JP7360240B2 - Method for evaluating the degradability of chemical substances, and test containers and oxygen consumption measurement devices used in the method - Google Patents

Description

The present invention relates to a method for evaluating the degradability of chemical substances, and a test container and an oxygen consumption measuring device used in the method.

The purpose of the Act on the Examination and Regulation of Manufacturing, etc. of Chemical Substances (Chemical Substances Control Act) is to prevent environmental pollution by chemical substances that may impair human health or interfere with the habitat and growth of animals and plants. It is a law that

Under Japan's Chemical Substances Control Law, chemical substances are evaluated in stages such as degradability, concentration, and toxicity, and if a chemical is determined to be well degradable in the degradability evaluation, it is prohibited to use it without further evaluation. It becomes possible to manufacture and import. The current degradability evaluation is a test equivalent to OECD Test Guideline (hereinafter referred to as OECD TG) 301C. Additionally, current testing equipment is available and OECD TG301F, which is being implemented overseas, will be introduced from FY2018. The test equivalent to the current OECD TG301C sometimes underestimated the degradative activity. Furthermore, in OECD TG301C and OECD TG301F, the test requirement is that the difference between the maximum and minimum decomposition values is less than 20%, and an evaluation method with less variation in results is required.

Non-Patent Document 1 reports research results regarding the evaluation of the degradability of such chemical substances. In the section “4.5.1.4 Effect of test volume in enhanced biodegradation screening tests” of Non-Patent Document 1, the influence of the test solution on the degradability test is investigated, and the test was conducted with the activated sludge concentration at 30 mg SS L ^-1 . Degradability tests have been conducted using 1L, 500 mL, 100 mL, 50 mL, and 10 mL solutions. As a result, it has been shown that the degree of decomposition of the chemical substance reaches a plateau at 500 mL, and the coefficient of variation regarding decomposition also reaches its minimum at 500 mL. Therefore, based on these results, it is considered that testing for the degradability of chemicals should not normally be performed using test solutions with volumes exceeding 500 mL.

Timothy James Martin, The Influence of microbial inocula on biodegradation outcome towards enhanced regulatory assessments, Newcastle University, 2014, Ph.D. thesis, 114-119

An object of the present invention is to provide an evaluation method in which the decomposition activity of a chemical substance is improved and the results are less variable, as well as a test container and an oxygen consumption measuring device used in the method.

The current test method under the Chemical Substance Control Law stipulates that the test liquid volume is 300 mL at the specified concentrations of chemical substances and activated sludge, making it difficult for the chemical substances to decompose compared to the actual environment. The new degradability testing method under the Chemical Substance Control Law stipulates the concentrations of chemical substances and activated sludge, but does not stipulate the amount of test liquid.

As a result of extensive research to achieve the above objective, the inventors of the present invention have found that, in a new degradability test method under the Chemical Substances Control Law, the decomposition of chemical substances can be improved by increasing the amount of test liquid to over 1000 mL. We obtained the knowledge that it is possible to promote

The present invention has been completed based on these findings and further studies, and provides a method, a test container, and an oxygen consumption measuring device for evaluating the decomposability of the following chemical substances.

Item 1. A method for evaluating the degradability of chemical substances, the method comprising:
(1) incubating a test solution containing a chemical substance and 25-35 mg/L activated sludge with a volume of more than 1000 mL at a temperature of 20-24 °C;
evaluation methods, including
Item 2. Item 2. The method according to Item 1, wherein the test solution has a volume of 1200 mL or more.
Item 3. Item 3. The method according to Item 1 or 2, wherein the test solution has a volume of 1500 mL or more.
Item 4. The method according to any one of paragraphs 1 to 3, wherein the concentration of the chemical substance in the test solution is 20 to 120 mg/L.
Item 5. (2) a step of calculating the degree of decomposition from the amount of oxygen consumed during the culture process in step (1);
5. The method according to any one of paragraphs 1 to 4, further comprising:
Item 6. The method according to any one of claims 1 to 5, wherein the test solution comprises an emulsifier and/or a carrier.
Item 7. The method according to item 6, wherein the emulsifier is a surfactant.
Item 8. The method according to Item 6 or 7, wherein the carrier is silica gel.
Item 9. 9. The method according to any one of Items 1 to 8, wherein the culture period in step (1) is within 30 days.
Item 10. Test vessels with a capacity greater than 1000 mL containing chemicals and activated sludge at 25-35 mg/L.
Item 11. Item 11. The test container according to item 10, wherein the test container has a capacity of 1500 mL or more.
Item 12. Item 12. The test container according to Item 10 or 11, wherein the test container has a capacity of 2000 mL or more.
Item 13. The test container according to any one of Items 10 to 12, comprising an emulsifier and/or a carrier.
Item 14. The test container according to item 13, wherein the emulsifier is a surfactant.
Item 15. The test container according to item 13 or 14, wherein the carrier is silica gel.
Section 16. An oxygen consumption measuring device comprising the test container according to any one of items 10 to 15.

