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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study to OECD Guideline, with sufficient substance identification analysis and analysis certificate.
Reason / purpose:
reference to same study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: Method C5 of 84/449/EEC OECD Guideline 301B, Modified Sturm Test
Principles of method if other than guideline:
No Deviations Reported
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
Empirical Formular - C18H16N2O4
Molecular Weight - 324
Physical State - White powder
Water solubility - 64 mg/L

Certificate of analysis was provided by the sponsor Batch Tested :910312

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A sample of activated sludge was collected on the day before the test from a sewage treatment works that treated predominantly domestic waste, and was aerated in the laboratory for four hours. A sample of the mixed liquor was then homogenised in a mechanical blender and allowed to settle
for thirty minutes. An aliquot of the supernatant was passed through a Whatman's GFC filter paper to remove coarse solids and the filtrate used as
the inoculum for the test.
Duration of test (contact time):
73 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 10 mg/L
Based on:
test mat.
Initial conc.:
ca. 20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Seven groups of four BOD bottles were filled with Mineral Salts Medium (MSM, Appendix 2), inoculum and test material and/or sodium benzoate
according to the schedule shown below, care being taken to avoid the introduction of air bubbles during preparation and transfer of media to

Group Additions to MSM (mg/l)
1 None
2 Inoculum
3 Inoculum + sodium benzoate (2)
4 Inoculum + test material (2)
5 Inoculum + test material (2) + sodium benzoate (2)
6 Inoculum + test material (10)
7 Inoculum + test material (10) + sodium benzoate (2)

Appropriate weights of BCI-MX were added, in glass ampoules, directly to the BOD bottles. Test concentrations of the reference substance were
prepared from an aqueous solution (1 g/l).

The concentrations of dissolved oxygen (DO) and thetemperatures of the contents of duplicate vessels from each group were measured, using a YSI dissolved oxygen meter fitted with a self-stirring DO/temperature bottle probe, at the start of the test and after incubation in darkness for five days
at 20·C.

The pH of each control, test and reference mixture was measured after oxygen and temperature measurement. The temperature of the incubator was measured at intervals during the test using a maximum-minimum thermometer.
Reference substance
Reference substance:
other: Sodium benzoate

Results and discussion

Preliminary study:
A five-day bacterial inhibition test was performed under the conditions of the Closed Bottle Test (EEC Procedure C6, Method
3010). This showed that BCI-MX at 2 and 10 mg/l did not inhibit degradation of the reference material sodium benzoate. In this
preliminary test, BCI-MX alone showed no evidence of biodegradation.
Test performance:
Cumulative C02 production in the control after 28 days (10.8 mgC02) was within the acceptable range for this assay system (recommended maximum 50 mgC02); cumulative C02 production after 73 days was 18.8 mgC02. These results confirm that the inoculum was viable and that the basic
validity criteria were met.
% Degradationopen allclose all
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: 10 mg/L (Test Substance)
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: 20mg/L (Test Substance)
% degradation (CO2 evolution)
Sampling time:
73 d
Remarks on result:
other: 10 mg/l (Test Substance)
% degradation (CO2 evolution)
Sampling time:
73 d
Remarks on result:
other: 20 mg/L( test Substance)
Details on results:
The test chemical does not reach the required Biodegradation level in 28 days to be considered readily biodegradable. Prelonged results indicate that at a concentration of 10mg/L that the substance is ultimately biodegradable. Reference substance results indicate that the inoculum was viable and
the test system was working satisfactorily.

BOD5 / COD results

Results with reference substance:
Sodium benzoate was degraded by 68% of its theoretical carbon dioxide production (TC02; 2.14 mgC02/m9) after nine days, 85%
after 28 days and 89% after 73 days.

Any other information on results incl. tables

Biodegradation results over the test period

 Day   Reference %TCO2  Test Substance 10mg  %TCO2     Test Substance 20mg  %TCO2


 2  27  1
 3  42  2
 6  58  4  1
 9  68  8  2
 14  78  14
 20  82  19
 27  84  28  6
 28  85  31  6
 37 86  39  9
 42 86  44  9
 48 86  49  11
 51 86  52   11
 56 86  58  13
 59 87  61  14
 64 87 67  16
 70 88  74  18
 72  89  78  19
 73 89  80  20

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
other: not readily biodegradable
The test can be concluded as reliable without restrictions. The test substance can be concluded to be ultimately biodegradable
Executive summary:

Test was conducted to appropriate OECD guideline, with GLP accreditation and satisfactoy analysis of the CO2 produced. An analytical certificate and sufficient substance identity were also provided. Preliminary toxicity to bacteria was tested in which no respiration inhibition and biodegredation was observed. Study protocol was also available.