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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 April 2018 to 08 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
other: “The Guidelines for the Testing of Chemicals” of Chemical Registration Center of the Ministry of Environmental Protection. The Guidelines for the Testing of Chemicals, Degradation and Accumulation. 301B Ready Biodegradability CO2 Evolution Test [M]
Version / remarks:
Second Edition Beijing: China Environmental Press. 2013, page 32-37.
Deviations:
no
Qualifier:
according to
Guideline:
other: National Standardization Technical Committee for the Management of Hazardous Chemicals. GB/T 21856-2008 Chemicals-Ready Biodegradability CO2 Evolution Test [S].
Version / remarks:
China Standard Press. 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Department of Science and Technology Standards of State Environmental Protection Administration. HJ/T 153-2004 Guidelines for Testing of Chemicals [S]
Version / remarks:
Beijing: China Standard Press. 2004.
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated: The activated sludge derived from a sewage treatment plant to the east of Suzhou city (a conventional treatment plant for domestic waste) was selected as source of inoculum.
- Collection and pre-treatment: The freshly sampled activated sludge was collected from the aeration basin and kept aerobic during transport. The activated sludge was filtered through fine filter sieve to remove coarse particles, washed using the test medium and settled for about 10 minutes, after which the supernatant was discarded. The sludge was re-suspended in test medium and the above operation was repeated 3 times. 10 mL activated sludge was sampled (4 replicates) and dried at 105 °C for 2 hours to determine the concentration of suspended solids, which was determined to be 8.5 g SS/L. The activated sludge was diluted with test medium to give the concentration of 4.0 g SS/L. The suspended sludge was kept aerated at 22 ± 2 °C until required for testing.
- Before the test, the activated sludge suspended solid was determined to be 4.4 g SS/L. From this result, the added amount of activated sludge was calculated.
Duration of test (contact time):
28 d
Initial conc.:
23 mg/L
Based on:
test mat.
Initial conc.:
ca. 10.41 mg/L
Based on:
other: C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The stock solution of medium was prepared using purified water and analytical grade reagents.
10 mL of stock solution A was firstly mixed with purified water, and then 1 mL of stock solution B, C and D was added, mixed and made up to 1 L with purified water. 48 L of test medium was prepared according to the above method. The pH value was measured to be 7.58-7.82 and adjusted to 7.47-7.53 with HCI.
Stock solution A: KH2PO4 8.502 g, K2HPO4·3H2O 28.509 g, Na2HPO4·12H2O 67.150 g and NH4Cl 0.500 g. The above reagents were dissolved and made up to 1000 mL with purified water. The pH was determined to be 7.40.
Stock solution B: CaCl2·2H2O 18.237 g. The reagent was dissolved and made up to 500 mL with purified water.
Stock solution C: MgSO4·7H2O 11.250 g. The reagent was dissolved and made up to 500 mL with purified water.
Stock solution D: FeCl3·6H2O 0.126 g. The reagent was dissolved and made up to 500 mL with purified water. One drop of HCl was added into stock solution D.
- Test temperature: 22 ± 2 °C
- Aeration rate: 30-100 mL/min (compressed air free from CO2 and oil)
- Suspended solids concentration: 30 mg SS/L
- The test was performed in diffused light.
- Preparation of Hydrochloric Acid Solution: 45 mL of hydrochloric acid was taken and made up to 10 L with purified water to obtain the hydrochloric acid solutions with the concentration of 0.05 mol/L. The concentration of hydrochloric acid solution was measured to be 0.0547 mol/L by titrimetric analysis.
- Preparation of Barium Hydroxide Solution: Ba(OH)2·8H2O was weighed and dissolved with purified water (the water was boiled and cooled to the room temperature before used) to obtain the barium hydroxide solutions with the concentration of 0.0125 mol/L. The solutions were filtered to keep the solution clear and distributed to the CO2 absorption bottles. The remaining solution was sampled to determine the concentration.

