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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.5 (Degradation: Biochemical Oxygen Demand)
GLP compliance:
Specific details on test material used for the study:
- Substance name: Methomyl
- Substance ID: DPX-X1179
- Lot#: 357
- Purity: 99.77%
Oxygen conditions:
Inoculum or test system:
other: activated sludge, predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: Pineham water treatment works, Milton Keynes, Buckinghamshire, England
- Storage conditions: Used immediately without storage
- Preparation of inoculum for exposure: On the day the test was set up, 1000 mL activated sludge was collected and aerated for 4 hours in the laboratory at ambient temperature. 500 mL was taken and homogenised for 2 minutes at medium speed in a blender. It was allowed to settle for up to 60 minutes until the solids have settled. Supernantant was decanted to obtain 200 mL for subsequent inoculation.
- Initial cell/biomass concentration: 10 ± 9E6 cells per mL
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
- Test temperature: 22 ± 1°C
- pH adjusted: No
- Continuous darkness: No

- Culturing apparatus: Bioreactor vessels
- Number of culture flasks/concentration: 4
- Method used to create aerobic conditions: CO2 scrubber connected to a pump to produce CO2 free air and linked to the stirred bioreactors containing the test substance/inoculum system
- Measuring equipment: Dohrmann DC90 carbon analyser
- CO2 absorbant: 0.0125 M of Ba(OH)2 solution was prepared by dissolving 4.0 g Ba(OH)2.8H2O per litre high purity water. 0.05 M NaOH was utilized if the analysis was made by DIC determination.
- CO2 trap: The bioreactors were linked to two CO2 absortion bottles (dreschels) each containing 300 mL of absorbant and arranged in series.

- Sampling frequency: Samples were taken at Days 3, 7, 12, 15, 21 from dreschel bottle. Samples were collected from the bioreactors at Day 0 and 27 for dissolved organic carbon analysis.
- Sample storage before analysis: Stored frozen (nominally -20°C)

- Negative control contained only the test medium and inoculum
Reference substance:
acetic acid, sodium salt
Test performance:
Mean DOC levels for negative control were 1.5 and 1.9 mg/L at Day 0 and 27, respectively
Key result
% degradation (DOC removal)
Sampling time:
28 d
Remarks on result:
other: 10 mg/L test substance
Key result
% degradation (DOC removal)
Sampling time:
28 d
Remarks on result:
other: 20 mg/L test substance
Details on results:
Negative biodegradation values were observed throughout the study at both the test substance concentrations. These results suggest an inhibitory effect towards the microbial inoculum, whereby the test treatment results in lower CO2 evolution with respect to the negative (background) controls. There was no significant loss of test substance (measured as DOC) in the bioreactors confirming the lack of total biodegradation.

Mean DOC levels for 10 mg/L test substance were 3.7 and 4.9 mg/L at Day 0 and 27, respectively
Mean DOC levels for 20 mg/L test substance were 8.9 at both Day 0 and 27
Theoretical carbon content at 10 mg/L was 3.7 mg/L and at 20 mg/L was 7.4 mg/L

The DOC levels from the test substance at the 10 mg/L level were lower than the theoretical value at the beginning of the study. Since biodegradation levels were calculated on the basis of measured initial DOC values, the apparent high negative values obtained for the degradation of the test substance at 10 mg/L may have resulted from an under estimation of the level of DOC in the bioreactor. The possibility is borne out since there is an apparent increase in DOC in the 10 mg/L test substance bioreactor at Day 27 compared to Day 0.
Results with reference substance:
28 day pecent biodegradation level for sodium acetate at 20 mg/L: 98.3%
Mean DOC levels were 4.1 and 1.3 at Day 0 and 27, respectively
Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The test substance was not readily biodegradable
Executive summary:

The ready biodegradability of the test substance was tested at concentrations of 10 and 20 mg/L according to the guidelines of OECD 301B (Modified Sturm Test). The objective of the study was to determine whether the test substance was readily biodegradable under the criteria of the guideline.

The test substance was exposed to activated sludge micro-organisms in a stirred aqueous minimal medium system. Carbon dioxide released from the test substance was assayed using a carbon analyser and the amount released was used to calculate the percent biodegradation.

At Day 28, negative levels of biodegradation were recorded for both test substance concentrations. The practical implications of this is that the test substance was somewhat inhibitory to the bacterial inoculum.

The test substance cannot therefore be termed readily biodegradable within the terms of OECD 301B.

Description of key information

Study Type

 Study Details Value  Guideline Reliability 
Ready Biodegradation Study

activated sludge micro-organisms in a stirred aqueous minimal medium system

Not Readily Biodegradable


EU Method C.5

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
other: minimal medium system

Additional information