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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Worlingworth sewage treatment works (Suffolk, UK)
- Method of cultivation / Preparation of inoculum for exposure: At the time of collection, the sludge was sieved (1 mm²) then transported to the laboratory and left stand for approximately 30 minutes to allow the sewage solids to settle. A portion of the supernatant was removed and the sludge aerated until required.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
Initial conc.:
17 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: mineral salts medium (MSM) according to guideline, prepared using tap-water that had been softened and treated by reverse osmosis and then purified; nominal resistivity ≥ 18 The pH wasadjusted to 7.4 ± 0.2 with 5M HCl. This water complies with the relevant standards (British Standard (BS) 3978, 1987; American Society for Testing and Materials (ASTM) D 1193-06) for classification as Grade 1 or Type 1 water for laboratory use.
- Test temperature: 20 to 22°C.
- pH: 7.5 to 7.6 at the start of the test and 7.1 to 7.5 at the end
- pH adjusted: yes, to 7.4 ± 0.2 with 5M HCl as required
- Suspended solids concentration: 30 mg/L
- Continuous darkness: not reported
- Other: The inoculum was added to the culture bottles 1 day before test initiation to allow a period of ageing.

- Culturing apparatus: glass culture bottles in a temperature controlled water bath
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: internal oxygen generating process
- Measuring equipment: Co-ordinated Environmental Services (CES) Ltd automated respirometer

- Sampling frequency: continuous monitoring of the cumulative amount of oxygen consumed; the cumulative oxygen demand made by each cell was printed at, typically, hourly intervals

- Inoculum blank (2 flasks): inoculated mineral salts medium (MSM)
- Reference (1 flask): inoculated MSM + sodium benzoate (50 mg O₂/L)
- Toxicity control (1 flask): inoculated MSM + test material (50 mg O₂/L) + sodium benzoate (50 mg O₂/L)
- Nitrification test (1 flask): inoculated MSM + test material (50 mg O₂/L) + Allylthiourea (ATU, 11.6 mg/L)
- Nitrification control (1 flask): inoculated MSM + ATU (11.6 mg/L)

Reference substance:
benzoic acid, sodium salt
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
In the nitrification controls the degradation of the test material was similar to that in uninhibited cultures.
In the presence of test material the degradation of sodium benzoate achieved 61% after 3 days indicating that the test substance was not inhibitory to the microbial inoculum.
Results with reference substance:
The reference substance sodium benzoate had achieved 60% of the ThOD after 4 days of incubation and 73% by Day 28.
Validity criteria fulfilled:
The results obtained for the rate of degradation of sodium benzoate (60% of its ThOD after 4 days) and for the cumulative amount of oxygen consumed by the control mixtures (28 and 37 mg O₂/L) fulfill the validity criteria for this test.
Interpretation of results:
under test conditions no biodegradation observed

Description of key information

Biodegradation in water: screening tests: biodegradation 0% (COD) in 28 days (OECD 301F, EU C.4-D)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information