TEST SYSTEM
- Culturing apparatus: 4 L brown flasks
- Number of culture flasks/concentration: 2
- About 2.9 L test medium were added in each 4 L brown flask. 20.5 mL of prepared activated sludge was added to give a concentration of 30 mg SS/L in the final 3 L of inoculated mixture. These inoculated mixtures were aerated with CO2-free air overnight to purge the carbon dioxide of system.`
- Test suspension: 69.13 mg and 69.16 mg of test material were directly added in flasks TS-1 and TS-2. The flasks were made up to 3 L with further test medium to give a final test material concentration of approximately 10.41 mg C/L in both vessels. Before adding the test material, the inorganic carbon concentration of the test suspension in flask TS-1 and TS-2 was measured to be 290.2 μg/L and 319.9 μg/L, respectively.
- Preparation of CO2 absorption bottle: Three 250 mL gas-washing bottles, each containing 150 mL of 0.0125 mol/L Ba(OH)2 solution were connected in series as CO2 absorption bottles, for each test flask to absorb the CO2 produced in the test flask.

MEASUREMENTS AND DETERMINATIONS
- Temperature: During the test, the temperature of test area was monitored by the temperature recorder. Stirring, aeration and temperature were checked every workday.
- CO2 Production: The analysis of CO2 was made on days 1, 2, 3, 4, 7, 9, 11, 14, 16, 19, 23, 25 and 29. On the days of CO2 determination, the CO2 absorption bottle closest to the test flask was disconnected and the others were moved forward in turn, then a new one with freshly prepared Ba(OH)2 solution was connected to the terminal. 50 mL of the absorption solution was taken from previously disconnected absorption bottle to titrate with hydrochloric acid solution. According to the change of the concentration of Ba(OH)2 solution to calculate the amount of CO2. On the 28th day, 1 mL of concentrated hydrochloric acid was added. Test flasks were aerated overnight to drive off the CO2 present in the test suspensions. On the 29th day, all CO2 absorption bottles were disconnected and titrated for the last analysis of evolved CO2.

CONTROL AND BLANK SYSTEM
- Inoculum blank: IC-1 and IC-2 flasks containing activated sludge and test medium only.
- Procedure control: 54.02 mg of reference material was added to PC flask containing activated sludge directly. The flask was made up to 3 L with further test medium to give a final concentration of 10.47 mg C/L.
- Toxicity control: 69.15 mg of test material and 54.10 mg of reference material were directly added in TC-1 flask containing activated sludge. The flask was made up to 3 L with further test medium to make the sum concentrations of the test material and reference material of 20.92 mg C/L.

DATA TREATMENT AND ANALYSIS
- Theoretical CO2 production (ThCO2):
Carbon content of reference material was calculated using the following equation:
% C= [(The number of carbon atoms in reference material x 12) / Molecular weight of reference material] x 100
Amount of theoretical CO2 of the test material was calculated using the following equation:
mg ThCO2= [( %C of test material ×44) / 12] × Test material added mg
Where,
12: Atomic weight of carbon, g/mol;
44: Molecular weight of CO2, g/mol.
ThCO2 of procedure control was calculated as above. Theoretical CO2 of toxicity control was sum of the amount of theoretical CO2 of test material and reference material.
The carbon content of reference material was 58.30 %, the ThCO2 of procedure control was 115.5 mg. The carbon content of test material was 45.17 %, the ThCO2 of test suspension were 114.5 mg and 114.5 mg, respectively, for the two replicates. The ThCO2 of toxicity control was 230.2 mg.

