Butyl propionate

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

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although the study was conducted according to OECD TG 201, EU Method C.3, TSCA 797.1500 and in accordance with the Principles of Good Laboratory Practice (GLP), the difference between the measured analytical concentrations and nominal concentrations were in excess of ± 45%.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Characterization of the test material used in this study was not conducted following GLP standards; however, a certificate of analysis identifying the test material purity was provided by the supplier.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Remarks:

same as above

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. EPA – TSCA 40 CFR 797.1500

Deviations:

no

Remarks:

same as above

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Butyl propionate

EC Number:

209-669-5

EC Name:

Butyl propionate

Cas Number:

590-01-2

Molecular formula:

C7H14O2

IUPAC Name:

butyl propanoate

Details on test material:

- Name of test material (as cited in study report): UCAR n-butyl propionate
- Substance type: esters
- Physical state: transparent colorless liquid
- Analytical purity: 99.92%
- Lot/batch No.: 0000369856

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 4.1, 10.2, 25.6, 64.0, 160, 400, and 1000 mg butyl propionate/liter test solutions
- Sampling method: The bulk dose solutions were sampled for analytical confirmation on day 0 of the study. On day 4, the three test solutions containing algae at each dose level were pooled to provide one composite algae-containing sample per dose level for analytical confirmation. The test solutions at each dose level containing no algae were sampled separately.

Test solutions

Vehicle:

no

Details on test solutions:

All test solutions were made via direct addition of butyl propionate to the test algal assay medium (AAM). Since the test material was a liquid, the weight of the test material added to the AAM was calculated on a weight-to-volume basis and was then converted to a volume using the specific gravity of the test material (0.8755 g/mL). Based on this, 2.3, 5.8, 14.6, 36.6, 91.3, 228, and 571 mL aliquots of butyl propionate were added to AAM in 0.5-L volumetric flasks for the 4.1, 10.2, 25.6, 64.0, 160, 400, and 1000 mg butyl propionate/L test solutions, respectively. The flasks were then brought to volume with AAM, stoppered, and inverted at least five times to mix thoroughly. The 1000, 400, 160, and 64.0 mg/L test solutions were sonicated for approximately 45, 25, 15, and 5 minutes, prior to bringing to volume, to aid in dissolution of the test material. The control test solution was test AAM with no test material added.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater alga
- Strain: axenic samples of Pseudokirchnerella subcapitata
- Source (laboratory, culture collection): originally received on 13 February 2003, from the University of Toronto Culture Collection at the University of Toronto, Toronto, Ontario, Canada. Stock cultures of this organism were maintained aseptically by weekly transfer into sterile AAM.
- Age of inoculum (at test initiation): The algal inoculum was prepared from a 3-day old stock culture of Pseudokirchneriella subcapitata. A Coulter Multisizer 3 was used to determine the algal density of the stock culture. This evaluation determined the aliquot of the culture required so that each test vessel would contain approximately 10,000 cells/ml (523 ml).
- Method of cultivation: Typical culturing conditions -
Organism: Pseudokirchneriella subcapitata (formerly know as Selenastrum capricornutum), a freshwater green alga
Temperature: 24 ± 2°C
Light (lux): 4300 ± 650
Photoperiod: Continuous
Medium: Algal assay medium (AAM) designated for the EPA algal assay bottle test
pH: Range: approximately 7.0-7.5
Culture Conditions: Axenic
Culture Volume: 200 ml
Culture Vessel: 500-ml Erlenmeyer flask
Culture Vessel Cap: Shimadzu closure

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

not applicable

Test temperature:

24.1 ± 0.1°C (22.8°C-24.2°C),

pH:

pH values ranged from 7.2 to 7.8 at test initiation, from 7.5 to 9.7 with algae at test termination, and from 7.2 to 7.4 without algae at test termination.

