Some standard content:
ICs 13.300;13. d20. 40
National Standard of the People's Republic of China
GB/T 21800-2008
Chemicals
Bioconcentration
Flow-through fish test
Chemicals-BioconcentrationFlow-through fish test2008-05-12Published
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of China
2008-09-01Implementation
GB/T 21800—2008
Terms and definitions
Information on test substance
Overview of methods
Apparatus
Test preparation
Test procedure
Quality assurance and quality control
Data and report
Appendix A (informative appendix)
Appendix B (informative appendix)
Appendix C (informative appendix)
Appendix D (informative appendix)
Appendix E (informative appendix)
References
Chemical properties of qualified dilution water
Recommended test fish
Prediction of duration of absorption and elimination phases Theoretical examples of sampling for bioconcentration tests of test substances with IgP 4 Calculation of exponential model for bioconcentration factor…10
GB/T 21800—2008bzxZ.net
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Guidelines for Testing Chemicals No. 305 (1996), "Bioaccumulation: Flow-through Fish Test" (English version).
This standard has been edited as follows:
The measurement units are changed to the legal measurement units of my country. Appendices A, B, C, D and E of this standard are informative appendices. This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251). The responsible drafting unit of this standard is the Chemical Registration Center of the Ministry of Environmental Protection. The participating drafting units of this standard are: Shanghai Testing Center, Shanghai Academy of Environmental Sciences, and Nanjing Institute of Environmental Sciences of the Ministry of Environmental Protection. The main drafters of this standard are: Yu Xiangyi, Mao Yan, Lu Ling, Zhao Huaqing, Dian Haowen, Shen Genyang, and Kong Deyang. 1 Scope
Chemicals
Biological accumulation flow-through fish test
GB/T 21800—2008
This standard specifies the method overview, test preparation, test procedures, quality assurance and quality control, data and report of the chemical biological accumulation flow-through fish test.
This standard is applicable to the test and evaluation of the biological accumulation of stable organic chemicals with 1gP values between 1.5 and 6.0: it is also applicable to the test and evaluation of the biological accumulation of highly lipophilic substances with 1gP values >6.0. 2 Terms and Definitions
The following terms and definitions apply to this standard. 2.1
Bioconcentration
The increase in the concentration of a test substance in the test organism (or specific tissue) relative to the concentration of the substance in the test medium. 2.2
Bioconcentration factor bioconcentration factor, Bc The ratio of the concentration of a test substance in the test organism (or specific tissue) to the concentration of the substance in the test medium at any time during the absorption phase of the test. BCF can also be directly calculated from the rate band number of the kinetic equation (K,/K,), recorded as BCFk. 2.3
Steady-state plaleau or steady-state The state when the curve drawn by the concentration of the test substance in the fish body against time tends to be parallel to the time axis (the change range is within ±20%). 2.4
Steady-state bioconcentration factor/steady-state bioconcentration factor stcady statebioconcentrationfactor, BCF The ratio of the concentration of a test substance in the test organism (or specific tissue) to the concentration of the substance in the test medium under steady state. 2.5
octanol-water partition coefficient, P..n-octanol-water partition coefficient
The ratio of the concentrations of a substance when it reaches equilibrium in the n-octanol-water two-phase medium. Usually expressed as P. 2.6
Absorption rate constant, K, the rate at which the concentration of the test substance in the fish body or its specific tissue increases after the test fish is exposed to the medium containing the test substance. Usually expressed as d-1.
