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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;
——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 13 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 13: Daphnia sp. acute immobilisation 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 daphnia sp. acute immobilisation test.
This part applies to daphnia sp. acute immobilisation test 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 effective concentration
concentration of the test substance, at which 50% of the test organisms are immobilised in the acute immobilisation test, expressed by EC50
Note: the unit is mg a.i./L.
2.2
immobilisation
phenomenon that the daphnia is not able to swim (but weak movement of appendages is allowed) within 15s when gently shaking the test vessel
2.3
test substance
substance to be tested
2.4
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]
2.5
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]
2.6
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]
2.7
active ingredient; a.i.
specific chemical ingredient that is biologically active in a pesticide product
[Definition 3.1, NY/T 1667.2-2008]
2.8
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
Prepare a series of test liquids with different concentrations, then observe the toxicity symptoms and immobilisation of test daphnia for consecutive 48h after transferring them into the test liquids, and calculate the EC50 (48h) and 95% confidence limit.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
Daphnia magna Straus is recommended; good culture conditions shall be maintained to make the reproduction of Daphnia magna Straus into parthenogenesis state. Non-firstborn daphnia born within 24h and cultured for more than 3 generations under laboratory conditions are selected. The test daphnia shall be healthy daphnia from the same maternal line, i.e., it does not show any signs of stress (e.g. high death rate, occurrence of male daphnia and winter eggs, delayed firstborn, abnormal body color, etc.).
4.1.2 Test substance
Formulation products, technical materials or pure pesticides shall be used as test substance. For insoluble pesticides, a small amount of organic solvents which are low in toxicity to daphnia sp. may be used, and in general, the amount thereof shall not exceed 0.1mL(g)/L.
4.1.3 Reference substance
Potassium dichromate (superior to analytically pure).
4.1.4 Main apparatus and instruments
Main apparatus and instruments are as follows:
——dissolved oxygen meter;
——pH meter;
——water hardness analyzer;
——electronic balance;
——pipette;
——containers made of glass or other chemically inert materials, etc.
4.1.5 Test water
Recombinant water is recommended for the culture, domestication and test of daphnia, and the use of other water shall meet the requirements of Annex A. The use of ISO standard diluted water, Elendt M4 and Elendt M7 nutrient solutions as recombinant water is recommended. See Annex B for the preparation method. Elendt M4 and Elendt M7 nutrient solutions shall not be used for tests of substances containing metal. During the test, the water quality shall be stable with a pH of 6.0~9.0 and dissolved oxygen of ≥3.0mg/L. For Daphnia magna Straus, the water hardness (calculated by CaCO3) is 140~250mg/L, which may be appropriately reduced for other daphnia sp.
4.1.6 Test conditions
Test temperature is 18℃~22℃; in the same test, the temperature change shall be controlled within ±1℃; lighting cycle (light-dark ratio) is 16h:8h or in fully dark conditions (especially test substances unstable under light). During the test, the test vessel shall not be inflated and the pH shall not be adjusted, and feeding shall not be carried out for the test daphnia.
4.2 Test operations
4.2.1 Pre-test
According to the conditions of formal test, expose the daphnia sp. to a large range of concentrations of the test liquids for 48h, with 5 daphnia larva placed at each concentration, and replicate may not be required to determine the concentration range of the formal test.
4.2.2 Formal test
Set at least 5 concentration groups at certain proportional spacing within the concentration range determined by the pre-test (geometrical difference shall be controlled within 2.2 times), and set a blank control group. If cosolvent is used, a solvent control group shall also be added. Set 4 replicates respectively for each concentration and control group, with at least 5 test daphnia each replicate and with a carrying capacity of not less than 2mL each daphnia. If the number for test concentration is less than 5, a reasonable explanation shall be given in the report. Observe and record the immobilisation number and abnormal symptoms or manifestations of test daphnia in each vessel at 24h and 48h after the start of the test.
4.2.3 Limit test
Set the upper concentration limit as 100mg a.i./L, i.e. the test daphnia inhibition rate shall not exceed 10% when the test substance reaches 100mg a.i./L, and there is no need to continue the test; otherwise formal test shall be carried out. If the solubility of the test substance is less than 100mg a.i./L, the maximum solubility is used as the upper concentration limit.
4.2.4 Reference substance test
The reference substance is adopted for reliability inspection in methodology so as to inspect whether the equipment, conditions and method of laboratory and quality of test organism meet the requirements. Reference substance test shall be carried out on a regular basis (at least quarterly). Potassium dichromate is adopted as reference substance.
4.2.5 Operational requirements
During the operation and test, daphnia shall not leave the water, and glass dropper is used to transfer them.
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 observe the median effective concentration EC50 of daphnia sp. at each observation time (24h and 48h); relevant statistical software may also be adopted for data analysis and calculation.
Foreword i
1 Scope
2 Terms and definitions
3 Test overview
4 Test methods
5 Test report
Annex A (Informative) Partial chemical properties of qualified test water
Annex B (Informative) Preparation of recombinant water
Annex C (Informative) Classification of toxicity levels of pesticides to daphnia sp.
Bibliography