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

- Nominal concentrations: 4.1, 10.2, 25.6, 64.0, 160, 400, and 1000 mg butyl propionate/liter test solutions
- Mean measured concentrations: 1.97, 4.51, 12.4, 30.2, 82.2, 152 and 434 mg butyl propionate/liter test solutions

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: closed
- Material, size, headspace, fill volume: 250 ml glass Erlenmeyer flask

- Initial cells density: ~ 10,000 cells/ml
- No. of vessels per concentration (replicates): 4 (3 with algae, 1 without algae)
- No. of vessels per control (replicates): 4 (3 with algae, 1 without algae)

GROWTH MEDIUM
- Standard medium used: yes Algal Assay Medium (AAM)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: de-ionized water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes (Environmental Growth Chamber)
- Photoperiod: continuous
- Light intensity and quality: continuous 8000 ± 1600 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal cell densities of the initial inoculum and test cultures were determined by electron particle counting using a Coulter Multisizer 3. In addition, at test termination morphological observations were done on a composited sample of the inoculated replicates at each test concentration. The cells were observed under a microscope (OlympusÒ BHB-DO System Microscope; 20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck) using a Bright Line Hemacytometer Counting Chamber.

TEST CONCENTRATIONS
- Range finding study: The dose levels selected for evaluating the effects of butyl propionate on the growth of Pseudokirchneriella subcapitata were based on the results from a series of probes. All of the probes were conducted at nominal butyl propionate concentrations of 0.100, 1.00, 10.0, and 100 mg/l. The first probe was conducted from 02 December to 06 December 2002. Percent inhibition compared to the control at 96 hours was 27, 4, 7, and 10% for the nominal 0.100, 1.00, 10.0, and 100 mg/l test levels, respectively. However, around the same time this probe was conducted, it was it was discovered that the algal culture was contaminated, so a second probe was scheduled after the new culture was established. The second probe was conducted 30 January to 03 February 2003. Percent inhibition compared to the control at 96 hours was -30, -48, 74, and 97% for the nominal 0.100, 1.00, 10.0, and 100 mg/l test levels, respectively.
Due to the inconsistency of the results between these probes, a third probe was scheduled. The third probe was conducted 03 March to 07 March 2003. Percent inhibition compared to control at 96 hours was -4, -8, -7, and 60% for the nominal 0.100, 1.00, 10.0, and 100 mg butyl propionate/l test levels, respectively. These results indicated that butyl propionate may be a difficult compound from which to get a good dose response test with Pseudokirchneriella subcapitata. The information derived from these tests was used to set the range of concentrations for the definitive test. Since there was not good agreement between probe results, it was determined to set the high dose in the definitive test at 1000 mg butyl propionate/l and test down from there at 60% cuts to encompass a greater range of concentrations.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

204 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

82.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

166 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

82.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

261 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

152 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

182 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

82.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Temperatures during the exposure period averaged ± standard deviation (range) 24.1 ± 0.1°C (22.8°C-24.2°C), light intensity averaged 7766 ± 435 lux (6830 lux-8590 lux), and pH values ranged from 7.2 to 7.8 at test initiation, from 7.5 to 9.7 with algae at test termination, and from 7.2 to 7.4 without algae at test termination.

Mean cell densities at 72 hours were 214.7, 228.5, 250.2, 187.0, 214.5, 187.4, 132.6, and 0.6338 x 10(4) cells/ml for the control, 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. Response relative to the controls ranged from 17% stimulation of growth to 100% inhibition of growth. The 72-hour calculated EC25 and EC50 values (95% confidence intervals) for cell density were 109 (72.4-164) and 175 (117-261) mg/l, respectively. Based on the Dunnett’s test, the 72-hour cell density was significantly less than the controls at test levels ³ 152 mg/l; therefore, the 72-hour NOEC value for cell
density was determined to be 82.2 mg/l.

