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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
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
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): laboratory treatment plant processing domestic sewage (Wupperverband)
- Storage conditions: not reported
- Storage length: not reported
- Preparation of inoculum for exposure:
- Pretreatment: none
- Concentration of sludge:
- Initial cell/biomass concentration:
- Water filtered: yes/no
Duration of test (contact time):
29 d
Initial conc.:
20 mg/L
Based on:
other: Total organic carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: not reported
- Additional substrate: not reported
- Solubilising agent (type and concentration if used): none
- Test temperature: not reported
- pH: not reported
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: 25 mg dry matter/L
- Continuous darkness: not reported

- Culturing apparatus:
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: aeration with CO2-free air
- Measuring equipment: not reported
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2: bottles filled with barium hydroxide solution or caustic soda

- Sampling method: continuous air flow through CO2 trap

- Inoculum blank: yes
- Toxicity control: no
Reference substance:
% degradation (CO2 evolution)
Sampling time:
2 d
% degradation (CO2 evolution)
Sampling time:
13 d
% degradation (CO2 evolution)
Sampling time:
29 d
Details on results:
The 10-day window was fulfilled.
Results with reference substance:
The reference substance aniline achieved a degradation of 67% within 29 days demonstrating the suitability of the sewage sludge.
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The substance is readily biodegradable.
Executive summary:

The ready biodegradability of the substance methyl benzoate was studied under GLP conditions in a carbon dioxide evolution test according to EU Method C.4-C over a period of 29 days. A defined volume of nutrient medium was inoculated with activated sludge from a treatment plant processing domestic sewage at a concentration of 25 mg dry matter/L. The test substance was added to this mixture at a concentration of 20 mg C/L corresponding to 28 mg/L substance. The bottles were aerated with CO2-free air. The biodegradation of the test substance was studied by determining the amount of CO2 generated during a testing period of 28 days. To this end, the air stream was directed through barium hydroxide solutions/caustic soda and the amount of CO2 was determined by titration of remaining hydroxide/caustic soda. The generated CO2 was related to the theoretical amount of CO2 released in case of complete oxidation of the test substance to obtain the fraction of substance that was biodegraded. Evolution of CO2 was surveyed over a period of 29 days. A fraction of 62% of the substance had been degraded at the end of the observation period. The 10-day window was fulfilled. A parallel degradation test was conducted with aniline, which resulted in a fraction of 67% of the reference substance being biodegraded showing the suitability of the activated sludge. In conclusion, the substance was considered as readily biodegradable according to the OECD criteria.

Description of key information

The substance was readily biodegradable (fulfilling the 10-day window) in a standard CO2 evolution study over a period of 28 days.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:

Additional information

The biodegradability of methyl benzoate was studied in a CO2 evolution test in accordance with EU Method C.4-C over a testing period of 29 days. The test was valid according to the criteria given in the testing guideline. A degradation of 62% of the test substance was found at the end of the testing period (degradation of the reference substance aniline was 69%). The 10-day window was fulfilled.