- Amount of CO2 produced:
The amount of CO2 produced from each test flask was calculated from the amount of base remaining in the absorption bottle. The amount of CO2 produced (mg) was calculated by:
CO2 produced(mg)= CHCl × (VF-VE) × 1.5 × 44
Where:
CHCl: Concentration of hydrochloric acid solution, mol/L;
VF: Volume of HCl solution titrated by freshly absorption solution, mL;
VE: Volume of HCl solution titrated by expired absorption solution, mL;
44: Molecular weight of CO2, g/mol.
The amount of CO2 produced from inoculums blank was calculated according to the above equation.
The amount of CO2 produced from test material and reference material (corrected for that derived from inoculum blank) were calculated by:
CO2 produced (mg)=CHCl × (VEI-VET) × 1.5 × 44
VET: Volume of HCl solution titrated by expired absorption solution of TS or PC or TC group, mL;
VEI: Mean volume of HCl solution titrated by expired absorption solution of inoculums blank, mL.

- Biodegradation Percentage:
The percentage biodegradation was calculated using the following equation:
% degradation= mg Cumulative CO2 production ×100 mg ThCO2

- Biodegradation Curve:
The degradation process was displayed by charting biodegradation percentage versus time.
Reference substance:
benzoic acid, sodium salt
Test performance:
Validity of the Test
The test met all the following validity criteria and the test was valid:
1) The difference of extremes of replicate values of the percentage degradation of the test material at the end of the test was 2.9 % (less than 20 %);
2) The percentage degradation of the reference material was 91.5 % on day 14 (more than 60 %);
3) The percentage degradation in the toxicity control was 41.8 % on day 14 (more than 25 %);
4) The total CO2 evolution in the inoculums blank at the end of the test was 37.02 mg/L (less than 40 mg/L);
5) At the beginning of the test, the inorganic carbon content of the test material suspension was 290.2 μg/L and 319.9 μg/L, which was less than 5 % of the total carbon.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
4.6
Sampling time:
28 d
Details on results:
- Temperature: During the test, the temperature of test area was in the range of 21.5-23.8 °C, satisfying test requirement (22 ± 2 °C).

- Titration of Barium Hydroxide Solution: The concentration of freshly prepared barium hydroxide solutions were titrated with hydrochloric acid solution.

- CO2 Production and Biodegradation Percentage: In the procedure control, the biodegradation percentage of reference material was 91.5 % on day 14, which was more than 60 %. The biodegradation percentage of toxicity control was 41.8 % on day 14, more than 25 %, which indicated the test material had no toxicity or inhibition to the inoculum. The mean biodegradation percentage of test material was 4.6 % on day 28, which was less than 60 %, and hence the test material cannot be considered to be readily biodegradable.
Results with reference substance:
In the procedure control, the biodegradation percentage of reference material was 91.5 % on day 14, which was more than 60 %.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of this study, the 28-day biodegradation of the test material was found to be 4.6 %. It cannot be considered to be readily biodegradable.
Executive summary:

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301B and other Chinese guidelines, under GLP conditions.

The objective of the study was to evaluate the ready biodegradability of test material using the CO2 evolution test. Activated sludge from a local sewage treatment plant was selected as inoculum and sodium benzoate as reference material. The test vessels were aerated by the passage of carbon dioxide-free air at a controlled rate and agitated at 22 ± 2 °C in diffused light for a period of 28 days. The amount of carbon dioxide produced was measured during the test. The amount of carbon dioxide produced from the test material (corrected for that derived from the blank inoculum) was expressed as percentage of the theoretical CO2.

In the procedure control, the biodegradation percentage of reference material was 91.5 % on day 14, which was greater than 60 %. The biodegradation percentage of the toxicity control was 41.8 % on day 14, which was greater than 25 %, indicating that the test material showed no toxicity or inhibition to the inoculum. The mean biodegradability percentage of test material was 4.6 % on day 28, which was less than 60 % and was not readily biodegradable in this study. The validity criteria of the test were all met.

