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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:
2006-06-05 to 2006-07-07
1 (reliable without restriction)
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted 1992-07-17
GLP compliance:
yes (incl. QA statement)
signed 2005-06-01
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: A sample of activated sludge was collected from Worlingworth sewage treatment works, which treats predominantly domestic waste.
Aliquots (25 ml) of a homogenised sample were filtered through dried (approximately 105°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.
Duration of test (contact time):
29 d
Initial conc.:
2.416 g/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Air-saturated ultrapure water was added to each of six, five-litre amber glass culture bottles followed by the volumes of each of the stock solutions to prepare three litres of mineral salts medium (MSM, see further below). Each culture bottle was then inoculated with activated sludge (30 mg solids/l) and a magnetic stirring bar was added. The bottles were stoppered then each was placed on an electrically-operated magnetic stirrer. The mixtures were aerated for three days with a supply of air that had been treated to remove carbon dioxide by passing it through cyclinders containing fused calcium chloride and Carbosorb AS.
On day 0 of the test, aqueous stock solutions of the test and reference substances were prepared (2.4156 and 1.720 g/l, respectively) and appropriate volumes (30 ml) were added to culture vessels to give a final nominal concentrations of 10 mgC/l. Each bottle was stoppered and the contents were mixed by swirling.
Ultrapure water (30 ml) was also added to the control cultures on Day 0 of the test in order to ensure similarity in volume additions to those containing the test substance.
The mixtures prepared for the test are as follows:
Bottle Nos. 1 & 2: Controls - mineral salts medium plus inoculum (30 mg solids/l)
Bottle No. 3: Reference- inoulated mineral salts medium plus sodium benzonate (10 mg C/l)
Bottle Nos. 4 & 5: Test substance (10 mg C/l) plus inoculated mineral salts medium
Bottle No. 6: Sodium benzonate (10 mgC/l) plus test substance (10 mg C/l) plus inoculated mineral salts medium
The PH of each culture was determined in situ and no adjustment was necessary.
The vessels contents were continuously flushed for 29 days with treated air. The air outlet from each vessel was connected to three Dreschel bottles in series, each containing 0.025 N nominal barium hydroxide (100 ml).
The residual concentrations of barium hydroxide in the bottles nearest to the test vessels were determined at intervals by duplicate titration of 20 ml samples with hydrochloric acid (0.05 N), using phenolphthalein indicator.

Following the removal of the first Dreschel bottle in a series, the second was connected to the vessel, and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.
The biogradation 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 biogradation of sodium benzoate achieved the pass level for ready biogradability (≥ 60%), the pH was measured and the treatment was terminated.
On Day 28 of the test, titrations were undertaken and the pH of each culture determined. Concentrated hydrochloric acid (1 ml) was then added to each vessel to drive off dissolved inorganic carbon.
The contents of the vessels were aerated overnight and the final titrations carried out on Day 29.
The rate of air flow through the apparatus was determined at intervals during the test.
The minimum and maximum temperature of the tet area and of a three-litre volume of water held in a culture bottle under the test conditions was recorded at intervals using a thermometer. The temperature of the three-litre volume of water is considered representative of temperature control for this system.

Mineral salts medium:
Stock solution 1:
Potassium dihydrogen phosphate: 8.50 g/l
di-Potassium hydrogen phosphate: 21.75 g/l
di-Sodium monohydrogen phosphate dihydrate: 33.40 g/l
Ammonium chloride: 0.50
Stock solution 2:
Magnesium sulphate heptahydrate: 22.50 g/l
Stock solution 3:
Calcium chloride dihydrate: 36.40 g/l
Stock solution 4:
Iron (III) chloride hexahydrate: 0.25 g/l
Reference substance:
benzoic acid, sodium salt
(AR grade) (Aqueous stock solution was prepared (1.720 g/l)
Preliminary study:
No data
Test performance:
Not applicable
% degradation (CO2 evolution)
Sampling time:
29 d
Details on results:
Cumulative CO2 production in the controls (62.2 and 62.7 mg CO2) was within the acceptable range for this assay system (recommended maximum for a three litre culture = 120 mgCO2).
Mean cumulative CO2 production by mixtures containing triethylmethylammonium tetrafluoroborate was negligible and equivalent to 2 % of the theoretical value (TCO2, 110.1 mgCO2) by the end of the test on Day 29.
The pH of each test and control mixture was 7.5 at the start of the test and ranged from 7.5 to 7.6 at the end.
Results with reference substance:
The degradation of sodium benzoate was rapid and had achieved 63% of its TCO2 after 7 days and 83% after 29 days.
The degradation of sodium benzoate was also rapid in the presence of triethylmethylammonium tetrafluoroborate and had achieved 60% of its TCO2 after 7 days. This test vessel was terminated on Day 7 of the test; the final pH of the culture was 7.6.

The rate of air flow during the test ranged from 30 to 75 ml/minute.

The temperature of a three-litre volume of water held under the test conditions ranged from 20.1 °C to 24°C during the test period.

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
The effect of test item triethylmethylammonium tetrafluoroborate on the activity of domestic activated sludge was investigated in an aerobic system according to OECD 301 B test (i.e. CO2 evolution test) on ready biodegradability. The mean cumulative CO2 production of the test item was negligible and equivalent to 2 % of the theoretical value of 110.1 mg CO2 by the end of the test on Day 29. The results obtained for the degradation of sodium benzoate (63% of its TCO2 after 7 days and 83% after 29 days) and for cumulative CO2 production by the control mixtures (62.2 and 62.7 mgCO2) fulfil the validity criteria for this test. Hence, the test item triethylmethylammonium tetrafluoroborate is not ready biodegradable.

Description of key information

Based on a reliable GLP- and guideline-conform key study on ready biodegradability (OECD 301 BCO2 evolution test) of triethylmethylammonium tetrafluoroborate, the mean cumulative CO2 production was negligible and equivalent to 2 % of the theoretical value at test end on day 29. Thus, triethylmethylammonium tetrafluoroborate is not considered to be readily biodegradable (Dickinson, 2006).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information