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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed journal
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Biodegradation study was carried out for evaluating the percentage biodegradability of test chemical 2-Nonanone.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Nonanone
- Molecular formula (if other than submission substance): C9H18O
- Molecular weight (if other than submission substance): 142.2402 g/mol
- Smiles notation (if other than submission substance): C(CCCCC)CC(C)=O
- InChI: 1S/C9H18O/c1-3-4-5-6-7-8-9(2)10/h3-8H2,1-2H3
- Substance type: Organic
- Physical state: Liquid
- Test chemical used was of analytical grade.
Oxygen conditions:
aerobic
Inoculum or test system:
other: Alcaligenes faecalis (Bacteria)
Details on inoculum:
- Laboratory culture: Alcaligenes faecalis (stock culture no. 9) was obtained from University Department of Microbiology, Ohio state.

- Method of cultivation: The cells were grown in a medium having the following composition:
Dextrose, 2.0 g; tryptone, 10.0 g; beef extract, 6.0 g; distilled water upto 1 liter.
Incubation was at 32°C for 48 hrs.

- Preparation of inoculum for exposure: Using aseptic techniques, the cells were harvested, washed four times with sterile distilled water, adjusted to 5,000 mg/l with sterile diluent. The suspension was blended for 10 sec in a sterile blender. 10 ml of the cell suspension were added to each Warburg flask.

- Concentration of sludge: 2,500 mg/l
Duration of test (contact time):
195 h
Initial conc.:
500 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Test temperature: 20°C
- pH: 7.0

TEST SYSTEM
- Culturing apparatus: Warburg flasks
- Measuring equipment: Respirometer
- Other: Oxidation was recorded as accumulative mg oxygen absorbed per liter of cell-substrate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: A flask containing 2,500 mg/l of test inoculum served as a control for measurement of endogenous respiration of the organism.
- Abiotic sterile control: Air oxidation or volatility of the substance was measured in a flask containing 500 mg/l of substrate in the mineral salts diluents. Corrections for air oxidation or volatility were made when necessary. These air oxidation flasks also served as a partial check of oxygen uptake by contaminants introduced in the unsterilized air or with unsterilized substrates.

Key result
Parameter:
% degradation (O2 consumption)
Sampling time:
192 h
Remarks on result:
other: After a long lag period, the organism was not able to utilize the test chemical 2-Nonanone. Thus the chemical 2-Nonanone remained uniformly resistant to oxidation by Alcaligenes faecalis.

When the substrate uptake curve fell below the curve for endogenous respiration (control), the result was interpreted as indicating some measure of inhibition of the organism by the substrate. It is assumed but not taken as proved that such an inhibitory compound is resistant to oxidation by Alcaligenes faecalis.

Validity criteria fulfilled:
not specified
Interpretation of results:
not readily biodegradable
Conclusions:
Biodegradation study was carried out for a period of 115 to 195 hrs evaluating the percentage biodegradability of test chemical 2 -Nonanone. After a long lag period, the organism was not able to utilize the test chemical 2-Nonanone. The chemical 2-Nonanone remained uniformly resistant to oxidation by Alcaligenes faecalis. Thus, the test substance 2-Nonanone was considered to be not readily biodegradable.
Executive summary:

Biodegradation study was carried out for a period of 115 to 195 hrs evaluating the percentage biodegradability of test chemical 2 -Nonanone. Oxygen uptake study was carried out in the Warburg constant volume respirometer on the ability of Alcaligenes faecalis to oxidize aerobically test chemical 2 -Nonanone at a temp. of 20°C and pH of 7.0. Alcaligenes faecalis was used as a test inoculum obtained from University Department of Microbiology, Ohio state. The cells were grown in a medium having the following composition: Dextrose, 2.0 g; tryptone, 10.0 g; beef extract, 6.0 g; distilled water upto 1 liter. Incubation was at 32°C for 48 hrs. Using aseptic techniques, the cells were harvested, washed four times with sterile distilled water, adjusted to 5,000 mg/l with sterile diluent. The suspension was blended for 10 sec in a sterile blender. 10 ml of the cell suspension were added to each Warburg flask. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively. Each flask contained 1.0 ml of 20% KOH solution in the center well. The reaction compartment of the flask held 20 ml of a mixture containing 500 mg/l of test chemical and 2,500 mg/l of inoculums, with mineral salts as the diluents. Incubation was at 20°C for 115 – 195 hrs. Aseptic techniques were used throughout the experiment. A flask containing 2,500 mg/l of test inoculum served as a control for measurement of endogenous respiration of the organism. Air oxidation or volatility of the substance was measured in a flask containing 500 mg/l of substrate in the mineral salts diluents. Corrections for air oxidation or volatility were made when necessary. These air oxidation flasks also served as a partial check of oxygen uptake by contaminants introduced in the unsterilized air or with unsterilized substrates. When the substrate uptake curve fell below the curve for endogenous respiration (control), the result was interpreted as indicating some measure of inhibition of the organism by the substrate. It is assumed that such an inhibitory compound is resistant to oxidation by Alcaligenes faecalis. After a long lag period, the organism was not able to utilize the test chemical 2 -Nonanone. The chemical 2 -Nonanone remained uniformly resistant to oxidation by Alcaligenes faecalis. Thus, the test substance 2 -Nonanone was considered to be not readily biodegradable.

