Some standard content:
CS13.300;13.020.40
National Standard of the People's Republic of China
GB/T 21812—2008
Chemicals
Honeybees, acute oral toxicity test
Chemicals-Honeybees, acute oral toxicity testIssued on 2008-05-12
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Administration of Standardization of the People's Republic of China
Digital anti-corrosion
Implemented on 2008-09-01
GB/T 21812—2008
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 213 (1998) Honeybees, acute oral toxicity test (English version).
This standard has been edited as follows:
- The scope section has been added;
: The measurement units have been changed to the legal measurement units of my country; - The terms and definitions in the appendix have been placed in the terms and definitions section of the main text. This standard was proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251). The responsible drafting unit of this standard is: China Inspection and Quarantine Scientific Research Institute. The participating drafting unit of this standard is: Ningbo Exit-Entry Inspection and Quarantine Bureau. The main contributors of this standard are: Zhou Xin, Chen Huiming, Wang Junbing, Yu Wenlian, Lin Zhenxing, Ma Zhongchun, Hao, Wang Lifeng, Sun Xin, Wang Jing. I
GB/T21812—2008
This standard is a laboratory test method developed to determine the acute oral toxicity of pesticides and other chemicals to adult worker bees. This standard is based on the European and Mediterranean Vegetation Protection Organisation (EPPO) Guidance document for the evaluation of adverse effects of vegetation protection products on honey bees (Apismettifera)17. It takes into account the recommendations for improving the EPPO test method presented at the Fifth International Symposium on Plant-Bee Relationships, held in Waggeningen (The Netherlands) in 1993. Other existing guidelines have also been considered, see Refs. [3], 4] and [5]. In the assessment of the toxicological properties of substances, acute oral toxicity tests on honey bees should be included when the chemicals are exposed to bees. Acute oral tests provide a basis for determining the inherent toxicity of pesticides and other chemicals to bees. The results of such tests can be used as a basis for the need for further evaluation. In particular, this standard can be used in a stepwise regression procedure for the evaluation of the toxicity of pesticides to honey bees to determine the need for experimental toxicity tests or field trials, see Ref. [6. A pesticide can be an active ingredient (AI) or a formulated product.
Reference substances are used to verify the sensitivity of bees and the accuracy of the test. 1 Scope
Acute oral toxicity test for chemicals on bees
GE/T 21812—2008
This standard specifies the scope, terms and definitions, test principles, test validity, test preparation, test procedures, data and reports of the acute oral toxicity test for chemicals on bees.
This standard is applicable to the determination of the oral toxicity of pesticides and other chemicals to adult worker bees. 2 Terms and definitions
The following terms and definitions apply to this standard. 2.1
Acute oral toxicityacuteoral toxicity The harmful effect of a single oral dose of the test substance within a test period of up to 96 hours. 2. 2
Dosedose
The total amount of the test substance consumed. The dose is expressed in terms of the mass of the test substance (g) or the mass of the test substance used for a single test animal (g/bee). When bees are fed en masse, the actual dose cannot be calculated, but it can be estimated using the average dose (total amount of test substance consumed in one cage/number of test bees). 2. 3
Oral lethal dose LD (mcdian etheral dose) is the single dose of a test substance that causes 50% mortality in animals when administered orally. LDs are expressed in "g/bee" For pesticides, the test substance can be an active ingredient (α.i.) or a formulation containing one or more active ingredients. 2.4
Mortality
The ratio of dead animals to the number of test animals.
3 Principle of the test
Adult worker bees consume the test substance by feeding on a sucrose solution. The bees are then fed the same diet without the test substance. The mortality is recorded for at least 48 h and compared with that of the control group. If the mortality rate continues to increase between 24h and 48h, while the mortality rate of the control group remains at an acceptable level of no more than 10%, the test period should be extended to a maximum of 96h. Calculate the LDs of 24h and 48h, as well as the I.Ds values of 72h and 96h when the test is extended. 4 Validity of the test
The test shall be valid only if it meets the following conditions: 1. The average mortality rate of the control group does not exceed 10% at the end of the test; 2. The LDso value of the reference substance meets the specified range. 5 Test preparation
5.1 Selection of bees
The test bees are required to be in good nutritional condition, healthy, have strong antiviral ability, and be of the same age. The test bees may not come from the same colony, but their family background should be clear and known. Collect them on the morning of the day of use or the night before the test, and keep them under experimental conditions until the start of test 1
GB/T 21812—2008
. Try to avoid collecting bees in spring or late autumn. If the test is conducted during this period, the shark bees should be allowed to eclode in an incubator and fed with pollen and sucrose solution for 1 week. If bees treated with antibiotics, disruptors or other chemical substances are used, they cannot be used for toxicity tests within 4 weeks after the last treatment.
