Method for carrying out chemical enhanced rapid biodegradation test through pressure-measuring respiration metering method and application of chemical enhanced rapid biodegradation test

Abstract

The invention relates to the field of chemical environment risk assessment, in particular to a method for carrying out a chemical enhanced rapid biodegradation test through a pressure measurement respiration measurement method and application of the method. The method specifically comprises the following steps: adding a tested chemical and activated sludge into a culture medium for culturing, measuring the oxygen consumption in a system, and calculating the biodegradation rate of the chemical according to the oxygen consumption. The method disclosed by the invention has the advantages of wide selection, comprehensive optimization conditions, easily obtained passing level, low test cost, rapidness, high efficiency and the like, and a more convenient method is provided for evaluating the biodegradability and durability of the chemical substances.

Description

Technical Field

[0001] The invention relates to the field of environmental risk assessment of chemicals, in particular to a method for conducting enhanced rapid biodegradation test of chemicals by manometric respirometry and its application. Background Art

[0002] Rapid biodegradability of chemicals refers to the biodegradability of the test substance (organic matter) when it comes into contact with inoculated microorganisms within a limited time. It is an important indicator for identifying the hazards of chemical substances and assessing environmental risks.

[0003] The six classic test methods introduced by the Organization for Economic Cooperation and Development (OECD) are widely used internationally, namely the dissolved organic carbon (DOC) reduction test (301A), the carbon dioxide (CO2) production test (301B), the improved MITI test (I) (301C), the closed bottle test (301D), the improved OECD screening test (301E) and the respirometry test (301F). The respirometry test (301F) has a wide range of applications, is easy to operate, and has fewer interference factors. It is an ideal method for determining the rapid biodegradability of chemicals. However, the respirometry test (301F) is usually carried out under specific environmental conditions, and has high requirements for temperature, microbial activity, etc., which may affect the universality and applicability of the test results.

[0004] In the existing technology, when a chemical shows that it is not rapidly biodegradable in a rapid biodegradation test, it is necessary to further carry out more screening degradation tests. Strengthening rapid biodegradation tests can identify whether the test substance has potential persistence in a relatively convenient and comprehensive way, and its testing cost is much lower than other screening tests such as simulation tests. Summary of the invention

[0005] The invention aims to provide an application of a method for conducting an enhanced rapid biodegradability test of chemicals by manometric respirometry, which is simple to operate, accurate, efficient and cost-saving.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for conducting an enhanced rapid biodegradation test of chemicals by manometric respirometry, wherein the test chemical and activated sludge are added to a culture medium for cultivation, and the oxygen consumption in the system is measured, and then the biodegradation rate of the chemical is calculated based on the oxygen consumption;

[0008] The formula for calculating the biodegradation rate of chemicals through oxygen consumption is:

[0009] BOD value divided by ThOD NH3 The biodegradation percentage is obtained by calculating the value:

[0010]

[0011] Further,

[0012] 1) Preparation of inoculation medium: suspend the activated sludge with known sludge concentration after pre-treatment in the culture medium so that the sludge inoculum concentration in the culture medium is 90 mg / L in terms of suspended solids, and set aside;

[0013] 2) Add the test chemical to the above-obtained inoculation medium so that the final concentration of the test chemical in the system is ThOD NH3 The concentration equivalent to 50-100 mg / L was used as the test group; meanwhile, the blank control was used without adding the test chemical.

[0014] 3) The test group and control group were placed in a biochemical incubator to maintain a constant temperature (21±1°C), and magnetic stirring and BOD value determination were started. During the test period, the BOD value was determined every 12 hours;

[0015] 4) The biodegradation rate is calculated using the BOD value measured during the above test period, and a rapid biodegradation curve is drawn based on time and degradation rate to obtain the biodegradation rate of the chemical;

[0016] The oxygen consumption (mg) (BOD value) of the test substance measured at different times is corrected by the blank control of the inoculum in the same period and divided by the amount of the test substance (mg) to obtain the BOD expressed in mg oxygen to mg test substance, that is:

[0017]

[0018] Furthermore,

[0019] S1: Prepare activated sludge inoculum: Collect sludge, filter with a fine sieve to remove coarse particles, let the sample stand, discard the supernatant and impurities floating on the surface, wash the sludge twice with test culture medium, and suspend the sludge with culture medium. Take 10mL of sludge, at least 3 replicates, weigh it after drying at 105℃ for 2±0.5 hours, and calculate the initial concentration of the sludge. Based on the results, prepare the sludge with culture medium so that the concentration is 3-5g / L in terms of suspended solids. The sludge will be aerated at 22±2℃ until used in the test (no more than 7 days). On the day of the test, take 10mL of sludge, at least 3 replicates, weigh it after drying at 105℃ for 2±0.5 hours, and calculate the sludge concentration (based on the results, determine the amount of activated sludge inoculum to be added in the test).

[0020] S2: Preparation of inoculation medium: Add the test medium to a 250 mL flask, then add an appropriate volume of activated sludge suspension to make the inoculum concentration in the 250 mL medium 90 mg / L in terms of suspended solids. This 250 mL inoculation medium will be added to each test bottle.

