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EC number: 202-625-6 | CAS number: 97-99-4
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Biodegradation in water: screening tests
Administrative data
Link to relevant study record(s)
- Endpoint:
- biodegradation in water: ready biodegradability
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from March 24, 2004 to May 17, 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: Biodegradability study of Chemical Substances by Microorganisms" stipulated in "Testing Methods relating to the New Chemical Substances" and "Ready Biodegradability: Modified MITI Test (I) (OECD Guideline 301C, July 17, 1992)
- Deviations:
- not specified
- GLP compliance:
- yes
- Remarks:
- Certificate is not included in the study report.
- Oxygen conditions:
- aerobic
- Inoculum or test system:
- other: Activated sludge,natural water and soil
- Details on inoculum:
- Site and Timing for Sediment sampling;
(1) Sampling sites;
• Fushikogawa sewage treatment plant (Sapporo-shi, Hokkaido), • Fukashiba sewage treatment plant (Kashima-gun, Ibaraki), • Nakahama sewage treatment plant (Osaka-shi, Osaka), • Ochiai sewage treatment plant (Shinjuku-ku, Tokyo)
(2)Sampling time; December 2003
Collected method;
Sludge collected at the site of respective sewage treatment plants as above mentioned.
Preparation of inoculum
To keep the homogeneity of the activated sludge, 10 L of filtrate, which is a mixture of 5 L of the filtrate of the mixed sludge collected from the sites described above and 5 L of the filtrate from the inoculum cultured for approximately three months(*1), was aerated(*2) in the culture tank while the pH of the mixture was adjusted to 7.0 ± 1.0.
*1 Ten litre filtrate of the mixed sludge solution collected from the above sites was cultured according to the procedure described in Section 2.4 below.
*2 Outdoor air was pre-filtered and used for aeration.
Cultivation
Approximately 30 minutes after ceasing aeration to the culture tank, supernatant corresponding to about one third of the whole volume was removed. De-chlorinated water was added to the remaining portion and total volume was adjusted to 10 L, and the mixture was re-aerated (for 30 minutes or more) and then 50 g/L synthetic sewage(*3) was added so that the concentration of synthetic sewage reached 0.1 wt% in the added de-chlorinated water. This procedure was repeated once every day, cultured and used as inoculum. Cultivation temperature was set at 25 ± 2oC.
*3 Synthetic sewage was prepared as follows: Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water so that each component concentration reached 50 g/L. pH of the solution was adjusted to 7.0 ± 1.0 with sodium hydroxide. - Duration of test (contact time):
- 28 d
- Initial conc.:
- 100 mg/L
- Based on:
- other: Test material (nominal)
- Parameter followed for biodegradation estimation:
- O2 consumption
- Details on study design:
- - Preparation of test solution
Test solutions were prepared as follows for 6 test vessels.
(1) Addition of the test substance and aniline
(a) Test vessel with water + test substance (n=1, vessel No.1)
Purified water (297 mL) was poured in a test vessel, to which 3 mL of the test substance solution was added to make 100 mg/L solution, and then its pH was measured. To prepare 10.0 g/L of the test substance solution, the test substance was accurately weighed using electronic analytical balance and was dissolved in purified water.
(b) Test vessel with sludge + test substance (n=3, vessel No.2, 3 and 4)
The basal culture medium (297 mL minus added amount of the inoculum (2.00 mL)) was placed in the test vessel, to which 3 mL of 10.0 g/L test substance solution was added to make the test substance concentration at 100 mg/L, and then pH of the solution was measured. To prepare 10.0 g/L of test substance solution, test substance was accurately weighed using electronic analytical balance and was dissolved in purified water.
(c) Test vessel with sludge + aniline (n=1, vessel No.6)
The basal culture medium (300 mL minus added amount of the inoculum (2.00 mL)) was placed in the test vessel, to which 29.5 μL [added amount: 30 mg = 29.5 μL × 1.022 g/cm3 (density)] of aniline was added using a micro syringe to make the concentration of aniline at 100 mg/L.
(d) Test vessel with sludge blank (n=1, vessel No.5)
The basal culture medium (300 mL minus added amount of the inoculum (2.00 mL)) was put in the vessel.
(2) Inoculation of the activated sludge
The prepared inoculum was added to each test solution of (b), (c) and (d) to make the concentration of the suspended solid 30 mg/L.
