GB/T 29763-2013 化学品 稀有鮈鲫急性毒性试验 GB/T29763-2013 标准压缩包解压密码：www.bzxz.net
本标准规定了稀有鮈鲫化学品急性毒性试验的方法,包括静态试验、半静态试验和流水式试验三种方式,受试物所需信息、原理、设备与材料、试验系统、试验程序、质量控制和结果报告。 本标准适用于测试稀有鮈鲫的化学品急性毒性。
本标准按照GB/T1.1—2009给出的规则起草。
本标准由全国危险化学品管理标准化技术委员会(SAC/TC251)提出并归口。
本标准起草单位:上海市检测中心、环境保护部化学品登记中心、中国科学院水生生物研究所、中国检验检疫科学研究院、环境保护部南京环境科学研究所、沈阳化工研究院有限公司安全评价中心。
本标准主要起草人:殷浩文、张京佶、赵华清、沈英娃、刘纯新、聂晶磊、王剑伟、陈会明、李晞、石利利、刘济宁、王蕾、蔡磊明、赵玉艳。
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GB/T27861 化学品 鱼类急性毒性试验

ICS13.300;11.100
National Standard of the People's Republic of China
GB/T29763—2013
Chemicals
Rare crucian carp acute toxicity test
Chemicals-Rare Minnow(Gobiocypris rarus)acute toxicity test2013-09-18 Released
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China China National Standardization Administration
Implemented on 2014-03-20
This standard Drafted in accordance with the rules given in GB/T1.1-2009. This standard is proposed and coordinated by the National Standardization Technical Committee for the Management of Hazardous Chemicals (SAC/TC251). GB/T29763—2013
This standard was drafted by: Shanghai Testing Center, Chemical Registration Center of the Ministry of Environmental Protection, Institute of Hydrobiology, Chinese Academy of Sciences, Chinese Academy of Inspection and Quarantine, Nanjing Institute of Environmental Sciences of the Ministry of Environmental Protection , Safety Evaluation Center of Shenyang Chemical Industry Research Institute Co., Ltd. The main drafters of this standard: Yin Haowen, Zhang Jingcai, Zhao Huaqing, Shen Yingwa, Liu Chunxin, Nie Jinglei, Wang Jianwei, Chen Huiming, Li, Shi Lili, Liu Jining, Wang Lei, Cai Leiming, Zhao Yuyan. 1Scope
Acute toxicity test of rare crucian carp chemicals
GB/T29763—2013
This standard specifies the acute toxicity test method for rare crucian carp chemicals, including static test and semi-static test Three methods of test and flow test, information required for the test object, principles, equipment and materials, test system, test procedures, quality control and result reporting. This standard is suitable for testing the acute toxicity of chemicals in rare abalone. 2 Normative reference documents
The following documents are essential for the application of this document. For dated reference documents, only the dated version applies to this document. For undated referenced documents, the latest version (including all amendments) applies to this document. GB/T27861 Chemicals Acute Toxicity Test for Fish 3 Terms and Definitions
The terms and definitions defined in GB/T27861 apply to this document. 4
The information required for the test substance
The information required for the test substance includes:
a) Solubility in water;
b) Vapor pressure;
e) Structural formula;
d) Purity;
stability in water and under light conditions; e
pKa value;
quantitative analysis method in aqueous solution;| |tt|| Partition coefficient of n-octanol-water (P);
Rapid biodegradation test results. || tt | The aqueous solution of the substance, with 96h as the test period, record the mortality of the test fish at 24h, 48h, 72h and 96h, and determine the concentration of the test substance at which 50% of the rare crucian carp dies, expressed as LCs. 6 Equipment and materials
6.1 Instruments and equipment
Instruments and equipment include:
GB/T29763-2013
Dissolved oxygen meter (1%):
Water hardness meter (1mg/L)
Temperature control system (0.1℃);
d) pH meter (0.1 pH);
Analytical balance (0.0001g);||tt ||An aquarium or water tank 1 made of inert materials with consistent specifications and appropriate volume; suitable for flow-type continuous distribution and dilution systems (such as metering pumps, diluters, etc.). g)
6.2 Materials and Reagents
Reagents used in standard dilution water include:
a) Calcium chloride;
b) Magnesium sulfate; ||tt| |c) Sodium bicarbonate;
d) Potassium fluoride.
