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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:
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
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted July 17, 1992
GLP compliance:
yes (incl. certificate)

Test material

Test material form:

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Inoculum of the aqueous phase of non-adapted activated sludge from a municipal sewage treatment plant.

- Pretreatment: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 3 days. 10 mL/L were used to initiate inoculation.

-Colony forming units in the test vessels: Approx. 10^7 - 10^8 CFU/L
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
30 mg/L
Based on:
test mat.
Initial conc.:
54.6 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Preparation of the test vessels: Based on the calculated oxygen demand, the test concentration of 30 mg/L, corresponding to an oxygen demand of 54.6 mg O2/L in the vessel, was selected. The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control and functional control) or were prepared directly in brown glass bottles (test item and toxicity control).

The test medium, consisting of the required volumes of mineral medium stock solutions, demineralised water and 10 mL/L inoculum, was prepared in a measuring flask. The test item was weighed out and was transferred directly into the brown glass bottle with 250 mL test medium. Accordingly, the test and refrence item for the toxicitty control were weighed out and transferred directly into the brown glass bottle. For the functional control, the reference item was weighed out and transferred into a 250 mL measuring flask with demineralised water. The required volumes of mineral medium stock solutions and 10 mL/L inoculum were added, then the flask was filled up with demineralised water. The test solution was placed in the brown glass bottle.

A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with OxiTop measuring heads and the measuring system was activated.

Measurements: The temperature in the incubator was documented continuously throughout the test. At test start the pH-values of the prepared solutions in the measuring flasks (inoculum control and functional control) and the prepared solutions in the separate replicates (test item and toxicity control), were determined. At test end the pH-values of the test solutions in the brown glass bottles were determined. The oxygen consumption was determined in the incubation vessels by the OxiTop® measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
Results of the Functional Control:
The pass level for ready biodegradation (≥ 60% degradation) was reached within 2 days. The validity criterion that the degradation should be ≥ 60% after 14 d was fulfilled. Stages of Biodegradation: The adaptation phase changed to degradation phase within 1 day (degradation > 10%) and the biodegradation came to a maximum of 95% on day 28.

Results of the Toxicity Control:
In the toxicity control the biodegradation achieved 51% after 14 days. After 28 days the biodegradation came to 56%. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.
% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
The test item replicates did not reach the 10% level (beginning of biodegradation); no biodegradation was determined.
After a test period of 28 days, the test item is classified as not readily biodegradable within the 10-day window.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
After a test period of 28 days, the test item is classified as not readily biodegradable within the 10-day window.
Executive summary:

After a test period of 28 days, the test item is classified as not readily biodegradable within the 10-day window.