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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
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf
- Laboratory culture: -
- Method of cultivation: -
- Storage conditions: -
- Storage length: 3 d
- Preparation of inoculum for exposure: -
- Pretreatment: The sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use
- Concentration of sludge: 4120 mg dw suspended solids/L
- Initial cell/biomass concentration: -
- Water filtered: yes
- Type and size of filter used, if any:
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: organic carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: standard medium
- Additional substrate: -
- Solubilising agent (type and concentration if used): -
- Test temperature: 19.7 – 21.3 °C
- pH: 7.6 (test item), 7.1-7.8 (controls)
- pH adjusted: no
- CEC (meq/100 g): -
- Aeration of dilution water: purified (by activated charcoal), CO2-scrubbed, moistened air
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:

- Culturing apparatus: 2000 mL SCHOTT-flasks
- Number of culture flasks/concentration: 2 (nominal 20 mg organic carbon/L)
- Method used to create aerobic conditions: aeration
- Method used to create anaerobic conditions: -
- Measuring equipment: carbon analyser TOC multi N/C 2100S
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: NaOH, 0.25 M solution
- Other:

- Sampling frequency: day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29 (10 samples)
- Sampling method: 1 mL sampling
- Sterility check if applicable: -
- Sample storage before analysis: -
- Other:

- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1
- Other: 2 blanks containing mineral medium only

Reference substance:
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Results with reference substance:
Degradation of the positive control was 61 % after 9 days.
Interpretation of results:
not readily biodegradable
All validity criteria were met.
Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation was not observed. Both replicates of the test item showed very good correspondence.
Therefore, the test item 2,2'-Azobis(2,4-dimethylvaleronitrile) can be considered as “not readily biodegradable”. Degradation missed 60 % within 28 days, though. Therefore, the test item is considered as “not ultimately biodegradable within 28 days”, either.
No observations were made which might cause doubts concerning the validity of the study outcome.
Executive summary:

The test item 2,2'-Azobis(2,4-dimethylvaleronitrile) was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 29.5 mg 2,2'-Azobis(2,4-dimethylvaleronitrile)/L) in test medium following OECD 301B and EU-Method C.4-C.


Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control was 61 % after 9 days.


The following data were determined for the test item 2,2'-Azobis(2,4-dimethylvaleronitrile):

10-day-window:                                  not detected

degradation at the end of 10-day-window none

degradation at the end of the test                  8 %

pass level following guideline: 60 % at the end of 10-day-window for pure substances

respective 60 % at the end of the test for mixtures


Therefore, when applying the 10-day-window, the test item 2,2'-Azobis(2,4-dimethylvaleronitrile) is not readily biodegradable following OECD 301B and EU C.4-C respectively. As degradation missed 60 % in the course of the test, 2,2'-Azobis(2,4-dimethylvaleronitrile) is considered as not ultimately biodegradable, either.

Description of key information

Based on the findings in the available screening study the test item is considered to be not readily biodegradable.

Key value for chemical safety assessment

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

Additional information