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 12 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 12: Fish 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 fish.
This part applies to fish acute toxicity 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 lethal concentration
concentration of the test substance, at which 50% of the test organisms die in the acute toxicity test, 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 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
static test
test liquid is not replaced during the test
2.8
semi-static test
test liquid is replaced at regular intervals (e.g. 24h) during the test to keep the concentration of the test liquid not lower than 80% of the initial concentration
2.9
flow-through test
test liquid is continuously updated during the test
2.10
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
There are three methods for determining fish acute toxicity: static test, semi-static test and flow-through test. Appropriate method shall be selected depending on the nature of the test substance. Different concentrations of the test substance liquids are prepared separately. The toxicity symptoms and death count of test fish are observed and recorded every day during 96h test period to calculate LC50 and 95% confidence limits at 24h, 48h, 72h and 96h.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
Juvenile fishes of Brachydanio rerio, Cyprinus carpio, Oncothynchus mykiss, Oryzias latipes or Gobiocypris rarus are recommended as fish species; see Annex A for the specific full length and suitable water temperature. If other fishes are used as test materials, the corresponding pre-culture and test conditions meeting their physiological requirements shall be used and explanation shall be given.
The test fish shall be healthy and disease-free and be uniform in size. Before the test, the fishes shall be pre-cultured for 7d ~ 14d under the same environmental conditions as the test. During pre-culture period, the fishes are fed once or twice a day, lit for 12h ~16h every day, and feces and food residues are removed in time. Feeding is stopped 24h before the 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 fish may be used, and 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;
——electronic balance;
——thermometer;
——acidometer;
——glass containers meeting the maximum carrying capacity;
——measuring cylinders, etc.
4.1.5 Test water
Tap water that has been stored and dechlorinated for more than 24h (if necessary, treated with activated carbon) or diluted water marked with the formula is used as test water. The water hardness is between 10mg/L and 250mg/L (calculated as CaCO3), pH is between 6.0 and 8.5, and the dissolved oxygen is not less than 60% of the oxygen saturation value in air. See Annex A for the test water temperature (single test temperature is controlled at ±2℃).
4.2 Test operations
4.2.1 Selection of test method
Static test, semi-static test or flow-through test may be selected according to the characteristics of the pesticide. If static or semi-static test is used, it shall be ensured that the concentration of the test substance in the test liquid during the test is not lower than 80% of the initial concentration. If the concentration of the test substance in the test liquid has a deviation of more than 20% during flow-through test, the actual concentration of the test substance in the test liquid shall be checked and the result calculated, or flow test is used to stabilize the concentration of the test substance in the test liquid.
4.2.2 Pre-test
A number of concentrations at a large difference are set according to the formal test conditions. At least 5 fishes are used for treatment without replicate; the toxicity symptoms and death conditions of the test fish in 96h (or 48h) are observed and recorded. The highest total survival concentration and the lowest total lethal concentration for the test fish are determined by pre-test, and the concentration of the formal test is set within this range.
4.2.3 Formal test
5 to 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, and a blank control group is also set. If solvent cosolvent is used, a solvent control group shall be added. At least 7 fishes are placed in each group without replicate, and it shall be ensured that the number of fish used in each group is the same. The toxicity symptoms and death counts of the test fishes are observed and recorded at any time within 6h after the start of the test and at 24h, 48h, 72h and 96h. If the fish has no visible movement by touching the fish tail with a glass rod, the fish is deemed to be dead and shall be removed in time. The test liquid temperature, pH and dissolved oxygen are measured and recorded daily.
4.2.4 Limit test
The upper effective concentration limit is set as 100mg a.i./L, that is, if the test fish does not die when the concentration of the test substance reaches 100mg a.i./L, it is not necessary to continue the test. If the solubility of the test substance is less than 100mg a.i./L, the maximum solubility is used as the upper concentration limit.
4.3 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 fish at each observation time (24h, 48h, 72h and 96h); data statistics software may also be adopted for analysis and calculation.
4.3.2 Karber method
The LC50 and 95% confidence limits of fish at 24h, 48h, 72h and 96h are obtained by Karber method. LC50 is calculated using Equation (1):
logLC50＝Xm-i(∑P-0.5) (1)
Where,
Xm——the logarithm of the highest concentration;
i——the logarithm of the ratio of two adjacent concentrations;
∑P——the sum of the death rate of each group (in decimal).
95% confidence limit is calculated using Equation (2):
95% confidence limit = logLC50±1.96SlogLC50 (2)
Standard error is calculated using Equation (3):
(3)
Where,
p——the death rate of one group;
q——1-p;
n——the number of fishes in each concentration group.
4.3.3 Linear interpolation method
The linear scale coordinates are used to plot the curve for concentration of the test substance versus the death rate, and the LC50 at 50% death rate is calculated.
4.3.4 Probit graphical method
A graph with the logarithm concentration as X-coordinate and the probit corresponding to the death rate as Y-coordinate is plotted in the semi-logarithmic paper. A straight line is drawn on the graph based on the measured values by visual method, and the logarithm concentration at 50% lethality is obtained from the straight line to estimate LC50.
Foreword i
1 Scope
2 Terms and definitions
3 Test overview
4 Test methods
5 Test report
Annex A (Informative) Body length and suitable temperature conditions for test fish
Annex B (Informative) Classification of toxicity levels of pesticides to fish
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 12 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 12: Fish 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 fish.
