This standard specifies the inherent biodegradability of chemicals: an overview of the method, test preparation, test procedures, quality assurance and quality control, data and reports for the Zahn-Whellens test. This standard applies to the determination of the inherent biodegradability of chemicals that are soluble in water, non-volatile, non-strongly adsorbable, not lost due to foaming of the solution, and have no inhibitory effect on microorganisms at the test concentration. GB/T 21816-2008 Zahn-Whellens test for inherent biodegradability of chemicals GB/T21816-2008 Standard download decompression password: www.bzxz.net
This standard specifies the inherent biodegradability of chemicals: an overview of the method, test preparation, test procedures, quality assurance and quality control, data and reports for the Zahn-Whellens test. This standard applies to the determination of the inherent biodegradability of chemicals that are soluble in water, non-volatile, non-strongly adsorbable, not lost due to foaming of the solution, and have no inhibitory effect on microorganisms at the test concentration.
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 302B (1992) "Zane-Whellens Test" (English version).
This standard has been edited as follows:
--- Change the measurement unit to the legal measurement unit of China.
Appendix A of this standard is an informative appendix.
This standard is proposed and managed by the National Technical Committee for the Management of Hazardous Chemicals (SAC/TC251). The
responsible drafting unit of this standard: Chemical Registration Center of the Ministry of Environmental Protection.
Participating drafting units of this standard: Nanjing Institute of Environmental Sciences of the Ministry of Environmental Protection, Safety Evaluation Center of Shenyang Institute of Chemical Industry, Shanghai Testing Center.
The main drafters of this standard: Liu Chunxin, Lu Ling, Nie Jinglei, Liu Jining, Shi Lili, Hou Songmei, Zhao Huaqing.

ICS 13. 300;13,020,40
National Standard for the Benefit of the People
GB/T 21816—2008
Chemicals
Inherent biodegradability
Zahn-Wellens test
Chemicals---Inherent -hiodegradability-Zahn-Wellens test080927000241
Published on 2008-05-12
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Standardization Administration of China
Implemented on 2008-09-01
October 2008
GB/T 21816-2008
This standard adopts the English version of the Zane-Whirens Test of the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 302B (1992).
This standard has made the following editorial changes:
The measurement unit is changed to the legal measurement unit of my country. Appendix A of this standard is an informative appendix:
This standard is proposed and managed by the National Technical Committee for the Management of Dangerous Chemicals (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, Safety Evaluation Center of Shenyang Institute of Chemical Industry, and Shanghai Inspection Center.
The main drafters of this standard: Liu Chunxin, Lu Ling, Hao Jinglei, Liu Jining, Shi Lili, Hou Songmei, and Zhao Huaqing. I
Inherent biodegradability of chemicals
Zane-Whellens test
GB/T 21816--2008
This standard specifies the method overview, test preparation, test procedures, quality assurance and quality control, data and report of the inherent biodegradability test of chemicals.
This standard is applicable to the determination of the inherent biodegradability of chemicals that are soluble in water (the mass concentration in water is not less than 50 μg/L, measured in DOC), non-volatile, non-strongly adsorbable, not lost due to solution foaming, and have no inhibitory effect on microorganisms at the test concentration. 2 Terms and Definitions
The following terms and definitions apply to this standard.
In lieu of biodegradability, the biodegradability potential of the test substance in long-term contact with the inoculum under optimal test conditions. 2.2
Primary biodegradation, the process in which the chemical structure of the test substance changes under biological action, resulting in the loss of its characteristics. 2.3
Total organic carbon, the total amount of organic carbon in the test medium (including solution and suspension). 2.4
Dissolved organic carbon, DOC, the content of organic carbon in the solution, usually measured by the organic carbon content in the liquid after filtering through a 0.4 um filter membrane, or the organic carbon content in the supernatant after centrifugation at 4 000 r/min for 15 minutes. 2.5
Chemical oxygen Under strong acid and heating conditions, the oxidant consumed by a certain amount of dichromate to oxidize the reducing substances in the water sample can be expressed as the number of grams of oxygen consumed per gram of the test substance (mg/mg). 3 Test substance information
a) Organic content;
b) Solubility in water;
e) Vapor pressure;
d) Foaming property;
e) Microphysical properties:
f) Adsorption;
g) Structural formula.
