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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 B (Ready Biodegradability: CO2 Evolution Test)
not specified
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
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 (e.g. location, sampling depth, contamination history, procedure): Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany.
- Method of cultivation: The inoculum was collected on March 2nd 2010 from the aeration tank of the plant and aerated in the laboratory until use. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. The blank controls were aerated with carbon dioxide free air for about 24 hours at 22 ± 2 °C to reduce the content of inorganic carbon.
- Preparation of inoculum for exposure: The activated sludge suspension was washed once with tap water. Therefore the aeration was stopped and the sludge was allowed to settle. The supernatant was discarded and the remaining sludge suspension was filled up with tap water. The sludge concentration was adjsuted to 6 g/L dry weight.
- Concentration of sludge: Aliquots of 7.5 mL were added to the test vessels resulting in a test concentration of sludge of 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: test medium according to test guideline OECD 301 B
- Test temperature: 22 ± 2 °C
- pH: 7.32 - 7.4
- Continuous darkness: yes

- Culturing apparatus: 2 L incubation bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with carbon dioxide free air
- Measuring equipment: Shimadzu TOC-analyzer (TOC-5000A)
- Details of trap for CO2 and volatile organics if used: CO2 was captured by passing evolved CO2 through two scrubbing bottles filled with 100 mL of 0.05 mol sodium hydroxide solution

- Sampling frequency: Twice a week TIC in the first trap scrubbing bottle was measured

- Inoculum blank: 2 Blanks
- Abiotic sterile control: no
- Toxicity control: 1 Inhibition control
- Other: 1 Reference assay
Reference substance:
% degradation (CO2 evolution)
>= 90 - <= 100
Sampling time:
28 d
Details on results:
- The selected test concentration was tested in an additional inhibition control test assay and no toxic effects to the microorganisms were observed. 76 % biodegradation has been observed for the inhibition control after 14 d.

- Measured DIC-concentrations in the blank controls at begin of exposure (mean value): 0.7 mg/L
- Amount of produced CO2 in the blank controls at the end of exposure (mean value): 20.0 mg/L
- Deviation of Degree of Biodegradation of the test substance in the plateau phase should be <20%: Yes
- Degree of Biodegradation of the reference substance should be >60% CO2/ThCO2 after 14 days: Yes
- Degree of Biodegradation e in the inhibition control >25 % CO2/ThCO2 after 14 days: Yes
- (DIC) in the blank control at begin of exposure at 20 mg/L TOC test concentration should be <1 mg/L: Yes
- Amount of produced CO2 in the inoculum blank (“blank controls”) at the end of exposure (mean value) should be < 70 mg/L: Yes
Results with reference substance:
Degree of Biodegradation of the reference substance after 14 days % CO2/ThCO2: 92 % CO2/ThCO2

Tab. 1: Results

Duration of the adaptation phase:

9 days

Degree of Biodegradation of the test substance at the end of the ten-day window

about 90 % CO2/ThCO2

Degree of Degradation DOC of the test

substance at the end of exposure

90-100 % DOC removal

Degree of Biodegradation of the test substance at the end of exposure, mean value

90-100 % CO2/ThCO2

Degree of Biodegradation of the reference substance after 14 days % CO2/ThCO2

92 % CO2/ThCO2

Degree of Biodegradation in the inhibition control after 14 days

76 (% CO2/ThCO2)



Both test substance assays and the inhibition control assay were acidified by adding 2 mL of concentrated hydrochloric acid after 28 days of exposure. The blank control- and reference substance assays were not acidified, since they were still required for other ongoing studies.

The amounts of released carbon dioxide in the traps of the test assays both with and without acidification were determined on day 29 and added to the amount calculated from day 28.

The required pass level for the assessment of the test substance as ready biodegradable was reached at day 19 after start of exposure. Therefore the abandonment of the acidification of the blank control- and reference substance assays had no influence upon the evaluation of the test result.


ThCO2= Theoretical amount of formed Carbon dioxide,

DIC = Dissolved Inorganic Carbon

Validity criteria fulfilled:
please refer to "Details on results"
Interpretation of results:
readily biodegradable
The test substance is readily biodegradable (according to OECD criteria).
Executive summary:

In this guideline (OECD 301 B) study conducted with GLP certification, the test material (EC 231-272-0) was determined to be readily biodegradable

Description of key information

 Study conducted to recognised training guidelines with GLP

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

In 2010 BASF SE conducted a ready biodegradability test for the test susbtance according to OECD 301 B guidelines under GLP regulations. Evolving CO2 was captured in sodium hydroxide solution and TIC was measured at least twice a week. After a lag phase of approximately 9 days the degradation degree reached about 90% at the end of the 10 -day window. Therefore, the test substance is readily biodegradable according to OECD criteria.

A supporting study according to EU Method C.6 (Degradation: Chemical Oxygen Demand) was conducted using the test material before the final distillation (Merck, 1989). After 28d a biodegradation of 59% was observed.