- Apparatus and conditions of cultivation
(1) Apparatus for test solution cultivation
Closed system oxygen consumption measuring apparatus
Thermostatic bath and measuring unit: Asahi Techneion Co., Ltd.
Data processor: Asahi Techneion Co., Ltd.
Test vessel
300 mL culture bottle (improved culture bottle)
Carbon dioxide gas absorbent
Soda lime, No.1 (for carbon dioxide absorption, Wako Pure Chemical Industries, Ltd.)
(2) Ambient conditions
Cultivation temperature 25 ± 1oC
Cultivating duration 28 days (under protection from light)
Stirring method Rolling stir by magnetic stirrer
(3) Test room 511 Coulometer Room - Reference substance:
- aniline
- Remarks:
- (Special grade, Lot No. SO-32360, Showa Chemicals Inc. )
- Key result
- Parameter:
- % degradation (O2 consumption)
- Value:
- 92
- Sampling time:
- 28 d
- Details on results:
- The test substance was biodegraded by microorganisms under the condition of this study.
- Results with reference substance:
- Percentage biodegradation of aniline after 7 and 14 days obtained by BOD were 70% and 75%, respectively.
Therefore, the validity of the condition of this study was confirmed. - Validity criteria fulfilled:
- yes
- Interpretation of results:
- readily biodegradable
Reference
Table 1 Analytical result after 28 days
|
Water + test material |
Sludge + test material |
Theoretical amount |
|||
I |
II |
III |
IV |
|
||
BOD* |
mg |
2.9 |
56.1 |
57.4 |
54.9 |
61.2 |
Residual volume and ratio of DOC* |
mgC |
17.7 |
0.5 |
0.3 |
0.4 |
17.6 |
% |
101 |
3 |
2 |
2 |
- |
|
Volume and ratio of the residual test substance(GC) |
mg |
28.7 |
0 |
0 |
0 |
30.0 |
% |
96 |
0 |
0 |
0 |
- |
*The values presented in (Sludge + test substance) are values obtained by subtracting the measurements for the sludge blank from measurements of the sludge + test substance.
Table 2 Percentage degradation after 28days
Method |
Percentage biodegradation (%) |
|||
II |
III |
IV |
Average |
|
Result by BOD |
92 |
94 |
90 |
92 |
Result by DOC |
97 |
98 |
98 |
98 |
Result by GC |
100 |
100 |
100 |
100 |
Table 3: BOD (mg) and calculated % biodegradation for abiotic control, test substance, reference substance and inoculum blank
Type of suspension |
7d |
14d |
21d |
28d |
||||
BOD (mg) |
(%) biodeg |
BOD (mg) |
(%) biodeg |
BOD (mg) |
(%) biodeg |
BOD (mg) |
(%) biodeg |
|
|
|
|
|
|
|
|
||
I (water + test substance) |
0.3 |
- |
0.5 |
- |
1.9 |
- |
2.9 |
- |
|
|
|
|
|
|
|||
II (sludge + test substance) |
13.4 |
19 |
49.9 |
75 |
61.7 |
88 |
65.3 |
92 |
|
|
|
|
|
|
|||
III (sludge + test substance) |
42.6 |
67 |
60.4 |
92 |
64.2 |
92 |
66.6 |
94 |
|
|
|
|
|
|
|||
IV (sludge + test substance) |
11.3 |
15 |
48.2 |
72 |
60.9 |
87 |
64.1 |
90 |
|
|
|
|
|
|
|||
V (sludge + aniline) |
1.9 |
- |
4.2 |
- |
7.6 |
- |
9.2 |
- |
|
|
|
|
|
|
|||
VI (sludge blank) |
64.8 |
70 |
71.6 |
75 |
75.1 |
75 |
77.2 |
75 |
|
|
|
|
|
|
Description of key information
Biodegradation in water: screening tests: 92% biodegradation in 28 days (O2 consumption)
Key value for chemical safety assessment
- Biodegradation in water:
- readily biodegradable
Additional information
92% biodegradation in 28 days (O2 consumption) was determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.
-9% biodegradation in 60 days for degradation in anaerobic digesting sludge was determined in a reliable study conducted according to an appropriate test protocol, but not according to GLP (Battersby and Wilson 1989). This result indicates that aerobic microorganisms are primarily responsible for the degradation of the substance observed in the key study.
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