Butyronitrile

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Butyronitrile

EC Number:

203-700-6

EC Name:

Butyronitrile

Cas Number:

109-74-0

Molecular formula:

C4H7N

IUPAC Name:

butanenitrile

Details on test material:

Purity Analysis: the test substance was determined to be 99.9% (area) pure using gas chromatography with flame ionization d~tection (GC/FID).
Structure Confirmation: the structure of the test substance was confirmed to be consistent with the proposed structure using gas chromatography with mass spectroscopy detection (GC/MS).
CAS No.: 109-74-0
SRlDNo.: T-11
Physical State and Appearance: Clear colorless liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Temperature, light intensity, and shaker speed (rpm) values were recorded at test start and after 24, 48, and 72 hours of exposure. The pH of solutions were measured at test start and test end.

Cell counts were performed after 24, 48, and 72 hours of exposure using a Coulter Counter. Prior to performing cell counts the Coulter Counter was calibrated using hemacytometer counts. Aliquots were collected for counting by swirling the flasks to achieve a uniform cell suspension and then removing approximately 4.0 mL of solution.

The concentrations of test substance in the exposure solutions were verified at test start and after 24, 48, and 72 hours of exposure. Samples were collected using the same methods used to collect samples for cell counts. Test substance exposure concentrations were verified using gas chromatography with flame ionization detection (CG/FID).

Test solutions

Vehicle:

no

Details on test solutions:

Sterile algal growth medium was prepared using high quality distilled water. The pH of the medium was measured and adjusted to 7.5 (±0.1) using 0.1 N NaOH prior to its use. This medium was used to prepare the test substance exposure solutions as well as the growth medium controls. The test substance exposure solution was prepared by direct addition of the test substance (volumetrically) to algal growth medium. The amount oftest substance added was adjusted to account for its specific gravity of 0.794 g/mL at 20°C. Approximately 0.151 mL (120 mg) of the test substance was added to 600 mL of algal growth medium using a "gas tight" Hamilton syringe. The nominal concentration of the test substance exposure solution was
200 mg/L.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test alga, Selenastrum capricornutum SF -3148, was obtained from the American Type Culture Collection, Rockville, MD, USA on 3 July 1996 and was subsequently cultured in the testing facility. A 4-day culture, passage 3 in liquid algal medium, was used as the source of test algae. Several passages were performed prior to the test in order to confirm exponential growth under the conditions of the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24°C

pH:

The pH values ranged from 7.4 to 7.6 in the test substance exposures and from 7.4 to 7.5 in the control exposures.

Nominal and measured concentrations:

The concentration of the test substance exposure solution was determined to be 206.0 mg/L at test start. The mean concentration of the test substance exposure solutions after 72 hours of exposure was determined to be 85.7 mg/L. The results of the analyses indicate that the test substance concentration was unstable under conditions of the test (58% loss), however a mean concentration of 133.4 mg/L was maintained throughout the study. Loss of test substance concentration during the study was most likely due to volatilization from the exposure solution.

Details on test conditions:

The test vessels, sterile 250-mL Erlenmeyer flasks, were conditioned by adding 1-2 milliliters of the appropriate test solution, swirling the flask to coat the sides, and then removing the solution by inverting it into a waste beaker. A total of eight flasks were conditioned. Five flasks were conditioned with test substance exposure solution and three were conditioned with algal growth medium. The test substance exposure solutions (flasks #3-5), the algal growth medium control exposure solutions (flasks #6-8), and the light/dark test substance control exposure solutions (flasks # 1 and #2) were prepared by adding 100 mL of the appropriate solution to each flask. The test substance and algal growth medium control exposure solutions (flasks #3-8) were then inoculated with aliquots of the algal stock culture. The light/dark control exposure solutions (flasks #1 and #2) were not inoculated with algae. The density of algae cells in the algal stock culture was determined to be 2.58x10e6 cells/mL by performing a hemacytometer count. The test substance exposure solutions (flasks #3-5) and the algal growth medium control solutions (flasks #6-8) were inoculated with 388 uL of algal stock culture to achieve an initial algal cell density of 1x10e4 cells/mL. The flasks were secured with foam stoppers and transferred to an incubator in random order where they were maintained at a temperature of24°C, shaken at 100 rpm, and illuminated at 747 (±5.5) footcandles(± standard deviation) throughout the study.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 133.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 133.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 133.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Any other information on results incl. tables

Algal Cell Counts (ln values)

Exposure Time (hours)	Control	133.4 mg/L
0	9.21	9.21
24	10.59	10.53
48	12.27	12.31
72	13.71	13.76

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The inhibition of growth of the alga, Selenastrum capricornutum, following exposure to the test substance was determined in a 72-hour growth inhibition limit test. Cultures of algae were exposed to the test substance at a nominal concentration of 200 mg/L over several algal generations. The stability of the test substance under conditions of the test was determined by analyzing the exposure solutions at test start and after 24, 48, and 72 hours of exposure. The concentrations of the test substance in the exposure solutions were analyzed using gas chromatography with flame ionnization detection (GC/FID). The results of the analyses indicate that the test substance concentration was unstable under conditions of the test (58% loss), however a mean concentration of greater than 100 mg/L was maintained throughout the study. The exposure concentration was calculated as the geometric mean of the test substance solutions analyzed at test start and at 24-hour intervals from test start. The mean analyzed concentration of the test substance exposure solution was determined to be 133.4 mg/L. Cell counts were performed after 24, 48, and 72 hours of exposure using a Coulter Counter. The results of the cell counts indicate that algae in the test substance exposure solution exhibited normal growth in comparison to the growth of algae in the control (growth medium). Two measures of growth (biomass and growth rate) were used to determine the effects of the test substance on the algae. Inhibition of biomass (EbC50) and growth rate (ErC50) were both estimated to be> 133.4 mg/L as greater than 50% inhibition in growth and/or biomass was not achieved in this test. The study did not calculate a NOEC for the limit test, but based upon the reported cell counts an NOEC of ≥133.4 mg/L can be empirically derived.