Depuration (loss) rate constant, K, the rate at which the concentration of the test substance in the fish body or its specific tissue decreases after the test fish is transferred from the medium containing the test substance to the medium without the test substance. Usually expressed as d-1. 3 Information on the test substance
a) Solubility in water:
b) Partition coefficient between n-octanol and water (P.);) Hydrolyzability:
GB/T 21800—2008
Determination of the phototransformation of chemicals in water under sunlight or simulated sunlight and under the illumination conditions of bioconcentration tests; Surface tension (if there is no P);
Vapor pressure;
Results of rapid biodegradability tests:
Toxicity of the test substance to the test fish, such as LCs values; Suitable analytical methods (with known accuracy, precision and sensitivity); Detailed information on sample preparation and storage:
Analytical detection of the test substance in water and in fish; k)
When the test substance is labeled with 14C, understand the percentage of impurities carried by radioactive substances. 4 Method Overview 4.1 Principle The bioconcentration test consists of the following steps: Exposure (during the absorption phase) of the test subject to two conditions that have been demonstrated to have reached the concentration of The absorption has not reached the equilibrium state yet. After the absorption reaches a stable state, the absorption reading can be omitted, unless the absorption of the test substance is in the absorption process. In addition to the regular measurement and inspection during the entire test, a blank bioconcentration system should be set up! The model for calculating the bioconcentration factor F> (see Appendix EC) is used to calculate the bioconcentration factor BCF based on the measured concentration of the test substance in fish. The value is generally expressed in
(viscera), and the specific group
wet weight can be used to express
instrument (such as
, should be #
the concentration of the substance in the test fish
4.2 Reference substances
No reference substances are recommended.
Instruments
Dissolved oxygen meter:
Water hardness meter,
pH meter;
Analytical balance:
TOC analyzer,
Test substance analyzer Instruments for sample pretreatment: continuous preparation and distribution system; water quality measuring instruments; aquarium tools made of chemically inert materials for the stage and stage containing the test substance; k) temperature control equipment. Content. The remaining stage. The stage is generally 28 d, unless the
phase can be extended and the steady state time can be extended. For example, the longest is 60 days.
, the elimination phase begins. The elimination phase must not be measured. Unless the two groups of test substances with different susceptibility are obviously incompatible, a more complex collection rate constant should be used. The elimination rate constant is used to produce the test substance in the edible part (fish meat) and the inedible part (P>3). When determining the test substance, the edible part (fish meat) and the inedible part (P>3) should be used. 21800—2008
6.1.1 Selection of endangered species
The criteria for selecting fish species: easy access, suitable size and easy to raise in the laboratory. Also include entertainment, commercial, ecological value, relative sensitivity and successful selection.
The recommended test fish species are shown in Appendix B: other fish species can also be used, but the test conditions should be adjusted accordingly, and the reasons for the selection of fish species and the test methods should be stated in the report.
6,1.2. Fish culture
The test fish should be cultured in water at the same temperature as the test for more than 2 weeks, and fed with feed equivalent to 1% to 2% of the fish body weight every day. The fat and total protein content of the feed should be measured. It is recommended to measure the content of pesticides and heavy metals. From the beginning of the culture of the test fish to 48 hours, record the mortality rate and judge according to the following standards: a) The mortality rate of the test fish within 7 days is greater than 10%: discard the whole batch of fish; b) The mortality rate of the test fish within 7 days is between 5% and 10%: culture for another 7 days c) The mortality rate of the test fish within 7 days is less than 5%: accept the current batch of fish: If the mortality rate of the test fish exceeds 5% within the second 7 days, discard the whole batch of fish.
Deformed or diseased fish cannot be used as test fish and should be discarded. No disease prevention and treatment should be given to the fish during the test and two weeks before the test. If adult fish are used in the test, it should be reported whether the test fish is male or female or a mixture of both. If male and female fish are mixed in the test, there should be no significant difference in the fat content of the two. Male and female fish should be kept together. 6.2 Water for acclimation
The acclimation water is generally derived from natural water of the same quality and without pollution. The test fish species can survive, grow and reproduce in the acclimation water and do not show any abnormal appearance or behavior during the acclimation period. The water characteristics should at least include pH, hardness, particulate matter, total organic carbon, ammonium and nitrite content, alkalinity and salinity (for marine fish only). Appendix A recommends the maximum concentration values of some parameters for freshwater and seawater.