5.2 Test cages
Use cages that are easy to clean and well ventilated. Any suitable material can be used, such as stainless steel, wire, plastic, disposable wooden cages, etc. The size of the test cage should be adapted to the number of bees to provide sufficient space. 5.3 Handling and warming conditions
Treatments and observations during the day (under light): A sucrose solution with a final concentration of 500 mg/L is used as food. After the test dose is given, food can be provided without restriction, and the food intake of each cage is recorded. Feeding is carried out using a glass tube with a length of 50 mm, a width of 10 tntm, and a bottom opening with a diameter of 2 mm.
5.4 Preparation of honey bees
Randomly distribute the collected honey bees to the test cages placed in the experimental case. Starve the honey bees for 2 hours before the test to ensure that the food content in the abdomen of the honey bees is the same at the beginning of the test. Replace the dying honey bees with healthy ones before the test. 5.5 Preparation of test substance dose
If the test substance is a chemical that is easily miscible with water, it can be directly dispersed in a 500g/L sucrose aqueous solution. For products with technical requirements and some substances with low hydrolysis, organic solvents, dispersants or emulsifiers with low toxicity (such as acetone, dimethyl sulfoxide, trimethylformamide) can be used as co-solvents. The degree of co-solvent can be determined according to the hydrolysis of the test substance, and the co-solvent concentration of each concentration group should be consistent. However, the concentration of the auxiliary agent should generally not exceed 1%. Appropriate solvent controls should be prepared, that is, when co-solvents and dispersants are used, two groups of solution controls should be prepared separately, one group is an aqueous solution control, and the other group is a sucrose solution control containing solvent, and the solvent concentration is the same as that in the test group. 6 Test Procedure
6. 1 Test group and control group
The setting of test concentration and the number of parallel tests should meet the statistical requirements for determining LD under 95% confidence limit. Usually, when conducting tests, it is required to set at least 5 concentrations arranged in geometric series within the concentration range containing LLDs, and the ratio of concentration group intervals should not exceed the range specified in Chapter 4. In addition, the setting of dilution factors and the number of test substance concentrations should also consider the correlation of the slope of the toxicity curve expressed as density versus mortality, as well as the statistical analysis method selected for result analysis. A preliminary test should be carried out first to determine the concentration range for the formal test. ||t t||Minimum requirement: Three parallels are set for each concentration group, and 10 bees are placed in each test container. The control group should have at least 3 parallels, each with 10 bees, and should be carried out simultaneously with the treatment group. The control group should also include a co-solvent or vehicle control. 6.2 Reference substance
The reference substance should be included in the test series. The reference substance concentration should be selected to cover at least 3 doses of the expected LDsc value. Each dose has 3 parallels, each with 10 shark bees. The preferred reference substance is dimethoate, and its reported 24h acute oral toxicity LDs values range from 0.10μB (ai)/squid to 0.35g (ai)/bee, see reference [7]. Other reference substances may be acceptable if there are sufficient data to demonstrate the expected dose response.
6.3 Piaolu
6.3.1 Dosage treatment
In each bee test group, 100μL to 200μL of sucrose solution containing a certain concentration of the test substance 500g/L is added. For substances with low solubility, a larger volume is required. If the test substance is low in solubility, low in toxicity or has a low concentration in the preparation, a higher proportion of sucrose solution is required. The consumption of treated food in each group is monitored. The food is usually consumed within 3h to 4h. Once the food is consumed, the food container is removed from the cage and replaced with food containing only sugar, without restriction. For some test substances, at the higher test doses, the bees refuse to eat, resulting in little or no food consumption. After up to 6h, the remaining treated food is replaced with sucrose solution. Estimate the amount of processed food consumed (i.e., test the volume or mass of processed food remaining). 2
6.3.2 Test conditions
GB/T 21812--2008
The bees should be kept in a dark laboratory at a room temperature of 25℃±2℃. The relative humidity should also be recorded during the test, usually 50%~70%
6.3.3 Duration
The test starts after the administration of 500 g/L sucrose aqueous solution containing the test substance, and the test lasts for 48 hours. If the mortality rate continues to increase by more than 10% after the first 24 hours, the test needs to be extended to a maximum of 96 hours. At the same time, the mortality rate of the control group shall not exceed 10%. 6.4 Observation
Record the mortality rate 4h, 24h, and 48h after the start of the test (i.e., after the addition of the dose). If the observation time needs to be extended, further evaluation shall be carried out at intervals of 24 hours, up to a maximum of 96 hours. The mortality rate of the control group shall not exceed 10%. Estimate the amount of food consumed by each group. Comparison of the food consumption rates of the treated and untreated groups over a given 6 h period can provide information on the palatability of the treated food. Record any abnormal behavior observed throughout the test. 6.5 Limit Tests
In cases where a test substance is expected to be of low toxicity, a limit test should be conducted to determine whether the LD is greater than the limit value, i.e. 100 ug (ai) bees. The test requirements are the same as the above procedures. If mortality occurs during the limit test, a full test must be conducted. If sublethal effects are observed, they should be recorded.
