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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March 2016 - 28 April 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 guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
equivalent or similar to guideline
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 guideline
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

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
Appearance: off-white powder
Test item storage: at room temperature
Specific details on test material used for the study:
Solubility in water: not available
Stability in water: stable

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', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Storage length: not indicated
- Preparation of inoculum for exposure: before use, the sludge was allowed to settle (40 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Pretreatment: 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: 3.7 g suspended solids per litre in the concentrated sludge.
- Water used: tap-water 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: 21.9 - 22.7°C.
- pH: start: 7.5 - 7.6; end: 7.4 - 7.7
- pH adjusted: no
- Aeration of dilution water: continuously during the test period
- Suspended solids concentration: 10 mL of supernatant liquid per litre (see 'Details on inoculum' section for details)
- Continuous darkness: yes

- Culturing vessels: 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; at a rate of approx. 30-100 mL/min)
- Measuring method: 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.
- Details of trap for CO2: 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 CO2 produced in each bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. 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.

- 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.
- 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 99.6%

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The ThCO2 of the test item was calculated to be 2.99 mg CO2/mg; the ThCO2 of the reference item (sodium acetate) was calculated to be 1.07 mg CO2/mg.
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of test substance (0% in both bottles tested).
In the toxicity control, more than 25% biodegradation occurred within 14 days (28%, 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).
For details containing full results, see attached background material (Tables.jpg).

BOD5 / COD results

Results with reference substance:
70% within 14 days

Any other information on results incl. tables

Acceptability of the test:

1. The positive control item was biodegraded by at least 60% within 14 days (i.e. 70%).

2. The difference of duplicate values for %-degradation of the test item was always less than 20% (i.e. ≤0%).

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (e.g. 60.9 mg CO2 per 2 litres of medium, corresponding to 30.45 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 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 0% (in the duplicate bottles tested) after 28 days of incubation. Therefore, the test substance is not considered to be biodegradable.
Executive summary:

In a 28 day during test performed according to OECD guideline 301B (modified Sturm test) and GLP principles, 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. The test item was tested in duplicate and showed 0% biodegradation in both bottles tested. Therefore, it is designated as not readliy biodegradable. All the criteria for acceptability of the test were met and the study is considered reliable without restrictions.