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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:
From 21 Oct 2003 to 26 mar 2004
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This is a GLP study following OECD guidelines.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:

Test material


Study design

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): water treatment plant of Evreux (France). Inoculum was collected on 21 October 2003.
- Preparation of inoculum for exposure: the inoculum was prepared by initially removing the biggest particles and sieving accross a filter paper (porosity 4-7 μm). The inoculum was used immediately after sieving (no preconditioning period).
- Pretreatment: none
- Concentration of sludge: In order to obtain a final concentration of approximately 10E4 bacteria per liter in all test suspensions, inoculum was diluted 20-fold with mineral medium and 0.238 mL/L of (diluted) inoculum were added to each test vessel.
- Initial cell/biomass concentration: ca 84x10E4 cells/mL of bacteria (determination with a Malassez cell counter).
- Water filtered: yes
- Type and size of filter used, if any: filter paper (4-7µm)
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
BOD was measured on day 0, 3, 7, 10, 14, 17, 21, 24, and 28.

- Composition of medium: reconstituted water (OECD and EEC recommended) was prepared using deionised water and analytical grade reagents.
- Test temperature: between 21°C and 24°C.
- pH: was measured before the beginning of the test in the mineral medium: 7.31
- pH adjusted: no
- Aeration of dilution water: Dilution water was aerated for at least 20 min prior the test.
- Continuous darkness: yes

- Culturing apparatus: closed glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration, no more data
- Measuring equipment: O2 measurements

SAMPLING: no data

- Inoculum blank: yes (2 replicates)
- Abiotic sterile control: no
- Toxicity control: yes (2 replicates)
- Other: group containing the reference item(3 mg/L) and inoculum (procedure control, 2 replicates)

Reference substance
Reference substance:
acetic acid, sodium salt
3 mg/L

Results and discussion

Preliminary study:
no preliminary study was performed
Test performance:
All validity criteria were respected:
• dissolved oxygen depletion in the inoculum blank did not exceed 1.5 mg/L after 28 days (0.72 mg/L),
• dissolved oxygen was ≥ 0.5 mg/L in all test suspension replicates during the test,
• biodegradation values of test item replicates deviated by less than 20% at the end of the 10-day window (the 10 days immediately following the attainment of 10% biodegradation),
• biodegradation in the reference test was 68% after 14 days then it was at least 60% within this period,
• biodegradation in the toxicity control was 58% (based on ThOD) after 14 days then it was at least 25% within this period.
% Degradationopen allclose all
% degradation (O2 consumption)
Sampling time:
0 d
Remarks on result:
other: test initiation
% degradation (O2 consumption)
Sampling time:
7 d
Remarks on result:
other: 10d-window already started
% degradation (O2 consumption)
Sampling time:
14 d
Remarks on result:
other: end of 10d-window
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
The 10-day window (the 10 days immediately following the attainment of 10% biodegradation) of the test item started on the 4th day. Biodegradation totalled 47% (mean of the two replicates) at the end of this 10-day window on the 14th day and 72% over the test period.
No significant inhibition of the inoculum due to toxicity of the test item was noted since the toxicity control reached 58% of the ThOD within 14 days. Biodegradation in this control totalled 71% after 28 days.

BOD5 / COD results

Results with reference substance:
The reference item degraded normally under the test conditions. Biodegradation in the reference test was 68% after 14 days then it was at least 60% within this period.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable, but failing 10-day window
The author concluded to the inherent biodegradability of the substance with 72% of the substance degraded after 28 days.
Executive summary:

This study (CIT, 2004) aimed to assess the ready biodegradability of the substance in a closed bottle test following recommended guidelines (OECD 301D).

The inoculum used for this test was a non-adapted inoculum collected from a domestic water treatment plant. After filtration it was diluted into OECD mineral medium to reach 10E4 cells/mL of bacteria. Mineral medium was aerated prior testing in the test room.

Inoculum blank, toxicity control, procedure control and test vessels were measured for O2 consumption in duplicate in bottles on day 0, 3, 7, 10, 14, 17, 21, 24, and 28. The reference substance used was acetate sodium.

O2 concentrations, biodegradation values in replicate at the end of the 10d-window, toxicity control and procedure control criteria were fulfilled and thus validated the test. The 10d-window started on day 4 and 47% of the substance degraded after 14 days of test. After 28 days of test, 72% of the substance degraded. The substance was therefore found inherently biodegradable.