Registration Dossier

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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD guideline

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:

Test material

Details on test material:
Batch number: 98 043 300
Appearence: white powder
Packaging: plastic bag
Quantity received and date: approximately 750 g received on April 2nd, 1998
Storage: in darkness at room temperature
Identification number for the study

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Organism, strains: a mixture of different strains of micro-organisms found in activated sludge.
Activated sludge from a municipal sewage treatment plant receiving little or no industrial effluent (from La Fouillouse - Loire -FRANCE)
Preparation of inocumum: on arrival at the laboratory the activated sludge was aerated for 5 hours 10 minutes; 5000mL of the mixture were sampled and homogenized for 2 minutes with magnetic stirring.
It was then allowed to settle for 2 hours; 1% inoculation of each test flask with the supernatant (concentrated by centrifugation and filtred) was then made the same day.
It was evaluated by surface agar counting with Millipore samplers MT00 00025 (72 hours at 25 °C): it was, at the minimum, between 550 000 and 770 000 micro-organisms per millilitre of inoculum.
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 15.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
inorg. C analysis
CO2 production by back titration
Details on study design:
The test was conduced in darkness
Measurement and experimental analyses
-determination of CO2 production by back titration for each assay every 1 to 3 days for the first 10 days, and then every 2 to 5 days until the end of the test (28 days); the titration of residual Ba(OH)2 was performed in control, reference and test media at the start and the end of the test.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Test performance:
Biodegradation in the toxicity medium on the 14th day: 46 %
Biodegradation of the test substance at the end of the 10-day window: 38% (graphical estimation)
Biodegradation of the test substance in 28 days: 62%
% Degradation
% degradation (CO2 evolution)
ca. 62
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
>80% after 28 days

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
other: not readily biodegradable
The test substance is not toxic for the inoculum
The test substance is not "readily" biodegradable in our experimental conditions
Executive summary:

In compliance with:

EEC directive 92/69 - method C4 and OECD guideline 301B


-carbon dioxide evolution test (modified sturm test) was performed for the evaluation of "ready" biodegradability of a test substance in an aerobic agueous medium

-the test was performed using the concentration of 10 mg of TOC (total organic carbon) per litre of test medium (2 test flasks)

-the released carbon dioxide (CO2), trapped in barium hydroxyde (Ba(OH)2) and incorporated as barium carbonate (BaCO3), was measured every 1 to 3 days over a 10 -day period and then every 2 to 5 days until the end of the tes (28 days)

-a control assay, without test substance, was performed in the same conditions (2 control flasks)

-an assay using reference substance (sodium acetate) was also performed in the same conditions (2 reference flasks)

-an assay to check the toxicity of the test substance, using a reference substance (sodium acetate) and test substance, was performed during a 28 -day period.

-the degradation of the test substance was evaluated comparing the total mean amount of CO2 produced by the test media (corrected for that derived from the control media), with the total amount of CO2; that the test substance could have theoretically produced (based on the carbon content of the test substance: 0.6450 mg TOC per mg test substance)

-the quality criteria for the test, notified in the protocols and relative to the biodegradation of the reference substance, the difference of CO2 production from the two test replicates, and to the total production of CO2 in the control assay, were respected.


Biodegradation of the test substance after the 10 -day window (graphically): 38%

biodegradability of the test substance after 28 days: 62%


The test substance is not toxic for the inoculum.

The test substance is not "readily" biodegradable in the experimental conditions