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
Study period:
05-nov-2008 to 04-dec-2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
equivalent or similar to
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Name of test material (as cited in study report): ASA404 C7
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 99.9%
- Lot/batch No.: 072301
- Expiration date of the lot/batch: 18 March 2009
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure:
Before use, the sludge was allowed to settle (31 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium. - Concentration of sludge: 10 ml supernatant/l of mineral medium
- Initial cell/biomass concentration: not determined
- Water filtered: no
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
35.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium:
1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.
- Test temperature: 21.4 to 22.8 °C
- pH: 7.6 to 7.8
- pH adjusted: no
- Aeration of dilution water: not before the test, the test is aerated continously
- Suspended solids concentration: The concentration of suspended solids was 3.8 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
- Continuous darkness: yes

- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions:
A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol,) was used as pH-indicator.On the 28th day, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

- Sampling frequency: Titrations were made on day: 2,5,7,9,14,19,23,27 and 29
- Sampling method: Titration of whole volume of C)2-absorber

- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
% degradation (CO2 evolution)
Sampling time:
29 d

BOD5 / COD results

Results with reference substance:
In the toxicity control more than 25% biodegradation occurred within 14 days (36%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
ASA404 C7 was not readily biodegradable under the conditions of the modified Sturm test presently performed