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Environmental fate & pathways

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)
Version / remarks:
July 1992
according to guideline
other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"
Version / remarks:
GLP compliance:
yes (incl. QA statement)
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): municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Method of cultivation / Preparation of inoculum for exposure: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. After treatment the concentration of suspended solids (SS) was determined to be 4.2 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 12.5 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
38.5 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: mineral medium according to testing guidelines; mineral components were dissolved in tap-water (purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon)
- Test temperature: 22 - 23 °C
- pH: 7.6 at the start of the test and 7.5 at the end
- pH adjusted: the pH was adjusted from 7.7 or 7.8 to 7.6
- Suspended solids concentration: 12.5 mg/L
- Continuous darkness: yes

- Culturing apparatus: 2-liter brown coloured glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

- Sampling frequency:
inoculum blank and test item: Exposure Day 2, 5, 8, 13, 15, 19, 23, 28 (before acidification), and 29 (after driving off residual CO2)
procedure and toxicity control: Exposure Day 2, 5, 8, 13 (before acidification) and 15 (after driving off residual CO2)
- Sampling method: The CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
- Sample storage before analysis: Samples were immediately analyzed.

- Inoculum blank: 2 flasks containing inoculated mineral medium
- Reference: 1 flasks containing inoculated mineral medium and sodium acetate (12 mg TOC/L)
- Toxicity control: 1 flask containing inoculated mineral medium, test material and sodium benzoate (12 mg TOC/L, each)

Weighed amounts of the test item were added to the 2 litre test bottles containing medium with microbial organisms and mineral components. To this end, small watch glasses were used to transfer the weighed amounts of test item to the respective test bottles. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms. Furthermore, the test medium was swirled around daily, since the test item tended to float on the water surface.
Reference substance:
acetic acid, sodium salt
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
see background material "S-600 - Biodegradation.pdf"
Results with reference substance:
Average biodegradation of the reference item sodium acetate was >60 % by Exposure Day 8, thus confirming suitability of the activated sludge (see background material "S-600 - Biodegradation.pdf").
Validity criteria fulfilled:
Validity criteria for IC/TC ratio, degradation in positive and toxicity controls and difference between test item replicates were met. CO2 evolution in the controls was < 40 mg/L on day 29.
Interpretation of results:
under test conditions no biodegradation observed

Description of key information

Biodegradation in water: screening tests: aerobic biodegradation 0% (CO2-evolution) in 28 days (OECD 301B, ISO 10634)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:

Additional information