According to the present invention, the decomposition activity of chemical substances can be improved. Furthermore, according to the present invention, it is possible to reduce variations in evaluation results and obtain stable results.

As a result of the improved decomposition activity of the present invention, when evaluating the degradability of a chemical substance that is originally easily degradable, the degradability of the chemical substance is not underestimated and determined to be difficult to decompose, and the degradability of the chemical substance is not underestimated. If the chemical substance is determined to be persistent, it will be possible to manufacture and import the chemical substance without carrying out the subsequent evaluation that is required under the Chemical Substance Control Law if it is determined to be persistent. It becomes possible to reduce the unreasonable costs required.

2 is a graph showing oxygen consumption (mg) in a decomposition test (300 mL and 900 mL) of 2-ethylanthraquinone. It is a graph showing the oxygen consumption (mg) in a decomposition test (3900 mL) of 2-ethyl anthraquinone. 2 is a graph showing the degree of decomposition (%) (after correction) from the amount of oxygen consumed in a decomposition test of 2-ethyl anthraquinone. FIG. 2 is a diagram showing a chromatogram of a 2-ethylanthraquinone standard solution at 100 mg/L. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 300 mL biodegradation zone-1 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 300 mL biodegradation zone-2 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 900 mL biodegradation zone-1 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 900 mL biodegradation section-2 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 3900 mL biodegradation zone-1 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 3900 mL biodegradation zone-2 after 28 days of culture. 1 is a graph showing the decomposition degree (%) of a test substance from chemical analysis in a decomposition test of tris(2-ethylhexyl) trimellitate. 2 is a graph showing the amount of oxygen consumed (mg) in a decomposition test (300 mL) of 2-ethylanthraquinone. 2 is a graph showing the amount of oxygen consumed (mg) in a decomposition test (1500 mL) of 2-ethylanthraquinone. FIG. 2 is a diagram showing a chromatogram of a 2-ethylanthraquinone standard solution at 101 mg/L. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 300 mL biodegradation zone-1 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 300 mL biodegradation zone-2 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 1500 mL biodegradation zone-1 after 28 days of culture. [2-Ethylanthraquinone] FIG. 2 is a diagram showing a chromatogram in 1500 mL biodegradation section-2 after 28 days of culture. It is a graph showing the oxygen consumption (mg) in a decomposition test (300 mL) of 4-fluorophenol. This is a graph showing the amount of oxygen consumed (mg) in a decomposition test (3900 mL) of 4-fluorophenol. FIG. 2 is a diagram showing a chromatogram of a 4-fluorophenol standard solution at 101 mg/L. [4-Fluorophenol] This is a diagram showing a chromatogram in 300 mL biodegradation section-1 after 28 days of culture. [4-Fluorophenol] A diagram showing a chromatogram in 300 mL biodegradation section-2 after 28 days of culture. [4-Fluorophenol] A diagram showing a chromatogram in 300 mL biodegradation zone-3 after 28 days of culture. [4-Fluorophenol] A diagram showing a chromatogram in 3900 mL biodegradation zone-1 after 28 days of culture. [4-Fluorophenol] A diagram showing a chromatogram in 3900 mL biodegradation zone-2 after 28 days of culture. [4-Fluorophenol] This is a diagram showing a chromatogram in 3900 mL biodegradation zone-3 after 28 days of culture.

The present invention will be explained in detail below.

Note that in this specification, the term "comprise" includes the meanings of "essentially consist of" and "consist of".

The method for evaluating the degradability of chemical substances of the present invention is as follows:
(1) Incubating a volume of test solution greater than 1000 mL containing chemicals (e.g., 20-120 mg/L) and activated sludge 25-35 mg/L at a temperature of 20-24°C. ,
It is characterized by including.

The test vessel of the invention is also characterized in that it has a capacity of more than 1000 mL, containing a chemical (eg, 20-120 mg/L) and 25-35 mg/L activated sludge. The oxygen consumption measurement device of the present invention is characterized by comprising the test container.

The method for evaluating the degradability of chemical substances of the present invention is basically a test for the degree of decomposition of chemical substances by microorganisms corresponding to OECD TG301F, or the test conditions of OECD TG301F are modified as necessary.

