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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:
4 April 2016 - 4 August 2016
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
other: ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test.
Version / remarks:
according to
other: ISO International 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".
Version / remarks:
GLP compliance:

Test material

Test material form:
solid: particulate/powder
Details on test material:
- Appearance: off-white powder
- Storage condition of test material: At room temperature
Specific details on test material used for the study:
Solubility in water: 5.4 mg/L (20°C)

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, 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 Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: The freshly obtained sludge was preconditioned to experimental conditions by continuous aeration until further treatment.
- Storage length: not indicated
- Pretreatment: Before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium. The day before the start of the test (day -1) mineral
components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Concentration of sludge: 3.4 g suspended solids/L of concentrated sludge
- Water filtered: Tap-water was purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
12 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: mineral medium according to OECD 301
- Test temperature: 21.4 - 22.9 °C
- pH: start: 7.6; end: 7.5 - 7.8
- pH adjusted: yes, using 1 M HCl (Merck, Darmstadt, Germany)
- Aeration of dilution water: continuously
- Continuous darkness: yes

- Culturing apparatus: 2 litre glass brown coloured bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with synthetic air (CO2 < 1 ppm)
- 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. 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.
- Measuring equipment: 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). Phenolphthalein (1% solution in ethanol) was used as pH-indicator.

- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made
over a period of at least 14 days.
- Sampling method: removing the CO2-absorber (nearest to the test bottle) for titration
- Sample storage before analysis: not applicable

- Inoculum blank: yes, 2 replicates
- Positive control: yes, 1 replicate
- Toxicity control: yes, 1 replicate

Reference substance
Reference substance:
acetic acid, sodium salt
Purity: 99.1%; 12 mg/L TOC

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: Result is based on the mean value of duplicate bottles tested (5% and 11%).
Details on results:
The relative biodegradation values calculated from the measurements performed during the test period revealed 5% and 11% biodegradation of test item (based on ThCO2), for the duplicate bottles tested.

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

Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve (see attached illustration).

BOD5 / COD results

Results with reference substance:
61% biodegradation within 14 days.

Any other information on results incl. tables

Validity criteria:

1. The positive control item was biodegraded by at least 60% (61%) within 14 days.

2. The difference of duplicate values for %-degradation of the test item was always less than 20% (≤6%).

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (39.3 mg CO2 per 2 litres of medium, corresponding to 19.7 mg CO2/L).

4. The Inorganic Carbon content (IC) of the test item (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, IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L)

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

Applicant's summary and conclusion

Validity criteria fulfilled:
For details on validity criteria see 'Any other information on results' section.
Interpretation of results:
under test conditions no biodegradation observed
Under the conditions of the modified Sturm test, according to OECD 301B, the test item was found to biodegrade for 5% and 11% (for the duplicate bottles tested) after 28 days of incubation. Therefore, it is not considered to be readily biodegradable.
Executive summary:

In a test performed according to OECD 301B (modified Sturm test) and GLP, the test item did not reach the pass level of 60% for ready biodegradability, neither within the 10 -d window nor after 28 days of incubation. Therefore, it is designated as not readily biodegradable. All the criteria for acceptability of the test were met, so the study is considered valid without restrictions.