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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:
2014-02-05 to 2014-03-18
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
according to
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
GLP compliance:
yes (incl. certificate)

Test material

Specific details on test material used for the study:
Designation: Art. 139301
Synonym: HTM-163
Appearance: pale yellow powder without visible impurities
Released until: August 15, 2016

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant, Rossdorf, Germany.

- Pretreatment:
The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated for two days. This suspension was used for the experiment.

- Concentration of sludge: 28.7 mg/L
- Water: deionised water
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
100 mg/L
Based on:
test mat.
Initial conc.:
286.8 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium:
Analytical grade salts were added to deionised water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with deionised water to 1000 mL volume; The pH-value was 7.4.
b) 22.5 g MgSO4 x 7 H2O filled up with deionised water to 1000 mL volume
c) 36.4 g CaCl2 x 2 H2O filled up with deionised water to 1000 mL volume
d) 0.25 g FeCl3 x 6 H2O filled up with deionised water to 1000 mL volume In order to avoid precipitation of iron hydroxide in the stock solution
d) after storage and before use, one drop of concentrated HCl per litre was added.

50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with deionised water.

- Additional substrate: -
- Solubilising agent (type and concentration if used): -
- Test temperature: 22°C ± 1°C,
- pH: 7.5
- pH adjusted: no
- Suspended solids concentration: 28.7 mg/L
- Continuous darkness: yes
- Other:

- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Manometric Test System
- Method used to create anaerobic conditions: Manometric Test System
- Measuring equipment: Manometric Test System
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: Potassium hydroxide solution (45%)
- Other:

- Sampling frequency: daily
- Sampling method: Manometric Test System
- Sterility check if applicable: -
- Sample storage before analysis: -
- Other: -

- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
Results of the Functional Control
The reference item sodium benzoate was sufficiently degraded to 82% after 14 days and to 85% after 28 days of incubation.

Results of the Toxicity Control
In the toxicity control containing both, the test item and reference item, 29% biodegradation was noted within 14 days
and 31% biodegradation was determined after 28 days of incubation.
% Degradation
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
no biodegradation was observed

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.
Executive summary:

The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.