Some standard content:
ICs 13.300,3.020.40
National Standard of the People's Republic of China
GB/T21831--2008
Chemicals
Ready biodegradability : Closed bottle test
Chemicals--Ready biodegradability : Closed bottle testIssued on 2008-05-12
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaAdministration of Standardization of the People's Republic of China
Implementation on 2008-09-01
GB/T21831--2008
This standard adopts the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 301D (1992) "Closed Bottle Test" (English version).
This standard has been edited as follows:
Change the measurement unit to the legal measurement unit of my country. Appendix A, Appendix B, Appendix C, Appendix D and Appendix E of this standard are informative appendices. This standard is proposed and managed by the National Technical Committee for Hazardous Chemicals Management Standardization (SAC/TC251). The responsible drafting unit of this standard: Chemical Registration Center of the Ministry of Environmental Protection. The participating drafting units of this standard: Nanjing Institute of Environmental Sciences of the Ministry of Environmental Protection, Shanghai Academy of Environmental Sciences, and Safety Evaluation Center of Shenyang Institute of Chemical Industry.
The main drafters of this standard: Sun Jinye, Liu Chunxin, Hou Lin, Liu Jining, Shi Lili, Shen Genyang, and Yang Hairong. I
1 Scope
Ready biodegradability of chemicals:
Closed bottle test
CB/T21831—2008
This standard specifies the ready biodegradability of chemicals: closed bottle test method overview, test preparation, test procedures, quality assurance and quality control, data and reports.
This standard is applicable to the testing of the ready biodegradability of water-soluble or poorly soluble, non-volatile, volatile or adsorbed chemicals.
2 Terms and Definitions
The following terms and definitions apply to this standard. 2.1
Ready biodegradability The biodegradability of the test substance when it is in contact with the inoculum within a limited time. 2.2
Biochemical oxygen demand, BoDThe amount of oxygen consumed by microorganisms to decompose organic matter, which can be expressed as the number of grams of oxygen consumed per gram of test substance (mg/mg). 2.3
Chemical oxygen demand, CoDThe amount of oxidant consumed by a certain amount of diosinate to oxidize reducing substances in a water sample under strong acid and heating conditions, which can be expressed as the number of grams of oxygen consumed per milligram of test substance (mg/mg). 2.4
Theoretical oxygen demand, ThoDThe total amount of oxygen required for the complete oxidation of the test substance calculated based on the molecular formula, which can be expressed as the number of milligrams of oxygen consumed per gram of test substance (mg/mg).
Jag phase
The period from the start of the test to the degradation rate reaching 10%. 2.6
10-d windo
Ten-day observation period
10-day test period after the biodegradation rate reaches 10%. 2.7
Degradation phase
The period from the end of the stagnation period to the time when the degradation rate reaches 90% of the maximum degradation rate. 3 Test substance information
Molecular formula;
b) Solubility in water;
c) Vapor pressure:
GB/T 21831-2008
d) Structural formula:
e) Purity;
f) Proportion of main components;
g) Adsorption;
h) Microbial toxicity.
4 Introduction to the method
4.1 Principle
Usually, a necessary amount of inoculum is inoculated into the test medium containing 2 mg/L~5 mg/L of the degradable substance, and the test substance is kept in a constant temperature and dark environment to completely fill the sealed bottle. The condensation rate is calculated based on the consumption of dissolved oxygen in 28 days. When the test substance is biodegraded, the degradation rate is expressed as a simple fraction of ThOD or COD after the inoculum is corrected. The amount of oxygen consumed is compared with a blank control in parallel tests. 4.2 Reference substance
This standard recommends amine (neoamine)
Description.
5 Test standard
5.1 Equipment
Sodium acetate or sodium benzoate is used as a reference substance. If other substances are used, they should be stated in the test report. a) Stoppered BOf bottle (150 mL~3
b) Water or culture
e) Beaker (2
d) Dissolved oxygen
5.2 Inoculum
5.2.1 Selection of inoculum
The inoculum can be collected and kept in a safe state during transportation. The optimal amount of inoculum
(±1℃);
or sewage treatment
. The inoculum can also be determined by steps such as sample sedimentation and shaking, or by turning over the water.