Chemical substances (test substances) whose degradability should be evaluated are not particularly limited, and include various new or existing chemical substances that need to be submitted to registration tests under the Chemical Substances Control Law. If the test substance is solid and does not dissolve in water to the specified concentration, it can be pulverized as much as possible.

The concentration of the test substance in the test solution is usually 80 to 120 mg/L, preferably 90 to 110 mg/L, particularly preferably 100 mg/L. In the case of a test substance that inhibits microorganisms, the concentration of the test substance in the test solution is usually 20 to 40 mg/L, preferably 27 to 33 mg/L, particularly preferably 30 mg/L.

As the activated sludge, activated sludge or return sludge from an aerobic reaction tank (for example, near the outlet) of a sewage treatment plant that mainly treats domestic wastewater under the jurisdiction of a local government is preferably used. After collecting sludge from a sewage treatment plant, it is desirable to remove large particles using a sieve as necessary and maintain aerobic conditions until it is used for testing. If activated sludge is not used on the day of collection, it can be maintained in aerobic conditions at approximately 22°C and used for 7 days after collection. As a general rule, activated sludge must not be enriched with test substances or reference substances (aniline, sodium acetate, or sodium benzoate).

The concentration of activated sludge in the test solution is usually 25 to 35 mg/L, preferably 28 to 32 mg/L, particularly preferably 30 mg/L. The concentration of activated sludge can be measured, for example, according to method 14.1 of JIS K0102-2013.

The test solution used is one in which the test substance and activated sludge are added to the basic culture medium. As the basic culture medium, water to which various inorganic salts are added can be used, and examples of the inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, ammonium chloride, Examples include calcium chloride, magnesium sulfate, and iron(III) chloride. Furthermore, examples of the pH adjustment solution used to adjust the pH of the basic culture medium include phosphoric acid, sodium hydroxide solution, and the like.

As the basic culture medium, preferably the following basic culture medium is used. In addition, the mass shown below is a preferable value, and can also be changed if necessary.
"Prepare the following solutions A, B, C, and D. Add water to 10 mL of solution A, 1 mL of solution B, 1 mL of solution C, and 1 mL of solution D to make 1 L. Use as basic culture medium.
A liquid:
Potassium dihydrogen phosphate (KH ₂ PO ₄ ) 8.50 g
Dipotassium hydrogen phosphate (K ₂ HPO ₄ ) 21.75 g
Disodium hydrogen phosphate dihydrate (Na ₂ HPO ₄・2H ₂ O) 33.40 g
Or disodium hydrogen phosphate dodecahydrate (Na ₂ HPO ₄・12H ₂ O) 67.21 g
Ammonium chloride (NH ₄ Cl) 0.50 g
Dissolve the above in water to make 1 L. Also adjust the pH to 7.4.
B liquid (equivalent to C liquid specified in 21 of JIS K0102-2013):
Calcium chloride (CaCl ₂ ) 27.50 g
or calcium chloride dihydrate (CaCl _{2.2H 2} O) 36.40 g
Dissolve the above in water to make 1 L.
C liquid (equivalent to B liquid specified in 21 of JIS K0102-2013):
Magnesium sulfate heptahydrate (MgSO ₄ 7H ₂ O) 22.50 g
Dissolve the above in water to make 1 L.
D liquid (equivalent to D liquid specified in JIS K0102-2013 21):
Iron(III) chloride hexahydrate (FeCl ₃ 6H ₂ O) 0.25 g
Dissolve the above in water to make 1 L. This solution should be prepared immediately before use. ”
Before adding activated sludge to the basal culture medium, adjust the pH of the solution to 7.4 ± 0.2 if necessary.

Furthermore, if the test substance is poorly water-soluble, the test solution may further contain auxiliary substances (solvents, emulsifiers, carriers, etc.) for the purpose of improving contact between the test substance and microorganisms. Auxiliary substances are preferably emulsifiers and carriers, more preferably surfactants and silica gel. As the auxiliary substance, it is desirable that it is biodegradable and does not inhibit microorganisms.

As the surfactant, any of nonionic surfactants, cationic surfactants, amphoteric surfactants, and anionic surfactants can be used without particular restrictions, and among them, those with low antibacterial activity are preferred. preferable. Examples of surfactants with low antibacterial properties include nonionic surfactants, and specific examples of nonionic surfactants include polyoxyethylene (POE) sorbitan fatty acid esters (Tween (registered trademark), etc.), POE-polyoxypropylene (POP) block copolymers (poloxamers, etc.), POE-POP block copolymer adducts of ethylene diamine, propylene glycol fatty acid esters, POE castor oil, POE alkylphenyl ethers, POE alkyl ethers, POE/POP alkyl ethers, Examples include sorbitan fatty acid ester, sucrose fatty acid ester, alkyl polyglycoside, propylene glycol fatty acid ester, polyethylene glycol monostearate, and glycerin fatty acid ester.