[0021] S3: Prepare the inoculum blank control test bottle: A test bottle containing only 250 mL of inoculum medium is used as the inoculum blank control, with two replicates, IC-1 and IC-2.

[0022] S4: Prepare the test bottle: weigh the test substance and add it directly to the test bottle. The final concentration of the test substance is ThOD NH3 The concentration of the test sample in the test bottle is set up in duplicate (test suspension, marked as TS-1, TS-2, TS-3).

[0023] S5: Prepare the reference test bottle: weigh the reference and add it directly into the test bottle. The concentration of reference sodium benzoate is equivalent to 50-100 mg / L in ThOD. Set one replicate (program control, marked as PC) for the reference test bottle.

[0024] S6: Determination of oxygen consumption: Use BOD meter to measure oxygen consumption. After the test bottle is prepared, it is loaded into the BOD meter system, and then placed in the induction stirring base, maintained at a constant temperature (21±1℃) in the biochemical incubator, and magnetic stirring and BOD value determination are turned on. During the 60-day test period, the BOD value of each test bottle is automatically recorded by the instrument every 12 hours. At the end of the incubation period, the BOD value is read.

[0025] S7: Data processing: The calculation of BOD value and biodegradation rate are shown in Formula 1 and Formula 2 respectively, and then the biodegradation curve is drawn through time and degradation rate.

[0026] Formula 1: The oxygen consumption (mg) of the test substance after a certain period of time is corrected by the blank control of the inoculum in the same period and divided by the amount of the test substance (mg) to obtain the BOD expressed in mg oxygen to mg test substance, that is:

[0027]

[0028] Formula 2: BOD value divided by ThOD NH3 The biodegradation percentage is obtained by calculating the value:

[0029]

[0030] The culture medium is prepared by first adding stock solution A into water, and then sequentially adding stock solution B, stock solution C and stock solution D, and then adjusting the volume to 1L, and adjusting the solution pH to 7.4±0.2; wherein the volume ratio of stock solution A, stock solution B, stock solution C and stock solution D is 10:1:1; the stock solution A is prepared by adding 0.85g of potassium dihydrogen phosphate, 2.175g of dipotassium hydrogen phosphate, 3.34g of disodium hydrogen phosphate dihydrate, and 0.05g of ammonium chloride per 100mL of water;

[0031] Stock solution B is 3.64 g of calcium chloride dihydrate per 100 mL of water;

[0032] Stock solution C is 2.25 g of magnesium sulfate heptahydrate per 100 mL of water;

[0033] Stock solution D is prepared by adding 0.025 g of ferric chloride hexahydrate to every 100 mL of water.

[0034] The concentration of the activated sludge inoculum when mixed with the test chemical in step 2) is 90 mg / L in terms of suspended solids.

[0035] The activated sludge is fresh activated sludge discharged from a sewage treatment plant that treats domestic sewage.

[0036] The culture was carried out at 21°C±1°C.

[0037] An application of the method, the method is applied to chemical hazard identification and environmental risk assessment.

[0038] The chemical to be tested is suitable for new chemical substances that are poorly soluble in water and easily adsorbed.

[0039] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0040] The manometric respirometry enhanced rapid biodegradation test method provided by the present invention has the advantages of reliable method, simple operation, accurate data, low detection cost, etc., fills the gap in the operation method of enhanced rapid biodegradation test, and is a test method with good promotion significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The biodegradation curve diagram provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments. The described embodiments are only a part of the embodiments of the method of the present invention, not all embodiments.

[0043] Example 1

[0044] Activated sludge from a municipal sewage treatment plant was collected as inoculum, and the biodegradation rate of the test cellulose was determined using the method of the present invention.

[0045] 1) The fresh activated sludge collected from the aeration tank of the domestic sewage treatment plant showed that the bacterial flocs were round, closed and compact under a microscope. At the same time, there were only a small amount of filamentous bacteria in the sludge, and almost no free bacteria. This showed that the sludge was easy to coagulate and concentrate, had good performance, and could be used for the test;

[0046] 2) Pretreatment of activated sludge: Filter the sludge after microscopic examination with a fine sieve to remove coarse particles, let the sample stand, discard the supernatant and impurities floating on the surface, wash the sludge twice with test culture medium, and suspend the sludge with culture medium. Take 10mL of sludge, replicate 4 times, dry it at 105℃ for 1.5 hours and weigh it, and the initial concentration of the sludge is calculated to be 7.52g / L. According to the results, the sludge is prepared with culture medium so that the concentration is 3g / L in terms of suspended solids. The sludge will be aerated at 20.9-22.0℃ for 2 days until it is used in the test. On the day of the test, take 10mL of sludge, replicate 4 times, dry it at 105℃ for 1.5 hours and weigh it, and the sludge concentration is calculated to be 3.30g / L, thus determining the amount of activated sludge inoculum added in the test to be 6.82mL;

[0047] 3) Preparation of inoculation medium: 6.82 mL of pre-treated activated sludge was added to the culture medium for suspension, so that the concentration of sludge inoculum in the culture medium was 90 mg / L in terms of suspended solids, and then the suspension was set aside;

[0048] 4) Add the test substance cellulose to the above-obtained inoculation medium so that the final concentration of cellulose is ThOD NH3 The test group was set up with three replicates, and the blank control group was set up with two replicates.