5.2.2 Pretreatment of inoculum
Neutralized sewage
If necessary, aerate for 5d to 7d at the test temperature.
5.3 Test water
Secondary effluent. Collect a fresh sample of clear liquid or filtrate in a quasi-aerobic state until 0.1 mL to 5 mL of filtrate is added. . The optimal amount of inoculum should be determined by preliminary tests using high-purity ionized water or stored water that removes volatile substances (such as alcohol) to ensure that the organic carbon content does not exceed 10% of the test substance concentration. The same batch of water should be used for the same test. 5.4 Culture medium
5.4.1 Test culture medium stock solution
Prepare the following stock solutions using analytically pure reagents: a) Phosphate buffer: weigh 8.50g potassium dihydrogen phosphate (KHzPO.), 21.75g potassium dihydrogen phosphate (KzHPO,), 33.40g disodium hydrogen phosphate dihydrate (CNaHPO.·2HzO) and 0.5 ammonium dinitride (NH,Cl), dissolve in water and make up to 1L with a pH of 7.4. Calcium chloride bath: weigh 27.50g anhydrous calcium chloride (CaCl) or 36.40g calcium chloride dihydrate (CaCl2HzO), dissolve in water b)
, and make up to 1L.
Magnesium sulfate solution: weigh 22.50g magnesium sulfate heptahydrate (MgS0·7H,0), dissolve in water, and dilute to 1L. c
Ferric chloride solution: weigh 0.25g ferric chloride hexahydrate (FeCl·6H,0), dissolve in water, and dilute to 1L. Add 0.05mL concentrated hydrochloric acid or 0.4g/LEDTA disodium salt buffer solution for storage. 2
If precipitation occurs in the above stock solution, it needs to be re-prepared. GB/T21831—2008
5.4.2 Preparation of test culture medium
Take 1mL each of phosphate buffer, calcium chloride solution, magnesium sulfate solution and ferric chloride solution in 800mL test water and dilute to 1L. 6 Test Procedure
6.1 Group Design
a) Generally, the following groups need to be set up in the test: a test group containing the test substance and the inoculum;
a blank control group containing only the inoculum; a process control group containing the reference substance and the inoculum. b) If necessary:
a generic control group containing the test substance, the reference substance and the inoculum (see Appendix A). 6.2 Test Substance Stock Solution
If the solubility of the test substance or the reference substance in water exceeds 1/L, weigh 18-10g, dissolve it in test water and make up to 1L. Otherwise, add the test substance directly to the test medium to ensure that the test solution is homogenized. 6.3 Test Operation
After the test medium is aerated at high intensity for at least 20 minutes, it is allowed to stand at the test temperature for 20 hours. The test medium used in the test is prepared from the same batch, and the test medium should be kept free of bubbles during the test operation. Place the fully aerated test medium into the BOD bottle, about 1/3 of the volume. Add appropriate amounts of test substance and reference substance stock solution (or add in other ways, see Appendix B) to each BOD bottle. The final concentration of the test substance in the test flask is generally not more than 10 mg/mL. To ensure the activity of the inoculum, the dissolved oxygen concentration in the BOD bottle must not be less than 0.5mg/L, usually limit the concentration of the test substance to less than 2mg/L, and the approximate maximum concentration can be estimated from the ThOD (mg/rag) of the test substance. For compounds that are not easily degradable and have low ThOD, the concentration can be set between 5mg/L and ~~~~10mg/L. If the test substance is found to be microbially toxic to the inoculum, a toxicity control must be set up, and the test substance and reference substance are added at the same time so that the final concentration of the test substance is equivalent to that of other test groups. Add the inoculum to all test bottles, insert the hose to the bottom 1/4 of the beaker containing the test culture medium (not from the bottom) to draw the test culture medium into each BOD bottle, so that all BOD bottles are completely filled. When the test substance is a poorly soluble substance, the method of addition is shown in Appendix B. Ensure that the solution is mixed evenly by stirring, tap gently to remove bubbles, plug the bottle stopper, ensure that there are no bubbles attached, and culture in the dark at 20 degrees. During the 28-hour culture period, at least two bottles from each bottle group are sampled for dissolved oxygen measurement at fixed time intervals (at least once a week). Sampling once a week to measure dissolved oxygen can determine the removal percentage during the 14-day observation period. If you want to determine the removal percentage during the ten-day observation period, you need to sample every 3 to 4 days. For nitrogen-containing test substances, it is necessary to correct the oxygen consumption of nitrification. While measuring the dissolved oxygen concentration, take samples from the BOY bottle to measure the nitrite and nitrate content.