The contents of the emulsifier and carrier in the test solution are not particularly limited and can be selected as appropriate. Emulsifiers can be used alone or in combination of two or more.

The lower limits of the test solution volume are, for example, 1100 mL, 1200 mL, 1300 mL, 1400 mL, 1500 mL, 1600 mL, 1700 mL, 1800 mL, 1900 mL, 2000 mL, 2100 mL, 2200 mL, 2300 mL, 2400 mL, 2500 mL, 2600 mL, 2700 mL, 2800 mL, 2900 mL, 3000 mL, 3100 mL, 3200 mL, 3300 mL, 3400 mL, 3500 mL, 3600 mL, 3700 mL, 3800 mL, 3900 mL, etc. It will be done. By setting the volume of the test solution to such a volume, it is possible to improve the decomposition activity of the chemical substance and reduce the variation in the evaluation results in the degradability evaluation of the chemical substance.

For example, the upper limit of the test solution capacity is 6500 mL, 6400 mL, 6300 mL, 6200 mL, 6100 mL, 6000 mL, 5900 mL, 5800 mL, 5700 mL, 5600 mL, 5500 mL, 5400 mL, 5300 mL, Examples include 5200 mL, 5100 mL, 5000 mL, 4900 mL, 4800 mL, 4700 mL, 4600 mL, 4500 mL, 4400 mL, 4300 mL, 4200 mL, 4100 mL, 4000 mL, 3900 mL, etc. It is thought that by increasing the size and bringing it closer to the actual environment, the decomposition activity will improve and it will be possible to reduce the variation in results.

There are no particular restrictions on the test container for holding the test solution, and an appropriate container can be selected from ready-made products made of glass or plastic, custom-made products, homemade products (for example, culture bottles), etc. can be used. In the present invention, a test container with a specific capacity (for example, a capacity exceeding 1000 mL) means a test container having a size that can accommodate a specific volume of test solution, and the capacity of the test container is Corresponds to the volume of the solution.

In the evaluation of the degradability of chemical substances, in addition to the test solution mentioned above, a reference substance (aniline, sodium acetate, or sodium benzoate) was added to the basic culture medium to which activated sludge was added at a concentration of 100 mg/L ( If necessary, prepare a base culture medium with activated sludge added (add auxiliary substances (appropriate amounts) if necessary), etc.

Cultivation is usually carried out for a certain period of time under light-shielded conditions with sufficient stirring. The temperature conditions for culturing are usually 20-24°C, preferably 21-23°C. The culture period is usually 30 days or less, preferably 29 days or less, more preferably 28 days or less, and usually 10 days or more, preferably 14 days or more, and more preferably 21 days. That's all. The culture period is most preferably 28 days.

During culturing, the amount of oxygen consumed during the culturing process (oxygen consumption) is usually measured. The oxygen consumption amount can be measured using, for example, an oxygen consumption amount measuring device. The oxygen consumption measuring device is a device for measuring biochemical oxygen consumption (BOD), and various commercially available products (eg, coulometer) can be used.

After culturing for a certain period of time, if necessary, the remaining test substance and changes are subjected to analysis and their amounts are measured. If the test substance is dissolved in water, the remaining amount of dissolved organic carbon is also measured. Also, measure the pH of the test solution if necessary.

At the end of the test (incubation), the validity of the test can be judged, for example, by the following criteria: the maximum and minimum degree of decomposition in the test solution to which the test substance, activated sludge and basic culture medium have been added. The difference in values is less than 20%, the oxygen consumption of the product using only the basic culture medium with activated sludge added is 60 mg/L or less, and the degree of decomposition of the reference material determined from the oxygen consumption of the product using the reference material. The test is considered valid when the rate reaches 60% after 14 days.

The method of evaluating the degradability of a chemical substance of the present invention preferably further includes the step of (2) calculating the degree of decomposition from the amount of oxygen consumed during the culture process (oxygen consumption) in step (1).

The degree of decomposition (%) from the amount of oxygen consumed can be calculated using the following formula.
Resolution (%) = (BOD－B) / TOD ⁽¹⁾ ×100
BOD: Biochemical oxygen consumption (measured value) of the test substance (mg)
B: Oxygen consumption (measured value) (mg) of basic culture medium inoculated with activated sludge
TOD: Theoretical oxygen consumption (calculated value) required when the test substance is completely oxidized (mg)
⁽¹⁾ If the test substance containing nitrogen is decomposed, calculate the TOD according to the degree of nitrification.

