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:
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 14: Alga growth inhibition 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 alga growth inhibition test.
This part applies to alga growth inhibition 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 test substance that makes the yield or growth rate of test organism to be decreased by 50% compared with that of control group in the growth inhibition test, which is expressed by EC50. In this part, the median effective concentration obtained by calculating the inhibition percentage of alga yield is expressed by EyC50, while that obtained by calculating the inhibition percentage of alga growth rate is expressed by ErC50
Note: the unit is mg a.i./L.
2.2
average growth rate
logarithm of unit yield of alga within certain exposure duration, expressed by μ
2.3
yield
increasing amount of unit biomass of alga within certain exposure duration, i.e. the amount of unit biomass at the end of exposure deducted by the unit biomass at the beginning of exposure, expressed by Y
2.4
test substance
substance to be tested
2.5
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]
2.6
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]
2.7
formulation product
product stable in use and made of technical material (or technical concentrate) of pesticides and auxiliaries
[Definition 3.1, NY/T 1667.2-2008]
2.8
active ingredient; a.i.
specific chemical ingredient that is biologically active in a pesticide product
[Definition 3.1, NY/T 1667.2-2008]
2.9
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
In this test, prepare a series of test liquids with different concentrations using a test substance, then observe the growth inhibition of test alga for consecutive 72h after mixing the test liquids with alga liquid, and calculate the median effective concentration EC50 (72h) and 95% confidence limit.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
For test alga, Chlorella vulgaris, Desmodesmus subspicatus or Pseudokirchneriella subcapitata are recommended.
4.1.2 Test substance
Pesticide formulation product, technical material or pure pesticides is adopted. For insoluble pesticides, a small amount of organic solvents, emulsifiers or dispersing agents which are low in toxicity to alga 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:
——acidometer;
——hemocytometer;
——spectrophotometer;
——microscope;
——artificial climate box;
——high-pressure steam sterilization pot;
——glassware, etc.
4.1.4 Culture medium
It is recommended to culture Desmodesmus subspicatus in 4# aquatic culture medium, culture Pseudokirchneriella subcapitata in BG11 culture medium and culture Chlorella vulgaris in BG11 culture medium or SE culture medium, and the formula for such culture mediums are given in Annex A. The name, formula and other information of any other culture medium (if used) shall be provided simultaneously; where other alga is used, suitable culture medium shall be selected and the name and formula thereof shall also be provided.
4.1.5 Test conditions
The test environment temperature is 21℃~24℃ (with temperature for single test controlled at ±2℃); the illumination is continuous and uniform with illumination intensity difference kept within ±15% and with light intensity of 4440lx~8880lx。
4.2 Test operations
4.2.1 Pre-culture of test alga
Inoculate the test alga into a conical flask with culture medium by using aseptic method for culture under the conditions specified in 4.1.5. Inoculate once every 96h and repeat the inoculation for 2~3 times to ensure the alga basically reaching the synchronous growth stage so as to serve as test alga. The growth of algae is inspected by observing with a microscope each time the inoculation is performed.
4.2.2 Pre-test
Set several groups of concentrations at a large difference according to the formal test conditions so as to calculate the lowest concentration of test substance to inhibit the growth of test alga and the highest concentration not inhibiting the growth of test alga, hereby set the concentration for formal test within such range.
4.2.3 Formal test
Set 5~7 concentration groups at certain proportional difference within the concentration range determined by the pre-test (geometrical difference shall be controlled within 3.2 times), and set a blank control group. If cosolvent is used, a solvent control group shall also be added, and 3 replicate are set for each concentration group. The test observation period lasts for 72h, and sample every other 24h. Accurately count the algae cell number with a hemocytometer under a microscope or directly determine the absorbance of alga with a spectrophotometer. When counting with a hemocytometer, the same sample shall be counted for at least twice. If the difference of counting results is greater than 15%, the counting shall be repeated. Select the appropriate technical method based on the properties of test substances.
4.2.4 Limit test
Set the upper concentration limit as 100mg a.i./L, that is, the alga is not affected if the test substance reaches 100mg a.i./L. If the solubility of the test substance is less than 100mg a.i./L, the upper solubility limit will be used as the test concentration. Set at least 6 replicates for the control group and treatment group, and conduct difference significance analysis for the concentration group and control group (e.g. t inspection).
4.2.5 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. The green alga is tested by using reference substance (at least twice a year); 3,5-dichlorophenol and potassium dichromate are recommended.
4.3 Data processing
4.3.1 Inhibition percentage of yield
The inhibition percentage of alga yield in treatment group is calculated using Equation (1):
(1)
Where,
Iy——the inhibition percentage of yield in treatment group, %;
Yc——the unit biomass of alga determined by blank control group, pcs/mL if it is expressed by cell number;
Yt——the unit biomass of alga determined by treatment group, pcs/mL if it is expressed by cell number.