7 Quality Assurance and Quality Control
a) At the end of the stable period, the test, or the end of the ten-day observation period, the maximum difference in degradation rate between the tests is less than 20% b) At 14 o'clock in the test, the degradation rate of the control of the substance program (as a percentage of ThOD) is not less than 60%; c) The dissolved oxygen loss of the blank control of the inoculum after 28 days is not higher than 1.5Ⅱg/L; d) During the test, the residual concentration of dissolved oxygen in the test bottle is not less than 0.5mg/L. 8 Data and Reporting
8.1 Data Processing
First, subtract the oxygen consumption of the corresponding inoculum blank control from the oxygen consumption of the test substance bottle at each time point, and divide it by the concentration of the test substance 3
GB/T 21831—2008
(mg/L), and get the BOD expressed as oxygen/test substance (mg/ng). Then divide this BOD by ThOD or COD to get the biodegradation percentage. See formula (1),
where:
oxygen consumption of the test substance, in milligrams (mg); oxygen consumption of the blank control, in milligrams (mg); amount of the test substance added, in milligrams (mg). The biodegradation percentage can be obtained from formula (2) or formula (3). p
where:
D---biodegradation rate, which is expressed in this article.
Note that the two methods may produce different
ThOD and
Use appropriate
ThOD (H4
Correct the oxygen for nitrification
8. 2 Result reporting
For best results, see Appendix D
The test report should contain
the following content:
a) Physical and basic physicochemical
identification data of the test substance.
b) Test conditions and sampling conditions; ratio of sewage to water; test period; the options adopted; e) Reasons and explanations for changes in the preparation method of the suspended liquid and the method of preparing the suspended liquid.
Fill in the data in "Data
(see Energy E):
-Any observed inhibition;
Any observed abiotic degradation,
Analytical data of chemical substances (if applicable);
Analytical data of degradation products of the test substance (if applicable): (1)
Known or estimated nitrification Select the concentrations of nitrate and nitrite during the test period Degradation curve of the test substance and the reference substance: including the stop period, degradation period and ten-day observation period: stable period, the percentage of degradation at the end of the test and (or) the end of the ten-day observation period. d) Discussion of results
Appendix A
(Informative Appendix)
Treatment of growth inhibition of inoculum by test substance A1Treatment of growth inhibition of inoculum by test substance GB/T 21831—2008
When the test substance shows no biodegradability in the rapid biodegradation test: In order to determine whether it is due to the inhibitory effect of the test substance on the inoculum or the inertness of the test substance, it is recommended to adopt the following measures: a) Use similar or identical inoculum in the microbial toxicity test and the biodegradation test b) Microbial toxicity test shear
Growth inhibition test
Biodegradation test
Below EC2
Use the following methods alone or in combination: sludge cough inhibition test, BOD and (or) microorganisms to avoid the test substance inhibiting the activity of the inoculum, it is recommended that the test substance concentration be set to 1/10 (or EC%), it can be judged that the test substance has no inhibitory effect on the inoculum; when the test substance is connected to the EC of the substance or uses the "test concentration" for compound materials evaluation, the test results: the test substance concentration is fast, it is recommended to use the closed bottle method to test the inoculum of the test substance, and a higher test concentration can be set during the test. 2008
B.1 Treatment of poorly soluble test substances
Appendix B
(Informative Appendix)
Treatment of poorly soluble test substances
For biodegradability tests of test substances that are poorly soluble in water, special attention should be paid to the following issues: Since homogeneous liquids rarely have sampling problems, appropriate methods should be used to homogenize solid substances to avoid errors caused by non-homogeneity. When it is necessary to collect a few milligrams of representative samples from a mixture or a test substance containing large amounts of impurities, special care should be taken; during the test, Use a variety of stirring methods. Pay attention to the stirring force that is sufficient to make the test substance separate, but not too strong to cause overheating and excessive foaming!
