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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction).
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
equivalent or similar to
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
The study was extended to 112 days
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Nieuwgraaf in Duiven, The Netherlands

- Culture: The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 200 mg Dry Weight (DW)/L of activated sludge was aerated for one week.
- Concentration of sludge: The sludge was diluted to a concentration of 2 mg/L in the BOD bottles

Duration of test (contact time):
0 - 112 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: The nutrient medium of the Closed Bottle test contained per litre of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HP04, 33.4 mg Na2HPO4.2H20, 22.5 mg MgSO4.7H20, 27.5 mg CaCI2, 0.25 mg FeCI3.6H20. Ammonium chloride was omitted from the medium to prevent nitrification. Due to this omission the pH of the medium decreased slightly. The decrease of the pH does not affect the biodegradation in the Closed Bottle test.
- Test temperature: ranged from 20 to 22°C (24°C for one day; minor deviation from the study plan).
- pH: 7 at Day 0, 7.3 at Day 28
- pH adjusted: no
- Aeration of dilution water: no
- Continuous darkness: yes

- Culturing apparatus: 250 to 300 mL BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration:
6 bottles containing only inoculum
10 bottles containing test substance, silica gel and inoculum
10 bottles containing silica gel and inoculum
6 bottles containing sodium acetate and inoculum (positive control)
- Measuring equipment: oxygen electrode and meter
- Test performed in closed vessels due to significant volatility of test substance: yes
- Other: The test substance is a poorly soluble substance in water and therefore the test substance was first dissolved in dichloromethane (1 g/L). The test substance in dichloromethane (0.56 mL) was added to 2 g silica gel (100-200 mesh) weighed in a glass petri dish. The solvent was allowed to evaporate by placing the petri dish in a ventilated hood for 3 hours, and the entire contents were then transferred to the BOD bottle. During the test period, the test compound should have been released slowly from the silica gel. Although no additional oxygen consumption was expected, controls with silica gel were carried out as well. This technique was described in an ISO document.

- Sampling frequency: Day 0, 7, 14, 21, 28, 42, 56, 84 and 112
- Sampling method: The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of Day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle.

- Inoculum blank: yes, 6 bottles containing only inoculum
- Controls: 10 bottles containing silica gel and inoculum, 6 bottles containing sodium acetate and inoculum (positive control)

Reference substance:
acetic acid, sodium salt
other: The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).
Sampling time:
28 d
other: The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).
Sampling time:
112 d
Details on results:
Inhibition of the degradation of a well-degradable compound. e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. A slight inhibition of the endogenous respiration of the inoculum by the test substance was not detected. Therefore, no inhibition of the biodegradation due to the “high” initial concentration of the test compound is expected.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 83%.

Time (days)

Biodegradation (%)


























Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Not readily biodegradable
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Executive summary:

In order to assess the biotic degradation of the test substance, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined at a concentration of 2.0 mg/L in the Closed Bottle test performed according to slightly modified OECD, EEC and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. The test was prolonged because the pass level was not reached at Day 28.

The tests substance was not biodegraded in the Closed Bottle test (28 days and prolonged) and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test. permit an accurate administration of the test compound. The test substance was biodegraded 7% in the Closed Bottle test at Day 28 and should therefore not be classified as readily biodegradable.

Description of key information

No biodegradation study with the test substance is available. A biodegradation study with 2,2’-azobis(isobutyronitrile) was used to fulfill the data gap for the test substance.The underlying hypothesis for the read-across is that the two substances are expected to have similar degradation pathway, where the initial transformation is nitrile hydrolysis. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

In a ready biodegradability study with 2,2‘-azobis(isobutyronitrile), the test substance was degraded 7% after 28 days and 13% after 112 days. The substance is therefore not considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information