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GB/T 31270 consists of 21 parts under the general title Test guidelines on environmental safety assessment for chemical pesticides:
——Part 1: Transformation in soils;
——Part 2: Hydrolysis;
——Part 3: Phototransformation;
——Part 4: Adsorption/desorption in soils;
——Part 5: Leaching in soil;
——Part 6: Volatility;
——Part 7: Bioconcentration test;
——Part 8: Degradation in water-sediment systems;
——Part 9: Avian acute toxicity test;
——Part 10: Honeybee acute toxicity test;
——Part 11: Silkworm acute toxicity test;
——Part 12: Fish acute toxicity test;
——Part 13: Daphnia sp. acute immobilisation test;
——Part 14: Alga growth inhibition test;
——Part 15: Earthworm acute toxicity test;
——Part 16: Soil microorganism toxicity test;
——Part 17: Trichogramma acute toxicity test;
——Part 18: Amphibian acute toxicity test;
——Part 19: Effects on non-target plants;
——Part 20: Livestock short-term dietary toxicity test;
——Part 21: Macro-crustacean toxicity test.
This is Part 10 of GB/T 31270.
This part is developed in accordance with the rules given in GB/T 1.1-2009.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be held responsible for identifying any or all such patent rights.
This part was proposed by and is under the jurisdiction of the Ministry of Agriculture of the People's Republic of China.
Test guidelines on environmental safety assessment for chemical pesticides -
Part 10: Honeybee acute toxicity test
1 Scope
This part of GB/T 31270 specifies the basic requirements for materials, conditions, operation, quality control, data processing, test reports, etc. for honeybee acute oral/contact toxicity test.
This part applies to honeybee acute oral/contact toxicity test for the registration of chemical pesticides. It may be used as a reference for other types of pesticides.
This part does not apply to volatile and insoluble chemical pesticides.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
median lethal dose
dose of the test substance, at which 50% of the test organisms will be killed in the acute contact/oral toxicity test, , expressed by LD50
Note: the unit is in μg a.i./bee.
2.2
test substance
substance to be tested
2.3
chemical pesticide
pesticide made from chemical substances through artificial synthesis, some of which are synthetic bionic pesticides made through imitation, structural transformation and innovation by taking the active substances in natural products as the matrix
Synonym: synthetic organic pesticide.
[Definition 2.3.1, NY/T 1667.1-2008]
2.4
technical material
final product consisting of the active ingredients and impurities obtained during the manufacturing process, which shall not contain visible foreign substances or any additives, and if necessary, a small amount of stabilizer may be added
[Definition 2.5.1, NY/T 1667.2-2008]
2.5
formulation product
product stable in use and made from technical materials (or technical concentrate) of pesticides and auxiliaries
[Definition 2.4, NY/T 1667.2-2008]
2.6
active ingredient; a.i.
biologically active ingredient (with a specific chemical structure) in pesticide products
[Definition 3.1, NY/T 1667.2-2008]
2.7
reference substances
chemical substance or mixture used in a test to confirm or deny certain characteristics of a test substance or to judge the effectiveness of a test system
3 Test overview
3.1 Method overview
The honeybee acute toxicity test may be carried out via acute oral toxicity test or acute contact toxicity test, the selection of which is subjected to the regulations for pesticide registration management and other regulations.
3.2 Acute oral toxicity
Disperse test substance of different doses in the sucrose solution to feed the adult worker bees, and measure the consumption of the liquid, and after the liquid is completely consumed, feed the bees with the sucrose solution containing no test substance. Record the toxicity symptom and the number of dead honeybees every day during the 48h test period, and calculate the values of LD50 and their 95% confidence limits at 24h and 48h.
3.3 Acute contact toxicity
Anesthetize the bees, and drip the test liquid of different concentrations on the mesonotum of the honeybees under test. Transfer the honeybees into the test cage after the solvent is volatilized, and feed them with an appropriate amount of sucrose solution that is soaked by absorbent cotton. Record the toxicity symptom and the number of dead honeybees every day during the 48h test period, and calculate the values of LD50 and their 95% confidence limits at 24h and 48h.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
The adult worker bees from Italy (Apis mellifera L.) are recommended for the test, and the honeybees used for the test shall be collected in the early morning or the night before. The tests shall not be carried out in early spring and late autumn, and if this is inevitable, the honeybees for the test shall be fed with honeycomb pollen for a week in the test environment; and the honeybees shall not be used for the test within four weeks after receiving antibiotics and mite-resistant drugs. The honeybees used for the test shall be healthy and uniform in size. Honeybees used in acute oral toxicity test shall be starved for 2h before the test.
4.1.2 Test substance
Pesticide formulation product, technical material or pure product is adopted. Insoluble test substances can be dissolved with a small amount of organic solvents, emulsifiers or dispersing agents which are less toxic to honeybees.
4.1.3 Main apparatus and instruments
Main apparatus and instruments are as follows:
——test cage;
——electronic balance;
——artificial climate chamber;
——micro-applicator;
——feeder, etc.
4.1.4 Test conditions
The test shall be performed in the dark at the temperature of 25℃±2℃ and relative humidity of 50%~70%.
4.2 Test operations
4.2.1 Acute oral toxicity
4.2.1.1 Pre-test
Arrange 4~5 dose groups at a large dose difference according to the conditions of the formal test, and perform pre-test to work out the maximum dose of the test substance causing no death of the honeybee and the minimum dose of the test substance causing total death of the honeybees.
4.2.1.2 Formal test
Arrange 5~7 dose groups, with the dose difference set at certain proportion (geometrical difference shall be controlled within 2.2 times) based on the concentration range determined by the pre-test, allocate at least 10 honeybees for each group and set a blank control group; if organic solvent is used to facilitate the dissolution, a solvent control group shall be set. Introduce the honeybees in the storage cage into the test cage, then add 100μL~200μL of 50% (mass concentration) sucrose aqueous solution containing test substance of different concentrations in a feeder (such as a centrifuge tube and a syringe), and determine the consumption of the liquid in each group. Once the liquid is completely consumed (which usually takes 3h to 4h), take out the food container and feed the honeybees with sucrose solution containing no test substance (with no limit in amount). For some test substances, if the honeybees refuse to eat at a higher test doses, resulting in little or no food consumed, the determination of food consumption may be carried out with a time extension of up to 6h (i.e., by determining the remaining volume or weight of the processed food). Three replicates are set for the control group and each treatment group. Observe and record the toxicity symptom and the number of dead honeybees after they are treated for 24h and 48h. In case the death rate of the control group is less than 10%, if the difference in death rate of the treated honeybees was more than 10% after 24h and 48h, the observation time shall be extended up to 96h.
Foreword i
1 Scope
2 Terms and definitions
3 Test overview
4 Test methods
5 Test report
Annex A (Informative) Classification of toxicity levels of pesticides to honeybees
Bibliography
