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Diss Factsheets

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
Study period:
15 Jan 2019 - 14 Feb 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
July 17, 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:

Test material

Constituent 1
Chemical structure
Reference substance name:
(S)-2-(5-Oxo-3-propyl-1,2-dihydropyrrol-1-yl) butyramide
Cas Number:
Molecular formula:
(S)-2-(5-Oxo-3-propyl-1,2-dihydropyrrol-1-yl) butyramide
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Off-white powder
- Storage condition of test material: In refrigerator protected from light

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Concentration of suspended solids: 3.6 g/L in the concentrated sludge, 11 mg suspended solids/L in the test vessels.
- Preparation of inoculum for exposure: Before use the sludge was washed with mineral medium.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
12 mg/L
Based on:
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: According to OECD 301B
- Test temperature: 22-23 °C
- pH: at start: 7.5; t = 14 d: 7.8 and 7.7 (procedure and toxicity control, resp.); t = 28 d: 7.6 (blank controls and test solutions)
- pH adjusted: no
- Continuous darkness: yes
- Continuous aeration and stirring: yes
- Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2 for test suspension, 2 for inoculum blank, 1 for procedure control, 1 for toxicity control.
- Method used to create aerobic conditions: aeration with synthetic air (mixture of oxygen (ca. 20%) and nitrogen (ca. 80%), CO2 < 1 ppm) at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min)
- Details of trap for CO2: 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.

- Inoculum blank: containing only inoculum
- Procedure control: containing reference item and inoculum
- Toxicity control: containing test item, reference item and inoculum

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. On the day of testing weighed amounts of the test item were added to the 2-litres test bottles containing medium with microbial organisms and mineral components.

- Sampling frequency: Titrations were made on day 2, 5, 8, 12, 15, 19, 23 and 29 for the inoculum blank and test item. Titrations for the procedure and toxicity control were made on day 2, 5, 8, 12 and 15.
- Sampling method: titration of Ba(OH)2 in the gas scrubbing bottles.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
29 d
Remarks on result:
other: HCl added on the 28th day (last CO2-measurement on the 29th day).
Details on results:
- ThCO2 of the test item was calculated to be 2.17 mg CO2/mg.
- ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
- Biodegradation test item: Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item (1% and 0%, based on ThCO2).
- Toxicity control: More than 25% biodegradation occurred within 14 days (39%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- The tempreature and pH during the exposure period were within the prescribed range of the study plan.

BOD5 / COD results

Results with reference substance:
The reference substance showed a normal biodegradation curve and reached > 60% (84%) biodegradation within 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
see 'Overall remarks'
Interpretation of results:
not readily biodegradable
The substance was not readily biodegradable in the CO2 Evolution Test, performed according to OECD guideline 301 B and GLP principles.
Executive summary:

In a CO2 Evolution Test performed according to OECD guideline 301B and GLP principles, the substance was evaluated for its ready biodegradability. The test was performed in an aerobic aqueous medium containing activated sludge over a period of 28 days. The substance was tested in duplicate at a target concentration of 20 mg/L, corresponding to 12 mg TOC/L. Two inoculum blanks, one procedure control (sodium acetate) and one toxicity control (substance plus sodium acetate) were included. Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item. Since 39% biodegradation occurred in the toxicity control within 14 days, the substance was assumed not to inhibit microbial activity. All acceptability criteria for the test were met and the study is therefore considered to be valid. Based on the obtained results in this test, the substance is concluded to be not readily biodegradable.