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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
Study period:
04 Oct - 01 Nov 2001
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. QA statement)
Niedersächsisches Umweltministerium, Hannover, Germany
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant, Hildesheim, Germany
- Laboratory culture: no
- Pretreatment: Activated sludget was filtered with a folded filter. The first 200 mL of the filtrate was not used. The second filtrate was used to initiate inoculation.
- Colony forming units: 10E+04 - 10E+06 CFU/L
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
test mat.
Initial conc.:
7.53 mg/L
Based on:
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: according to guideline
- Additional substrate: no
- Test temperature: 20.3 - 21.0 °C
- pH: inoculum control: 7.11 (beginning), 5.05 (end); functional control: 7.08 (beginning), 6.56 (end); test item: 7.00 (beginning), 6.38 (end); toxicity control: 7.04 (beginning), 6.48 (end)
- pH adjusted: no
- Aeration of dilution water: One day before the test started the demineralized water for the test medium was aerated until oxygen saturation and left at room temperature for at lest 20 h.

- Culturing apparatus: BOD bottles with glass stoppers (volume: 300 mL)
- Number of culture flasks/concentration: 10
- Measuring equipment: Oxygen concentration was measured using an Oximeter "WTW" Oxi 530
- Test performed in closed vessels due to significant volatility of test substance: closed without air bladders
- Other: All test solutions were prepared by adding 0.2 mL of the inoculum filled up with test medium.

- Sampling frequency: on day 0, 7, 14, 21 and 28

- Inoculum blank: yes, 10 bottles
- Abiotic sterile control: no
- Toxicity control: yes, 10 bottles
- Other: reference substance: yes, 10 bottles
Reference substance:
acetic acid, sodium salt
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
The ThOD of the test item was 2.51 mg O2/mg test item.
Results with reference substance:
The reference substance was degraded to 87% after 28 d (O2 consumption). The degradation was > 60% after 14 d. Thus, the result is valid.

In the toxicity control the degradation achieved 63% after 14 d. Thus, the substance is not considered to be inhibitory to the inoculum. The oxygen demand in the inoculum control was 1.73 mg O2/L after 28 d. The slightly higher oxygen demand in the inoculum control after 28 d had no impact on quality and integrity of the study.

Table 1: Degradation of the reference substance, test item and in the toxicity control

Sampling day

Reference substance

Test item

Toxicity control

Degradation in %

Degradation in %

Degradation in %






















Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The substance is readily biodegradable in the 14-day-window and after 28 days according to OECD 301 D.

Description of key information

Readily biodegradable: 69% after 28 d (O2 consumption, OECD 301D; 14-day window criterion was met)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One GLP guideline study is available, which assessed the ready biodegradability of the test substance according to OECD guideline 301 D. Activated sludge was collected from a local municipal sewage treatment plant treating predominantly domestic sewage. The non-adapted activated sludge was inoculated with the test substance at a nominal concentration of 3.0 mg/L, corresponding to a ThOD of 7.53 mg O2/L and incubated for 28 d. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage ThOD and was calculated for day 7, 14, 21 and 28. A mean degradation of 71% on day 7, 70% on day 14, 85% on day 21 and 69% on day 28 was recorded. The pass level was reached within the 14-day window. Thus, the substance is considered to be readily biodegradable according to the OECD criteria.
The biodegradation of the functional control reached 87% after 28 d confirming the suitability of the test system. The toxicity control showed no toxic effects on bacteria at the tested concentration of 1.5 mg/L (63% degradation after 14 d).