GB/T 21816—2008
4 Method Overview
A.1 Principle
A certain amount of inoculum is added to the test medium containing the test substance, and the test solution is stirred and aerated for 28 days at 20℃～25℃ in scattered light or darkness. Samples are collected at regular intervals, and the DOC or COD content in the samples is determined. The biodegradation rate is expressed as the removal rate of DOC or COD (after correction of the null value). The biodegradation rate of the test substance is plotted against the corresponding time points, which is the biodegradation curve. If the test substance undergoes biodegradation, 4.2 Reference substances www.bzxz.net
In order to detect the activity of activated sludge, each test needs to be set up in parallel with substances with known biodegradability as reference substances. This standard recommends ethylene glycol, diethylene glycol, dodecyl disodium tartrate or aniline (activated by distillation) as reference substances. If these reference substances are used, the DOC or COD removal rate of these substances within 14 days should not be
5 Test preparation
5.1 Equipment
a) Glass cylindrical container (
5 cm~10
Rotate (you can also put a cotton chamber for compressed air to pass through 1cm at the bottom of the container to check for impurities, centrifuge (
d) pH meter
e) DOC measurement
5.2 Inoculum
#Higher than 1000
The meter and COD measurement
can be rinsed twice by water from a normally operating water treatment plant. 1α
Guest heart 3mi
Possibly diverse strains,
Different times
Treat according to the above method. The sewage flooding product should be tested by the sample to test the activity of the sludge. 5.3 Test water Use a removal substance (preferably C) with low, one batch of water for each series of tests. 5.4 Culture medium 5.4.1 Test culture medium stock solution A water-free active emulsion with a diameter of 0.2m~0.45 OD less than 25 r
ink will be active
cable stirrer waist above the bottom of the container
power stirrer to replace the agitator in addition to the agitator, but also need to teach a pipe, to breathe in air:
pure is a transmission, oil-free and organic
fresh activated sludge sample tank, with culture medium or white. Under special circumstances, in order to obtain the best river water, etc.) to get the end sample to mix, and in the culture medium to blast until use, with the high purity of the reference material Remove the high-purity water or steamed stuffing water. In order to reduce the voidage value, the organic carbon content in the water should be relatively low. Use analytically pure reagents to prepare the following solutions:
a) Phosphoric acid solution: weigh 8.50g potassium dinitrogen phosphate (KHPO.), 21.75g ​​potassium dihydrogen phosphate (K,HPO), 33.40g dihydrogen phosphate (Na2HPO4·2HO) and 0.5g ammonium chloride (NH,CI) and dissolve them in water to make up to 1L, and the pH value is 7.4.
b) Calcium chloride solution: weigh 27.50g anhydrous calcium chloride (CaCl2) or 36.40g monohydrate calcium chloride (CaCl2·2H2O), dissolve them in water, and make up to 1L.
Magnesium sulfate solution: weigh 22.50 g of magnesium sulfate heptahydrate (MgSO47H2SO4), dissolve it in water, and make up to 1 L. )
d) Ferric chloride solution: weigh 0.25 g of ferric chloride hexahydrate (FeCl26H2O4), dissolve it in water, and make up to 1 L. Add 0.05 mL of concentrated hydrochloric acid or 0.4 g/L of disodium salt of EDTA to buffer and store. If precipitation occurs in the above stock solution, it needs to be freshly prepared. 2
5. 4. 2 Preparation of test medium
GB/T 21816—2008
Mix 10 mL of phosphate buffer in 5.1.1 with 800 mL of test water, and then add 1 mL each of calcium chloride solution, magnesium sulfate solution and ferric chloride solution to make up to 1 L. 6 Experimental Procedure
6.1 Group Design
Usually, the following groups need to be set up in the experiment: a) Test group containing the test substance and inoculum
b) Empty control group containing only the inoculum of the inoculum; c) Process control group containing the reference substance and inoculum6.2 Experimental Procedure
Before the experiment begins, the regulatory effect of the test substance on the active substance at the test concentration should be determined by the method of language interpretation. If an inhibitory effect is found, the concentration of the test substance should be reduced to a level that does not produce an inhibitory effect
Add part
0mL wall
100 mg/L or COD/100 g/I.
(such as DOC) ratio
2.5: 1
The amount of the recipient and the technical
set up one or two only to connect the material and the dark program control in parallel. If it is necessary to obtain non-scattered light or
test bottle placed at 20 ℃,
suspended pollutant
interval detection pH value,
in the test and
clear, culture 28
with NaOI
h+0.5h tablets
at least 4 times sampling; the degradation process in the second
28 days sampling, the ladder of the body ... At the same time, the data on the solution are collected. Purify the solution with pure humid air for a period of time, adjust the pH value to 6.5, ensure that the content contains 50mg/L-g/L of DOC inoculum (intentionally), and the volume of the inoculum and the test medium is 1 L-5. At the same time, set up a sterilized, uninoculated test solution with the test substance instead of the test substance. Stir the test gas and ensure that the test concentration is not lower than 1mg/L for a certain period of time. The type of instrument is the test time. If the test is completed at the end of the test, the test time is 15 minutes.
For example, samples are collected from the last two days of the 1st to 27th day period, and DOC or COD analysis must be performed additionally (e.g., every day) to determine whether the activated sludge is in the process of oxidization.
