of water, and make up to 1000 ml with water. Can be stored for 6 months. 6.2.14 Stock solution of sodium bicarbonate. ρ (NaHCO3) = 2.52 g/L. Weigh 2.52 g of sodium bicarbonate (6.2.4), dissolve in a small amount of water, and make up to 1000 ml with water. Can be stored for 6 months. 6.2.15 Potassium chloride stock solution. ρ (KCl) = 0.22 g/L. Weigh 0.22 g of potassium chloride (6.2.5), dissolve in a small amount of water, and make up to 1000 ml with water. Can be stored for 6 months. 6.2.16 Standard dilution water. Calcium chloride stock solution (6.2.12), magnesium sulfate stock solution (6.2.13), sodium bicarbonate stock solution (6.2.14) and chlorine Potassium stock solution (6.2.15) Mix the four stock solutions with 25 ml each, and make up to 1,000 ml with water.

7 instruments and equipment

7.1 Refrigerated sampling box. 2 ℃ ～ 8 ℃; 7.2 pH meter. The measuring range is 0-14, and the minimum division is 0.1 pH unit; 7.3 Dissolved oxygen analyzer. The measurement range is 0 mg/L ～ 20 mg/L, and the minimum division is 0.1 mg/L; 7.4 Inverted microscope or stereo microscope. the minimum magnification is 30 ×; 7.5 Refrigerator. 2 ℃ ～ 8 ℃ in refrigerating room; ≤-18 ℃ in freezer; 7.6 Constant temperature incubator or constant temperature room. the temperature can be adjusted to 26 ℃ ± 1 ℃; 7.7 Zebrafish breeding system. including systems for water purification, storage, water supply, water quality control and recycling; 7.8 Multiwell cell culture plate. 2.5ml ～ 5ml per well or other cell culture plates with the same function. Commercially available 7.9 Spawning box. inert material, recommended size. 20 cm × 10 cm × 11 cm (see Appendix C), can be selected for sale; 7.10 Thermometer. 0 ℃ ～ 50 ℃; 7.11 Plastic dropper. 5 ml, caliber greater than 3 mm; 7.12 Utensils and equipment commonly used in general laboratories.

8 samples

8.1 Collection and storage of samples Use brown glass bottles (or polypropylene, polytetrafluoroethylene) with a capacity of 1,000 ml or more according to the nature of the sample and monitoring needs Ene, polyethylene container) Water samples shall be collected in accordance with the requirements of HJ 91.1, HJ/T 91 or HJ/T 164. Pour the inner wall of the bottle slowly, leaving no gap between the bottle and the stopper. After the water sample is collected, immediately transport and store it in the dark at 2 ℃ ～ 8 ℃, and carry out the test as soon as possible. After 48 h. For long-term storage of water samples, they should be returned to the laboratory for freezing (≤-18 ° C) as soon as possible after collection. Mix the water sample thoroughly before storage, and dispense the water sample in a volume of 500 ml to 700 ml per 1000 ml container. The storage period should not exceed Over 2 months. 8.2 Sample preparation 8.2.1 Temperature Freeze-preserved water samples should be thawed in a water bath at a temperature not exceeding 25 ° C, or refrigerated at 2 ° C-8 ° C overnight. Accept Before the test, the test water sample is placed in a constant temperature incubator or a constant temperature room (7.6). For testing. 8.2.2 pH Determine the pH of water samples according to GB/T 6920 method. When pH value is < 6.5 or pH value> 8.5, use hydrochloric acid solution (6.2.8) or sodium hydroxide solution (6.2.9) to adjust the pH value of the water sample to 6.5 ～ 8.5, and reduce the amount of acid-base adjusting solution To reduce the effect on the concentration of water samples. Measure and record the pH value of the water sample after adjustment, and perform water sample measurement according to the subsequent steps. When the effect of pH value needs to be reflected in the test results or adjusting the pH value will cause physical degradation or chemical reaction of the water sample In this case, the pH of the water sample is not adjusted. According to the actual needs of water sample measurement, test comparison can also be carried out on water samples before and after pH adjustment. 8.2.3 Dissolved oxygen Determine the dissolved oxygen concentration of the water sample according to the HJ 506 method to ensure that it is used for each dilution of water in the exposure test (9.3) step The initial dissolved oxygen concentration in the sample is not less than 4 mg/L (about 50% saturation). For oxygenation, the dissolved oxygen concentration should not exceed the corresponding Saturated dissolved oxygen value. 8.3 Sample dilution 8.3.1 Pre-experiment When the concentration is difficult to predict, a pre-test is required, and a dilution ratio of 10 is used to select 3 consecutive concentrations of diluted water samples. Test with 5 fish eggs at a concentration of 9.3 ～ 9.6 to determine that the fish egg mortality rate is 100% ～ 0% and the concentration of the diluted water sample is large. To the corresponding range. 8.3.2 Dilution when measuring LID In order to ensure the highest dilution multiple of water sample fish eggs, the survival rate of ≥90%, and the lowest dilution multiple of water sample fish eggs, if possible 90%, generally choose 4 ~ 5 in succession (select 2 ~ if the approximate correspondence between fish egg survival rate and water sample concentration is known 3) Dilution multiple water samples. Refer to Table 1 for water sample dilution. Table 1 Dilution of water samples Dilution Number D Raw water samples after dilution Total number of water samples (%) Dilute water sample Composition of diluted water sample (ml) Required standards Dilution water Required water sample volume Resulting dilution Water volume 1 100.0 1x diluted water sample (ie raw water sample) 0 Raw water sample.200.200 2 50.0 2 times diluted water sample 100 raw water sample 100.200 3 33.0 3 times diluted water sample 140 Raw water sample 70 210 4 25.0 4 times diluted water sample 100 2 times diluted water sample 100.200 6 16.7 6 times diluted water sample 100 3 times diluted water sample 100.200 8 12.5 8 times diluted water sample 100 4 times diluted water sample 100.200 12 8.3 12-fold diluted water sample 100 6-fold diluted water sample 100.200 16 6.2 16 times diluted water sample 100 8 times diluted water sample 100.200 24 4.2 24 times diluted water sample 100 12 times diluted water sample 100.200 32 3.1 32-fold diluted water sample 100 16-fold diluted water sample 100.200 8.3.3 Dilution when measuring EC50 To ensure that the mortality rate of the maximum concentration of the diluted water sample is 100% or close to it, the mortality rate of the minimum concentration of the diluted water sample is 0% or close to it, generally 5 consecutive concentrations of diluted water samples are selected according to a certain dilution ratio (generally ≤ 2). Above the standard dilution water used for sample dilution, the water temperature should be balanced to 26 ℃ ± 1 ℃ according to step 8.2.1 before emptying and empty. The air is aerated until the dissolved oxygen concentration reaches the air saturation value.