7 Data and Reporting
7.1 Data
Data should be presented in tabular form, giving the data for each treatment group, control group, and reference substance group, the number of bees used, the mortality rate at each observation time, and the number of bees with abnormal behavior [see references [8] and 9]. Use appropriate statistical analysis methods to analyze the mortality data of bees, draw dose-effect curves for each observation time (i.e. 24 h, 48 h, 72 h, 96 h), calculate the slope of the curve and the mean lethal dose (LD) at 95% confidence limits, see references [9] [10]. If the treated food is not completely consumed, the consumption of the test substance in each group should be determined. L.D5a is expressed as the amount of test substance consumed by each bee, unit/bee. 7.2 Test report
The test report must include the following information:
7.2.1 Test substance:
. 1. Physical properties and corresponding physicochemical characteristics (such as water solubility, vapor pressure); 1. Chemical identification data, including molecular formula, structural formula, purity (such as for pesticides, the characteristics and concentration of the active ingredient). 7.2.2 Test organisms:
- Scientific name, age, collection method, collection period, information on the test honey bee colonies that were not collected, including health status, pretreatment, etc. 7.2.3 Test conditions:
- Temperature and relative humidity of the test chamber:
- Conditions of the test container, including type, size and material; Preparation of test solutions and stock solutions (if a co-solvent is used, give the concentration): Test settings, for example, the concentration and number of test containers used, the number of control groups. For each test: include the concentration and control, the number of replicates and the number of honey bees in each cage. 7.2. 4 Results;
If a preliminary test was conducted, its concentration range; - Raw data on mortality at each observation time; - Dose-effect curve at the end of the test: - - LDsa values of the test substance and the reference substance at 95% confidence limits at each observation time;3
GB/T 21812—2008
-Statistical analysis method for determining LDsc;
-Mortality rate of the control group;
Other biological effects observed or detected, such as abnormal behavior of bees (including the situation that bees refuse to eat when the test substance reaches a certain dose), food consumption rate of the treatment group and the control group; Relevant deviation from the operating method and other relevant information. 4
参考文献
GB/T21812—2008
[1]J EPPO (1992), Guideline on Test Methods for Evaluation the Side-Effects of Plant Prutec-tion Products on Hancybces (No. 170). Bullctin OEPP/EPPO Bulletin, 22, 203-215.[2] Harison, EG (1993). Proceedings of the Fifth International Symposium on the Hazardsof Pesticidcs to Bees, October 26-28, 1993, Plant Protection Service, Wageningen, The Netherlands.Report JUBBS, 14pp+Appendices 1gopp.[3] SETAC (1995), Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesti-cides. Edited by Dr. Mark R, Lynch, Published by SETAC-Eurape,Belgium. March 1g95. [4] Stute, K. (1991). Land-und Forstwirtschaft (BBA),Braunschwcig,Germany.[5] US EPA(1995). Honey Bce Acute Contact Taxicity Test (OPPTS850.3020). EcologicalEffects Test Guidelinces, EPA 71 2-C-95-147, Washington DC, United States ol Ametica.[6] EPPO/Council of Europc. (1993). Decision-Making Scheme for the Environmental RiskAssessmcnt of Plant Protection Products-Honeybees. EPPO bulletin. vol. 23, No. 1, 151-165.March 1993.
[7] Gough, HJ, MeIndoe, EC, Lewis, GB (1994).Ihe use af dimethpate as a referenccampound in laboralary acute toxicity tests on honry bees (Apis mellifera L. ) 198l-1992. Journal ofApicultural Research, 22, 119-125.[8] Litchfield, JT and Wilcaxon, F. (1949). A simplified method of evaluating dose-effect experiments. Jour.Pharmacol. and Exper. Ther.,96,99-113.[9] Finney, DJ (1971). Probit Analysis. 3rd ed,, Cambridge, Landon and New York.[ io] Abbott, WS (1925). A method far computing the cffertiveness of an insecricide. Jour.Econ.Entomol.,18,265-267.
GB/T 21812-200B
People's Republic of China
National Standard
Acute oral toxicity test for bees
Chemicals
GB/T 21812 -2008 wwW.bzxz.Net
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