7 Test procedure
7.1 Preparation
7.1.1 Test container
The test container should be made of chemically inert materials and have no significant adsorption to the test substance. It is recommended to use Teflon materials, stainless steel or glass tubes, and reduce plastic hoses to a minimum. For substances with high adsorption coefficients, silanized glass is required. Used containers must be discarded.
7.1.2 Test dilution water
The source and quality of dilution water should be the same as that of aquaculture water. During the entire test period, the quality of the test water should remain stable, the pH value should be maintained at 6.0~8.5, and the pH value change range should be maintained within the range of ±0.5. The dilution water should be sampled and analyzed regularly, including the determination of heavy metals (such as Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (such as Ca2~, Mg+, Na, K+CI-SO.-, pesticides (such as total organic phosphorus and total organochlorine pesticides), total organic carbon and suspended solids.
The maximum acceptable mass concentration of natural particulate matter in the dilution water is 5rmg/L (dry matter intercepted by a 0.45μm filter membrane), and the maximum acceptable value of total organic carbon is 2IⅡB/L (see Appendix A). During the entire test, the organic carbon from other sources in the test container should not exceed the organic carbon from the test substance. If a co-solvent is used, it should not exceed 10mg/L (±20%). 7.1.3 Preparation of test substance solution
Prepare a stock solution of the test substance at an appropriate concentration. It is recommended to prepare the stock solution by simply mixing or stirring the test substance in the dilution water. Co-solvents or dispersants should be used as little as possible or with caution. 3
GB/T 21800—2008
agent. The cosolvents that can be used are ethanol, methanol, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, dimethylformamide and triethylene glycol. The dispersants that can be used are Cremophor RH40 (ethoxylated hydrogenated sesame oil), Tween 80 (sorbitan monooleate ester), 0.01% methylcellulose and HCO-40 (ethoxylated hydrogenated sesame oil): At least 5 times the volume of water in the test tank should be replaced every day. The flow rate of the stock solution and dilution water should be checked 48 hours before the start of the test and at least once during each test. Check the water flow rate of each test tank to ensure that the water flow rate does not change by more than 20% within each test or between tests.
7.2 Test operation
7.2.1 Preliminary test
Optimize the test conditions, such as the concentration of the test substance, the duration of the absorption phase and the clearance phase. 7. 2. 2 Experimental Design Fish should be exposed to at least two concentrations of the test substance. Normally, the higher (or highest) concentration should be 1% of the acute VCs and should be at least 10 times the method detection limit of the test substance in water. The highest test concentration can also be estimated by dividing the 96h LC by the appropriate acute and chronic ratio. If possible, other concentration series can be selected. If the VCs are limited to 1% and the lower limit of analysis, a concentration greater than 1% should not be used. When an organic solvent is used in the experiment, the solubility of the solvent should be determined, and at least one of the dilution water controls should be set up. 7.2.3 Exposure Conditions 7.2.3. 1 Absorption step The duration of the absorption phase or the octanol-water separation characteristics can be predicted (see Appendix C). The duration of the absorption phase should be 2. The absorption phase should be prolonged. 7.2.3.2 Elimination phase duration The duration of the elimination phase can be measured as the absorption phase. If the duration of the elimination phase is too long, for example, the absorption phase is too long, for example, the absorption phase is too long. Consider the use of 4 labeled test substances. Any concentration should be the same in all tests. For example, when the test substance concentration is less than 10% of the cumulative concentration, the steady state or the absorption phase is broken ... If stability has not been reached after 28a, a shorter method is used.
explanation), if 95% clearance is achieved, then the clearance phase time can be shortened (for example, a longer clearance time is needed to determine the clearance rate constant. The clearance phase time should depend on the concentration of the test substance in the fish. According to 7.2.3.3 Number of test fish
The number of test fish required for each test concentration should ensure that at least 4 layers of fish are sampled each time. If a larger statistical sample is required, more fish will be needed.