9 Test procedure

9.1 Mating The breeding of zebrafish fingerlings (6.1.1) began in the evening before the test. The washed and dried spawning box (7.9) was placed in the inner tank. Outer tank, insert the partition, add about 2/3 tank of standard dilution water (6.2.16), select the body length and fish age are equivalent, sexual development characteristics are obvious Show male, male and female species of healthy and active fish. Add 1 female and 2 males to the left and right compartments, cover the cover, and Place in a constant temperature incubator or constant temperature room (7.6) in the dark overnight. Follow the above steps to breed at least 3 groups of fish. 9.2 Spawning and initial screening After the next day's light starts, open the lid of the spawning box (7.9), extract the inner tank, discard the water in the outer tank, and put it back in the inner tank Carefully add standard dilution water (6.2.16) along the inner tank wall, and the water surface should be about 2 species wide from the bottom of the inner tank to avoid injury. Harmful fish. Remove the baffle and let the male and female breeding fish mate and lay eggs. After 30 minutes, check the spawning situation of each type of fish and collect the layed eggs. The corresponding fish eggs in the eggs are washed in the crystallizing dish with standard dilution water (6.2.16) and placed in a constant temperature incubator or constant temperature room (7.6) After being left to stand for 45 minutes in the middle, the crystallizing dish is contrasted with a black background, and the side is observed by light. Randomly choose fish eggs 20 in each crystallizing dish. Grains identify fertilized fish eggs according to the method in Appendix D.1 to avoid harming the fish eggs. Count the fertilization rate of fish eggs produced by each group of fish, to The three groups of fish eggs with a fertilization rate ≥70% should be selected and mixed for later use. 9.3 Exposure test Add the prepared diluted water samples of different concentrations to the porous cell culture plate (7.8) according to the test layout set in Appendix E. 2 ml per well. Take 60 ml to 70 ml of each of the above diluted water samples in different culture dishes, and use plastic droppers (7.11) in the spare fish eggs. Take 20 round, plump, complete tablets each into the above petri dish, and place each petri dish in an inverted microscope or a stereo microscope (7.4) Observe the next one by one, select the 4-cell stage to 128-cell stage (see Appendix A), and remove the cells Fish eggs with obvious abnormalities during division (such as asymmetry, vesicles, or no egg membrane) or damaged egg membrane After adding water samples Add 1 fertilized egg to the corresponding well of the porous cell culture plate (7.8), and cover the cover. The above operation process should be completed within 60 minutes after the fish eggs (9.2) stand for 45 minutes. Place the above porous cell culture plate (7.8) in a constant temperature incubator or a constant temperature chamber (7.6) according to the water temperature and light in Appendix B. The 48 h exposure test was performed under the conditions. 9.4 Fish egg microscopy to determine the end of the test The end points of the test were egg coagulation, somite formation, tail separation, and no heartbeat. The determination method is as follows (see Appendix D.2). a) Egg condensation The contents of the coagulated fish eggs are completely opaque and hard in texture. The coagulated fish eggs are opaque And a gloomy state. b) Somite is not formed In the middle and posterior part of the outer side of the yolk sac of the fish egg yolk sac, those with no somite are not formed. c) The tail is not separated In normal developing fish eggs, the tail of the embryo will elongate and separate from the yolk sac. If not, the tail is not separated. If After 48 hours, the degree of embryo tail separation was not significantly different from that at 24 hours, and it was also determined that the tail was not separated. d) No heartbeat The zebrafish egg embryo heart is located between the yolk sac and the embryo's head. Indicates no heartbeat. Place the porous cell culture plate (7.8) of the above exposure test under an inverted microscope or a stereo microscope (7.4) according to the table 2 Require microscopy of fish eggs. After the fish eggs were exposed for 24 h and 48 h, if any of the test endpoints occurred, the fish eggs were judged dead, and each diluted water sample was counted. Survival and mortality of fish eggs. Table 2 Observation requirements for fish eggs microscopic examination Test endpoint egg coagulum somite did not form a tail without separation without heartbeat Observation method microscope or naked-eye microscope microscope Observation time 24 h exposure- 48 h Note. “” means observe, judge and record, “-” means this observation is not needed. 