[0049] 5) Prepare the reference test bottle: weigh the reference and add it directly into the test bottle. The concentration of reference sodium benzoate is equivalent to 50 mg / L in ThOD. Set one replicate for the reference test bottle (program control);

[0050] 6) The test group and the control group were placed in a biochemical incubator to maintain a constant temperature (20.8-21.7° C.), and magnetic stirring and BOD value determination were started. During the 60-day test period, the BOD value was determined every 12 hours;

[0051] 7) The biodegradation rate is calculated using the BOD value measured during the above test period, and a biodegradation curve is drawn based on time and degradation rate (see Figure 1 ), and then obtain the biodegradation rate of chemicals;

[0052] The oxygen consumption (mg) (BOD value) of the test substance measured at different times is corrected by the blank control of the inoculum in the same period and divided by the amount of the test substance (mg) to obtain the BOD expressed in mg oxygen to mg test substance, that is:

[0053]

[0054] The results of the determination show that the average biodegradation rate of the test cellulose in the method of the present invention is 59% after 28 days, and 69% after 60 days, reaching a pass level of 60% at the end of the test. Compared with the average biodegradation rate of 48% of the test cellulose in the conventional 301F method (inoculation concentration is 30 mg / L in terms of suspended solids, and the test period is 28 days), the biodegradation rate is greatly improved. Therefore, the purpose of strengthening rapid biodegradability is effectively achieved by the method of the present invention. At the same time, the degradation rate of the program control group in the test is more than 60% in 14 days, indicating that the test is effective.

[0055] The above contents are further detailed descriptions of the present invention in combination with specific / preferred implementations, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can also make several substitutions or modifications to these described embodiments without departing from the concept of the present invention, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for conducting an enhanced rapid biodegradation test of chemicals by manometric respirometry, characterized in that: The test chemicals and activated sludge are added to the culture medium for cultivation, and the oxygen consumption in the system is measured, and then the biodegradation rate of the chemicals is calculated based on the oxygen consumption; The formula for calculating the biodegradation rate of chemicals through oxygen consumption is BOD value divided by ThOD NH3 The biodegradation percentage is obtained by calculating the value:

2. The method for conducting an enhanced rapid biodegradation test of a chemical by manometric respirometry according to claim 1, characterized in that: 1) Preparation of inoculation medium: suspend the activated sludge with known sludge concentration after pre-treatment in the culture medium so that the sludge inoculum concentration in the culture medium is 90 mg / L in terms of suspended solids, and set aside; 2) Add the test chemical to the above-obtained inoculation medium so that the final concentration of the test chemical in the system is ThOD NH3 The concentration equivalent to 50-100 mg / L was used as the test group; meanwhile, the blank control was used without adding the test chemical. 3) The test group and control group were placed in a biochemical incubator to maintain a constant temperature (21±1°C), and magnetic stirring and BOD value determination were started. During the test period, the BOD value was determined every 12 hours; 4) The biodegradation rate is calculated using the BOD value measured during the above test period, and a biodegradation curve is drawn based on time and degradation rate to obtain the biodegradation rate of the chemical; The oxygen consumption (mg) (BOD value) of the test substance measured at different times is corrected by the blank control of the inoculum in the same period and divided by the amount of the test substance (mg) to obtain the BOD expressed in mg oxygen to mg test substance, that is:

3. The method for conducting an enhanced rapid biodegradation test of a chemical by manometric respirometry according to claim 2, characterized in that: The culture medium is Add stock solution A to water first, then add stock solution B, stock solution C and stock solution D in sequence, and then adjust the volume to 1L, and adjust the solution pH to 7.4±0.2; wherein, the volume ratio of stock solution A, stock solution B, stock solution C and stock solution D is 10:1:1; the stock solution A is prepared by adding 0.85g of monopotassium phosphate, 2.175g of dipotassium hydrogen phosphate, 3.34g of disodium hydrogen phosphate dihydrate, and 0.05g of ammonium chloride per 100mL of water; Stock solution B is 3.64 g of calcium chloride dihydrate added to every 100 mL of water; Stock solution C is 2.25 g of magnesium sulfate heptahydrate per 100 mL of water; Stock solution D is prepared by adding 0.025 g of ferric chloride hexahydrate to every 100 mL of water.

4. The method for conducting an enhanced rapid biodegradation test of a chemical by manometric respirometry according to claim 2, characterized in that: The activated sludge is fresh activated sludge discharged from a sewage treatment plant that treats domestic sewage.

5. The method for conducting an enhanced rapid biodegradation test of a chemical by manometric respirometry according to claim 2, characterized in that: The culture was carried out at 21°C±1°C.

6. An application of the method according to claim 1, characterized in that: The method is applied to the identification of chemical hazards and environmental risk assessment.

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