Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
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;
——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 11 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 standard 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 11: Silkworm 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 acute toxicity test of chemical pesticides on silkworm with leaf-dipping method and fumigation method.
This part applies to silkworm acute toxicity tests with leaf-dipping method and fumigation method for the registration of chemical pesticides. It may be used as 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 concentration
concentration of the test substance, at which 50% of the test organisms die in the toxicity test with leaf-dipping method, expressed by LC50
Note: the unit is mg a.i./L.
2.2
test substance
substance to be tested
2.3
chemical pesticide
pesticide made of chemical substances through artificial synthesis, for some, the active substances in natural products are used as the mother body, and then subjected to imitation, structural transformation and innovation, they are bionic synthetic pesticides
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 of technical material (or technical concentrate) of pesticides and auxiliaries
[Definition 2.4, NY/T 1667.2-2008]
2.6
active ingredient; a.i.
specific chemical ingredient that is biologically active in a pesticide product
[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 silkworm acute toxicity test is carried out by leaf-dipping method or fumigation method, and the relevant methods are selected according to the regulations of pesticide registration management and other regulations.
3.2 Leaf-dipping method
The mulberry leaves are dipped into different concentrations of pesticide liquid, and dried to feed the silkworm. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed after 24h, 48h, 72h, and 96h; at the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 is calculated.
3.3 Fumigation method
The test based on the pesticide application conditions in simulation chamber shall be carried out on the fumigation test device or fumigation chamber meeting the test requirements. The relevant parameters of fumigation test device or the fumigation chamber shall be designed according to the recommended dosage on the premise of meeting the test requirements. The test substance is quantitatively burned (or electrically heated) in the test device or the fumigation chamber. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
The test silkworm (Bombyx mori) species are selected from Jingsong × Haoyue, Chunlei × Zhenzhu, Suju × Minghu, or other representative lines. The second-instar silkworm is used as materials for toxicity test.
4.1.2 Test substance
Pure pesticides, technical materials or formulation products shall be used as test substance. For insoluble pesticides, a small amount of organic solvents, emulsifiers or dispersing agents which are low in toxicity to silkworm may be used, and the amount thereof shall not exceed 0.1mL(g)/L.
4.1.3 Main apparatus and instruments
Main apparatus and instruments are as follows:
——artificial climate chamber;
——electronic balance;
——culture dish;
——fumigation box, etc.
4.1.4 Test conditions
Newly-hatched silkworm shall be fed at test temperature of (25±2)℃ and relative humidity of 70%～85%.
4.2 Test operations
4.2.1 Leaf-dipping method
4.2.1.1 Pre-test
3~5 concentration groups are set at a large difference according to the conditions of the formal test; the highest total survival concentration and the lowest total lethal concentration for the silkworm are calculated through pre-test.
4.2.1.2 Formal test
5~7 concentration groups are set based on certain proportional difference (geometrical difference shall be controlled within 2.2 times) within a certain range of concentration determined by the pre-test, each group consists 20 silkworms and blank control is set; if solvent cosolvent is used, a solvent control group shall be added. 3 replicates are set for the control group and each concentration group. The second-instar silkworms are cultured in a culture dish, and the mulberry leaves are completely dipped into different concentrations of the pesticide liquid for 10s, and dried for use by the silkworms. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed at 24h, 48h, 72h, and 96h. At the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 and 95% confidence limit are calculated.
If the insect growth regulator is adopted as test substance, and the death rate of silkworm increases by more than 10% between 72h and 96h, the observation time shall be extended until the death rate increases by less than 10% within 24h.
4.2.1.3 Limit test
The upper concentration limit is set as 2000mg a.i./L, that is, if the silkworm does not die when the concentration of the test substance reaches 2000mg a.i./L, it is not necessary to continue the test. If the solubility of the test substance is less than 2000mg a.i./L, the maximum solubility is used as the upper concentration limit.
4.2.2 Fumigation method
The fumigation test can be carried out in a fumigation box or other satisfactory test device. The test substance is quantitatively burned (or electrically heated) in the test device. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms. 9 replicates are set for each treatment group and a blank control (3 replicates) is set at the same time. The food intake situation of silkworm (reduced or refusal), discomfort symptoms (escape, head-up, shaking head, headbang, twisting, struggling, emesis, etc.) and death conditions are observed and recorded.
4.3 Data processing
4.3.1 Selection of statistical analysis method
Karber method, linear interpolation method or probit graphical method may be adopted to calculate the median lethal concentration LC50 of pesticide toxicity on silkworm in leaf-dipping method at each observation time (24h, 48h, 72h and 96h); data statistics software may also be adopted for analysis and calculation.
