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:
2006-10-16 to 2007-05-25
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) and EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test) without deviations.

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)
The testing facility indicated that the protocol was followed without deviation.
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
The testing facility indicated that the protocol was followed without deviation.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
: Certificate issued by the United Kingdom GLP monitoring authorities.

Test material

Details on test material:
- Name of test material (as cited in study report): VRT-753136
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: white powder
- Analytical purity: 99.4% w/w by High Performance Liquid Chromatography (HPLC)
- Impurities (identity and concentrations): individual unknown components were detected at 0.03 area%; isopropanol (residual solvent) at 300 ppm
- Composition of test material, percentage of components: not applicable
- Isomers composition: not applicable
- Purity test date: 2006-05-25
- Lot/batch No.: 25515
- Expiration date of the lot/batch: May 2008
- Stability under test conditions: no data
- Storage condition of test material: at room temperature
- Other: no data
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Study design

Oxygen conditions:
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: A sample of activated sludge was collected from the aeration tank of Worlingworth (UK) sewage treatment works, which treats predominantly domestic waste.
- Method of cultivation: no data
- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure: Aliquots (25 mL) of a homogenized sample were filtered through dried (approximately 105 deg C) and pre-weighed Whatman GF/C filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/L.
- Laboratory culture: not applicable
- Pretreatment: no
- Concentration of sludge: 30 mg solids/L
- Initial cell/biomass concentration: no data
- Water filtered: yes
- Type and size of filter used, if any: Whatman GF/C filter papers (pore size not indicated)
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
10 other: mgC/L
Based on:
other: chemical structure of the test substance
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: The mineral salts medium (MSM) was prepared according to the guidelines. The dilution water used to prepare stock and test solutions of mineral salts and aqueous solutions of the test and reference substances was tap water that had been softened and treated by reverse osmosis and then purified (nominal resistivity, >= 18
- Additional substrate: none
- Solubilising agent: none
- Test temperature: 21-23 deg
- pH: 7.5-7.6
- pH adjusted: yes, with 5N HCl
- CEC (meq/100 g): no data
- Aeration of dilution water: the dilution water was air-saturated
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no data; test performed in amber glass bottles
- Other: no data

- Culturing apparatus: 5 L amber glass culture bottles
- Number of culture flasks/concentration:
- bottles 1 and 2: controls - mineral salts medium plus inoculum;
- bottle 3: reference substance - inoculated MSM plus sodium benzoate (10 mgC/L);
- bottles 4 and 5: test substance (10 mgC/L) plus inoculated MSM; and
- bottle 6: sodium benzoate (10 mgC/L) plus test substance (10 mgC/L) plus inoculated MSM
- Method used to create aerobic conditions: The vessels contents were continuously flushed for 29 days with treated air at 35-90 mL/min
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: The amount of produced CO2 was measured by duplicate titration with hydrochloric acid (0.05 N) using phenolphthalein indicator.
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The air outlet from each vessel was connected to three Dreschel bottles in series, each containing 0.025N, nominal barium hydroxide (100 mL). The residual concentrations of barium hydroxide in the bottles nearest to the test vessels were determined at intervals by titration. Following the removal of the first Dreschel bottle in a series, the second was connected to the test vessel, and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.
- Other: no data

- Sampling frequency: 10 samples over the test period
- Sampling method: duplicate titration of 20 mL samples
- Sterility check if applicable: not applicable
- Sample storage before analysis: no data
- Other: no data

- Inoculum blank: yes (bottles 1 and 2)
- Abiotic sterile control: none
- Toxicity control: The biodegradation of the reference substance in the mixture containing the test and reference substance was calculated to confirm that the test substance was not inhibitory to the activity of the microbial inoculum. When the level of biodegradation of sodium benzoate achieved the pass level for ready biodegradability (>= 60%), the pH was measured and the treatment was terminated.
- Other: The reference substance was tested in the presence of the test substance and the inoculum to examine any inhibitory effects on the microbial inoculum.

- No statistical analyses were performed.
Reference substance
Reference substance:
other: sodium benzoate

Results and discussion

Preliminary study:
- The result of a preliminary solubility trial showed that the test substance was sufficiently soluble to allow the preparation of an aqueous stock solution.
Test performance:
- The pH of test and control mixtures ranged from 7.5 to 7.6 at the start and the end of the test.
- The rate of air flow during the test ranged from 35 to 90 mL/minute.
- The temperature of a 3 L volume of water held under the test conditions ranged from 21.3 deg C to 22.6 deg C during the test period.
% Degradation
other: CO2 production
<= 3
Sampling time:
29 d
Details on results:
- Cumulative CO2 production in the controls (73.7 and 72.1 mgCO2) was within the acceptable range for this assay system (recommended maximum for a 3 L culture = 120 mgCO2).
- The degradation of sodium benzoate was rapid and had achieved 63% of its TCO2 after 6 days and 85% after 29 days.
- The degradation of sodium benzoate was also rapid in the presence of the test substance and had achieved 62% of its TCO2 after 6 days. This test vessel was terminated on day 6 of the test; the final pH of the culture was 7.5.
- Mean cumulative CO2 production by the mixtures containing the test substance was negligible and had achieved, at most, 3% of the TCO2 by the end of the test on day 29.

BOD5 / COD results

Results with reference substance:
Not applicable

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

The mean cumulative CO2 production by mixtures containing the test substance at 10 mgC/L was negligible having achieved, at most, 3% of the TCO2 by the end of the test on Day 29. Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. Therefore, the test substance cannot be considered to be readily degradable.
Executive summary:

Not applicable