When testing, test fish with similar body size should be selected, and the smallest fish weight should be less than two-thirds of the largest fish weight. All test fish should be of the same age and from the same source. Accurately record the mass and age of the test fish. It is recommended to take samples and weigh the test fish before the test. 7.2.3.4 Carrying capacity
The recommended carrying rate is 0.1g/(d·L)1.0g/(d·L) of fish weight (wet weight). If the concentration fluctuation of the test substance can be kept within 20% of the soil and the dissolved oxygen concentration is not less than 60% of the saturation value, the carrying capacity can be increased. When selecting an appropriate carrying capacity, the normal growth environment required by the fish species should be considered. 7.2.3,5 Feeding
During the test period, use the same feed as during the rearing period. Feed the fish with feed equivalent to 1% to 2% of their body weight every day. It is recommended to take samples and weigh the test fish before the test. It can be based on The mass of the fish in each test shall be estimated based on the weight of the fish sampled most recently. The mass of the fish in the test shall not be weighed.
GB/T21800—2008
Within 30min60min after feeding each day, use a siphon to remove the remaining bait and fish excrement in the test tank. Keep the test tank as clean as possible during the entire test to ensure that the concentration of organic matter is as low as possible. 7.2.3.6 Light and temperature
During the test, the light cycle should be 12h~~16h, and the temperature should be controlled within ±2℃ of the most suitable temperature for the test fish species (see Appendix B). 7.2.4 Frequency of water quality measurement
Test The dissolved oxygen, TOC, pH and temperature in all test containers should be measured at all times. The total hardness and salinity should be measured in the control group and the higher or highest test concentration group. During the absorption phase, the dissolved oxygen should be measured at least 3 times, namely at the beginning of the test, in the middle of the absorption phase and at the end. The clearance phase should be measured once a week. TOC should be measured before the test fish are put into the test (24h~48h before the absorption phase), at least once a week during the absorption phase and the clearance phase. PH should be measured at the beginning and end of each phase. Hardness should be measured once for each test. The temperature should be measured daily, and the temperature in at least one test container should be monitored continuously. 7.2.5 Sampling and analysis of test fish and water
7.2.5.1 Sampling schedule for test fish and water
Water samples should be collected from the test tank for test substance concentration determination before adding the test fish, during the absorption phase, and during the clearance phase. The frequency of water sampling should be no less than that of fish sampling and should be carried out before feeding. Fish samples should be collected at least 5 times during the absorption phase and at least 4 times during the clearance phase. In some cases, it is difficult to calculate a sufficiently accurate BCF value with only these few samples, so it is recommended to increase the number of samplings in both phases (see Appendix D). Appendix D gives an example of a sampling schedule. Using other estimates of P to calculate the exposure time for 95% absorption, a cabinet-related schedule can also be obtained.
Sampling should be continued during the absorption phase until steady state occurs or 28 days of the test period (whichever is shorter). If steady state has not been reached after 28 days, sampling should continue until steady state is reached or 60 days, whichever is shorter. Before the start of the clearance phase, the test fish should be transferred to a container of clean water.
7.2.5.2 Sampling and sample preparation
Water samples for analysis should be collected from the middle area of the test tank using an inert material tube according to the siphon principle. The container should be kept as clean as possible and the content of total organic carbon should be tested during the absorption and removal stages. Each time fish samples are taken, an appropriate number of test fish (at least 4) are taken from the test tank, the test fish are quickly rinsed with water, the surface water is sucked dry, and the fish are quickly killed in the most humane way and weighed. Fish and water samples should be determined as soon as possible after collection to avoid degradation or other losses, and the absorption and removal rates should be approximately calculated. When samples cannot be measured immediately, appropriate methods should be used to preserve the samples. The method of preserving the specific test substance, storage time and extraction method should be mastered before the start of the test.
