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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:
20 February - 20 March 2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and 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)
according 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): Aquapel® 203
- Substance type: yellow fatty liquid
- Physical state: liquid
- Analytical purity: 89.9%
- Lot/batch No.: G12FY067
- Expiration date of the lot/batch: 12 June 2008
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): 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.
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Storage length:
- Preparation of inoculum for exposure:
- Pretreatment: Before use, the sludge was allowed to settle (83 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium
- Initial cell/biomass concentration: The concentration of suspended solids was 4.4 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
12 mg/L
Based on:
other: TOC
Initial conc.:
31 other: mg/2L
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: 21.3 - 22.9°C.
- pH: 7.5 - 7.8
- pH adjusted: yes
- Suspended solids concentration: 4.4 g/l in the concentrated sludge
- Continuous darkness: yes
- Other:

- 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).
The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume). The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
- Method used to create aerobic conditions: During the test period the test media were aerated and stirred continuously.
- Measuring equipment: 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), Merck, Darmstadt, Germany).
- Details of trap for CO2 and volatile organics if used:
Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2 were connected in series to the exit air line of each test bottle. Synthetic air (CO2 < 1 ppm): A mixture of oxygen (21%) and nitrogen (79%) 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).
The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate.

- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. 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, Merck, Darmstadt, Germany) was used as pH-indicator.
On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCl (37%, Merck, Darmstadt, Germany) was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: reference substance was Sodium acetate

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
Because the theoretical calculation of the CO2 production was not possible a sample of Aquapel® 203 was taken for TOC analysis.
The TOC content of Aquapel® 203 was determined to be 78%. Based on the TOC content the ThCO2 of Aquapel® 203 was calculated to be 2.87 mg CO2/mg.
The ThCO2 of Sodium Acetate was calculated to be 1.07 mg CO2/mg.

% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The relative degradation values calculated from the measurements performed during the test period revealed 54 and 52% degradation of Aquapel® 203, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

BOD5 / COD results

Results with reference substance:
In the toxicity control more than 25% degradation occurred within 14 days (48%, 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:
other: not readily biodegradable
Aquapel® 203 was not readily biodegradable under the conditions of the modified Sturm test presently performed.