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Diss Factsheets

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:
24 October 2007 - 21 November 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
17 July 1992
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
not specified
Details on test material:
- Details on test material are confidential

Study design

Oxygen conditions:
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge: sludge was collected from 10 different locations in Japan (4 sewage treatment plants, 3 rivers, 1 lake and 2 bays) in September 2007.
- Method of collection from sewage plant: return sludge was collected. Method of collection from rivers, lake and sea: surface water and surface soil which was in contact with the atmosphere were collected.
- Method of cultivation: After ceasing aeration for approx. 30 minutes, supernatant of the sludge mixture corresponding to about 1/3 of the whole colume was removed. Dechlorinated water 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 at a temperature of 25±2°C
- Preparation of inoculum for exposure: the filtrate (5 L) of the supernatant of the activated sludge cultivated 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 with prefiltered open air after the pH value was adjusted to 7.0 +/- 1.0.
- Concentration of sludge: 3970 mg suspended solids/L
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: according to OECD 301C
- Additional substrate: no
- Test temperature: 25±1°C
- pH: not measured due to volatility of the test item
- pH adjusted: no, only the pH of the initial inoculum was adjusted
- Aeration of dilution water: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Other: each test solution was stirred with a magnetic stirrer

- Culturing apparatus: closed system oxygen consumption measuring apparatus
- Number of culture flasks/concentration: 3 bottles for the test item + sludge, 1 bottle for the other vessels (see below)
- Measuring equipment: automatic data sampler
- Test performed in closed vessels due to significant volatility of test substance: yes

- Sampling frequency and method: BOD was measured automatically with a data sampler, HPLC was used at the end of the test to analyse the amount of test item left in the system.
- Other: appearance of the test solution was observed once a day

- Blank control: yes, mineral medium only (1 bottle)
- Abiotic control: yes, test item in water (1 bottle)
- Toxicity control: no
- Other: reference control (1 bottle, reference item in medium)
Reference substance
Reference substance:
(100 mg/L)

Results and discussion

% Degradationopen allclose all
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: regarded as 0% because the calculated average value was calculated to be negative
% degradation (test mat. analysis)
Sampling time:
28 d
Details on results:
- Degradation percentages in individual bottles were -6, -6 and -4% (based on BOD) and 0, 1 and 1% (based on HPLC).

BOD5 / COD results

Results with reference substance:
Aniline was degraded for 75% after 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
See 'overall remarks'
Interpretation of results:
not readily biodegradable
Based on a biodegradation study (MITI test), performed according to OECD guideline 301 C and GLP principles, no biodegradation of Indene was observed after a 28 day test duration.
Executive summary:

In a MITI test, performed according to OECD guideline 301 C and GLP principles, Indene was assessed for its biodegradation over a period of 28 days. Sludge was collected from ten different sites and inoculated for a period of 3 months. This inoculum was used to prepare the mineral medium for the study. The test item was tested in mineral medium containing sludge at a concentration of 100 mg/L in triplicate next to one replicate of test item in water, the reference item (aniline) in mineral medium and one control blank containing only mineral medium. During the test the amount of consumed oxygen was measured and at the end of the test period the amount of remaining test item was analysed by using HPLC. Based on the BOD the average % degradation was calculated to be 0% for the 3 replicates. Based on HPLC analysis, the average % degradation was calculated to be 1% for the 3 replicates. The acceptability criteria were met and the study was considered to be valid. Based on the results, Indene is considered to be not readily biodegradable.