All reagents should be of analytical grade and prepared with distilled or deionized water with a conductivity less than or equal to 10μS/cm. 7 Test system
7.1 Selection of test method
7.1.1 Before the test, the appropriate test method should be determined based on the chemical stability of the test object in water, so as to select the required container and "Device".
2 During the test, the concentration of the test substance in the aqueous solution is maintained within ±20% of the initial measured concentration. The static test method can be used. When the test substance in the aqueous solution is unstable, the concentration When the change exceeds ±20%, a semi-static test method or a flow test system should be used to maintain the concentration of the test substance
7.2 Rare crucian carp
7.2.1 Select
test. The fish species is rare crucian carp (Gobiocyprisrarus), with a total length of 30mm and 10mm. It should be kept under fish farm and laboratory conditions (pH 6.0~8.5, temperature 21℃~25C: light 12h~16h per day; dissolved oxygen concentration) The fish should be acclimated and raised under conditions that do not exceed 80% of the air saturation value. It should be ensured that the fish used for one test are of the same batch, with consistent specifications, reliable sources, and no obvious deformities. 7.2.2 Temporary raising and breeding ||tt ||7.2.2.1 Rare animals should be kept in the laboratory for at least 12 days before being used for testing. The holding conditions can be consistent with the individual breeding conditions. They should be tamed for at least 7 days in an environment that meets the following conditions. 2.2
Water: the same quality as the test water;
Light: 12h16h per day;
Temperature: 21℃~25℃;
Dissolved oxygen concentration: not lower than air 80% of the saturation value: e
feeding, 3 times a week or every day until 24 hours before the start of the test. According to the physical and chemical properties of the sample, select a suitable test container, such as glass, etc. Choose the test method and load. 2
7.2.2.3 48 hours after the start of domestication, record the mortality rate and handle it according to the following conditions: GB/T29763-2013
a) The mortality rate is less than 5 within 7 days. %, can be used for testing. b) If the mortality rate is between 5% and 10% within 7 days, continue to raise it for 7 days. If death does not occur again, the batch of test fish can be used for testing. e) If the mortality rate is greater than 10% within 7 days, discard it. The whole group of fish. 7.3 The total hardness of the test water
should be standard dilution water (see Appendix A), tap water with aeration and dechlorination, or high-quality water. Calculate CaCO) pH is 6.0~8.57.4 Preparation of test substance
7.4.1 Dissolve the test substance in test water to prepare a test substance stock solution, and dilute the stock solution to prepare a test with a certain concentration Solution.
7.4.2 The stock solution of poorly soluble substances can be prepared by ultrasonic dispersion or other suitable physical methods. When necessary, organic solvents, emulsifiers and dispersants with low toxicity to fish can be used. A solvent control test group should be added, and the solvent concentration in all test containers should be the same and should not exceed 100 mg/L or 100 μL/L. 7.4.3 The pH of the test substance stock solution should be adjusted before adding it to make it close to the pH of the test water. When adjusting the pH of the stock solution, the concentration of the test substance should not change significantly, and chemical reactions or precipitation should not occur. HCI and NaOH should be used for adjustment. 7.4.4 If the pH of the water changes significantly after adding the test substance (exceeding 6.0 to 8.5), prepare the test solution again. 8 Test procedures
8.1 Exposure conditions
Duration: 96h
b) Carrying capacity: The total wet weight of fish per liter of water in static and semi-static test systems does not exceed 1.0g, running water The load capacity of the test system can be appropriately increased.