This part applies to fish acute toxicity 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 lethal concentration
concentration of the test substance, at which 50% of the test organisms die in the acute toxicity test, 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 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
static test
test liquid is not replaced during the test
2.8
semi-static test
test liquid is replaced at regular intervals (e.g. 24h) during the test to keep the concentration of the test liquid not lower than 80% of the initial concentration
2.9
flow-through test
test liquid is continuously updated during the test
2.10
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
There are three methods for determining fish acute toxicity: static test, semi-static test and flow-through test. Appropriate method shall be selected depending on the nature of the test substance. Different concentrations of the test substance liquids are prepared separately. The toxicity symptoms and death count of test fish are observed and recorded every day during 96h test period to calculate LC50 and 95% confidence limits at 24h, 48h, 72h and 96h.
4 Test methods
4.1 Materials and conditions
4.1.1 Test organism
Juvenile fishes of Brachydanio rerio, Cyprinus carpio, Oncothynchus mykiss, Oryzias latipes or Gobiocypris rarus are recommended as fish species; see Annex A for the specific full length and suitable water temperature. If other fishes are used as test materials, the corresponding pre-culture and test conditions meeting their physiological requirements shall be used and explanation shall be given.
The test fish shall be healthy and disease-free and be uniform in size. Before the test, the fishes shall be pre-cultured for 7d ~ 14d under the same environmental conditions as the test. During pre-culture period, the fishes are fed once or twice a day, lit for 12h ~16h every day, and feces and food residues are removed in time. Feeding is stopped 24h before the 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 fish may be used, and 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;
——electronic balance;
——thermometer;
——acidometer;
——glass containers meeting the maximum carrying capacity;
——measuring cylinders, etc.
4.1.5 Test water
Tap water that has been stored and dechlorinated for more than 24h (if necessary, treated with activated carbon) or diluted water marked with the formula is used as test water. The water hardness is between 10mg/L and 250mg/L (calculated as CaCO3), pH is between 6.0 and 8.5, and the dissolved oxygen is not less than 60% of the oxygen saturation value in air. See Annex A for the test water temperature (single test temperature is controlled at ±2℃).
4.2 Test operations
4.2.1 Selection of test method
Static test, semi-static test or flow-through test may be selected according to the characteristics of the pesticide. If static or semi-static test is used, it shall be ensured that the concentration of the test substance in the test liquid during the test is not lower than 80% of the initial concentration. If the concentration of the test substance in the test liquid has a deviation of more than 20% during flow-through test, the actual concentration of the test substance in the test liquid shall be checked and the result calculated, or flow test is used to stabilize the concentration of the test substance in the test liquid.
4.2.2 Pre-test
A number of concentrations at a large difference are set according to the formal test conditions. At least 5 fishes are used for treatment without replicate; the toxicity symptoms and death conditions of the test fish in 96h (or 48h) are observed and recorded. The highest total survival concentration and the lowest total lethal concentration for the test fish are determined by pre-test, and the concentration of the formal test is set within this range.
4.2.3 Formal test
5 to 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, and a blank control group is also set. If solvent cosolvent is used, a solvent control group shall be added. At least 7 fishes are placed in each group without replicate, and it shall be ensured that the number of fish used in each group is the same. The toxicity symptoms and death counts of the test fishes are observed and recorded at any time within 6h after the start of the test and at 24h, 48h, 72h and 96h. If the fish has no visible movement by touching the fish tail with a glass rod, the fish is deemed to be dead and shall be removed in time. The test liquid temperature, pH and dissolved oxygen are measured and recorded daily.
4.2.4 Limit test
The upper effective concentration limit is set as 100mg a.i./L, that is, if the test fish does not die when the concentration of the test substance reaches 100mg a.i./L, it is not necessary to continue the test. If the solubility of the test substance is less than 100mg a.i./L, the maximum solubility is used as the upper concentration limit.
4.3 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 fish at each observation time (24h, 48h, 72h and 96h); data statistics software may also be adopted for analysis and calculation.
4.3.2 Karber method
The LC50 and 95% confidence limits of fish at 24h, 48h, 72h and 96h are obtained by Karber method. LC50 is calculated using Equation (1):
logLC50＝Xm-i(∑P-0.5) (1)
Where,
Xm——the logarithm of the highest concentration;
i——the logarithm of the ratio of two adjacent concentrations;
∑P——the sum of the death rate of each group (in decimal).
95% confidence limit is calculated using Equation (2):
95% confidence limit = logLC50±1.96SlogLC50 (2)
Standard error is calculated using Equation (3):
(3)
Where,
p——the death rate of one group;
q——1-p;
n——the number of fishes in each concentration group.
4.3.3 Linear interpolation method
The linear scale coordinates are used to plot the curve for concentration of the test substance versus the death rate, and the LC50 at 50% death rate is calculated.
4.3.4 Probit graphical method
A graph with the logarithm concentration as X-coordinate and the probit corresponding to the death rate as Y-coordinate is plotted in the semi-logarithmic paper. A straight line is drawn on the graph based on the measured values by visual method, and the logarithm concentration at 50% lethality is obtained from the straight line to estimate LC50.
Contents of GB/T 31270.12-2014
Foreword i
1 Scope
2 Terms and definitions
3 Test overview
4 Test methods
5 Test report
Annex A (Informative) Body length and suitable temperature conditions for test fish
Annex B (Informative) Classification of toxicity levels of pesticides to fish
Bibliography
