Registration Dossier

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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted at a GLP-accredited testing facility

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. certificate)

Test material


Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge microorganisms was obtained on 2 April 2007 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough (Leicestershire, UK). This plant predominantly treats domestic sewage.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
An amount (1500mg) of test material was dispensed in approximately 3l of culture medium and dispersed by high shear mixing (ca. 7400rpm for 40 minutes). Undissolved material was removed by filtration through a 0.2µm Sartorius Sartopore filter, with the initial 500ml being discarded to precondition the filter, to give a saturated solution of 66.13mg carbon/l (by DOC analysis). An aliquot (453.7ml) of this saturated solution was dispersed in inoculared culture medium, and the volume adjusted to 3l to give a final concentration of 10mg C/l.

A standard material, sodium benzoate, was evaluated at 10mg C/l, as well as a toxicity control which contained both sodium benzoate and the test substance both at concentrations equivalent to 10mg C/l.

The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove excess dissolved organic carbon (DOC). The washed sample was maintained under continuous aeration at a temperature of approximately 21°C and used on the day of collection. The suspended solids content of the sludge was carried out by filtering a 100ml sample of the washed sludge by suction through pre-weighed GF/A filter paper (washed three times with deionised reverse-osmosis water and dried in an oven) using a Buchner funnel. The funnel was rinsed three times with 10ml deionised reverse osmosis water and further filtered for 3 minutes. The filter paper was dried in an oven (ca. 105°C for at least one hour) and allowed to cool before weighing. The process was repeated until a constant weight was acheived. The suspended solids concentration was 2.2g/l prior to use.

The culture medium was as recommended in the OECD guideline.

Approximately 24h prior to the addition of the test and standard materials 5l culture vessels were filled with 2400ml culture medium and 40.9ml inoculum, to give a final concentration of 30mg suspended solids per ml, and aerated overnight. The following test preparations were made in duplicate (unless otherwise stated) and the final volume made up to 3l with culture medium:
1. A control of inoculated culture medium.
2. The standard material at 10mg C/l.
3. The test material at 10mg C/l.
4. The test material and standard material at 20mg C/l (total) to act as a toxicity control (one vessel only).

The test was carried out at ca. 21°C in darkness. The culture vessles were sealed and CO2-free air was bubbled through the solution at a rate of approximately 40ml/min and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

The CO2 produced by degradation was collected in two 500ml Dreschel bottles containing 350ml of NaOH (0.05M). The solutions were prepared using purified de-gassed water.

Samples (2ml) were taken from the first CO2 absorber vessel on days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28, and 29, and from the second CO2 absorber vessel on days 0 and 29. On day 28, 1ml of concentrated HCl was added to each vessel to drive off any inorganic carbonates that had formed. The vessels were resealed, aerated overnight and final samples taken from the absorbers on day 29. The samples were analysed for CO2.

Samples (20ml) were taken from each vessel on days 0 and 28, filtered through Gelman 0.45µm Acrocap filters (ca. 5ml discarded) prior to DOC analysis.

The pH of the test solutions was determined on day 28, prior to the addition of HCl, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Results taken for DOC analysis from the preliminary investigational work indicated that the test material did not absorb to filter matrices or to activated sewage sludge. For the purpose of the study, the samples taken for DOC analysis were therefore filtered to remove the suspended solids without the loss of test material.
Test performance:
Total CO2 evolution i the test vessels on day 28 was 39.61mg/l and therefore satisfied the validation criteria laid down in the OECD test guideline.
The IC content of the test material suspension in the mineral medium at the start of the test was below 5% of the TC content and therefore satisfied the validation criteria laid down in the OECD test guideline.
The difference between the values for CO2 evolution at the end of the test for replicate vessels was <20% and therefore satisfied the validation criteria laid down in the OECD test guideline.
The reference substance attained 96% biodegradation after 14 days, and 114% after 28 days, thereby confirming the suitability of the inoculum and test conditions.
The toxicity control attained 54% degradation after 14 days and 52% after 28 days, confirming that the test material was nbot toxic to the sewage micro-organisms used in the test.
IC analysis of the samples from the second absorber vessel confirmed that there was no significant carry-over of CO2 into the second absorber vessels.
% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The test material attained 40% degradation after 28 days and therefore cannot be considered to be readily biodegradable according to the criteria laid down in OECD Guieline No 301B

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
other: not meeting criteria for readily biodegradability, but showed limited biodegradation under the conditions of the test
The test substance attained 40% biodegradation over 28 days. Accordingly it does not meet the criteria for ready biodegradability, but it may be inherently biodegradable.