Emulsifiers can be used to make the test substance in a stable separation state, but the emulsifier should not decay, degrade, or foam in the inoculum under the test conditions;
The requirements for solvents are the same as those for emulsifiers;
For solid test substances, emulsifiers must not be added to solid carriers, but carriers can be used for oily test substances; When auxiliary substances such as emulsifiers, solvents and carriers are used, auxiliary substance blank controls should be set up. +
C.1 Theoretical oxygen consumption (ThoD)
Appendix C
(Informative Appendix)
Calculation and determination of relevant parameters
G/T 21831—2008
When the elemental composition is determined or known, the theoretical oxygen consumption can be calculated. For compounds C, HnClN, NanO.P, S,, ThOD is usually calculated based on the degradation mode of forming ammonium salts. The theoretical oxygen demand is formula (C.1) 162c±
If the cumulative prediction or confirmation is
, the oxygen demand is formula (C.2)
In formula (C. 1) and
(C2):
2m+ 3s+
nitrification, ThODno should be calculated based on the degradation mode of nitrate formation. Its theoretical value is 3s+
carbon in each biological molecule, 3s+
hydrogen in each biological molecule, 3s+
chlorine in each biological molecule, 3s+
nitrogen in each biological molecule, 3s+
phosphorus in each molecule, 3s+
sodium in each molecule, 3s+
oxygen in each biological molecule (g/mg) (4
-(C,2)
If nitrification does not occur completely, ThOD is corrected by measuring the concentrations of nitrate and nitrite. C.2 Chemical oxygen demand (COD)
Chemical oxygen demand of soluble organic matter in water can be determined by other methods. Chemical oxygen demand is usually best determined by other methods, namely the closed system Kelkenberg method with pressure balance. This method can be used to determine compounds that are difficult to determine by conventional methods, especially for the test of difficult substances. However, this method cannot determine substances such as pyrimidines. If the concentration of potassium dichromate is increased from 0.0026mol/L to 0.0416mol/L, 5ng~10mg of substances added by weight can be easily determined.This will satisfy the determination of chemical oxygen demand for poorly soluble substances in water. GB/T 21831--2008
Appendix D
(Informative Appendix)
Correction of oxygen consumption for nitrification
In the respirometer method, the oxygen consumed in the ammonium oxidation process will significantly affect the determination of the test oxygen consumption. When the oxygen consumption method is used to test the biodegradability of nitrogen-free test substances, even if the ammonium nitrogen contained in the medium in the test and control bottles is randomly oxidized, the error of the result without considering nitrification is very small (less than or equal to 5%); however, for nitrogen-containing test substances, if the oxygen consumed by the oxidation of ammonium to nitrate and nitrite is not corrected, the test results will be seriously distorted. When complete nitrification occurs or ammonium is converted to nitrate, the oxidation process is shown in formula (D, 1).
NH,+2O→NO-+2H++H,0
Oxidation of 14 nitrogen to nitrate will consume 648g of oxygen, that is, the oxygen consumed to generate nitrate is the increase in nitrate nitrogen multiplied by 4.57. If incomplete nitrification occurs, the oxidation process is shown in equations (D.2) and (D.3): NH ++3/2O→ NO-+ 2H+-+ HO
NO,-+1/2O,→NO,
Oxidation of 14 nitrogen to nitrite will consume 48g of oxygen, that is, the conversion factor is 3.43. Depending on the type of microorganism present in the root, the above two reactions are continuous equilibrium reactions, that is, the nitrite content may increase or decrease, and in the latter case, an equivalent amount of nitrate is generated. In this way, the oxygen consumption for the generation of nitrate is the increase in the content of nitrite nitrogen multiplied by 4.57, while the oxygen consumption for the generation of nitrite is the increase in the content of nitrite nitrogen multiplied by 3.43. If only the total nitrogen that is nitrified is determined, its oxygen consumption should preferably be calculated as the increase in the content of oxidized nitrogen multiplied by 4.57. The biodegradation rate is calculated as the corrected oxygen consumption for oxidative oxidation divided by the theoretical oxygen consumption without consumption (ThODB, see Appendix C for calculation).
