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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:
7th March 2006 - 3rd May 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
29 Dec 1992
GLP compliance:

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Light pink tinted powder
Specific details on test material used for the study:
- Source and lot/batch No.of test material: supplied by Syngenta, CTL, Alderley Park, Macclesfield, Cheshire, SK10 2TJ, UK on behalf of AstraZeneca
- Expiration date of the lot/batch: not specified
- Purity test date: 8th May 2006

- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

- Treatment of test material prior to testing: stored at ambeint temperature in container it was recieved in
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Buckland Sweage Treatment Works, Newton Abbot, Devon, UK. This works treats Sewage of predominantly domestic origin.
- Laboratory culture: not specified
- Method of cultivation: not specified
- Storage conditions: At the laboratory the activated sludge was kept aerated at room tempareture and the pH mainted at 7.0 +/- 1.0
- Storage length: not specified
- Preparation of inoculum for exposure: not specified
- Pretreatment: 5 days prior to test start, activated sludge was centrifuged, washed and resuspended in the test medium and the solids concentration determined. This sludge was then diluted in medium, added to test bottle and stirred until required for use.
- Concentration of sludge: 30 mg/l
- Initial cell/biomass concentration: not specified
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The experimental design included inoculum blanks and two types of control, toxicity controls and positive controls. The inoculum blanks contained organisms with no UL125 or reference substance (sodium benzoate), the purpose of which was to demonstrate that there was no other carbon source in the test mediaum. The toxicity controls comprised a mixture of UL125 and sodium benzoate, both at nominal 100 mg/l. In the event that the test substance failed to degrade during the 28 day test period, the purpose of the toxicity control was to indicate wether the failure was caused by the test substance inhibiting the inoculum. The positive controls contanied inoculum and sodium benzoate to demonstate the viability of the inoculum.

The test was conducted using activated sludge as the inoculum. Following pre-conditioning, the test bottles were set up for the test. Each set of bottles was prepared in triplicate (Inoculum blank, 100mg/l sodium benzoate positive control, 100mg/l UL125, 100mg/l UL125 + 100 mg/l sodium benzoate toxicity control). In addition, an extra UL125 bottle and toxicity control bottle were also prepared for the determination of pH at day 0.

The units containing Ul125 were prepared by directly weighing the required quantity of UL125 into the relevant test bottles. sodium Benzoate was dosed as a 1000 mg/l stock solution, prepared by dissolving sodium benzoate in deionised water to give a clear and colourless solution.

Oxygen update was recoreded at 112 minute intervals during the 28 day experimental period. Oxygen uptake values were corrected for the inoculum blank. the biodegredation was calculated as a percentrage of the theoretic oxygen demand for the substance under test and as a percentage of the theoretical oxgen demand for the reference substance.

- Composition of medium: The test medium was made up according to OECD and EU guidelines and contained the following nutrients per litre of deinonised water:
85mg of KH2PO4
217.5 mg of K2HPO4
334mg of Na2HPO4.2H2O
5mg of NH3CL
22.5mg of MgSO4.7H2O
36.4 mg of CaCl2.2H2O
0.25mg of FeCL3.6H20
- Additional substrate: UL125 and Sodium Benzoate
- Solubilising agent (type and concentration if used): not specified
- Test temperature: 22+/- 2 degrees centigrade
- pH: monitored, see results
- pH adjusted: The pH of the test medium was measured prior to use and adjusted as necessary to 7.4 +/- 0.2
- CEC (meq/100 g):
- Aeration of dilution water: n/a
- Suspended solids concentration: 30 mg/l
- Continuous darkness: yes - dark glass bottle
- Other:

- Culturing apparatus:
- Number of culture flasks/concentration: 4 - Innoculum blank, 100mg/l of sodum benzoate (positive control), 100mg/l UL125, 100mg/l UL125 +100mg/l sodium benzoate (toxicity control)
- Method used to create aerobic conditions: use of Oxitop respirometer
- Method used to create anaerobic conditions: n/a
- Measuring equipment: the measurement of oxygen uptake was conducted in the oxitop respirometers (Wissenschaftlich-Technische wekstatten, GmbH, Germany). Oxygen uptake was measured as a decrease in pressure. the oxitop controller collected the pressure values from the measuring tops and calculated the BOD (mg O2 per litre)
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: CO2 produced by microbila respiration was absorbed by KOH solution placed in a sealed cup in the neck of each bottle.
- Other:

- Sampling frequency: every 7 days
- Sampling method: pressure value
- Sterility check if applicable: n/a
- Sample storage before analysis:
- Other:

- Inoculum blank: yes
- Abiotic sterile control: containing 100mg/l of sodium benzoate
- Toxicity control: 100mg/l sodium benzoate + 100mg/l UL125
Reference substance
Reference substance:
other: Sodium benzoate

Results and discussion

Test performance:
the measured COD value of sodium benzoate was 1.74 O2 g-1 of substance. The COD obtained for sodium benzoate compares favourable with its theoretical oxygen demand of 1.66g O2 g-1 thus demonstrating the validity of the analytic technique.
% Degradation
Key result
% degradation (O2 consumption)
< 5
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
% degradation after 7 d
% degradation after 14 d
% degradation after 21 d
% degradation after 28 d

BOD5 / COD results

BOD5 / CODopen allclose all
1.53 g O2/g test mat.
Remarks on result:
other: UL125
1.41 g O2/g test mat.
Remarks on result:
other: UL125
Results with reference substance:
Points of degradation plot (reference substance):
57 % degradation after 7 d
62 % degradation after 14 d
66 % degradation after 21 d
67 % degradation after 28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Experimental values, test substance day 7-28: <5% biodegradation