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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 November 2007 - 15 February 2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
according to guideline
other: "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (November 21, 2003; No. 1121002, MHLW; November 13, 2003, No. 2, METI; No. 031121002, MOE, Japan)
GLP compliance:

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): S-500
- Substance type: yellow powder
- Physical state: solid
- Lot/batch No.: MF1456-2345
- Stability under test conditions: stable (confirmed by IR)
- Storage condition of test material: dark storage place at room temperature

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
On-site sludge sampling was carried out at the following ten locations in Japan:
Fushikogawa city sewage plant (Sapporo-shi, Hokkaido), Fukashiba industrial sewage plant (Kamisu-shi, Ibaraki), Nakahama sewage treatment plant (Osaka-shi, Osaka), Ochiai treatment plant (Shinjuku-ku, Tokyo), Kitakami River (Ishinomaki-shi, Miyagi), Shinano River (Niigata-shi, Niigata), Yoshino River (Tokushima-shi, Tokushima), Lake Biwa (Otsu-shi, Shiga), Hiroshima Bay (Hiroshima-shi, Hiroshima) and Dokai Bay (Kitakyushu-shi, Fukuoka).
From sewage plants, return sludge was collected. From rivers, lake and sea, surface water and surface soil which was in contact with the atmosphere were collected.

- Method of cultivation: approx. 30 min after ceasing aeration of the sludge mixture, supernatant corresponding to about one third of the whole volume was removed. Dechlorinated water was added to the remaining portion so that the total volume reached 10 L. This mixture was aerated for 30 min or more, and then a predetermined amount of synthetic sewage (glucose, peptone and potassium dihydrogen phosphate were dissolved in purified water to obtain 5 g/L of the solution for each component. The pH was adjusted to 7.0+/-1.0 with NaOH) was added to the mixture so that the concentration of the synthetic sewage was 0.1% in the volume of dechlorinated water added. This procedure was repeated once every day.
- Storage conditions: 25+/-2°C
- Storage length: activated sludge was cultivated for 19.5 hours after the synthetic sewage had been added
- Preparation of inoculum for exposure: the filtrate (5 L) of the supernatant of the activated sludge cultivated about for 3 months was mixed with the mixed filtrate (5 L) of the supernatant of a sludge collected newly at each location. The mixed filtrate (10 L) was aerated after the pH value of the mixture was adjusted to 7.0+/-1.0
- Concentration of sludge: 3580 mg/L suspended solid in activated sludge
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
- Composition of medium: each 3 mL of solutions A, B, C and D (prescribed in Japanese JIS K0102-1998 Section 21) were made up to 1 L with purified water and the pH adjusted to 7.0.
- Test temperature: 25 +/- 1 °C
- pH: 7.0
- pH adjusted: yes
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

- Culturing apparatus: closed system oxygen consumption measuring apparatus
- Number of culture flasks/concentration: 3 (sludge + test item), 1 (water + test item)

- Sampling frequency: test solution only after 28 days (end of test), BOD on days 7, 14, 21, and 28.

- Inoculum blank: yes (i.e. control blank)
- Toxicity control: sludge + 100 mg/L aniline
- Other: water blank (only purified water)
Reference substance
Reference substance:

Results and discussion

Preliminary study:
not applicable
Test performance:
The test item was not dissolved and the test solution was colorless before and after the test.
% Degradationopen allclose all
% degradation (test mat. analysis)
Sampling time:
28 d
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: based on BOD
Details on results:
Residual amount of test item after 28 days (by HPLC): 28.8 mg in control, 29.8, 29.8 and 27.8 mg in test vessels. Theoretical amount 30 mg.
Amount of water-insoluble aluminium after 28 days (by AAS): 153 µg in control, 164, 169 and 150 µg in test vessels. Theoretical amount 180 µg (soluble and insoluble).
Amount of water-soluble aluminium after 28 days (by AAS): 6.5 µg in control, 3.1, 0 and 5.2 µg in test vessels.
Amount of water-insoluble calcium after 28 days (by AAS): 350 µg in control, 256, 286 and 245 µg in test vessels.

BOD5 / COD results

Results with reference substance:
Percentage biodegradation of aniline by BOD: 64% after 7 days and 78% after 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
Some of aluminium part and calcium part of the aluminium salts and calcium salts in the constituents of the test item were eliminated under the conditions of this study. Organic compounds produced by the elimination were not new converted products but had the same structural formulae as some constituents of the test item. Therefore, it was concluded that the test item was not biodegraded by microorganisms.