Further, the degree of decomposition can also be calculated by direct quantification. Direct quantification can be performed using total organic carbon analyzers and other analyzers. Examples of analysis methods using a total organic carbon analyzer and other analyzers are shown below.

(When using a total organic carbon analyzer)
Take an appropriate amount of the test liquid from the test container, centrifuge it at approximately 40,000 m/s ² for 15 minutes or filter it (0.45 μm), take an appropriate amount from the supernatant liquid or filtrate, and remove the total organic Quantify the remaining dissolved organic carbon using a carbon analyzer.

(When using other analyzers)
After performing appropriate pretreatment such as extraction and concentration of the contents in the test container with a solvent suitable for the test substance, etc., perform quantitative analysis using analytical equipment. In this case, as a general rule, the analysis is performed in accordance with the general rules for analysis methods (high performance liquid chromatography, gas chromatography, spectrophotometry, mass spectrometry, atomic absorption spectrometry, etc.) stipulated by JIS.

The degree of degradation (%) from direct quantification can be calculated using the following formula:
Test substance decomposition degree (%) = (B-A) / B×100
A: Residual amount of test substance (measured value) after completion of degradation test (mg)
B: Amount of test substance added (mg)

The standard for determining good degradability is that the degree of decomposition is 60% or more and that no decomposition products are generated. Furthermore, those that are not easily degradable are determined to be difficult to decompose.

According to the present invention, the decomposition activity of chemical substances can be improved. Furthermore, according to the present invention, it is possible to reduce variations in evaluation results and obtain stable results. As described above, as a result of the improved decomposition activity of the present invention, when evaluating the degradability of chemical substances that are originally easily degradable, the degradability is not underestimated and judged to be difficult to decompose, and the original degradability is not underestimated. By determining that the chemical substance is easily degradable, it becomes possible to manufacture and import the chemical substance without carrying out the subsequent evaluation that is required under the Chemical Substances Control Law if it is determined to be difficult to decompose. Therefore, it is possible to reduce unreasonable costs required for registration.

Examples are given below to explain the present invention in more detail. However, the present invention is not limited to these Examples in any way.

Test example /test method 1. Microorganisms Activated sludge from a sewage treatment plant was used as the microorganism source. The concentration of activated sludge was measured according to the method of JIS K0102 (1993).

2. Test substances 2-ethylanthraquinone, tris(2-ethylhexyl) trimellitate, and 4-fluorophenol were used.

3. Test device Oxygen consumption was measured over time using an oxygen consumption measuring device (coulometer) that can maintain a constant temperature and has a stirring function. When a test substance is decomposed aerobically, oxygen is consumed, so the degree of decomposition of the test substance can be evaluated by measuring the cumulative amount of oxygen consumption.

4. Test conditions A decomposition test was conducted using a coulometer under the following conditions.
Test substance concentration: 100 mg/L
Activated sludge concentration: 30 mg/L
Temperature: 22±1℃
Stirring: Use the stirrer included in the coulometer Light: Block out Duration: 28 days

5. Test area <biodegradation area>
Basic culture medium ^*1) with test substance and activated sludge added. In the following tests I to III, silica gel was used as a carrier, and the test substance was added in a state ^*2) adsorbed on the silica gel (n=2). No silica gel was added in the IV test (n=3).

<Control area>
Basic culture medium ^*1) with activated sludge added (n=1). In the following tests I to III, the same amount of silica gel ^*2) (test substance not adsorbed) as in the biodegradable area was added. No silica gel was added in the IV test.

^*1) Preparation of basic culture medium (according to OECD TG 301F)
Solutions A, B, C, and D shown below were prepared. Water was added to 10 mL of solution A, 1 mL of solution B, 1 mL of solution C, and 1 mL of solution D to make 1 L, and this was used as the basic culture medium.
A liquid:
Potassium dihydrogen phosphate (KH ₂ PO ₄ ) 8.50 g
Dipotassium hydrogen phosphate (K ₂ HPO ₄ ) 21.75 g
Disodium hydrogen phosphate dodecahydrate (Na ₂ HPO ₄・12H ₂ O) 67.21 g
Ammonium chloride (NH ₄ Cl) 0.50 g
The above was dissolved in water to make 1 L. The pH was also adjusted to 7.4.
B liquid:
Calcium chloride (CaCl ₂ ) 27.50 g
The above was dissolved in water to make 1 L.
C liquid:
Magnesium sulfate heptahydrate (MgSO ₄ 7H ₂ O) 22.50 g
The above was dissolved in water to make 1 L.
D liquid:
Iron(III) chloride hexahydrate (FeCl ₃ 6H ₂ O) 0.25 g
The above was dissolved in water to make 1 L. Note that this solution was prepared immediately before use.