Under the conditions of this study, the 28-day biodegradation of the test material was found to be 4.6 %. It cannot be considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April 2018 to 17 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms (301 C)
Version / remarks:
Stipulated in the "Testing Methods For New Chemical Substances" (March 31 2011, No . 0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No. 5 Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No.110331009, Environmental Policy Bureau,Ministry of Environment Japan; latest revision, March 29, 2018, No.0329-13, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; No.
20180326-1, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 1803293, Environmental Policy Bureau, Ministry of the Environment, Japan)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lake and inland sea, and return sludge from sewage plants). Activated sludge, which was prepared and was controlled in the laboratory according to the test methods followed was used in the study.
- The activated sludge, which was cultivated for 19.5 hours after feeing with the synthetic sewage, was used. The synthetic sewage was prepared according to the following method: glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0 ± 1.0.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The basal culture medium (5L) was prepared at the same proportion as the following method: purified water was added to each 3 mL aliquot of solutions A, B, C and D described in the test guidelines, in order to prepare 1 L of solution. The pH of this solution was then adjusted to 7.0.
- Test temperature: 25 ± 1 °C (measured values: 24.9 – 25.0 °C)
- Each solution was kept stirring
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: glass vessels
- Number of culture flasks/concentration: 3
- In each test vessel, 30 mg of the test material was accurately weighed with an electronic analytical balance and added to the basal culture medium [the volume subtracting the volume (2.59 mL) of activated sludge from 300 mL], so that the concentration of the test material reached 100 mg/L.
- Measuring equipment: Closed system oxygen consumption measuring apparatus (No. CM-067)
- Details of trap for CO2 and volatile organics if used: Soda lime No.1 was used for the absorption of carbon dioxide.

OBSERVATION AND MEASUREMENT
- Observation of test solution: At the start and end of the incubation, appearances of the test solution (water and test material), the test solutions (sludge and test material) and the test solution (control blank) were observed. During the incubation period, appearance of the mentioned test solutions were observed once a day.
- Measurement of the biochemical oxygen demand (BOD): During the incubation period, BOD of all test solutions were measured continuously with a closed system oxygen consumption measuring apparatus.

CONTROL AND BLANK SYSTEM
- Inoculum blank (sludge, n=1): In one test vessel, the basal culture medium [the volume subtracting the volume (2.59 mL) of activated sludge from 300 mL] was added.
- Test control (water and test material, n=1): In one vessel, 30 mg of the test material was accurately weighed with an electronic analytical balance and added to 300 mL of purified water so that the concentration of the test material reached 100 mg/L.
- Toxicity control (sludge and aniline, n=1): in one test vessel, 29.5 µL (30 mg) of aniline was taken out with a micro syringe and added to the basal culture medium [the volume subtracting the volume (2.59 mL) of activated sludge from 300 mL], so that the concentration of the aniline reached 100 mg/L.
Reference substance:
aniline
Test performance:
VALIDITY OF THE TEST
- Difference between extremes of replicate values of percentage biodegradation should be <20 %. The values in the test were 7 % (BOD) and 1 % (HPLC).
- Percentage biodegradation of aniline by BOD should be >40 % after 7 days and >65 % after 14 days. The values in the test were 74 % (7 days) and 95 % (14 days).
- BOD of the control blank should be ≤60 mg/L (≤18 mg). The value in the test was 32 mg/L.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Remarks:
(HPLC)
Value:
0
Sampling time:
28 d
Details on results:
APPEARANCES OF THE TEST SOLUTIONS
At the start of incubation:
- Water and test material: The test material was not dissolved. The test solution was colourless.
- Sludge and test material: The test material was not dissolved. The test solution was colourless.
- Control blank: Insoluble compound except the sludge was not observed. The test solution was colourless.
At the end of incubation:
- Water and test material: Insoluble compound was observed. The test solution was colourless. Vessel 1: pH 6.9.
- Sludge and test material: Insoluble compound in addition to the sludge was observed. Growth of the sludge was not observed. The test solution was colourless. Vessel 2, 3 and 4: pH 7.2.
- Control blank: Insoluble compound except the sludge was not observed. The test solution was colourless. Vessel 5: pH 7.3.

ANALYTICAL RESULTS
- See Table 1.

PERCENTAGE BIODEGRADATION
- See Table 2.