If more information is needed about the behavior of the activated sludge, the sludge can be re-exposed to the test medium, that is, stirring and aeration are stopped to allow the activated sludge to settle, the supernatant is discarded, the culture is cultured to the original volume, stirred for 15 minutes, and the above operation is repeated. Alternatively, the activated sludge can be separated by centrifugation instead of sedimentation. The sludge treated above needs to be re-tested. If the amount of treated sludge does not meet the requirement of 0.2 g/L to 1 g/L (thick weight), fresh sludge can be added to it. 6.4 Analytical method
Filter the collected sludge supernatant samples (including the test blank control group and the process control group) and discard the first 5 ml of the filtrate. Carefully cleaned filter paper or filter membrane should be used for filtration, and it must be ensured that the filter paper or filter membrane used neither releases nor adsorbs organic compounds. If it is uncertain, the filter membrane should be rinsed three times with deionized water or distilled water at about 60°C to remove soluble organic matter. The purified filter membrane can be stored in water. Difficult-to-filter sludge can be separated by centrifugation or other suitable separation techniques. DOC or COD determination is performed on the samples after filtration or separation by appropriate methods, and repeated twice. Primary biodegradability requires specific methods (such as ultraviolet spectroscopy) to analyze the test substance. If the sample cannot be analyzed on the day of sampling, the sample can be stored at 2°C~4°C for 48h or at 18°C ​​for a longer period of time, but it is not recommended to store it for a long time. 7 Quality Assurance and Quality Control
7.1 If the removal rate of the reference substance reaches 70% within 14 days or the DOC and COD in the test suspension are gradually removed every day and every week (which indicates the occurrence of biodegradation), the test is effective. h
GB/T 21816-~2008
7.2 The determination range of DOC is usually 0.5 mg/L~1 mg/L (in terms of C), and the determination range of COD is generally 15 mg/L (in terms of O). 8 Data and Reporting
8.1 Data Processing
Calculate the biodegradation rate at time t using formula (1): 0,
Wu:
D,——biodegradation rate at time t, expressed in %: LLE
cA....3h±0.5h incubation period after the DOC or COD value of the test suspension, in grams per liter (mg/L); c: the average value of DOC or COD in the test suspension at time t, in grams per liter (mg/L); C——3h±0.5h incubation period, the average value of DOC or COD in the blank control, in milligrams per liter (mg/L): ca-time: the average value of DOC or COD in the blank control, in milligrams per liter (mg/L). (1)
The calculation method of the biodegradation rate of the reference material is the same as above. Draw a biodegradation process curve (as shown in Appendix A) and record all results in the data table.
In some cases, complete or significant degradation occurs within 3 h after the start of the test, and the difference between the blank and the test solution is unexpectedly small, indicating that physicochemical adsorption plays a role. In this case, the value after 3 h can be compared with the initial value calculated from the amount of test substance and the value measured before the addition of the supplement to obtain additional information. If the exact distinction between biodegradation (or partial degradation) and adsorption is to be known, further tests are required. It is more appropriate to use suspended degraded sludge as an inoculum for rapid respirable biodegradation tests.
When the degradation rate of the test substance is very low or even zero, it may be due to the inhibitory effect of the test substance on the microorganisms. In this case, microbial toxicity tests at the test concentrations should be carried out to exclude this possibility. 8.2 Report of results
The test report should include the following:
a) Test substance
The physical properties of the substance and the relevant physicochemical characteristics; identification data of the test substance.
b) Source of inoculum;
-Concentration:
C) Test conditions
Analytical methods;
-Procedure control and control compounds: Reasons for program changes and explanations.
d) Results
Biodegradation curve:
Suitability evaluation:
Biodegradability after a certain day of static test (biodegradability at the end of the 28-day test or when the biodegradation is completely degraded in less than 28 days);
-Active sewage adsorption (the significant difference between the DOC or COD result measured at the first sampling 3 hours after the start of the test and the DOC or COD calculated by the amount of the test substance added to the root): -The degradation period (d) determined from the biodegradation curve, the degradation period (d) and the degradation end point reached after a certain day. e) Conclusion and discussion.
Product Chemical Companion
Appendix A
(Informative Appendix)
Examples of biodegradation
Examples of biodegradation and sludge formation are shown in Figures A.1 and A.2, respectively. 100
Activated sludge
Time/
Example of biodegradation
Degradation period
Example of sludge formation
Protein sludge
Degradation period
Example of sludge formation
GB/T 21816-2008
Vinyl alcohol
Time/d
GB/T 21816---2008
References
[i] Zahn R, and Welens H. (lo74). biologischen Abba-ubarkeit von Produkten and Abwasserinhaltsslofen. Chenikcr Zcitung 98, 228-232.[2 Schefer W.end Walchli O.(1980). 209.L3 Reynolds, L, et al (1987). Evaluation of the taxicity of substances tu be assessed for hiode-gradability.Chemosphere 16,2259.[4] DIN 38409, Teil 3; 1983 Bestimmung des gelosten organischen Kohlenstuffgehaltes(DOC).
[5] ISO 6060;1986 Water Quality-Determination of Cheinical Oxygen Demand.L6] OECD (1984). Test Guideline 209, Paris. [7]
1sO 8192:l086 Watcr Quality-Test for inhibition of oxygen consumption by activated
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