^*2) Method of adsorbing the test substance to silica gel
Silica gel for column chromatography (particle size: 15 μm to 500 μm) recommended by ISO 10634 guidance (ISO, 1995) was used. The method for adsorbing the test substance onto silica gel was as follows. 1800 mg of the test substance was dissolved in about 150 mL of chloroform, and 24 g of silica gel was added thereto and stirred for 2 hours. Thereafter, in order to volatilize the chloroform, it was treated with evaporation and a vacuum oven set at 45° C. for about 14 days. In order to calculate the amount of the test substance adsorbed per unit weight of silica gel, the test substance was extracted from the treated silica gel with a solvent and chemically analyzed.

In addition, the control group was treated using the above-mentioned treatment method, with silica gel and chloroform set at the same ratio, and without the addition of the test substance.

In addition, in the following tests I and II, since the chloroform used for adsorption remained in the silica gel, the amount of oxygen consumption increased even in the control group, which required correction. In tests I and II, chloroform was volatilized by placing silica gel in a glass container (eggplant flask) in a vacuum oven, but in test III, silica gel was spread on a vat to increase the surface area and volatilize. , the residual amount of chloroform decreased rapidly, and the amount of oxygen consumed by chloroform was almost eliminated, making correction unnecessary.

6. Test capacity
The biodegradation area and control area were tested using containers for 300 mL, 900 mL, 1500 mL, and 3900 mL.

7. Evaluation method
(1) Method of calculating degree of decomposition (%) from oxygen consumption The following formula was used.
Resolution (%) = (BOD－B) / TOD ×100
BOD: Oxygen consumption amount (measured value) in biodegradation zone (mg)
B: Oxygen consumption (measured value) in control area (mg)
TOD: Theoretical oxygen consumption (calculated value) required when the test substance is completely oxidized (mg)
[See (i) to (iii) below for calculation method]
When evaluating the degree of decomposition, when using the results of test plots with different capacities, the oxygen consumption was corrected according to the capacity.

<TOD calculation method>
Complete oxidation reaction equation: Test substance + a O ₂ → b CO ₂ + c H ₂ O
Amount of O ₂ per 1 mg of test substance = a × O ₂ molecular weight / molecular weight of test substance
TOD = _O2 amount per 1 mg of test substance x test substance concentration (mg/L) x test volume (L)
(i) In the case of 2-ethylanthraquinone Complete oxidation reaction formula: C ₁₆ H ₁₂ O ₂ + 18 O ₂ → 16 CO ₂ + 6 H ₂ O
Amount of oxygen per 1 mg of test substance = 18 O ₂ / C ₁₆ H ₁₂ O ₂ = 575.98 / 236.27 = 2.44
TOD (e.g. at 300 mL): 2.44 x 100 mg/L x 0.3 L = 73.2 mg
(ii) In the case of tris(2-ethylhexyl) trimellitate Complete oxidation reaction formula: C ₃₃ H ₅₄ O ₆ + 43.5 O ₂ → 33 CO ₂ + 27 H ₂ O
Amount of oxygen per 1 mg of test substance = 43.5 O ₂ / C ₃₃ H ₅₄ O ₆ = 1391.95 / 546.78 = 2.55
TOD (e.g. at 300 mL): 2.55 x 100 mg/L x 0.3 L = 76.5 mg
(iii) In the case of 4-fluorophenol Complete oxidation reaction formula: C ₆ H ₅ FO + 6.75 O ₂ → 6 CO ₂ + 2.5 H ₂ O + F
Amount of oxygen per 1 mg of test substance = 6.75 O ₂ / C ₆ H ₅ FO = 215.99 / 112.10 = 1.93
TOD (e.g. at 300 mL): 1.93 x 100 mg/L x 0.3 L = 57.9 mg

(2) Method of calculating test substance decomposition degree (%) from chemical analysis (HPLC) The following formula was used.
Test substance decomposition degree (%) = (B-A) / B×100
A: Residual amount (measured value) of the test substance in the biodegradation area after 28 days of culture (mg)
B: Amount of test substance added (mg)
Degradation product production rate (%) = C / D ×100
C: Amount of decomposition products produced in the biodegradation zone after 28 days of culture ^* (measured value) (mg)
D: Added amount of test substance (mg)
^* Quantitative determination assuming that the test substance and decomposition product have the same extinction coefficient.