4.3.2 Inhibition percentage of growth rate
The inhibition percentage of alga growth rate in treatment group is calculated using Equation (2):
(2)
Where,
Ir——the inhibition percentage of alga growth rate in treatment group, %;
μc——the average growth rate of blank control group;
μt——the average growth rate of treatment group. Thereof, μ is calculated using Equation (3):
(3)
Where,
μj-i——the average growth rate between time points i and j;
Xi——the unit biomass of alga at time point i, pcs/mL if it is expressed by cell number;
Xj——the unit biomass of alga at time point j, pcs/mL if it is expressed by cell number.
4.3.3 Median effective concentration
4.3.3.1 Selection of statistical analysis method
Calculate median effective concentrations EyC50 and ErC50 respectively based on inhibition percentages of alga yield and alga growth rate. The alga data are analyzed with appropriate statistical software and the median effective concentrations and 95% confidence limits at each observation time (24h, 48h and 72h) are calculated.
4.3.3.2 Karber method
EC50 and 95% confidence limits of alga at 24h, 48h and 72h may be obtained by Karber method.
EC50 is calculated using Equation (4):
logEC50=Xm-i(∑P-0.5) (4)
Where,
Xm——the logarithm of the highest concentration;
i——the logarithm of the ratio of two adjacent concentrations;
∑P——the sum of the inhibition rate of each group (in decimal).
95% confidence limit is calculated using Equation (5):
95% confidence limit=logEC50±1.96SlogEC50 (5)
Standard error is calculated using Equation (6):
(6)
Where,
p——the inhibition rate of one group;
q——1-p;
n——the growth rate or yield of each concentration group.
4.3.3.3 Linear interpolation method
The linear scale coordinates are used to plot the curve for inhibition percentage versus the test substance concentration, and the EC50 at 50% immobilisation is calculated.
4.3.3.4 Probit graphical method
A graph with the logarithm concentration as X-coordinate and the probit corresponding to the inhibition percentage as Y-coordinate is plotted in the semi-logarithmic paper. A straight line is drawn on the graph based on the measured values with visual method, and the logarithm concentration at 50% immobilisation is obtained from the straight line to estimate EC50.
Foreword i 1 Scope 2 Terms and definitions 3 Test overview 4 Test methods 5 Test report Annex A (Informative) Formula of culture medium Annex B (Normative) Classification of toxicity levels of pesticides to alga 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 14 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 14: Alga growth inhibition 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 alga growth inhibition test.
This part applies to alga growth inhibition 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 test substance that makes the yield or growth rate of test organism to be decreased by 50% compared with that of control group in the growth inhibition test, which is expressed by EC50. In this part, the median effective concentration obtained by calculating the inhibition percentage of alga yield is expressed by EyC50, while that obtained by calculating the inhibition percentage of alga growth rate is expressed by ErC50
Note: the unit is mg a.i./L.
2.2
average growth rate
logarithm of unit yield of alga within certain exposure duration, expressed by μ
2.3
yield
increasing amount of unit biomass of alga within certain exposure duration, i.e. the amount of unit biomass at the end of exposure deducted by the unit biomass at the beginning of exposure, expressed by Y
2.4
test substance
substance to be tested
2.5
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]
2.6
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]
2.7
formulation product
product stable in use and made of technical material (or technical concentrate) of pesticides and auxiliaries
[Definition 3.1, NY/T 1667.2-2008]
2.8
active ingredient; a.i.
specific chemical ingredient that is biologically active in a pesticide product
[Definition 3.1, NY/T 1667.2-2008]
2.9
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
In this test, prepare a series of test liquids with different concentrations using a test substance, then observe the growth inhibition of test alga for consecutive 72h after mixing the test liquids with alga liquid, and calculate the median effective concentration EC50 (72h) and 95% confidence limit.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
For test alga, Chlorella vulgaris, Desmodesmus subspicatus or Pseudokirchneriella subcapitata are recommended.
4.1.2 Test substance
Pesticide formulation product, technical material or pure pesticides is adopted. For insoluble pesticides, a small amount of organic solvents, emulsifiers or dispersing agents which are low in toxicity to alga 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:
——acidometer;
——hemocytometer;
——spectrophotometer;
——microscope;
——artificial climate box;
——high-pressure steam sterilization pot;
——glassware, etc.
4.1.4 Culture medium
It is recommended to culture Desmodesmus subspicatus in 4# aquatic culture medium, culture Pseudokirchneriella subcapitata in BG11 culture medium and culture Chlorella vulgaris in BG11 culture medium or SE culture medium, and the formula for such culture mediums are given in Annex A. The name, formula and other information of any other culture medium (if used) shall be provided simultaneously; where other alga is used, suitable culture medium shall be selected and the name and formula thereof shall also be provided.