Description of key information

Biodegradation study was carried out for a period of 115 to 195 hrs evaluating the percentage biodegradability of test chemical 2 -Nonanone. Oxygen uptake study was carried out in the Warburg constant volume respirometer on the ability of Alcaligenes faecalis to oxidize aerobically test chemical 2 -Nonanone at a temp. of 20°C and pH of 7.0. Alcaligenes faecalis was used as a test inoculum obtained from University Department of Microbiology, Ohio state. The cells were grown in a medium having the following composition: Dextrose, 2.0 g; tryptone, 10.0 g; beef extract, 6.0 g; distilled water upto 1 liter. Incubation was at 32°C for 48 hrs. Using aseptic techniques, the cells were harvested, washed four times with sterile distilled water, adjusted to 5,000 mg/l with sterile diluent. The suspension was blended for 10 sec in a sterile blender. 10 ml of the cell suspension were added to each Warburg flask. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively. Each flask contained 1.0 ml of 20% KOH solution in the center well. The reaction compartment of the flask held 20 ml of a mixture containing 500 mg/l of test chemical and 2,500 mg/l of inoculums, with mineral salts as the diluents. Incubation was at 20°C for 115 – 195 hrs. Aseptic techniques were used throughout the experiment. A flask containing 2,500 mg/l of test inoculum served as a control for measurement of endogenous respiration of the organism. Air oxidation or volatility of the substance was measured in a flask containing 500 mg/l of substrate in the mineral salts diluents. Corrections for air oxidation or volatility were made when necessary. These air oxidation flasks also served as a partial check of oxygen uptake by contaminants introduced in the unsterilized air or with unsterilized substrates. When the substrate uptake curve fell below the curve for endogenous respiration (control), the result was interpreted as indicating some measure of inhibition of the organism by the substrate. It is assumed that such an inhibitory compound is resistant to oxidation by Alcaligenes faecalis. After a long lag period, the organism was not able to utilize the test chemical 2 -Nonanone. The chemical 2 -Nonanone remained uniformly resistant to oxidation by Alcaligenes faecalis. Thus, the test substance 2 -Nonanone was considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Total 3 studies (2 from peer reviewed journal and 1 study from secondary source) for the target compound 2-Nonanone(CAS No. 821-55-6) were reviewed for the biodegradation end point which are summarized as below:

In a key study from peer reviewed journal (C. V. Marion and G. W. Malaney, 1963) for target chemical2-Nonanone(CAS No. 821-55-6),biodegradation study was carried out for a period of 115 to 195 hrs evaluating the percentage biodegradability of test chemical 2 –Nonanone (CAS no. 821-55-6). Oxygen uptake study was carried out in the Warburg constant volume respirometer on the ability of Alcaligenes faecalis to oxidize aerobically test chemical 2 -Nonanone at a temp. of 20°C and pH of 7.0. Alcaligenes faecalis was used as a test inoculum obtained from University Department of Microbiology, Ohio state. The cells were grown in a medium having the following composition: Dextrose, 2.0 g; tryptone, 10.0 g; beef extract, 6.0 g; distilled water upto 1 liter. Incubation was at 32°C for 48 hrs. Using aseptic techniques, the cells were harvested, washed four times with sterile distilled water, adjusted to 5,000 mg/l with sterile diluent. The suspension was blended for 10 sec in a sterile blender. 10 ml of the cell suspension were added to each Warburg flask. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively. Each flask contained 1.0 ml of 20% KOH solution in the center well. The reaction compartment of the flask held 20 ml of a mixture containing 500 mg/l of test chemical and 2,500 mg/l of inoculums, with mineral salts as the diluents. Incubation was at 20°C for 115 – 195 hrs. Aseptic techniques were used throughout the experiment. A flask containing 2,500 mg/l of test inoculum served as a control for measurement of endogenous respiration of the organism. Air oxidation or volatility of the substance was measured in a flask containing 500 mg/l of substrate in the mineral salts diluents. Corrections for air oxidation or volatility were made when necessary. These air oxidation flasks also served as a partial check of oxygen uptake by contaminants introduced in the unsterilized air or with unsterilized substrates. When the substrate uptake curve fell below the curve for endogenous respiration (control), the result was interpreted as indicating some measure of inhibition of the organism by the substrate. It is assumed that such an inhibitory compound is resistant to oxidation by Alcaligenes faecalis. After a long lag period, the organism was not able to utilize the test chemical 2 -Nonanone. The chemical 2 -Nonanone remained uniformly resistant to oxidation by Alcaligenes faecalis. Thus, the test substance 2 -Nonanone was considered to be not readily biodegradable.

