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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-03-05 to 2012-07-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
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 - 4 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 5 days. 10 mL/L were used to initiate inoculation.

-Colony forming units in the test vessels: Approx. 1E7 - 1E8 CFU/L
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Initial conc.:
57.6 mg/L
Based on:
ThOD
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 57.6 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 reference item for the toxicity 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:
benzoic acid, sodium salt
Test performance:
Results of the Functional Control:
The pass level for ready biodegradation (≥ 60 % degradation) was reached within 3 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 2 days (degradation > 10 %) and the biodegradation came to a maximum of 97 % on day 26.

Results of the Toxicity Control:
In the toxicity control the biodegradation achieved 50 % after 14 days. After 28 days the biodegradation came to 53 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
The 1st test item replicate did not reach the 10 % level (beginning of biodegradation); no biodegradation was determined. The 2nd test item replicate reached the 10 % level after 8 days. The 60 % pass level was not reached within 28 days. Temporarily, a minor increase of the biodegradation could be observed for about one week, which decreased afterwards to the former level. This is caused by the calculation procedure (BOD values of the control are subtracted from those of the test item replicates) and faint temperature fluctuations.
After a test period of 28 days, the test item is classified as not readily biodegradable within the 10-day window.

Results

The mean oxygen depletion in the inoculum control was 7.1 mg O2/L on day 28. The pass level > 60 % was reached within 3 days in the functional control and the biodegradation reached a maximum of 97 % degradation on day 27. In the toxicity control, containing both test and reference item, 50 % degradation occurred within 14 days and after 28 days the biodegradation came to 52 %. The degradation of the reference item was not inhibited by the test item. The 1st test item replicate did not reach the 10 % level (beginning of biodegradation); no biodegradation was determined. The 2nd test item replicate reached the 10 % level after 8 days. The 60 % pass level was not reached within 28 days. Temporarily, a minor increase of the biodegradation could be observed for about one week, which decreased afterwards to the former level. This is caused by the calculation procedure (BOD values of the control are subtracted from those of the test item replicates) and faint temperature fluctuations. The validity criteria of the guideline are fulfilled.

 

Conclusion

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

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
After a test period of 28 days, the test item is classified as not readily biodegradable within the 10-day window.
Executive summary:

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Manometric Respiratory Test according to OECD 301F under GLP. The test item was tested at a concentration of 30 mg/L, corresponding to an oxygen content of 57.6 mg O2/L. The oxygen concentration was measured with OxiTop® measuring system. The mean oxygen depletion in the inoculum control was 7.1 mg O2/L on day 28. The pass level > 60 % was reached within 3 days in the functional control and the biodegradation reached a maximum of 97 % degradation on day 27. In the toxicity control, containing both test and reference item, 50 % degradation occurred within 14 days and after 28 days the biodegradation came to 52 %. The degradation of the reference item was not inhibited by the test item. The 1st test item replicate did not reach the 10 % level (beginning of biodegradation); no biodegradation was determined. The 2nd test item replicate reached the 10 % level after 8 days. The 60 % pass level was not reached within 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached in a 10-d-window within the 28 days period of the study.

Description of key information

After a test period of 28 days, the test item is classified as not readily biodegradable within the 10-day window (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Manometric Respiratory Test according to OECD 301F under GLP (reference 5.2.1-1). The test item was tested at a concentration of 30 mg/L, corresponding to an oxygen content of 57.6 mg O2/L. The oxygen concentration was measured with OxiTop® measuring system. The mean oxygen depletion in the inoculum control was 7.1 mg O2/L on day 28. The pass level > 60 % was reached within 3 days in the functional control and the biodegradation reached a maximum of 97 % degradation on day 27. In the toxicity control, containing both test and reference item, 50 % degradation occurred within 14 days and after 28 days the biodegradation came to 52 %. The degradation of the reference item was not inhibited by the test item. The 1st test item replicate did not reach the 10 % level (beginning of biodegradation); no biodegradation was determined. The 2nd test item replicate reached the 10 % level after 8 days. The 60 % pass level was not reached within 28 days. In conclusion, the test item is not readily biodegradable as the pass level of 60 % degradation was not reached in a 10-d-window within the 28 days period of the study.