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Biodegradation in water: screening tests

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biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-11-03 - 2010-12-08
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
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): a mixed population of activated sludge sewage sludge micro-organisms was obtained from the aeration stage of the Seven Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Storage conditions: maintained under continuous aeration
- Preparation of inoculum for exposure: a sample of the activated sewage sludge was washed twice by setllment and resuspension in clulture medium to remove any excessive amounts of dissolvd organic carbon. A sub-sample of the washed sewage sludge was then removed and the SS concentration determined
- Pretreatment: due to shortage of filters samples were not filtered or centrifuged to remove the sewage sludge solids
- Concentration of sludge: 180 mg ss/L
- Initial cell/biomass concentration: 100 mg C/L
- Water filtered: no
- Type and size of filter used, if any: no filter used
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
- Test temperature in the BOD bottles: 20-24°C
- pH: 7.4
- pH adjusted: yes (pH of stock solutions was adjusted to approximate neutrality before use)
- Aeration of dilution water: yes
- Suspended solids concentration: 300 mg SS/L
- Continuous darkness: yes

- Dilutions water: reverse osmosis, purified deionised water
- Additional substrate: none
- Test substance was added as aqueous solution (2000mg/L)

- Culturing apparatus: 3 L glass culture vessels each containing 2 L of solution
- Number of culture flasks/concentration:
- Measuring equipment: samples were analysed for DOC using a Shimadzu TOC-Vcph TOC analyser
- Test performed in closed vessels due to significant volatility of test substance: yes

- Sampling frequency: samples taken for analysis at 0, 3 hours. 1, 2, 7, 10, 14, 21, 27 and 28 days
- Duplicate vessels from each concentration were finished at each sampling occasion and used for DOC, temperature and pH measurement.

- Control: yes (2 replicates)
- Reference item: yes (2 replicates)
- Abiotic steril control: yes, using sodium azide (100 mg/L)
- Toxicity control: yes, using both the test substance and diethylene glycol at 200 mg C/L
Reference substance:
diethylene glycol
100 mg/L
Preliminary study:
The mean % carbon content for the test item was 65.97 and the test item did not adsorb to activated sewage sludge
Test performance:
- Diethylene glycol had been degraded to 100% of its DOC after 14 d and achieved a max of 100% after 28 days.
- In the presence of the test substance, diethylene glycol had been degraded to 58% after 14 days
- the pH alues did not fall below 6.9 in any culture vessel during the study
- aerobic conditions in the culture vessels were maintained throughout the study as DO remained at approx. 6.1 mg O2/L or above in all culture vessels
- These results indicate that the inoculum was viable and that the test substance was not significantly inhibitory.
Key result
% degradation (DOC removal)
Sampling time:
28 d
Remarks on result:
other: Maximum value
Details on results:
The results obtained from the abiotic test vessel showed no significant abiotic degradation/loss of test item over the test period
Validity criteria fulfilled:
Interpretation of results:
not inherently biodegradable
The test item attained 9% degradation after 28 days. The results obtained from the abiotic test vessel showed no significant abiotic degradation/loss of test item over the test period. A figure of 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralisation may be regarded as evidence for ultimate biodegradation. The test item can therefore be considered not to have exhibited any evidence of inherent, primary biodegradation under the experimental conditions in this study.
biodegradation in water: ready biodegradability
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:

Description of key information

The key study was performed equivalent to OECD guideline 302B under GLP conditions. Only 9 % degradation was observed with the test substance after 28 days (based on DOC removal), whereas the reference substance (di ethylene glycol) showed up to 100 % degradation after 14 days. Therefore, it can be concluded that the test results are valid. In conclusion, a 28-day modified Zahn-Wellens test showed that the substance is not biodegradable under the specified test conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One study describing an inherent biodegradability test was identified. This was a study of Huntsman (2011), which is an OECD 302 B study (Zahn-Wellens test), which is an inherent biodegradability test, and according to ISO 7827, which is an ultimate biodegradation guideline. The study concludes that the test substance is not biodegradable under test conditions. Less than 10 % biodegradation was observed. As the study is performed according to GLP, this study was rated K1 and assigned as key study.