·result
I. 2-Ethylanthraquinone (300 mL to 3900 mL)
[Decomposition degree calculated from oxygen consumption]
Figures 1 and 2 show the amount of oxygen consumed (mg) by activated sludge. As a result, it became clear that the amount of oxygen consumed in the 900 mL biodegradation area was greater than that in the 300 mL biodegradation area, and the oxygen consumption in the 3900 mL biodegradation area was greater than in the 900 mL biodegradation area. This trend was also observed in the control area. This is due not only to the fact that the decomposition of 2-ethylanthraquinone was promoted by increasing the test volume, but also to the residual amount of chloroform used when adsorbing the test substance onto the silica gel.

In Figure 1, the 300 mL biodegradable area ([1] and [2] in the figure), the 300 mL control area ([5] in the figure), and the 900 mL biodegradable area ([3] and [3] in the figure). [4]) and the 900 mL control area ([6] in the figure), as well as the 3900 mL biodegradation area and the control area in Figure 2, the oxygen consumption before the decomposition of 2-ethylanthraquinone rapidly progresses. Since the amount was larger in the control area, it is thought that more chloroform remained in the control area. Therefore, the decomposition degree calculated using the method in 7.(1) was a negative value, and the bottom value was around -15%. Therefore, we decided to correct the resolution by adding 15%.

In addition, in the 3900 mL control group, the upper limit for oxygen consumption measurement was reached around the 12th day, so oxygen consumption could not be measured for the entire 28 days. The oxygen consumption in the 900 mL control group ([6] in Figure 1) was about three times that of the 300 mL control group ([5] in Figure 1), and the oxygen consumption in the 3900 mL control group (Figure 2) was also 12 days. The oxygen consumption was approximately 13 times that of the 300 mL control group. Therefore, the oxygen consumption in the 300 mL control section at each time point was multiplied by 13 to correct the value for the 3900 mL control section.

Figure 3 was created with the above corrections, and shows that the decomposition in the 900 mL test area started after the 21st day (900 mL-2 in Figure 3) and that the degree of decomposition started earlier than in the 300 mL test area. There are cases where the concentration has started to increase (900 mL-1 in Figure 3), and in both 3900 mL test areas (3900 mL-1 and 3900 mL-2 in Figure 3), the degree of decomposition was reached before 21 days. increased, and decomposition was accelerated.

[Calculating the degree of decomposition of the test substance from chemical analysis]
After 28 days, acetonitrile was added to the test solution at a ratio of 3:2, a portion of it was collected and centrifuged to precipitate activated sludge and silica gel, and the supernatant was analyzed using HPLC as described below. Chemical analysis was conducted under the following conditions, and the degree of decomposition of the test substance was evaluated using the method described in 7.(2) using the results of the standard solution. The results are shown in Figures 4-10.

For 300 mL (Figures 5 and 6) and 900 mL (Figures 7 and 8), there were variations in the degree of decomposition of the test substance and the production rate of decomposed products between the two series (Table 1). On the other hand, when the volume was increased to 3900 mL, the degree of decomposition of the test substance reached 100% in both cases, and the production rate of decomposed products was also about the same (Figure 9, Figure 10, Table 1). This also suggests that increasing the amount of test solution accelerates the decomposition of the test substance and reduces variation.

・HPLC analysis conditions Column: L-column ODS (2.1 mm×150 mm)
Mobile phase: B conc. 50.0%→(3.1%/min)→100%
A: DW containing 0.1% formic acid
B: Acetonitrile containing 0.1% formic acid Detection: 254 nm, Column temperature: 40℃
Flow rate: 0.2 mL/min, injection volume: 10 μL

II. Tris(2-ethylhexyl) trimellitate (300 mL to 3900 mL)
After 28 days, tetrahydrofuran was added to the test solution at a ratio of 3:2, a portion of it was collected and centrifuged to precipitate activated sludge and silica gel, and the supernatant was subjected to the HPLC method described above. Chemical analysis was performed under the analytical conditions, and the degree of decomposition of the test substance was evaluated using the method described in 7.(2). The results are shown in FIG. Compared to 300 mL, the 900 mL test area reduced the variation between two replicates. Additionally, the degree of decomposition of the test substance was improved in the 3900 mL test area compared to 900 mL. The degree of decomposition calculated from the amount of oxygen consumed is used as a standard for degradability, but the degree of decomposition of the test substance calculated by chemical analysis also shows similar behavior, so here it was evaluated by chemical analysis.

