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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:
1 (reliable without restriction)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
ISO 14593
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl nitrate
EC Number:
EC Name:
2-ethylhexyl nitrate
Cas Number:
Molecular formula:
2-ethylhexyl nitrate
impurity 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
Sewage sludge microorganisms from a domestic wastewater treatment plant. Sample filtered through coarse filter paper, filtrate purged with CO2 free air for approximately 1 hour while maintaining pH at 6.5 using concentrated orthophosphoric acid. The pH was then adjusted to 7.6 with sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to the removal of the supernatant for use in the study.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
36.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
107 mL of inoculated culture medium added to each of 25 replicate test vessels which were then sealed using Teflon lined silicon septa and aluminum crimp caps. After sealing 4.0 μl of test material was injected through the septum of each vessel to give test material concentration of 36.5 mg/L (20 mg carbon/L). 29 control vessels prepared as above without the addition of the test material.

Replicates: Duplicates for the control, standard material, test material and toxicity control at each sampling point (5 replicates were available for day 28 analysis). Headspace to liquid ratio was 1:2.

Temperature of incubation: 20+1 °C

Study initiation: Test cultures provided with CO2 free air and placed on a rotary shaker and mixed at 150 rpm for the study duration.

Sampling: Days 2, 6, 8, 10, 14, 16, 20, 22, 24, 28 (after acidification on day 28)

Concentration of test substance: 36.5 mg/L (20 mg carbon/L)

Controls: Toxicity, blank and positive controls used per guideline. Positive control was sodium benzoate added to the control vessel at a loading of 20 mg C/L. Both test substance and reference material were added to the toxicity controls to obtain a maximum concentration of 40 mg C/L

Study termination: An aliquot of concentrated orthophosphoric acid was injected through the septum of each vessel taken for analysis in order to reduce pH to <3. The vessels were then shaken at ~150 rpm for 1 hour at 20+1 °C prior to samples being withdrawn from the headspace for analysis.

Method of calculating biodegradation values: Percent biodegradation calculated as percent ratio of cumulative net carbon dioxide to theoretical carbon dioxide as determined from elemental analysis of test material.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The test substance was not considered readily biodegradable under the criteria that requires 60% biodegradation within 28 days, achieved within 10 days of reaching 10% biodegradation. The CO2 production from the reference chemical exceeded the 60% of theoretical necessary to consider the test valid. The degradation for the toxicity control was 34% as of day 28 therefore the test substance was not considered to be inhibitory at the concentration tested. The water solubility of this test material is relatively low and coupled with the volatile nature of the test material, this may have resulted in the majority of the test material being present in the headspace and not proportionally dissolving in the test media. The test material may not hav been bioavailable and this may have contributed to the lack of biodegradation.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not inherently biodegradable
The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable.