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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:
From 14 March 2012 to 12 April 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
according to
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Version / remarks:
according to
other: ISO Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995)
GLP compliance:

Test material

Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Off-white powder with lumps
- 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:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 5.0 g/L in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (34 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
12 mg/L
Based on:
Initial conc.:
18.5 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 (tap-water purified by reverse osmosis)
Stock solutions of mineral components:
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.
- Milli-Q water: Milli-RO water passed over activated carbon and ion-exchange cartridges (Millipore Corp., USA)
- Test temperature: between 21.6 and 22.5°C.
- pH at t=0 d: 7.5
- pH adjusted: no
- pH at t=28 d (test suspensions): 7.4
- pH at t=28 d (blank controls): 7.4
- pH at t=28 d (positive control): 7.7
- pH at t=28 d (toxicity control): 7.7
- Aeration of dilution water: Overnight prior to the start of the test. During the test the medium was aerated and stirred continously.
- 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: Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%), with CO2 < 1 ppm) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Details of trap for CO2: CO2 was trapped in barium hydroxide solution. 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. 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 blanks and test suspensions. Titrations for the positive and toxicity control were made over a period of 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, Merck) was used as pH-indicator. On day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) 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.

Since the substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 37.05 mg; test substance bottle B: 37.11 mg and toxicity control bottle: 37.02 mg). To this end, approx. 10 mL of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

- Sampling frequency: Titrations were made on day: 2, 5, 7, 9, 14, 19, 23, 27 and 29
- Sampling method: Titration of the whole volume of CO2-absorber

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

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
29 d
Remarks on result:
other: HCl added on the 28th day (last CO2-measurement on the 29th day).
Details on results:
- Theoretical CO2 production:
The ThCO2 of the substance was calculated to be 2.38 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

- Biodegradation:
The relative biodegradation values calculated from the measurements performed during the test period revealed 25% and 14% biodegradation of the test substance, for A and B, respectively (based on ThCO2). Thus, the criterion for ready biodegradability was not met.

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.

Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

BOD5 / COD results

Results with reference substance:
The positive control substance was biodegraded by at least 60% (73%) within 14 days.

Any other information on results incl. tables

Acceptability of the test:

- The positive control substance was biodegraded by at least 60% (73%) within 14 days.

- The difference of duplicate values for %-degradation of the test substance was always less than 20 (≤ 14%).

- The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (46 mg CO2 per 2 litres of medium, corresponding to 23 mg CO2/L).

- The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test substance, 12 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Under the conditions of the modified Sturm test the substance was determined to be not readily biodegradable.
Executive summary:

The ready biodegradation of the substance under the conditions of the carbon dioxide (CO2) evolution test (modified Sturm test) was investigated according to OECD guideline 301 B and GLP principles. A test concentration of 12 mg/L (as TOC) was tested in duplicate during 28 days. The relative biodegradation values calculated from the measurements performed during the test period revealed 25% and 14% biodegradation of the substance, for replicate A and B, respectively (based on ThCO2). In the toxicity control, the substance was found not to inhibit microbial activity. The study is considered to be reliable without restrictions. In conclusion, the substance was determined to be not readily biodegradable under the conditions of the modified Sturm test.