9.5 Controlled trials For each batch of water sample test, the control test shall be carried out according to the test layout of Appendix E and 9 steps. a) Microplate control The standard well-diluted water (6.2.16) was used as a water sample for the microplate control test. b) negative control Negative control tests were performed using standard dilution water (6.2.16) as the water sample. c) Positive control A 3,4-dichloroaniline working solution (6.2.11) was used as a water sample for the positive control test. The above 9 test steps all use standard dilution water (6.2.16) for breeding (9.1), spawning and initial screening of fish eggs (9.2) For microplate control and negative control tests (9.5), the water temperature should be equilibrated to 26 ° C ± 1 ° C according to 8.2.1, and aerated with air to The dissolved oxygen concentration should be at least 80% of the air saturation value. The temperature of the laboratory involved is controlled at about 26 ° C, in a constant temperature incubator or The temperature of the constant temperature chamber (7.6) is controlled at 26 ℃ ± 1 ℃. 10 Calculation and representation of results The lowest dilution factor for fish egg survival rate of 90% and above is the lowest non-effect dilution factor LID. LID results Should be an integer, such as LID = 2. The EC50 of the water sample can be calculated according to the method in Appendix G, and it can also be calculated by other methods such as the probability unit method and the linear interpolation method. The result is retained to one digit after the decimal point. 11 Effectiveness, sensitivity and precision 11.1 Effectiveness and sensitivity Six laboratories performed 6 replicate determinations on negative control water samples (6.2.16 standard dilution water). survival rates between 90% to 100%. Six laboratories performed 6 repeated tests on positive control water samples (6.2.11 reference substance 3,4-dichloroaniline working solution) Definition. Mortality is between 40% and 60%. 11.2 Precision Six laboratories respectively tested negative control water samples (6.2.16 standard dilution water), ρ = 1.3 mg/L, ρ = 3.7 mg/L, Low, medium and high concentration water samples with ρ = 6.3 mg/L (prepared with the positive reference material 3,4-dichloroaniline) Determination of acute toxicity of eggs, each water sample is measured on average 6 times. the relative standard deviations of the first three fish eggs survival rates in the laboratory were 0.0% to 5.8%, 4.5% to 8.5%, 9.7% to 18.1%, and the relative standard deviations between laboratories were 1.8%, 3.5%, 13.6%, repeatability limits of 12.9%, 15.5%, 17.1%, reproducibility limits of 12.7%, 16.8%, 23.4%; the latter fish eggs The relative standard deviation of mortality in the laboratory is 0.0% to 5.3%, the relative standard deviation of the laboratory is 1.5%, and the repeatability limit is 10.6%, the reproducibility limit is 10.6%. Six laboratories respectively treat domestic sewage (drainage from urban sewage treatment plants, LID = 2), industrial wastewater (containing organic chemicals Wastewater and heavy metal wastewater, LID = 4) The acute toxicity of zebrafish eggs was determined. Each water sample was measured on average 6 times. when When the sewage and industrial wastewater water samples are at the lowest non-effect dilution factor LID, The standard deviations were 5.3% to 5.5% and 5.3% to 5.8%, and the relative standard deviations between laboratories were 1.9% and 1.4%, respectively. The renaturation limits were 14.5% and 14.8%, and the reproducibility limits were 14.2% and 14.0%. Six laboratories tested zebrafish eggs for acute reference substance 3,4-dichloroaniline (EC50 = 3.1 mg/L) The determination of EC50 was performed 6 times in each laboratory. the relative standard deviation in the laboratory was 2.9% to 12.7%, and The standard deviatio...

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