7.2.5.3 Analytical methods
For specific test substances, it is necessary to check the accuracy and reproducibility of the analytical method, as well as the recovery rate of the test substance in water and fish. In addition, the test substance should not be detected in the dilution water. If necessary, the C and C values measured in the test should be corrected. 7.2.5.4 Determination of fish samples
When radioactive labeled substances are used in the test, the total amount of radioactive labeled substances (including parent and metabolites) can be determined, or the parent test substance can be determined separately after washing the sample. The main metabolite can also be identified at steady state or absorption stage (whichever is shorter). If the BCF value obtained based on the total amount of radioactive labeled residues is not less than 1000, it is necessary to identify whether the degradation rate of the total residue of the test substance in the fish tissue at steady state is not less than 10%, especially for special categories of test compounds such as pesticides. If the degradation rate of the total residue of the test substance in the fish tissue is not less than 10%, it is necessary to determine the degradation of the test substance in water. Usually, the content of the test substance in each fish should be determined. If this is not possible, the test fish with the same test concentration should be combined after each sampling, but this will not be able to perform mathematical and statistical analysis. If mathematical statistics are really needed, the sample size (number of test fish) should be fully considered in the experimental design.
BCF values can be expressed as a function of the total wet weight of the test fish or, for highly lipophilic substances, as a function of the fat content of the fish. If possible, the fat content of the fish sample should be determined each time it is sampled. The trichloromethane/methanol extraction method is recommended as the standard method. The results obtained by different test methods will not be exactly the same, so detailed steps of the method used should be given. If possible, the same sample extract should be used for fat determination and test substance determination, because the chromatographic analysis of the test substance determination usually requires the removal of fat. The difference in fat content (expressed in mg/kg wet weight) of the test fish at the end of the test and at the beginning of the test should not exceed ± 25%. The dry weight of the fish tissue should be reported to understand the ratio of fat content converted from wet weight to dry weight.
8 Quality Assurance and Quality Control
An effective test shall meet the following conditions: a) Temperature fluctuation is less than ±2°C;
6) Dissolved oxygen concentration is not less than 60% of the air saturation value; c) The phototransformation of the test object under the test lighting conditions is controlled, and ultraviolet radiation less than 290Hm is filtered out to avoid the test fish from being exposed to abnormal photochemical products;
d) During the absorption period, the concentration of the test object in the test container is maintained within the range of ±20% of the measured average value; e) Until the end of the test, the mortality rate or other adverse effects or diseases of the control and test group fish are less than 10%. When the test is extended for several weeks or months, the mortality rate or other adverse effects of the test fish in the two groups should be less than 5% per month and not more than 30% during the whole process.
9 Data and Report
9.1 Data Processing
Plot the concentration of the test substance in the fish body (or specific tissue of the fish body) against time on the coordinate paper to form a curve. If the curve has reached a stable state, that is, it has become an approximate asymptote with respect to the time axis, use formula (1) to calculate the biological control factor (BCFu) of the stable state:
Where:
C—-the average concentration of the test substance in the fish body tissue; C,—-the average concentration of the test substance in the test solution. C
When the stable state is not reached, the BCFs value of 80% or 95% of the stable state can also be calculated. (1
The kinetic enrichment factor (BCFx) can also be determined by the ratio of the absorption rate constant K, of a first-order exponential equation, and the elimination rate constant K, which is usually determined by an elimination curve (i.e., a line plotting the concentration of the substance in the fish body against time). The absorption rate constant K, can be calculated based on the given K value and the C value from the absorption curve, see Appendix E for details. A better way to obtain BCF: and K, K. is to use nonlinear parameter estimation software, otherwise the graphical method can be used to calculate K1 and K, values. If the elimination curve is obviously not a first-order exponential equation, more complex methods (see Appendix C) and biostatistical methods should be used. 9.2 Test report
