Propiononitrile

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: screening test, other

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

publication

Title:

etabolism of Acetonitrile and Propionitrile by Nocardia rhodochrous LL100-211

Author:

M. J. DiGERONIMO AND A. D. ANTOINE

Year:

1976

Bibliographic source:

DiGeronimo, M.J. & Antoine, A.D., 1976. Metabolism of acetonitrile and propionitrile by Nocardia rhodochrous LL100-21. Applied and Environmental Microbiology, 31(6), pp.900–906.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: the biodegradation potential of propionitrile by the fungi Nocardia rhodochrous was tested.
- Short description of test conditions: cultures were grown in a basal salts broth on a gyratory shaker for 1 day at 28°C.
- Parameters analysed / observed: growth (optical density), nitrile and metabolites in the filtrate

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Propiononitrile

EC Number:

203-464-4

EC Name:

Propiononitrile

Cas Number:

107-12-0

Molecular formula:

C3H5N

IUPAC Name:

propanenitrile

Specific details on test material used for the study:

obtained from Eastman Kodak, Rochester, N.Y., or from Fisher Scientific Co., Springfield, N.J. The nitriles used in this study contained less than 0.05% (wt/wt) Nes

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

other: basal salts broth (BSB)

Details on inoculum:

Cultivation and isolation of cultures:
The basal salts broth (BSB) contained (per liter): NaCl, 0.1 g; MgSO4 7H20, 0.2 g; K2HPO4, 1.03 g; KH2PO4, 0.75g, CaCl2, 25mg, ethylenediaminetetraaceticacid, 15 mg; ZnSO47H2O, 6.7mg; MnSO44H2O, 1.7mg; FeSO4-7H2O, 1.5mg;CoCl26H2O, 0.49mg; CuSO4, 0.47 mg; NaMoO4- 2H2O, 0.45 mg; pH 7.0. The MgSO4 *7H20 and CaCl2 were autoclaved separately and added aseptically to the autoclaved BSB. The isolate was obtained from barnyard soil by an enrichment culture technique using BSB (500ml/1,000-ml Erlenmeyer flask) containing 4% (wt/vol) acetonitrile as a source of both carbon and nitrogen. After a 10-day period of stationary incubation at 28°C, samples of the enrichment culture were plated on a basal salts agar (2%wt/vol) plus 4% (wt/vol) acetonitrile and incubated aerobically at 28°C. Colonies were selected and reinoculated into the BSB-acetonitrile medium for confirmation of its ability to grow on acetonitrile.

Duration of test (contact time):

1 d

Initial test substance concentration

Initial conc.:

10 g/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

other: nitrile concentration

Details on study design:

Substrate screening:
Cells were inoculated into 20-ml culture tubes containing 5 ml of BSB with the test compound (10 mg/ml). The growth control tubes contained 10 mg of sodium acetate per ml and 0.05 mg of ammonium sulfate per ml in place of a nitrile or amide. The inoculum was prepared by grwoing the isolate in BSB (100 ml/250 ml Erlenmeyer flask) containing 10 mg of acetonitrile per ml on a gyratory shaker, 200 rpm for 18h. The cells were harvested by centrifugation at 12 000 x g for 10 min at 5°C and resuspended in sterile 0.1 M potassium phosphate buffer (pH 7.2). A 5% (vol/vol) inoculum of this cell suspension and an incubation temperature of 28°C were used throughout the study.

Whole-cell studies:
For metabolic studies,cultures were grown on a gyratory shaker (200 rpm) in Fernbach flasks containing 1 liter of BSB and 10 mg of the nitrile per ml tested. Samples (5 ml) were removed from the culture broth periodically during the growth cycle. The sample was vacuum filtered through a 0.45-µm membrane filter (Millipore Corp., Bedford, Mass.) to remove the cells, and the filtrate was analyzed for nitrile concentration and for the presence of any detectabel metabolites. Growth was measured by optical density readings using a Coleman spectrophotometer (model 295) at 600 nm.

Cell-free studies:
Cell-free extracts were prepared by growing the isolate in 1-liter volumes of the BSB-acetonitrile (10 mg/ml) medium and harvesting the
cells in mid-log phase (18 h) by centrifugation with a steam-driven Sharples at 30,000 rpm. The packed cells (4 g of wet wt/liter of medium) were suspended in 2 ml ofcold 0.1 M potassium phosphate buffer (pH 7.2) per g of wet weight of biomass. The cells were disrupted in a Raytheon 10-Kc sonic oscillator for 10 min with 10 g of glass beads (no. 812 Cataphote, Cataphote Corp., Toledo, Ohio) for every 25 ml of cell suspension. Following sonification, the mixture was centrifuged at 4,300 x g for 3 min to remove the glass beads and unbroken cells. The supernatant fluid was then centrifuged at 35 000 x g for 15 min, which resulted in a clear supernatant fluid (typical protein concentration was 28 mg/ml). The supernatant fluid (crude extract) was stored at -25 °C. Assay of the crude extract for enzymatic activity was based on the detection of the substrates and products by GLC and colorimetric procedures. In the assay, 3.0 ml of 0.1 M potassium phosphate buffer (pH 7.2) containing 10 mg of acetonitrile per ml (0.244 mmol/ml) was equilibrated to 37°C, and 0.1 to 0.5 ml of the cell-free extract was added to start the reaction. The reaction was stopped by adding 1 ml of cold 10% trichloroacetic acid at various time intervals. The reaction mixture was then centrifuged at 3,000 rpm for 5 min (Sorvall, model no. GLC-1), and the supernatant fluid was analyzed.

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

96 h

% Degradation 2

Parameter:

% degradation (test mat. analysis)

Value:

11

Sampling time:

24 h

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Interpretation of results:

inherently biodegradable

Conclusions:

Complete biodegradation of propionitrile by Nocardia rhodochrous after 96 hours in synthetic medium was observed.