4.1.5 Test conditions
The test environment temperature is 21℃~24℃ (with temperature for single test controlled at ±2℃); the illumination is continuous and uniform with illumination intensity difference kept within ±15% and with light intensity of 4440lx~8880lx。
4.2 Test operations
4.2.1 Pre-culture of test alga
Inoculate the test alga into a conical flask with culture medium by using aseptic method for culture under the conditions specified in 4.1.5. Inoculate once every 96h and repeat the inoculation for 2~3 times to ensure the alga basically reaching the synchronous growth stage so as to serve as test alga. The growth of algae is inspected by observing with a microscope each time the inoculation is performed.
4.2.2 Pre-test
Set several groups of concentrations at a large difference according to the formal test conditions so as to calculate the lowest concentration of test substance to inhibit the growth of test alga and the highest concentration not inhibiting the growth of test alga, hereby set the concentration for formal test within such range.
4.2.3 Formal test
Set 5~7 concentration groups at certain proportional difference within the concentration range determined by the pre-test (geometrical difference shall be controlled within 3.2 times), and set a blank control group. If cosolvent is used, a solvent control group shall also be added, and 3 replicate are set for each concentration group. The test observation period lasts for 72h, and sample every other 24h. Accurately count the algae cell number with a hemocytometer under a microscope or directly determine the absorbance of alga with a spectrophotometer. When counting with a hemocytometer, the same sample shall be counted for at least twice. If the difference of counting results is greater than 15%, the counting shall be repeated. Select the appropriate technical method based on the properties of test substances.
4.2.4 Limit test
Set the upper concentration limit as 100mg a.i./L, that is, the alga is not affected if the test substance reaches 100mg a.i./L. If the solubility of the test substance is less than 100mg a.i./L, the upper solubility limit will be used as the test concentration. Set at least 6 replicates for the control group and treatment group, and conduct difference significance analysis for the concentration group and control group (e.g. t inspection).
4.2.5 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. The green alga is tested by using reference substance (at least twice a year); 3,5-dichlorophenol and potassium dichromate are recommended.
4.3 Data processing
4.3.1 Inhibition percentage of yield
The inhibition percentage of alga yield in treatment group is calculated using Equation (1):
(1)
Where,
Iy——the inhibition percentage of yield in treatment group, %;
Yc——the unit biomass of alga determined by blank control group, pcs/mL if it is expressed by cell number;
Yt——the unit biomass of alga determined by treatment group, pcs/mL if it is expressed by cell number.
4.3.2 Inhibition percentage of growth rate
The inhibition percentage of alga growth rate in treatment group is calculated using Equation (2):
(2)
Where,
Ir——the inhibition percentage of alga growth rate in treatment group, %;
μc——the average growth rate of blank control group;
μt——the average growth rate of treatment group. Thereof, μ is calculated using Equation (3):
(3)
Where,
μj-i——the average growth rate between time points i and j;
Xi——the unit biomass of alga at time point i, pcs/mL if it is expressed by cell number;
Xj——the unit biomass of alga at time point j, pcs/mL if it is expressed by cell number.
4.3.3 Median effective concentration
4.3.3.1 Selection of statistical analysis method
Calculate median effective concentrations EyC50 and ErC50 respectively based on inhibition percentages of alga yield and alga growth rate. The alga data are analyzed with appropriate statistical software and the median effective concentrations and 95% confidence limits at each observation time (24h, 48h and 72h) are calculated.
4.3.3.2 Karber method
EC50 and 95% confidence limits of alga at 24h, 48h and 72h may be obtained by Karber method.
EC50 is calculated using Equation (4):
logEC50=Xm-i(∑P-0.5) (4)
Where,
Xm——the logarithm of the highest concentration;
i——the logarithm of the ratio of two adjacent concentrations;
∑P——the sum of the inhibition rate of each group (in decimal).
95% confidence limit is calculated using Equation (5):
95% confidence limit=logEC50±1.96SlogEC50 (5)
Standard error is calculated using Equation (6):
(6)
Where,
p——the inhibition rate of one group;
q——1-p;
n——the growth rate or yield of each concentration group.
4.3.3.3 Linear interpolation method
The linear scale coordinates are used to plot the curve for inhibition percentage versus the test substance concentration, and the EC50 at 50% immobilisation is calculated.
4.3.3.4 Probit graphical method
A graph with the logarithm concentration as X-coordinate and the probit corresponding to the inhibition percentage as Y-coordinate is plotted in the semi-logarithmic paper. A straight line is drawn on the graph based on the measured values with visual method, and the logarithm concentration at 50% immobilisation is obtained from the straight line to estimate EC50.
Contents of GB/T 31270.14-2014
Foreword i
1 Scope
2 Terms and definitions
3 Test overview
4 Test methods
5 Test report
Annex A (Informative) Formula of culture medium
Annex B (Normative) Classification of toxicity levels of pesticides to alga
Bibliography