E.1 Laboratory
E.2 Date of start of test
E.3 Test substance
Name:
Concentration of inoculum stock solution:
Initial concentration in bottle:
ThOD Or COD:
E.4 Inoculum
Source:
Treatment method:
Pretreatment, if any:
Concentration in test mixture:
E.5 Determination
Test group
Blank containing only inoculum
Combined test substance and inoculum
Test group
Bottle number
Blank control mean
Appendix E
《Informative Appendix）
Closed bottle test data sheet
Oxygen determination results
Oxygen determination results
In; reference substance and toxicant control tests can use a similar format. E.6 Correction of nitrificationbzxZ.net
Table E.2 Results of correction of nitrification
Oxygen consumption of nitrification
(1) Nitrate concentration (in N)/(mg/L)
(2) Change of nitrate concentration (in N)/(mg/L)0
GB/T 21831—2008
Oxygen fluctuation after n days/(mg/L)
Cultivation time/a
GB/T 21831—2008
Oxygen consumption during degradation
(3) Equivalent oxygen content/(mg/L)
4) Nitrate concentration (in N liters)/(mg/L)5) Changes in nitrite concentration (in N>/(mg/L)(6) Equivalent oxygen content/(mg/L)
<7) Equivalent total oxygen content/(mg/L)
Oxygen consumption
Degradation rate determination results
(m, —a1)*
(m—a)
D,x100
GXThOD
Dm.*_ D, +D,
Here it is assumed that: mo) = s,
In the formula:
Usually blank test value:
Table E.2 (continued)
Table E.3 Results of degradation rate determination
Value of the test substance in bottle 1 on day 0;
Value of the test substance in bottle 2 on day 0. Incubation time/a
Loss after n days/(mg/L)
If g(and 2co> or a2(are not equal, use: (o>a)—(m)—m) and (2)—(m)—m) In the formula:
The average value of the blank test on day 1;
The value of the test substance in bottle 1 on day 2;
The value of the test substance in bottle 2 on day 3.
Do not take the average value when the results of repeated tests are quite different. E.8 Oxygen consumption of blank control
Oxygen consumption of blank control: (mb)-mbc2s) mg/L. This consumption is very important to the validity of the test and should be less than 1.5 mg/L.
Use (7) in E.6 to correct for any nitrification. 103 Results of degradation rate determination
Value of the test substance in bottle 1 on day 0;
Value of the test substance in bottle 2 on day 0. Incubation time/a
Loss after n days/(mg/L)
If g(and 2co> or a2(are not equal, use: (o>a)—(m)—m) and (2)—(m)—m) in:
mean value of blank test on day 1;
value of the test substance in bottle 1 on day 2;
value of the test substance in bottle 2 on day 3.
Do not take the mean value when the results of repeated tests are quite different. E.8 Oxygen consumption of blank control
Oxygen consumption of blank control: (mb)—mbc2s) mg/L. This consumption is not critical to the validity of the test and should be less than 1.5 mg/L. Use (7) in E, 6 to correct for any nitrification.3 Results of degradation rate determination
Value of the test substance in bottle 1 on day 0;
Value of the test substance in bottle 2 on day 0. Incubation time/a
Loss after n days/(mg/L)
If g(and 2co> or a2(are not equal, use: (o>a)—(m)—m) and (2)—(m)—m) in:
mean value of blank test on day 1;
value of the test substance in bottle 1 on day 2;
value of the test substance in bottle 2 on day 3.
Do not take the mean value when the results of repeated tests are quite different. E.8 Oxygen consumption of blank control
Oxygen consumption of blank control: (mb)—mbc2s) mg/L. This consumption is not critical to the validity of the test and should be less than 1.5 mg/L. Use (7) in E, 6 to correct for any nitrification.
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