Lighting: 12h~16h every day
Temperature: 21℃~25℃ and the temperature change during the test shall not exceed 2℃. d
Dissolved oxygen concentration: should not be less than 60% of the air saturation value. The concentration of the test substance should not change significantly during aeration. e) Road
f) Feeding: fasting.
g) Thousand Disturbance: Avoid disturbances that may affect fish behavior. 8.2 Pre-test
8.2.1 To determine the concentration range required for the formal test, a larger range of concentration series can be selected, such as 1000mg/L, 100mg/L, 10mg/L, 1mg/L, 0.1mg/ L. No less than 3 rare crucian carps will be released in each concentration group, and there will be no parallel groups. The test time is 96h. Record the number of dead fish in each container every day and remove the dead fish in a timely manner. 8.2.2 If the results of a preliminary test cannot determine the concentration range required for the formal test, the concentration range should be adjusted and the preliminary test should be conducted again. 8.3 Formal test
8.3.1 According to the preliminary test, at least 5 concentration groups should be set between the lowest concentration that causes all rare fish to die and the highest concentration that causes all rare fish to survive after 96 hours of unloading, and they should be arranged in a "geometric series". The concentration interval coefficient should be less than or equal to 2.2. At the same time, a blank control group should be set up. If a solvent is used, a solvent control group should be added, and the solvent concentration in all test containers should be the same. At least 7 rare abalone crucian carp should be used in each concentration group and control group.
3) Geometric series.
GB/T29763-2013
8.3.2 After the test solution is adjusted to the test temperature, fish are randomly taken out from the domesticated fish group and randomly and quickly placed in each test container. Fish that are improperly handled during transfer should be discarded. In the same test, all test fish should be grouped within 30 minutes. 8.3.3 During the test, the test solution should not be strongly aerated, and the dissolved oxygen content should be Higher than 60% of the air saturation value. No eating is allowed during the test.
8.3.4 Check the condition of the test fish at least after 24h, 48h, 72h and 96h: If a fish does not have any visible movement (such as the flapping of gills) and does not react after touching the tail stalk, it can be judged that the fish is dead. After observing and recording the number of dead fish, the dead fish should be taken out of the container. The condition of the fish in each test group should be observed 3h or 6h after the start of the test, and any abnormal phenomena of the test fish should be recorded (such as fish body flipping, loss of balance, weakened swimming ability and breathing function, pigmentation, etc.). 8.3.5 Measure the pH, dissolved oxygen concentration and temperature in all test containers at least once a day. Measure at the beginning and end of the test. 8.3.6 Before the start of the test, an effective chemical analysis method for the test substance in water should be established. During the test, the concentration of the test substance in water should be measured according to the following requirements,| |tt||a) For static test methods, the test substance concentration of the test solution in the test container should be measured at least at the beginning and end of the test. For semi-static test methods, the test substance concentration of the test solution in the test container should be measured at least at the beginning of the test, before and after each renewal, and at the end of the test.
c) For flow-through test methods, the test substance concentration of the test solution in the mother liquor and the test container should be measured at least at the beginning, middle and end of the test.
8.4 Limit test
When this standard is adopted, in order to prove whether LCe is greater than 100 mg/L (active ingredient concentration) or greater than the saturated liquid concentration of the test substance under the test system, a limit test with a test substance concentration of 100 mg/L (active ingredient concentration) or a saturated solution of the test substance under the test system can be carried out. The limit test should use at least 7 rare potassium fish, which is different from the fish used in the control group. The number of fish should be equal. If solvent is used, a solvent control group should be added and the solvent concentration in all test containers should be the same. If the test fish dies, the complete test should be carried out according to the test procedures of this standard. The observed sublethal effects (such as fish turning over, loss of balance, reduced swimming ability and respiratory function, pigment deposition, etc.) should be recorded. 9 Quality Control
An effective test should meet the following conditions:
a) At the end of the test, the mortality rate of the control group fish should not exceed 10%. If there are less than 10 fish in each group, the death of the control group should not exceed 1 fish:
b) During the test, the dissolved oxygen concentration of the test solution should not be less than 60% of the air saturation value; e) During the test, the conditions should be maintained as constant as possible. If necessary, a semi-static or flow-through test method should be used; d) During the test, the concentration of the test substance in the test solution should not be less than 80% of the prepared concentration. If it is less than 80% of the prepared concentration, the test results should be expressed in terms of the measured concentration.
Data and Report
10.1 Result Processing
10.1.1Use appropriate statistical methods to calculate the LCso value and 95% confidence limit for a certain exposure time. At the same time, draw a curve of test concentration versus mortality.
