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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference Type:
Biodegradation and bioaccumulation data of existing chemicals based on the Chemical Substances Control Law (CSCL)
MITI (Ministry of International Trade and Industry)
Bibliographic source:
Chemicals Inspection and Testing Institute (CITI, ed.); Japan Chemicals Industry Ecology Toxicology and Information Center

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
14 day-test
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): m-Nitrotoluene
- Analytical purity: no data

Study design

Oxygen conditions:
Inoculum or test system:
other: sludge sampling from different sewage plants, natural waterbodies and soil
Details on inoculum:
1. Sludge sampling sites and time

1.1. Time: in March, June, September, and December, sludge was sampled
1.2. Sites (Japan): 1. Fukogawa city sewage plant, 2. Fukashiba industry sewage plant, 3. Nakahama city sewage plant, 4. Ochiai city sewage plant, 5. Kitakami river, 6. Shinano river, 7. Yoshino river, 8. Lake Biwa, 9. Hiroshima bay, 10. Dookai bay
A mixture composed of city and industry sewage and river, lake and sea surface water and soil was tested.

2. Sludge sampling method

2.1. City sewage: Returned sludge from sewage plants was taken.
2.2. Rivers, lake and sea: Surface water and surface soil which were in contact with atmosphere were collected.

3. Method of cultivation

3.1. Mixing of fresh and old activated sludge: 5 L of the filtrate of the supernatant of old activated sludge was mixed with 500 mL of the filtrate of the supernatant of new sludge and cultured at pH 7.0 ± 1.0 under sufficient aeration using prefiltered open air.

3.2. Culture: About 30 minutes after ceasing aeration to the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Then the equal volume of dechlorinated water was added to the remaining portion and aerated again, followed by addition of synthetic sewage at aconcentration of 0.1% (w/v). This procedure was repeated once every day. The culturing was carried out at 25 ± 2 °C.

3.3. Control: During the cultivation, appearance of the supernatant, precipitability, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked and necessary adjustments were made, Microflora in the activated sludge was microscopically observed and sludge with no abnormal symptom was used for the test.

4. Inspection of activity

Activity of the sludge was inspected to use reference substance. And the relation between new and old activated sludge was taken account.

5. Adaptation

There is no specific statement about the adaptation of the used microorganisms but the sampling sides and culture conditions suggest not adapted microorganisms.

6. Concentration of sludge: 30 mg/L
Duration of test (contact time):
14 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test performed in open system: no
Preparation of basal culture medium (sludge):
- Suspended solids concentration: determined according to Method Japanese Industrial Standards (JIS) K 0102-1986-14.1
- Composition of basal culture medium: 3 mL each of four stock solutions, as described in JIS K 0102-1986-21, are diluted to 1000 mL with purified water
- pH of basal culture medium: 7.0
- pH adjusted: yes
Test system:
- Abiotic sterile control: 300 ml purified water + 30 mg test substance
- Test solution: 300 ml basal culture medium (sludge) + 30 mg test substance
- Reference: 300 ml basal culture medium (sludge) + 30 mg aniline
- Inoculum blank: 300 ml sludge
Test instruments and conditions:
- Culturing apparatus: Closed system oxygen consumption measuring apparatus (Coulometer: Ohkura Electric Co., Ltd.)
- Vessel size: 300 mL in volume
- absorbent for evolving carbon dioxide: Soda lime No .l (extra pure reagent, Wako Pure Chemical Industries, Ltd.).
- stirring method: test solution was stirred by a magnetic stirrer
- cultivating temperature: 25 ± 1°C
- reference substance: aniline
Analysis after termination of the test:
- test substance
- pH

Degradation (%) was obtained from the following equations:
BOD: Degradation (%) = (BOD-B)/ThOD x 100 BOD (mg):
BOD in (sludge + m-Nitrotoluene system)
B (mg): BOD in sludge blank
ThOD: theoretical oxygen demand
required when m-Nitrotoluene was completely oxidized.
Reference substance
Reference substance:

Results and discussion

% Degradation
% degradation (O2 consumption)
Sampling time:
14 d

Applicant's summary and conclusion

Interpretation of results:
other: not readily biodegradable
Executive summary:

In a modified MITI I test according to OECD guideline 301 C a non adapted mixed microbial inoculum mineralised 2 % of the initial test substance concentration within 14 days (MITI, 1992).

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