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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
July 1992
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
May 2008
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 aerated until required.
Duration of test (contact time):
27 d
Initial conc.:
58.6 mg/L
Based on:
other: ThODNO3
adressing potential nitrification
Initial conc.:
30.5 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 treated by reverse osmosis and then purified; nominal resistivity ≥ 18
- Test temperature: 21 to 22°C.
- pH: 7.5 at the start of the test and 7.4-7.6 at the end
- pH adjusted: the inoculum was added to the culture bottles 1 day before test initiation. The pH of this mixture was adjusted to 7.6 with 5M HCl.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: not reported

- Culturing apparatus: glass culture bottles in a temperature controlled water bath
- Number of culture flasks/concentration: 4
- Method used to create aerobic conditions: internal oxygen generating process
- Measuring equipment: Co-ordinated Environmental Services (CES) Ltd automated respirometer and associated software

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

- Inoculum blank (2 flasks): inoculated mineral salts medium (MSM)
- Reference (1 flask): inoculated MSM + sodium benzoate (50 mg O₂/L)
- Toxicity control: No inhibition assay was performed in this test as data from two previous tests had deemed this unnecessary. The test material was not inhibitory to the activated sludge, with percentage degradation achieving 60% by Day 4 in both cases.
Reference substance:
benzoic acid, sodium salt
Key result
% degradation (O2 consumption)
St. dev.:
Sampling time:
27 d
Remarks on result:
other: based on ThODnitrate
Details on results:
The test was ended on Day 27 due to a power failure. However, the degradation of the test substance had already passed the 60% level for more than three consecutive determinations.

For the individual results and graphs see attached document "Figure & Tables - Biodegradation.pdf".
Results with reference substance:
The reference substance sodium benzoate had achieved 61% of the ThOD after 3 days of incubation and 91% by Day 27 (see attached document "Figure & Tables - Biodegradation.pdf").

To compensate for the potential effect of nitrification on the test mixtures, and the associated oxygen demand caused by this, the calculation of the ThODnitrate (ThODNO3) was used instead of the ThODammonia (ThODNH3). Therefore, the ThODNO3 was retrospectively calculated and used instead of the ThODNH3. The new calculated ThOD/L (mgO2/L) of 58.6 mgO2/L still lies within the acceptable range of 50-100 mgO2/L for this test as outlined in the OECD 301F guideline and Directive (EC) 440/2008, C.4-D.

Validity criteria fulfilled:
rate of degradation of sodium benzoate > 60% of its ThOD by Day 14; cumulative amount of oxygen consumed by the control mixtures was between 20 and 30 mg O2/L; difference between replicates was ≤ 20% at the end of the 10-d window
Interpretation of results:
readily biodegradable

Description of key information

Biodegradation in water: screening tests: aerobic biodegradation 76% (ThOD) in 28 days (OECD 301F, EU C.4-D)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

[Type of water: freshwater]