 

Another supporting biodegradation study was carried out for evaluating the percentage biodegradability of test chemical 2 –Nonanone (CAS no. 821-55-6) (Robert M. Gerhold and George W. Malaney, 1962). Activated sludge was used as test inoculums obtained from 3 treatment plants of different sizes and designs and fed by different sewage systems. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively. Warburg constant temperature respirometer was used as a test vessel. The suspended solid conc. was adjusted to 2,500 mg/l by removal of supernatant liquid or by addition of tap water.The sludge was not washed. No mineral salts were added. The sludge suspension was blended for 10 sec and 20 ml were pipetted into 125 ml Warburg flasks containing the test substance (substrate).  A control flask for measurement of endogenous respiration was included with each run. Readings were made for 24 hr at 0.5 to 5 hr interval, depending on the rate of oxygen uptake. The experimental results were plotted as accumulative oxygen uptake corrected for endogenous respiration. Test chemical was observed to be more resistant to oxidation by activated sludge. Thus, the chemical 2-Nonanone was considered to be not readily biodegradable in nature.

 

In an additional supporting data from secondary source (Robert Murray Gerhold, 1962) for the target chemical2-Nonanone(CAS No. 821-55-6), biodegradation study was carried out for evaluating the percentage biodegradability of test chemical 2 -Nonanone. Activated sludge was used as test inoculums obtained from 3 treatment plants of different sizes and designs and fed by different sewage systems. Initial test substance conc. used for the study was 500 mg/l and conc. of the inoculum used was 2,500 mg/l, respectively. Test chemical (substrates) easily soluble in water were made up in 0.1 per cent concentration with distilled water and stored at 6°C until needed.Warburg constant temperature respirometer was used as a test vessel. They were modified 125 ml Erlenmeyer flasks fitted with 1.5 ml center-wells and female ground glass joints. Warburg flasks were cleaned by the following procedure: (a) flasks were rinsed once with tap water, and dried In the 103°C oven; (b) flasks were washed with two rinses of chloroform to remove fats and greases, then dried; (c) the flasks were submerged in potassium dichromate cleaning solution for 24 hr, rinsed In the same manner as the pipettes, and dried in an inverted position.Each flask received 10 ml of substrate solution or suspension delivered with a volumetric pipette. Next, 10 ml of blended sludge were added to each flask. The final concentration of substrate was 500 mg/liter. The final concentration of sludge solids was 2500 mg/liter. The control for endogenous respiration contained 10 ml of distilled water and 10 ml of adjusted sludge. Endogenous respiration was defined as the amount of accumulative O2uptake observed in the control flask containing sludge and distilled water. After 10-20 min of shaking for temperature equilibration the flasks were closed off to the atmosphere and shaken for 24 hr at 78 oscillations per min. From 9 to 16 readings were made during each experiment.The terms "percentage oxidized," or "percentage of oxidation," or "X per cent oxidized" mean the ratio of the amount of oxygen taken up by the sludge in the presence of that concentration of the substrate to the amount of oxygen required for complete oxidation of that concentration of substrate, i.e., oxidation to carbon dioxide, water, nitrate, and sulfate. This ratio is also referred to as the "percentage of total theoretical oxygen demand (ThOD)."Theoretical O2 uptake of the test chemical by activated sludge was determined to be1462 mg/l. No sufficient degradation of the test chemical was observed in 24 hr period. Thus, the chemical 2 -Nonanone was considered to be not readily biodegradable in nature.

 

On the basis of above results for target chemical2-Nonanone(Study 1 and 2 from peer reviewed journal and study 3 from secondary source), it can be concluded that the test substance2-Nonanonecan be expected to be not readily biodegradable in nature.