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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sewage sludge was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough (UK) which treats predominantly domestic sewage.

The activated sludge was washed by settlement and resuspension in culture medium to remove any excess dissolved organic carbon (DOC). The sludge was then aerated (continuous) in the laboratory at 21 degrees celsius and used on the day of collection. The amount of suspended solids in the sludge was determined by filtering a 100ml washed activated sludge sample using GF/A filter paper (weighed prior to use) and a Buchner funnel. The sludge was rinsed three successive times with deionised reverse osmosis water (10ml) and filtered. The filter paper was dried for at least an hour in an oven at 105 degrees celsius and allowed to cool before weighing. The process was repeated until a constant weight was determined. The concentration of suspended solids was 4.0 g/l prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
A preliminary study was carried out to determine adsorption of test substance to filters or activated sewage sludge. Samples were analysed for DOC using a Shimadzu TOC-5050A analyser. A 100 mg/l test substance stock solution was made by dissolving test material (100 mg) into culture medium (1000 ml), from this stock solution, 2 samples were taken for DOC analysis - one filtered through a Gelman 0.45 um AcroCap filter; the other sample was not filtered. A similar stock solution was prepared by dissolving test material (100 mg) into culture medium and then inoculating with 30 mg suspended solids/l before adjusting to a volume of 1000 ml. Two samples from this stock solution were taken for DOC analysis - one filtered through a Gelman 0.45 um AcroCap filter; the other sample was centrifuged and the supernatant extracted for analysis. Control samples were also prepared by adding suspended solids (30 mg ss/l) to culture medium and then filtering or centrifuging before DOC analysis.

For the definitive test, the following test preparations were prepared (in duplicate):
- A control, with inoculated culture medium
- Sodium benzoate with inoculated culture medium, giving a final concentration of 10 mg carbon/l
- Test material with inoculated culture medium, giving a final concentration of 10 mg carbon/l
- Test material, sodium benzoate and inoculated culture medium to give a final concentration of 20 mg carbon/l (toxicity control was prepared to assess the toxic effects of the test material to the sewage sludge micro-organisms. A solution of 13 mg test material/l and 17.1 mg sodium benzoate/l were used to create a 20 mg Carbon/l solution. The toxicity control was not conducted in duplicate)

Each test vessel contained a final concentration of 30 mg suspended solid/l. 24 hours before testing, culture medium and inoculum were added to test vessels and aerated with CO2-free air overnight. After this period, test material and sodium benzoate were added to the relevant vessels. All culture vessels were sealed and CO2-free air was bubbled through the solution at 40 ml/minute and continuously stirred (by magnetic stirrer). CO2 production by degradation was collected in Drechel bottles containing NaOH. The test was carried out at 21 degrees C in the dark.

Reference substance:
benzoic acid, sodium salt
Preliminary study:
The test material did not adsorb to filters or activated sewage sludge.
% degradation (CO2 evolution)
Sampling time:
28 d
% degradation (DOC removal)
Sampling time:
28 d
Details on results:
The CO2 evolution data satisfied the 10-day window criterion for ready biodegradability (DOC was evaluated only on Days 0 and 28).
Results with reference substance:
Sodium benzoate achieved 78% degradation after 14 days and 79% degradation after 28 days. This confirmed that the inoculum and test conditions used were suitable for this test.

The toxicity control achieved 80% degradation after 14 days, this confirmed that the test material was not toxic to sewage treatment micro-organisms used in the test.

DOC analysis: Analysis of the test material vessels revealed 100% degradation in both replicates. For sodium benzoate, 100% degradation was also observed in both replicates.

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
Under the conditions of this study, the test item is considered to be readily biodegradable achieving 72% degradation after 28 days and passing 60% within 10 days after reaching 10%.

Description of key information

The ready biodegradability of the test item was determined in an OECD 301B guideline test.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

In a OECD 301B guideline study with non-adapted activated sludge, 72% biodegradation was achieved after 28 days based on carbon dioxide production (60% being surpassed within 10 days after reaching 10%).

The test item can therefore be considered readily biodegradable.