RESULT OF QUALITATIVE ANALYSIS OF DEGRADATION PRODUCTS
- The qualitative analysis of degradation products was conducted by LC-MS (with a photo-diode array detector). As a result, one degradation product (D1) was detected and three components of the test material (T1 to T3) were increased or decreased in the test solutions (slude and test material). D1 could not be identified. There were no changes of the test material except those mentioned above in the test solutions (sludge and test material). On the other hand, no degradation products were detected and there was no change of the test material in the test solution (water and test material).

DISCUSSION
- Test solutions (sludge and test material):
The average percentage biodegradation by BOD was 0 %. The percentage residue of the test material was 100-101 % and growth of the sludge was not observed. These results showed that the test material was not biodegraded. In the determination of the test material by HPLC with a GPC column, the peak shape of the test material changed on the chromatograms. Considering the peak position of the molecular weight standard (Mw 1013), the low-molecular-weight (molecular weight <1000) components of the test material seemed to be transformed. In the qualitative analysis by LC-MS with a reverse phase column, one degradation product (D1) was detected. D1 could not be identified and the product amount was unknown. The polarity of D1 was considered to be higher than that of the test material because the retention time of D1 was shorter than that of the test material at region ϒ on the LC-MS chromatograms. In addition, three components of the test material (T1 to T3) were increased or decreased. There were no changes of the test material except those mentioned above. It was concluded from the above results that three components of the test material were increased or decreased and a degradation product was produced by microorganisms, and the test material and D1 were not biodegraded. The detected amount of DOC was 0.8 mgC. Then, as a result of the qualitative analysis for water-soluble components in the test solutions (in LC-MS analytical sample-2) by LC-MS, some components of the test material including T1 were detected. These results showed that a small amount of the test material was dissolved in the solutions.

- Test solution (water and test material):
The percentage residue of the test material was 100 % and there was no change in the peak shape of the test material on the HPLC chromatogram. There was no remarkable change of the test material in the qualitative analysis by LC-MS. These results showed that the test material was stable in water. The results of the determination of DOC and the qualitative analysis for water-soluble components by LC-MS were almost the same as those of the test solutions (sludge and test material). Therefore, it was considered that a small amount of test material was dissolved in the test solution (water and test material).
Results with reference substance:
74 % (7 days), 95 % (14 days) and 98 % (28 days) biodegradation was observed with the reference substance.

Table 1: Analytical Results of the Test Solutions

 

Water and Test Material

Sludge and Test Material

Theoretical Amount

Vessel No.1

Vessel No.2

Vessel No.3

Vessel No.4

BOD *3

mg

0.4

-2.2

-4.6

-0.2

61.5*4

Detected Amount and % Detection of DOC

mgC

0.6

0.8

0.8

0.8

13.3*4

%

4

6

6

6

-

Residual Amount *5 and % Residue of Test Material (by HPLC)

mg

29.9

30.1

30.3

30.3

30.0

%

100

100

101

101

-

Detection of Degradation Products (by LC-MS)

-

Not detected

See results of qualitative analysis of degradation products

-

*3: The value of the test solution (control blank) was subtracted from the values of the test solutions (sludge and test material).

*4: The theoretical amount was calculated from the molecular formula C71H149O16P7S14 which was one of the representative structural formulae of the test material

*5: The value of the test solutions (sludge and test material) included degradation products in addition to the test material.

 

Table 2: Percentage Biodegradation After 28 Days

 

Sludge and Test Material

Vessel No. 2

Vessel No. 3

Vessel No. 4

Average

Percentage Biodegradation by BOD

%

-4

-7

0

0 (-4) *6

Percentage Biodegradation by DOC

%

The percentage biodegradation by DOC was not calculated because the test material cannot be dissolved in water at the test concentration (100 mg/L) or more.