III. 2-Ethylanthraquinone (300 mL to 1500 mL)
[Decomposition degree calculated from oxygen consumption]
Figures 12 and 13 show the amount of oxygen consumed (mg) by activated sludge. One set in the 300 mL biodegradation area began to decompose rapidly after 15 days, but the other set did not start decomposing rapidly even after 28 days. On the other hand, in two sets of 1500 mL biodegradation plots in which the test concentration was the same but the test liquid volume was increased, rapid decomposition started within 28 days both after 10 and 22 days.

[Degradation degree calculated from chemical analysis]
After 28 days, acetonitrile was added to the test solution at a ratio of 3:2, a portion of it was collected and centrifuged to precipitate activated sludge and silica gel, and the supernatant was analyzed using HPLC as described above. Chemical analysis was conducted under the following conditions, and the degree of decomposition of the test substance was evaluated using the method described in 7.(2) using the results of the standard solution. The results are shown in FIGS. 14 to 18 and Table 2. As a result, there was a large difference in the degree of decomposition of the test substance at 300 mL, between 97% (Figure 15) and 5% (Figure 16), while at 1500 mL, it was 89% (Figure 17) and 100% (Figure 16). 18), the variation between the two series was reduced, and the average value of the degree of degradation was improved from 51% to 91% (Table 2).

IV. 4-Fluorophenol (without silica gel) (300 mL to 3900 mL)
[Calculation of degree of decomposition from oxygen consumption]
Figures 19 and 20 show the amount of oxygen consumed (mg) by activated sludge. Based on the amount of oxygen consumed by microorganisms (mg) when the test volume was 300 mL, it was found that rapid decomposition started after 12.5 to 14 days in the three biodegradation plots (Figure 19). On the other hand, with a test volume of 3900 mL, oxygen consumption increased rapidly after 12 to 13 days in all three series, and then air entered from the connection between the container and the lid in only one series, exceeding the theoretical oxygen demand of the test substance. (Figure 20). In the 300 mL test container, decomposition started by the 14th in all three replicates, and there was a 1.5 day difference in the start of decomposition among the three replicates, whereas in the 3900 mL test container, decomposition started by the 13th in all three replicates. Since there was only a one-day difference between the three successive periods after the start of decomposition, increasing the amount of test solution to 3900 mL showed that decomposition started earlier than with 300 mL, and the decomposition activity improved, even under conditions without the addition of silica gel, etc. At the same time, it was found that the variation between runs was also reduced.

[Degradation degree calculation from chemical analysis]
After 28 days, a portion of the test solution was collected and chemically analyzed under the following HPLC analysis conditions without treatment, and the degree of decomposition of the test substance was evaluated using the method in 7. (2) using the results of the standard solution. . The results are shown in Figures 21-27. As a result, the test substance disappeared in all test areas, so the residual amount of the test substance was considered to be 0 mg, and the degree of decomposition was calculated as 100%.

・HPLC analysis conditions Column: L-column ODS (3.0 mm×150 mm)
Mobile phase: B conc. 10.0%→(3.0%/min)→100%
A: DW containing 0.1% formic acid
B: Acetonitrile containing 0.1% formic acid Detection: 254 nm, Column temperature: 40℃
Flow rate: 0.2 mL/min, injection volume: 10 μL

Claims (11)

A method for evaluating the degradability of chemical substances, the method comprising:
(1) Incubating a test solution with a volume of 1100 mL or more containing a chemical substance and 25 to 35 mg/L activated sludge at a temperature of 20 to 24 °C;
evaluation methods, including 2. The method of claim 1, wherein the test solution has a volume of 1200 mL or more. The method according to claim 1 or 2, wherein the volume of the test solution is 1500 mL or more. The method according to any one of claims 1 to 3, wherein the concentration of the chemical in the test solution is between 20 and 120 mg/L. The method according to any one of claims 1 to 4, further comprising: (2) calculating the degree of decomposition from the amount of oxygen consumed during the culture process in step (1). The method according to any one of claims 1 to 5, wherein the test solution comprises an emulsifier and/or a carrier. 7. The method of claim 6, wherein the emulsifier is a surfactant. The method according to claim 6 or 7, wherein the carrier is silica gel. The method according to any one of claims 1 to 8, wherein the culture period in step (1) is within 30 days. A method for evaluating the degradability of chemical substances, the method comprising:
(1) incubating a test solution with a volume of more than 1000 mL containing a chemical substance, 25-35 mg/L activated sludge, and an emulsifier and/or carrier at a temperature of 20-24 °C;
evaluation methods, including Test vessels with a volume greater than 1000 mL containing chemicals, activated sludge at 25-35 mg/L, and emulsifiers and/or carriers.