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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:
Oct - Dec 2009
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
equivalent or similar to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
China Enviromental Science Press, 2004
according to guideline
other: Guidelines for the Testing of Chemicals HJ/T153-2004
GLP compliance:
test was performed according to Chinese standards

Test material


Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
The inoculum was derived from the secondary Shanghai Longhua Sewage Plant.

- Preparation of inoculum for exposure:
The inoculum was pre-conditioned by aerating the secondary effluent, withour other treatment or addition, for 5-7 days at the test remperature of 20 +/- 1°C.

- Pretreatment: no

- Concentration of sludge:
10(exp4) to 10(exp6) organisms/L

- Initial cell/biomass concentration:

- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
1. Preparation of Bottle
It was important that the bottles were thouroughly clean before use. First of all, add to the empty bottle 5-10 mL of the wash solution (e.g. 2.5 g iodine+ 12.5. g potassium iodide/L of 1% (w/v) sulfuric acid) shaking well to coat the bottle walls. Leave to stand for 15 min, pour off the solution and rinse thouroughly with tap water and finally deionised water. Stronlgy aerate mineral medium for at least 20 min and stand for 20 h at the test temperature. Determine the concentration of dissolved oxygen for control purposes.
Prepare parallel groups of BOD bottles for the determination of the test and reference substances in simultaneous experimental series. Add fully aerated mineral medium to BOD bottles so that they were one-third full, then add sufficient of the stock solutions of the test and reference substances to separate BOD bottles. The concentration of test substance was 2 mg/L. Inoculate the solutions in the BOD bottles with secondary effluent.

2. Number of Bottles
At least 10 containing the test substance and inoculum
At least 10 containing only inoculum
At least 10 containing reference compound and inoculum

3. Operations
Dispense each prepared solution or suspension immediately into the respective group of BOD bottles by hose from the lower quarter of the appropriate large bottle, so that all the BOD bottles were completely filled. Analyse the zero-time bottles immediately for dissolved oxygen by the electrode method. Stop the remaining replicate bottles ensuring that no air bubbles were enclosed, and incubate at 20°C in the dark. Each series must be accompanied by a complete parallel series for determination of inoculated blank medium. Withdraw duplicate bottles of all series for dissolved oxygen analysis every 7 days over the 28-day incubation.
Reference substance
Reference substance:
other: sodium lauryl sulfate

Results and discussion

% Degradationopen allclose all
Key result
% degradation (O2 consumption)
Sampling time:
28 h
% degradation (O2 consumption)
Sampling time:
7 d
% degradation (O2 consumption)
Sampling time:
28 d
% degradation (O2 consumption)
Sampling time:
21 d
Details on results:
The temperature range recored during the test was 19 to 21°C and was within the protocal specified range throughout the test. The result of the standard plate counts performed on the inoculum was 3.8 x .10(exp6) organisms/L. The measured dissolved oxygen concentration for test media prior to preparing the test mixtures was 9.02 mg O2/L.
The oxygen depletion of the control for inoculum was < 1.5 mg O2/L.
The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium lauryl sulfate. The average percent degradation on days 7, 14, 21 and 28 were 70.6%, 78.9%, 80.3% and 81.9%, respectively, which fulfilled the criteria for a valid test that a percent biodagardation of reference substance should be more than 60% after 14 days. Based on the ThOP of the test item (2.5 mg O2/mg), the average percent degradation on days 7, 14, 21 and 28 was 2.6%, 3.6%, 3.9% and 4.0%, respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The average percent degradation of the test item on days 7, 14, 21 and 28 was 2.6%, 3.6%, 3.9% and 4.0%, respectively.
Executive summary:

The biodegradability of the test substance was determined usng the CLosed Bottle Test Method. In the closed bottle test, inoculated mineral medium was dosed with a known amount of test substance as the nominal sole source of organic carbon. Degradation was followed by the analysis of dissolved oxygen over a 28 -day test period. The test contained an inoculum control group, a reference group and a treatment group. Each group comprised ten replicate test bottles. The inoculum control was used to measure the oxygen uptake of the inoculum and was not dosed with a carbon source. The reference bottles were doesed with sodium lauryl sulfate, a substance known to be biodegradable, at a nominal concentration of 2 mg/L. The treatment group test bottles were used to evaluate the test substance a nominal concentration of 2 mg/L. Measurements of dissolved oxygen were performed on two bottles from the control, reference, and treatment groups on days 0, 7, 14, 21 and 28.

The results indicated that the inoculum was active by degrading the reference substance, sodium lauryl sulfate, within the acceptable range (i.e. 60% within 14 days). The percent biodegradation of the test item was calculated as 2.6%, 3.6%, 3.9% and 4.0% on days 7, 14, 21 and 28.