Percentage Biodegradation of Test Material (by HPLC)

%

0

-1

-1

0 (-1) *6

*6: The average percentage biodegradation was regarded as 0 % because the calculated average value shown in parentheses was negative.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of this study, three components of the test material were increased or decreased and a degradation product was produced by microorganisms under the test conditions of this study. The test material and the degradation product were not biodegraded.
Executive summary:

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301C and other Japanese guidelines, under GLP conditions.

The test material at 100 mg/L and 30 mg/L activated sludge were incubated at 25 ± 1 °C for 28 days under dark conditions. Measurement of the biochemical oxygen demand (BOD) was performed with a closed system oxygen consumption measuring apparatus. Determination of the test material was performed using high-performance-liquid-chromatography (HPLC).

Under the conditions of this study, three components of the test material were increased or decreased and a degradation product was produced by microorganisms under the test conditions of this study. The test material and the degradation product were not biodegraded.

Description of key information

Yuping, 2018

Under the conditions of this study, the 28-day biodegradation of the test material was found to be 4.6 %. It cannot be considered to be readily biodegradable.

Taruki, 2018

Under the conditions of this study, three components of the test material were increased or decreased and a degradation product was produced by microorganisms under the test conditions of this study. The test material and the degradation product were not biodegraded.

Baolian, 2018

Under the conditions of this study, the 28-day biodegradation of the test material was found to be 0 %. It cannot be considered to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Yuping, 2018

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301B and other Chinese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to evaluate the ready biodegradability of test material using the CO2 evolution test. Activated sludge from a local sewage treatment plant was selected as inoculum and sodium benzoate as reference material. The test vessels were aerated by the passage of carbon dioxide-free air at a controlled rate and agitated at 22 ± 2 °C in diffused light for a period of 28 days. The amount of carbon dioxide produced was measured during the test. The amount of carbon dioxide produced from the test material (corrected for that derived from the blank inoculum) was expressed as percentage of the theoretical CO2.

In the procedure control, the biodegradation percentage of reference material was 91.5 % on day 14, which was greater than 60 %. The biodegradation percentage of the toxicity control was 41.8 % on day 14, which was greater than 25 %, indicating that the test material showed no toxicity or inhibition to the inoculum. The mean biodegradability percentage of test material was 4.6 % on day 28, which was less than 60 % and was not readily biodegradable in this study. The validity criteria of the test were all met.

Under the conditions of this study, the 28-day biodegradation of the test material was found to be 4.6 %. It cannot be considered to be readily biodegradable.

Taruki, 2018

The biodegradation of the test material was investigated in accordance with the standardised guideline OECD 301C and other Japanese guidelines, under GLP conditions.The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material at 100 mg/L and 30 mg/L activated sludge were incubated at 25 ± 1 °C for 28 days under dark conditions. Measurement of the biochemical oxygen demand (BOD) was performed with a closed system oxygen consumption measuring apparatus. Determination of the test material was performed using high-performance-liquid-chromatography (HPLC).

Under the conditions of this study, three components of the test material were increased or decreased and a degradation product was produced by microorganisms under the test conditions of this study. The test material and the degradation product were not biodegraded.

Baolian, 2018

The objective of the study was to evaluate the inherent biodegradability of test item

using the modified MITI test (II). Activated sludge was selected as inoculum and aniline as

the reference item. The test was performed under 25±2 oC and cultured for 28 days. The

biodegradation was expressed as a percentage of COD (corrected for uptake by inoculum

blank).

Test results:

In the procedural control, the biodegradation of reference item within 7d and 14d was

72.3%(more than 40%) and 87.3%(more than 65%).

Based on the result of chemical analysis (LC/MS/MS), the biodegradation percentage

was 51.1%. From this result, it was concluded that the component in the test item analyzed

in this test was not stable and converted to other products. These products seemed to be

not biodegraded by microorganism as the biodegradation percentage calculated based

BOD values did not increase.

Based on the result of BOD values, the mean biodegradation of test item on 28 day

was 0%, which was not more than 20%. Therefore, the test item was not inherently

biodegradable.