Mean cell densities at 96 hours were 492.7, 505.6, 515.1, 303.2, 480.7, 442.1, 383.7, and 3.063 x 10(4) cells/ml for the control, 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. Response relative to the controls ranged from 3% stimulation of growth to 99% inhibition of growth. The 96-hour calculated EC25 and EC50 values (95% confidence intervals) for cell density were 134 (62.8-204) and 239 (168-310) mg/l, respectively. Based on the Dunnett’s test, the 96-hour cell density was significantly less than the control test at the 12.4, 152, and 434 mg/l test levels. Percent inhibition based on the controls was -3, -5, 38, 2, 10, 22, and 99% for the 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. The results at the 12.4 mg/l test level (38% inhibition) were not consistent with the dose response. Since the responses at the next three higher test levels ranged from 0 to 22% inhibition, the response at the 12.4 mg/l test level was not considered treatment related; therefore, the 96-hour NOEC value for cell density was determined to be 82.2 mg/l.

Mean specific growth rates at 72 hours were 1.787, 1.806, 1.838, 1.741, 1.788, 1.742, 1.629, and -0.1577 day-1 for the control, 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. Response relative to the controls ranged from 3% stimulation to 109% inhibition of growth rate. The 72-hour calculated ErC50 value (95% confidence intervals) for specific growth rate was 204 (106-392). Based on the Dunnett’s test, the 72-hour specific growth rate was significantly less than the controls at test levels
³ 152 mg/l; therefore, the 72-hour NOEC value for specific growth rate was determined to be 82.2 mg/l.

Mean specific growth rates at 96 hours were 1.549, 1.556, 1.561, 1.428, 1.543, 1.523, 1.487, and 0.2625 day-1 for the control, 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. Response relative to the controls ranged from 1% stimulation to 83% inhibition of growth rate. The 96-hour calculated ErC50 value (95% confidence intervals) for specific growth rate was 261 (124 -> 434). Based on the Dunnett’s test, the 96-hour specific growth rate was significantly less than the controls at the 12.4 and 434 mg/l test levels. Percent inhibition based on the controls was 0, -1, 8, 0, 2, 4, and 83% for the 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. Similar to the 96-hour cell density results, the results at the 12.4 mg/l test level (8% inhibition) were not consistent with the dose response. Since the responses at the next three higher test levels ranged from 0 to 4% inhibition, the response at the 12.4 mg/l test level was not considered treatment related; therefore, the 96-hour NOEC value for specific growth rate was determined to be 152 mg/l.

Mean biomass area values at 72 hours were 3830, 4053, 4343, 3279, 3948, 3341, 2056, and 23.58 for the control, 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. Response relative to the controls ranged from 13% stimulation to 99% inhibition of biomass. The 72-hour calculated EbC50 value (95% confidence intervals) for biomass was 166 (157-177). Based on the Dunnett’s test, the 72-hour biomass was significantly less than the controls at the 152 and 434 mg/l test levels; therefore, the 72- hour NOEC value for biomass was determined to be 82.2 mg/l.

Mean biomass area values at 96 hours were 12295, 12839, 13502, 9138, 12267, 10871, 8228, and 43.94 for the control, 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434 mg/l test levels, respectively. Response relative to the controls ranged from 10% stimulation to 100% inhibition of biomass. The 96-hour calculated EbC50 value (95% confidence intervals) for biomass was 182 (121-274). Based on the Dunnett’s test, the 96-hour biomass was significantly less than the controls at the 152 and 434 mg/l test levels; therefore, the 96-hour NOEC value for biomass was determined to be 82.2 mg/l.

Microscopic evaluation of cells at each test concentration and the control revealed no abnormal observations.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

Standard statistical methods were employed

Any other information on results incl. tables

None

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 72-hour ErC50 (growth rate) was 204 mg/l and the 72-hour EbC50 (biomass) was 166 mg/l

Executive summary:

The purpose of this study was to assess the effects of butyl propionate on the growth of Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), a freshwater green alga. The freshwater alga was exposed to seven nominal test concentrations of 4.10, 10.2, 25.6, 64.0, 160, 400, and 1000 mg butyl propionate/l of algal assay medium, plus a medium control, over a 96-hour period. These nominal concentrations were equivalent to mean measured concentrations of 1.97, 4.51, 12.4, 30.2, 82.2, 152, and 434-mg butyl propionate/l, respectively; the medium control was less than the lowest level quantified (0.656 mg/l).

 

Treatment groups and the medium control group were set in triplicate, with an initial cell density of ~10,000 cells/ml. