CN108548902B - Reference substance for testing rapid biodegradability of chemicals and use method thereof - Google Patents
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Abstract
The invention discloses a reference substance for testing rapid biodegradability of chemicals and a using method thereof. The reference substance is 4-isopropyl phenol, 4-isopropyl phenol is used as a reference substance preparation program control group in a chemical biodegradation test, and a certain addition concentration is set. The 4-isopropyl phenol is easy to obtain, is difficult to degrade compared with the existing reference substance, has stable indication result, is easy to store and calculate the result, can effectively indicate the degradation activity of the inoculum, and ensures that the rapid biodegradation test result is accurate and reliable.
Description
Technical Field
The invention relates to the field of chemical environmental risk assessment, and relates to a reference substance for rapid biodegradability test of chemicals and a using method thereof.
Background
China is a large country for research, development, production and use of chemicals, and the wide use of chemicals brings potential environmental risks. Biodegradability data is key data for chemical hazard identification and also determines the persistence and exposure concentration of chemicals in the environment.
At present, the rapid biodegradability and the inherent biodegradability of chemicals are generally measured by a rapid biodegradability test method (301 series and 310 series) and an inherent biodegradability test method (302 series) established by the economic cooperation and development Organization (OECD). In the above-mentioned rapid biodegradability test method, different types of degradation inocula (such as activated sludge and secondary effluent) are used, and in order to indicate the degradation activity of the inocula and obtain effective test data, the method recommends using sodium benzoate, sodium acetate or aniline as reference preparation program control group, and under the same condition measuring the biodegradability of reference material, using the programThe sequence control group 14d biodegradation rate results indicate inoculum activity and test result validity. When the biodegradation rate of the program control group 14d reached a passing level, namely: DOC removal rates of up to 70% (301A and 301E), or the amount of carbon dioxide produced experimentally reached the theoretical amount of carbon dioxide produced (ThCO)2) 60% of (301B), or oxygen consumption up to 60% of the theoretical oxygen demand (ThOD) (301C, 301D and 301F), indicates that the inoculum is active and the test is valid, otherwise the test is considered invalid and the test is carried out again.
The most basic requirements as reference substances are as follows: (1) when the test medium does not contain the inoculum, no degradation should occur; (2) containing the inoculum, can be stably degraded. (3) Is easy to prepare.
However, OECD in the biodegradability test technical guide (OECD, OECD Series on the test guidelines program number 2, Detailed review paper on biodogradabilitytesting. Paris,1995, p 38) states that since sodium benzoate, sodium acetate or aniline are too easily degraded, it is almost impossible to achieve a pass level in the program control as a reference, and a case where the test is ineffective due to failure of the program control to achieve the pass level has never been seen. It is therefore advisable to sterilize the test containers or to find other reference substances which are relatively poorly degradable.
The laboratory adopts OECD rapid biodegradability-respirometry (301F), and the biodegradability of aniline, sodium benzoate and sodium acetate 3 reference substances under two test conditions of adding inoculum and not adding inoculum is measured. As a result, it was found that sodium benzoate and sodium acetate were also degraded without addition of inoculum (deionized water) (14 d degradation rate more than 60%), and sodium benzoate was completely degraded in 4 test media without addition of inoculum (deionized water, mineral water, tap water, sterilized water) for 28 days. Sodium benzoate and sodium acetate proved to be too degradable to be suitable as an indicator of inoculum activity.
Under the above test conditions, it was found that aniline was not degraded in deionized water to which no inoculation was added (degradation rate: 14%) at 14 days of the test, and was degraded in activated sludge as an inoculum (up to 100%), indicating that aniline was degraded to some extentThe activity of the inoculum may be indicated. However, aniline as a reference has the following disadvantages: (1) aniline is unstable and is easily oxidized by oxygen in the air to generate benzoquinone and azo salt, so that a new reagent needs to be frequently replaced and a test stock solution needs to be prepared again each time; (2) aniline contains nitrogen atoms and may produce ammonium ions (NH) after degradation3 -) Nitrate ion (NO) may be produced3 -) Or both, and therefore the degradation rate calculation cannot determine which theoretical oxygen demand (ThOD) to useNO3Is also ThODNH3) And uncertainty is brought to result calculation.
Disclosure of Invention
The invention aims to provide a reference substance for testing the biodegradability of chemicals and a using method thereof, so as to stably indicate the degradation activity and the test effectiveness of an inoculum and improve the quality of biodegradation test data.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the reference substance for the rapid biodegradability test of the chemicals is 4-isopropyl phenol.
The use method of the reference substance uses 4-isopropylphenol as a reference substance preparation program control group on the basis of an OECD rapid biodegradation test method (301 series methods), and comprises the following steps:
step one, setting a program control group, a test object treatment group, an inoculum blank control group and a toxicity control group according to an OECD rapid biodegradation test method, adding a reference object 4-isopropylphenol into a test culture medium of the program control group, and adding an inoculum to prepare the program control group without parallelism;
step two, periodically measuring one or more of oxygen consumption, dissolved oxygen reduction, carbon dioxide generation or DOC reduction of the program control group under the same culture condition with the test object treatment group until the test is finished within 28 days; and the biodegradation rate of the program control group is determined according to the percentage of oxygen consumption, dissolved oxygen reduction, carbon dioxide generation, DOC reduction and inoculum blank control group value in the program control group to the theoretical oxygen consumption, the theoretical carbon dioxide generation or the organic carbon according to the amount.
The concentration of 4-isopropyl phenol in the program control group is 1-100 mg/mL.
The inoculum is activated sludge or secondary effluent.
Specifically, the concentration of 4-isopropylphenol in the program control group is 12-50 mg/L (301A and 301E methods), 12-26 mg/L (301B method), 100 mg/L (301C method), 1-4 mg/L (301D method), 15-40 mg/L (301F method).
The inoculum concentration in the program control group was equal to the test substance treatment concentration. Wherein the 301A, 301B and 301F method is 3-30 mg of activated sludge/L, the 301E method is 0.5 mL of secondary effluent/L, the 301D method is 0.05-5 mL of secondary effluent/L, and the 301C method is 30mg of activated sludge/L;
according to the protocol requirements, program controls oxygen consumption (301C and 301F), dissolved oxygen reduction (301D), carbon dioxide production (301B and 301C), DOC depletion (301A) were measured periodically under the same culture conditions as the test article treatment until the end of the test for 28 days.
The biodegradation rate of the program control group is determined by the percentage of the oxygen consumption (301C and 301F) or the dissolved oxygen reduction (301D) or the carbon dioxide generation (301B) or the DOC reduction (301A) of the program control group subtracted by the value of the inoculum blank control group to the theoretical oxygen consumption or the theoretical carbon dioxide generation or the organic carbon.
The test effectiveness judgment method comprises the following steps: program control group degradation reached a pass level over a 10 day observation period (10 days since the biodegradation rate reached 10%).
The invention has the beneficial effects that:
(1) compared with the prior reference substances (sodium benzoate and sodium acetate), the 4-isopropyl phenol is not easy to degrade, is not degraded in a test system without inoculum and contains 30 mg.L-1The stable degradation in the test system of the activated sludge can effectively indicate the activity of the inoculum.
(2) Indicating that the results were stable. Comparison between methods and laboratories shows that 4-isopropyl phenol is used as a reference substance, and when inocula from the same source are adopted, rapid biodegradability test results of different test mechanisms are consistent; different inoculants and different testing methods are adopted, so that the quick biodegradability test results are consistent; with different sources of inoculum, consistent test results were obtained.
(3) Compared with the existing reference substance (aniline), 4-isopropyl phenol is not easy to decompose, reagents and prepared stock solution do not need to be frequently replaced, and the use is simple and convenient; 4-isopropyl phenol does not contain nitrogen atoms, the theoretical oxygen demand is fixed, and the calculation result is determined.
(4) The program control effectiveness criterion in the OECD rapid biodegradability test method is a 14 day pass level. The method provides that the test effectiveness standard reaches a passing level in a 10-day observation period. The effectiveness judgment standard is improved, the indication effect of the reference substance on the activity of the inoculum is intensified, and the result judgment of the reference substance is consistent with the result judgment of the tested chemical.
In general, 4-isopropyl phenol as a reference makes up the problem that the existing reference is easy to degrade and cannot effectively indicate the degradation activity of the inoculum, improves the quality of biodegradation test data, and provides a basis for scientific evaluation of the environmental risk of chemicals.
Drawings
FIG. 1 shows the biodegradation rate of 4-isopropylphenol (301D) measured by the same inoculum and different testing institutions.
FIG. 2 shows the results of the biodegradation rates of 4-isopropylphenol measured by inocula in different areas and by different testing institutions (301D).
FIG. 3 shows the results of the biodegradation rates of 4-isopropylphenol measured by inocula in different areas and by different testing institutions (301F).
Detailed Description
The present invention will be further described with reference to specific examples.
Example 1: degradation capability of three existing references
Activated sludge from a municipal sewage treatment plant was collected as an inoculum, and the method of OECD 301F was used to add no inoculum and no inoculum (activated sludge concentration of 30 mg. L)-1) Time of flightThe 28-day biodegradation rates of aniline, sodium benzoate and sodium acetate, all at 30 mg/L, were determined separately. The degradation rates of sodium benzoate and sodium acetate both reached passing levels (degradation rate over 60% in 28 days) without and with the addition of inoculum, indicating that sodium benzoate and sodium acetate are not suitable for reference to indicate inoculum activity. Only aniline did not degrade without the addition of inoculum (28 days degradation rate 0) and degraded with the addition of inoculum (28 days degradation rate over 60%).
The rapid biodegradability of sodium benzoate was determined by the 301F method, using deionized water, mineral water, tap water and sterilized water as test media, respectively, without any inoculum added. The results show that the biodegradation rate of sodium benzoate reaches 100% in deionized water, mineral water, tap water and sterile water to which no inoculum was added, and further that sodium benzoate does not indicate the degradation activity of inoculum at all.
Example 2: screening of 4-isopropylphenol
Activated sludge from a municipal sewage treatment plant was collected as an inoculum, and the inoculum was added (activated sludge concentration: 30 mg. multidot.L) and not added by the 301F method-1) Under the conditions, the biodegradability of 5 chemicals, namely 4-tert-octylphenol, 4-isopropylaniline, 4-nitroaniline, 4-isopropylphenol and ethylene glycol, with the addition concentration of 30 mg/L is measured. The results show that only ethylene glycol was degraded (28-day degradation rate of 90%) and that none of 4-tert-octylphenol, 4-isopropylaniline, 4-nitroaniline and 4-isopropylphenol was degraded (28-day degradation rate of 0) when no inoculum was present.
And at 30 mg.L-1In the activated sludge test, ethylene glycol and 4-isopropylphenol reached a rapid biodegradation pass level. 4-tert-octylphenol, 4-isopropylaniline and 4-nitroaniline did not reach the passing level.
The reference substance should not be biodegraded in the absence of the inoculum, but may be biodegraded in the presence of activated sludge or when the activity reaches a certain level. 4-isopropylphenol is suitable as a reference substance.
Example 3: stability indication of 4-isopropylphenol as reference substance
Different testing institutions uniformly use secondary effluent of east sewage treatment plant of Nanjing City as inoculum to carry out a 301D test for carrying out comparison among laboratories, wherein the addition concentration of 4-isopropyl phenol is 2 mg/L. The comparison result shows that when 4-isopropyl phenol is used as a reference substance and inocula from the same source are adopted, the quick biodegradability test results of different test mechanisms are consistent (in a 301D test, the 28D degradation rate is 60% -85%), and the figure is shown in figure 1.
Comparison between methods of carrying out the method by using secondary effluent (a 301D method is used in 2 laboratories, and the adding concentration of 4-isopropyl phenol is 2 mg/L) or activated sludge (a 301F method is used in 7 laboratories, and the adding concentration of 4-isopropyl phenol is 18.6-35.2 mg/L) of sewage treatment plants in different regions as inocula by different testing mechanisms shows that the quick biodegradability test results (28D degradation rate, 88% -89% of 301D and 93.5% -100% of 301F) are consistent by using the inocula (the activated sludge or the secondary effluent) in different regions and different test methods, and the results are shown in a graph 2 and a graph 3.
Example 4: determination of a test substance
6 treated 4-isopropyl phenol was used as a reference substance by the 301F method, and the biodegradability of a test substance was measured, and the test settings were as follows:
bottle 5 (program control): reference (40 mg/L) + inoculum (30 mg SS/L);
vial 6 (toxicity control): test substance (32.0 mg/L) + reference substance (40 mg/L) + inoculum (30 mg SS/L).
Turn on BOD analyzer (Oxitop)® Control 6, WTW, germany), the stirrer (magnetic stirrer containing a polytetrafluoroethylene coating) was started, the temperature was maintained at 22 ± 1 ℃ in the dark, and the oxygen consumption was measured daily. After 28 days, the oxygen consumption in 6 test bottles was read and the biodegradation rate was calculated.
The measurement result shows that: the program control group had a 90.5% (> 60%) biodegradation rate during the 10-day observation period; the toxicity control 14 days had a biodegradation rate of 54.3% (> 25%), the oxygen consumption of the test treatment was greater than 60% of the oxygen consumption of the inoculum blank during the test period, and the test had no inhibitory effect on the inoculum. In summary, this test is effective. Under effective test conditions, the biodegradation rate of the test substance 24d was 0%.
Claims (4)
1. A method for using a reference substance for testing the rapid biodegradability of chemicals is characterized in that 4-isopropylphenol is used as a reference substance preparation program control group on the basis of the OECD rapid biodegradation test method commonly adopted at home and abroad at present.
2. The use of the reference substance for rapid biodegradability test of chemicals according to claim 1, characterized by comprising the following steps:
step one, setting a program control group, a test object treatment group, an inoculum blank control group and a toxicity control group according to an OECD rapid biodegradation test method, adding a reference object 4-isopropyl phenol into a test culture medium of the program control group, and adding an inoculum;
step two, periodically measuring one or more of oxygen consumption, dissolved oxygen reduction, carbon dioxide generation or DOC reduction of the program control group under the same culture condition with the test object treatment group until the test is finished within 28 days; and the biodegradation rate of the program control group is determined according to the percentage of oxygen consumption, dissolved oxygen reduction, carbon dioxide generation, DOC reduction and inoculum blank control group value in the program control group to the theoretical oxygen consumption, the theoretical carbon dioxide generation or the organic carbon according to the amount.
3. The method for using the reference substance for rapid biodegradability of chemical products according to claim 2, wherein the concentration of 4-isopropyl phenol in the program control group is 1-100 mg/mL.
4. The method for using the reference substance for testing the rapid biodegradability of chemicals according to claim 3, characterized in that the inoculum is activated sludge or secondary effluent.
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| WO2005007587A1 (en) * | 2003-07-11 | 2005-01-27 | Universität Stuttgart | Method for the biological breakdown of effluent containing nitroaromatics |
| CN101626829A (en) * | 2007-03-21 | 2010-01-13 | Omya发展股份公司 | Process for the removal of endocrine disrupting compounds |
| CN104693427A (en) * | 2015-02-10 | 2015-06-10 | 海门市明阳实业有限公司 | Polylactic acid biodegradable material and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005007587A1 (en) * | 2003-07-11 | 2005-01-27 | Universität Stuttgart | Method for the biological breakdown of effluent containing nitroaromatics |
| CN101626829A (en) * | 2007-03-21 | 2010-01-13 | Omya发展股份公司 | Process for the removal of endocrine disrupting compounds |
| CN104693427A (en) * | 2015-02-10 | 2015-06-10 | 海门市明阳实业有限公司 | Polylactic acid biodegradable material and preparation method and application thereof |
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| 3.酚类化合物好氧生物降解的QSBR研究;何菲 等;《中国环境科学》;20010228;第21卷(第2期);第152-155页 * |
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