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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Sludge collected from sewage treatment works, which treats predominantly domestic waste, was aerated and activated for 8 days in the laboratory. Then aliquots (50 mL) of homogenized sludge were filtered through a dried (approximately 100°C) and pre-weighed sintered crucible. The filters were dried for at least one hour (approximately 100°C), allowed to cool, and re-weighed. The solids level in the sludge was determined and then an appropriate volume was used to inoculate control and test vessels to give final suspended solid’s concentrations of 30 mg/L.
Duration of test (contact time):
29 d
Initial conc.:
10 other: mg C/L
Based on:
IC (inorganic carbon)
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
PREPARATION OF TEST MEDIUM:
Solution A: was prepared by dissolving about 8.50 g of KH2PO4, 21.75 g of K2HPO4, 33.40 g of Na2HPO4.2H2O and 0.50 g of NH4Cl in 1 litre water (pH of this solution was 7.43).
Solution B: was prepared by dissolving about 36.40g of CaCl2.2H2O in 1 litre water.
Solution C: was prepared by dissolving about 22.50 g of MgSO4.7H2O in 1 litre water.
Solution D: was prepared by dissolving about 0.25g of FeCl3.6H2O in 1 litre water.
The medium was prepared by mixing 10 mL of solution A with 800 mL of water. Then 1 mL of solutions B, C, D were added and the solution was made up to 1 litre with water.

TEST CONDITIONS:
The pH measured for each test container at the beginning of the test ranged between 7.11-7.30 at the end of the test was 7.14-7.49. The rate of air-flow during the test ranged from 31-93 mL/minute.

TEST SYSTEM
- Culturing apparatus: Five-litre amber-glass culture bottles
- Number of culture flasks/concentration: Six. 30 mg solids/L
- Method used to create aerobic conditions: The mixtures were aerated overnight with a supply of compressed air at 31 - 93 mL/min which was filtered through a carbon trap to remove carbon dioxide.
- Test performed in open system: Each vessel was fitted with a stopper, holding an air inlet tube reaching approximately 10 cm below the liquid surface and an air outlet just below the stopper.

CONTROL AND BLANK SYSTEM
- Test item (total volume 3000 mL) – 51.42 mg of test item (10 mg C/L) plus inoculated mineral salts medium
- Controls (total volume 3000 mL) - mineral salts medium plus inoculum (30 mg solids/L)
- Reference (total volume 3000 mL) - inoculated mineral salts medium plus 51.48 mg of sodium benzoate (10 mg C/L)
- Inhibition mixture (total volume 3000 mL) -Sodium benzoate (10 mg C/L) plus test item (10 mg C/L) plus inoculated mineral salts medium
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
ThCO2
Value:
5.4
Sampling time:
29 d
Details on results:
The cumulative level of CO2 production in sample controls after 29 days was 98 mg of CO2, and was within the acceptable range (recommended maximum = 120 mg CO2 for a three-litre culture). These results confirm that the inoculum was viable and that the test was valid.
The degradation observed for sodium benzoate in the presence of the test substance after 14 days was 33.9%, which was above the pass level of >25% requirement within 14 days. Thus, the test substance is not considered inhibitory to the activity of the microbial inoculum.
Results with reference substance:
Sodium benzoate, biodegraded to 63% and 94% of its theoretical CO2 after 14 days and 29 days respectively
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
29 day ThCO2: 5.4%
Executive summary:

The present study was conducted to determine the ready biodegradability of the test substance using the CO2 evolution test (Modified Sturm test, Procedure EEC C.4-C, OECD Procedure 301B).

The biodegradability of test substance was studied by dissolving the test item in inoculated mineral salt medium at 10 mg carbon (C)/L. A control sample containing the inoculated mineral medium was studied in parallel to check the viability of the inoculum. The validity of the test was checked by monitoring the evolution of CO2 from the sodium benzoate reference item as a positive control at 10 mg C/L level. The potential inhibitory effect of the test item on the microbial activity of the inoculums was studied using a mixture containing the reference item at 10 mg C/L and the test item at 10 mg C/L.

Inoculums containing the reference item, inhibition mixture and the test item were aerated for 29 days with air that had been treated to remove carbon dioxide (CO2). The CO2 produced by each culture was trapped in a series of dreschel bottles containing barium hydroxide, which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration. The pH of control, reference, test mixtures and test plus reference mixture were measured at the start of the test and after 28 days.

The reference item sodium benzoate, biodegraded to 63% and 94% of its theoretical CO2 after 14 days and 29 days respectively, thereby confirming the validity of the test. The cumulative level of CO2 production in sample controls after 29 days was 98 mg of CO2, and was within the acceptable range (recommended maximum = 120 mg CO2 for a three-litre culture). These results confirm that the inoculum was viable and that the test was valid.

The degradation observed for sodium benzoate in the presence of the test substance (Inhibition) after 14 days was 33.9%, which was above the pass level of >25% requirement within 14 days as per the test guidelines. Thus, the test substance is not considered inhibitory to the activity of the microbial inoculum.

The percentage degradation by samples containing only test substance had not achieved its theoretical value (ThCO2, 110.1 mg CO2) at the end of test (Day 29). A test item is considered to be readily biodegradable if CO2 production is equal to or greater than 60% of the theoretical value (ThCO2) within 10 days of achieving the 10% level. The test substance reached a maximum mean of 5.4% ThCO2 by day 29. When the results of this study are considered in the context of the guideline criteria, the test substance is not readily biodegradable.

Description of key information

The test substance reached a maximum mean of 5.4% ThCO2 by day 29.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information