The test report must include the following information:
a) Test substance:
Physical properties and related physicochemical properties; Chemical property data, including organic carbon content; If radioactive labeled substances are used, the exact position of the labeled atoms and the percentage of relevant radioactive impurities. b)) Birth control:
scientific name, strain, source, pretreatment, feeding, age and individual size, etc. c) Test conditions:
--Test procedure used
Type, characteristics and photoperiod of light source used; +
GB/T 21800-2008
Test design, such as the size and number of test tanks, water change frequency, number of children, number of test fish per replicate, number of test substance concentrations, duration of absorption period and clearance period, frequency of sampling of fish and water; method for preparing test stock solution and frequency of stock solution updating (when solvent is used, its concentration, contribution rate to organic carbon content of test solution):
set test concentration, average concentration measured in test container and its standard deviation, analysis method! Source of dilution water, description of pretreatment, performance of test fish in water and water quality characteristics: pH, hardness, temperature, dissolved oxygen concentration, residual chlorine level (if measured), total organic carbon, suspended particulate matter, content of test medium and any other measurement results;
Water quality in the test container, pH, hardness, TOC, temperature, dissolved oxygen; detailed information on feeding, such as bait type, source, composition (including at least fat and protein content), feeding amount and rate, information on fish and water sample handling, including detailed preparation, storage, extraction, analytical procedures for test substances and fat content and sugar determination.
d) Results:
- Results obtained from preliminary tests;
Mortality of fish in the control group and each exposed group, abnormal behavior of fish observed; fat content of fish;
curve (including all test data) showing the test substance in the fish until the steady-state absorption and elimination stage: C and C values for all sampling times (including standard deviation and variation range), C value of the control group and background value (C is expressed in mg/g whole fish wet weight or fish special tissue such as fat apparent weight or mg/kg, C. expressed in mg/g or mg/kg);
Steady-state bioconcentration factor (BCF) and kinetic enrichment factor (BCF), if possible, absorption and elimination rate constants with 95% confidence limits (expressed in terms of relevant numbers, total fat content of fish or its special tissues), confidence limits and standard deviations, and calculation and data analysis methods for the concentration of each variable test substance; when radioactive labeled substances are used, any metabolites measured, if necessary: any abnormal conditions in the test, adjustments to the test procedures and other relevant information; other explanatory items.
e) Discussion of results:
The preliminary test results of the determination method study should avoid the occurrence of results such as "not detected under the detection limit of the method" as much as possible, because this will not be used to calculate the rate constant. GB/T21800—2008
The chemical properties of qualified dilution water are shown in A.1. Appendix A
(Informative Appendix)
Chemical properties of qualified dilution water
1Chemical properties of qualified dilution water
A 1
Particulate matter
Total organic carbon
Free ammonia
Total organophosphorus pesticides
Total organic ammonia pesticides and polychlorinated biphenyls
Total organochlorine
Limit concentration
5 mg/L
1 μg/L
10 μg/ L
50ng/L
50 ng/L
25ag/L
1 μg/L
1 μg/L
1 μg/L
1 pg/L
1 μg/L
1 μg/L
1 pg/L
100 ng/L
100 ng/L
B.1 Recommended test fish (B.1)
Test fish
Zebrafish (Brachydanio reria)
Rare yolk (Gobiocypris)
Swordtail fish (Xiphapor)
Fathead softmouth (Pimepha
Lithium fish (Cn
Oryzim
Guppy (Paec
Lotus sunfish (Li
Rainbow salmon (O)
Three-spined salmon (Gast
The above listed||t t||disease or parasite
source.
rochirus
chusmgkiss
ceats
easy to keep and common
, they can also
Appendix B
(Informative Appendix)
Recommended test fish
Table B_1 Recommended test fish
Test mattress leather range/center
GB/T 21800—2008
Test fish body length/cm
3. 0±0. 5
5,0 on 3. 0
13.0±1.0
.0±2.0
Estuarine fish species may be difficult to collect. In controlling diseases, the health of the test fish and its
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