Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

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 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Fresh activated sludge was used as the inoculum. The activated sludge was obtained from the Somerset-Raritan Valley Sewage Authority, Bridgewater, New Jersey on 13 February 2018, Day-1 of the test. This treatment facility treats pre-dominantly domestic sewage as specified in the guideline. There were no known contaminants in the fresh activated sludge believed to be present at levels high enough to have interfered with this study.
- Laboratory culture:
- Method of cultivation:
- Storage conditions: The whole unit was placed into an environmental chamber for 48 hours at an average temperature of 25.5C. Based on comparison of the density of colonies growing on the agar with the model density chart provided by the supplier, the microbial activity was determined to be 10^5 CFU/mL. The remaining decanted sludge supernatant was used for final preparation of the test medium on Day -1.
- Storage length:
- Preparation of inoculum for exposure:In order to achieve the required 3-5 g/L total suspended solids (TSS), four liters of sludge was allowed to settle and approximately 1000 mL of supernatant was removed and discarded. The remaining sludge was combined into a 8 liter carboy and aerated prior to analysis. To determine TSS, duplicate 10 ml aliquots of the activated sludge were filtered through pre-weighed Whatman 934-AH filters in a Buchner funnel and vacuum flask set up. The filters were placed in an aluminum pan and dried in an oven for one hour at an average temperature of 102C. After cooling the filters in a desiccator, the filters were re-weighed and the mean total suspended solids concentration was determined to be 3.47 g/L. Approximately one liter of the sludge was homogenized in a blender for two minutes at low speed. The homogenated sample was allowed to settle for 1 hour, after which the supernatant was decanted (avoiding carry-over of sludge solids). An aliquot of the supernatant was used to determine microbial activity using the Easicult®-TTC dip slides Lot No. 1791618. This was accomplished by removing the agar stick from the culturing tube, and dipping the agar into the supernatant aliquot. Excess supernatant was blotted off with a clean paper towel, and the agar stick was then placed back into the culture tube.
- Initial cell/biomass concentration: 10^5 CFU/mL
Duration of test (contact time):
28 d
Initial conc.:
38 mg/L
Based on:
test mat.
Remarks:
first test system replicate
Initial conc.:
39.2 mg/L
Based on:
test mat.
Remarks:
second replicate
Initial conc.:
41.8 mg/L
Based on:
test mat.
Remarks:
3rd replicate
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The test medium was prepared on Day -1. Fifteen liters of glass distilled water was collected in one carboy. 345 mL of glass distilled water was removed from the carboy before adding the following volumes of each mineral salts and 10 ml of inoculum per liter of water to obtain a final volume of fifteen liters: 1ml- Magnesium sulfate, 2.25%, 1 ml- Calcium chloride, 2.75%; 10.0ml -Phosphate buffer, pH 7.2, 1.0 ml-Ferric chloride, 0.025%, and 10.0ml of Activated Sludge Supernatant. The solution in the carboy was aerated with CO2-free air (air passed through an Ascarite II column to remove CO2), for 25 hours.
- Aeration of dilution water: yes
- Suspended solids concentration:
- Continuous darkness: yes/no


CONTROL AND BLANK SYSTEM
The test system was considered as one or any combination of the following in a flask: Test Substance or Positive Control Substance with Test Medium, Test Medium (containing the following; Mineral Salt Solutions, Activated Sludge Supernatant and Glass Distilled Water). The apparatus was a manometric respirometer, manufactured by Co-ordinated Environmental Service, Ltd. (Kent, England). The system is based on a proven oxygen generating process coupled to a sensor head. The sample is placed in a sample flask, which is then sealed to a sensor head with a CO2 trap and immersed in a temperature stabilized water bath. The CO2 trap contains a wick (fluted filter paper) and 5 mL of 2 N sodium hydroxide. For the duration of the experiment, the sample is stirred by a magnetically coupled stirrer. As the biodegradation process progresses, the microorganisms convert O2 to CO2. The CO2 is absorbed by the alkali CO2 trap and causes a net reduction in gas pressure within the sample flask. This pressure reduction is detected by the sensor head and triggers the electrolytic process. This generates oxygen and restores the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current are proportional to the amount of oxygen supplied to the microorganisms.
The test procedure evaluated the ready biodegradability of the test and positive control substances by microorganisms in water. The consumption of oxygen was determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas volume in the respirometer flask, or from the change in volume or pressure (or a combination of the two) in the apparatus. Evolved carbon dioxide was absorbed in a solution of 2N sodium hydroxide. The amount of oxygen taken up by the microbial population during biodegradation of the test or positive control substances (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of theoretical oxygen demand (ThOD).
The sodium benzoate was tested at 48.4 mg/L and administered to the respected test systems as an aliquot of an aqueous stock solution with glass fiber filter added to each test chamber. MRD-18-435 was tested at an average concentration of 39.7 mg by weighing the test substance on to a glass fiber filter and placing in each test chamber. Bias was minimized by preparing the test medium on a large volume basis. In addition, the test medium was aerated for approximately 24 hours to improve homogeneity and ensure random distribution of test organisms to all test systems. The initial pH of individual systems was not determined due to the poor solubility of the test substances in water; the pH of the test medium was 7.56.
Reference substance:
benzoic acid, sodium salt
Remarks:
A solution of sodium benzoate at about 12100 mg/L was prepared in glass distilled water. The pH was 7.4. A Shimadzu Total Organic Carbon analyzer determined the carbon content of the sodium benzoate stock solution.
Test performance:
The test substance met the 10-day window by biodegrading to an average of 17.23% by Day 3 and reached 61.58% degradation by Day 12, this substance is determined to be readily biodegradable.

Sodium benzoate biodegraded to >60% by Day 3. The average of the cumulative oxygen consumed in the blank systems was 17.05 mg/L after 28 days. Based on the study data and the passing of all validity requirements set forth in the OECD guideline (OECD, 1992), the test is considered valid.
Parameter:
% degradation (O2 consumption)
Value:
0.33
Sampling time:
2 d
Parameter:
% degradation (O2 consumption)
Value:
17.23
Sampling time:
3 d
Parameter:
% degradation (O2 consumption)
Value:
59.95
Sampling time:
11 d
Parameter:
% degradation (O2 consumption)
Value:
76.2
Sampling time:
21 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
83.5
Sampling time:
28 d
Results with reference substance:
Sodium benzoate biodegraded to >60% by Day 3 and 91.3% by day 28.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance degraded to 83.5% by day 28 and met the 10 day window, therefore it is readily biodegradable. The positive control, sodium benzoate degraded to >60% by day 3.
Executive summary:

The test substance is readily biodegradable as it degraded to >60% by day 12 and met the 10 day window. As the substance degraded to 83.5% it is expected that the substance mineralized to CO2 as the major metabolite.

Description of key information

n-Hexyl acrylate degraded to 83.5% in the OECD 301F ready biodegradability test, and can be considered to be rapidly biodegradable. Based on the extent of biodegradation, the test substance is shown to be extensively mineralized to CO2 by microorganisms from a domestic sewage treatment biological unit.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information