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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
yes (incl. certificate)

Test material

Test material form:
Specific details on test material used for the study:
Appearance: Colourless liquid
Batch number: OC15014 after rotavapor
Sample code: 109/BF-81/17
Composition provided by the Sponsor: NaCl (~ 4%) dry matter (9.3%)

The solvents, water and ethanol were completely evaporated from the extract to constant mass of solid reside, under vacuum from 300 to 1 torr and at temperature of 65-67°C. The resulted dry residue was dissolved in double distilled water and examined in biodegradation test.

Study design

Oxygen conditions:
Inoculum or test system:
sewage, domestic (adaptation not specified)
from the Sewage Treatment Plant ”Czajka” , Warsaw
Details on inoculum:
Inoculum as a sample of secondary efluent was taken from the Sewage Treatment Plant ”Czajka and was pre-conditioned by aerating, without other treatment or addition, for 7 days at the test temperature.
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 2 mg/L
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Expressed as a percentage of COD.
Details on study design:

Initial concentration of test item in medium, C : 2 mg/L as organic matter in dried solid
Initial concentration of reference item in medium, C : 2 mg/L
Volume of test solution in flask, V : 0.300 L
Test item COD : 1.275 ± 0.12 mg O2/mg test item
Reference item ThOD : 0.78 mg O2/mg reference item
Amount of effluent in 1 L of mineral solution : 0.2 ml/L
Initial concentration in toxicity control test : 2.0 mg/L test item + 2.0 mg/L reference item
Concentration of test item as organic matter in dried solid in stock solution : 81.45 g/L
Concentration of reference item in stock solution : 10 g/L

- Test temperature: 22 +/- 5°C
- Continuous darkness: yes

- Culturing apparatus: full closed bottle

Inoculum blanks: 18 flasks (two parallel samples for each oxygen concentration measurement) containing inoculum and mineral medium.
Procedure control: 18 flasks (two parallel samples for each oxygen concentration measurement) containing inoculum, reference item (2 mg/l) and mineral medium.
Test: 18 flasks (two parallel samples for each oxygen concentration measurement) containing inoculum, test item (2 mg/l) and mineral medium.
Toxicity control: 18 flasks (two parallel samples for each oxygen concentration measurement) containing inoculum, test item (2 mg/l), reference item (2 mg/l) and mineral medium.

STATISTICAL METHODS:The calculations and the graphs will be performed using SigmaPlot 9.0 software of SYSTAT Software, Inc
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Test performance:
Validity of test
1. The difference of extremes of replicate values of the removal of the test chemical at the plateau. at the end of the test is less than 20% and the percentage degradation of the reference compound has reached on the 2nd day the pass level (60%) for the limit of day 14.
2. In a toxicity test containing both the test item and a reference item. on the 14th test day the biodegradation (based on ThOD of reference item and on COD of test item) attained 58.9 % and the test item cannot be assumed to be inhibitory.
3. Oxygen depletion in the inoculum (0.91 mg/L) did not exceed the limit of 1.5 mg dissolved oxygen/litre after 28 days.
4. The residual concentration of oxygen in the test bottles did not fall below 0,5 mg/litre at any time.
% Degradation
Key result
% degradation (O2 consumption)
ca. 62.94
Sampling time:
28 d
Details on results:
oxygen depletion: % degradation (toxicity test, 28 days) : 71.53% (mean of the two bottles)

BOD5 / COD results

Key result
ca. 1.275 g O2/g test mat.
Results with reference substance:
oxygen depletion: % degradation (reference substance, 28 days) : 87.50% (mean of the two bottles)

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Executive summary:

The biodegradability attained 62.9 % within twenty-eight days.

The pass level for ready biodegradability of 60% of COD was reached on 24th day, but not in 10-day window, in which the biodegradability attained 49%.

Thus, the test item can not be considered as readily biodegradable. This negative result in a test for ready biodegradability does not necessarily mean that the chemical will not be degraded under relevant environmental conditions, but it means that the next level of testing, i.e. either a simulation test or an inherent biodegradability test, should be considered