10.1.2If the test data are not suitable for calculating LCs using the standard method, the highest concentration that does not cause mortality and the lowest concentration that causes 100% mortality can be used to estimate the LCs, that is, the geometric mean of these two concentrations. 10.1.3When conducting a limit test, the concentration used in the limit test should be reported, and it should be stated that the LCs are greater than the concentration taken in the limit test: 4
GB/T29763—2013
10.1.4During the test, if the actual concentration of the test substance is maintained at ±20% of the prepared concentration, the results can be expressed using the prepared concentration. If the measured concentration of the test substance cannot be maintained at ±20% of the prepared concentration or the LCso value of the test substance is near the boundary value of the hazard assessment classification (e.g., 1 mg/L, 10 mg/L, 100 mg/L), the measured concentration shall be used to express the results. 10.2 Test report
The test report shall at least include the following: a) Physical properties and related physicochemical characteristics of the test substance, basic information (see Chapter 4), etc. b)
Specifications, sources, health conditions, breeding conditions, etc. Test conditions
Test process: static, semi-static or flow-through, aeration, carrying capacity, etc. Water quality characteristics: dissolved oxygen, pH value, temperature, hardness, etc.; dissolved oxygen concentration, pH value, temperature in the test solution every 24 hours (in semi-static tests, the dissolved oxygen concentration, pH value, and temperature should be measured before and after renewal);
Methods for preparing stock solutions and test solutions; ·Concentration information of the test substance in the test solution; ·The number of fish used for each test concentration.
d) Test results
The highest concentration during the test at which no death occurred; the lowest concentration during the test that caused 100% death; the cumulative mortality rate of each concentration at each observation time; if possible, the LCso at each observation time and its 95% confidence limit. Concentration-mortality curve after the test; statistical method for determining LC value;
mortality rate of the control group;
factors during the test that may affect the test results; abnormal reactions of fish poisoning.
Result discussion
4) HJ/T154—2004 Guidelines for Hazard Assessment of New Chemical Substances. 5
GB/T29763—2013
A.1 Preparation of standard dilution water stock solution
Appendix A
(Informative Appendix)bzxZ.net
Preparation of standard dilution water
Calcium chloride solution: Dissolve 11.76gCaClz·2H.O in water and dilute to 1L. a)
Magnesium sulfate solution: Dissolve 4.93gMgSO.7H,0 in water and dilute to 1L Sodium bicarbonate solution: Dissolve 2.59gNaHCO, in water and dilute to 1L. Potassium chloride solution: Dissolve 0.23gKCI in water and dilute to 1L. A.2 Preparation of standard dilution water
Mix 25mL of each of the above four solutions and dilute to 1L with water. The total amount of calcium ions and magnesium ions in the solution is 2.5mmol/L, the concentration ratio of calcium ions to magnesium ions is 4:1, and the concentration ratio of sodium ions to potassium ions is 10:1. The acid capacity constant Ks in the solution is 0.8mmol/L
The dilution water should be aerated until oxygen saturation, and then stored for 2 days for standby use, and no aeration is required before use. 6
References
GB/T29763-—2013
[1] ISO764l-1 Water quality-Determination of the acute lethal toxicity of substances to afreshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)J-Part lStaticmethod,1996.
[2] ISO 7641-2 Water quality- Determination of the acute lethal toxicity of substances to afreshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)J-Part 2; Semi-staticmethod,1996.
[3] ISO 7641-3 Water quality-Determination of the acute lethal toxicity of substances to afreshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,Cyprinidae)J-Part 3:Flow-through method, 1996. [4] State Environmental Protection Administration Chemical Testing Methods (2004) 203 Fish Acute Toxicity Test [5] OECD SERIES ON TESTING AND ASSESSMENT Number 10. REPORT OF THE OECD WORKSHOP ON STATISTICAL ANALYS ISO FAQUATIC TOXICITY DATA, 1998. [6] Working Document Guidance Document on Aquatic Ecotoxicology in the context of the Directive 91/414/EEC, 2002 [7] OECD SERIES ON TESTING AND ASSESSMENT Number 23. GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, 2000.
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