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 silkworm
Bibliography
Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
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;
——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 11 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 standard 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 11: Silkworm 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 acute toxicity test of chemical pesticides on silkworm with leaf-dipping method and fumigation method.
This part applies to silkworm acute toxicity tests with leaf-dipping method and fumigation method for the registration of chemical pesticides. It may be used as 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 concentration
concentration of the test substance, at which 50% of the test organisms die in the toxicity test with leaf-dipping method, expressed by LC50
Note: the unit is mg a.i./L.
2.2
test substance
substance to be tested
2.3
chemical pesticide
pesticide made of chemical substances through artificial synthesis, for some, the active substances in natural products are used as the mother body, and then subjected to imitation, structural transformation and innovation, they are bionic synthetic pesticides
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 of technical material (or technical concentrate) of pesticides and auxiliaries
[Definition 2.4, NY/T 1667.2-2008]
2.6
active ingredient; a.i.
specific chemical ingredient that is biologically active in a pesticide product
[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 silkworm acute toxicity test is carried out by leaf-dipping method or fumigation method, and the relevant methods are selected according to the regulations of pesticide registration management and other regulations.
3.2 Leaf-dipping method
The mulberry leaves are dipped into different concentrations of pesticide liquid, and dried to feed the silkworm. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed after 24h, 48h, 72h, and 96h; at the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 is calculated.
3.3 Fumigation method
The test based on the pesticide application conditions in simulation chamber shall be carried out on the fumigation test device or fumigation chamber meeting the test requirements. The relevant parameters of fumigation test device or the fumigation chamber shall be designed according to the recommended dosage on the premise of meeting the test requirements. The test substance is quantitatively burned (or electrically heated) in the test device or the fumigation chamber. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
The test silkworm (Bombyx mori) species are selected from Jingsong × Haoyue, Chunlei × Zhenzhu, Suju × Minghu, or other representative lines. The second-instar silkworm is used as materials for toxicity test.
4.1.2 Test substance
Pure pesticides, technical materials or formulation products shall be used as test substance. For insoluble pesticides, a small amount of organic solvents, emulsifiers or dispersing agents which are low in toxicity to silkworm may be used, and the amount thereof shall not exceed 0.1mL(g)/L.
4.1.3 Main apparatus and instruments
Main apparatus and instruments are as follows:
——artificial climate chamber;
——electronic balance;
——culture dish;
——fumigation box, etc.
4.1.4 Test conditions
Newly-hatched silkworm shall be fed at test temperature of (25±2)℃ and relative humidity of 70%～85%.
4.2 Test operations
4.2.1 Leaf-dipping method
4.2.1.1 Pre-test
3~5 concentration groups are set at a large difference according to the conditions of the formal test; the highest total survival concentration and the lowest total lethal concentration for the silkworm are calculated through pre-test.
4.2.1.2 Formal test
5~7 concentration groups are set based on certain proportional difference (geometrical difference shall be controlled within 2.2 times) within a certain range of concentration determined by the pre-test, each group consists 20 silkworms and blank control is set; if solvent cosolvent is used, a solvent control group shall be added. 3 replicates are set for the control group and each concentration group. The second-instar silkworms are cultured in a culture dish, and the mulberry leaves are completely dipped into different concentrations of the pesticide liquid for 10s, and dried for use by the silkworms. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed at 24h, 48h, 72h, and 96h. At the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 and 95% confidence limit are calculated.
If the insect growth regulator is adopted as test substance, and the death rate of silkworm increases by more than 10% between 72h and 96h, the observation time shall be extended until the death rate increases by less than 10% within 24h.
4.2.1.3 Limit test
The upper concentration limit is set as 2000mg a.i./L, that is, if the silkworm does not die when the concentration of the test substance reaches 2000mg a.i./L, it is not necessary to continue the test. If the solubility of the test substance is less than 2000mg a.i./L, the maximum solubility is used as the upper concentration limit.
4.2.2 Fumigation method
The fumigation test can be carried out in a fumigation box or other satisfactory test device. The test substance is quantitatively burned (or electrically heated) in the test device. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms. 9 replicates are set for each treatment group and a blank control (3 replicates) is set at the same time. The food intake situation of silkworm (reduced or refusal), discomfort symptoms (escape, head-up, shaking head, headbang, twisting, struggling, emesis, etc.) and death conditions are observed and recorded.
4.3 Data processing
4.3.1 Selection of statistical analysis method
Karber method, linear interpolation method or probit graphical method may be adopted to calculate the median lethal concentration LC50 of pesticide toxicity on silkworm in leaf-dipping method at each observation time (24h, 48h, 72h and 96h); data statistics software may also be adopted for analysis and calculation.
Contents of GB/T 31270.11-2014
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 silkworm
Bibliography
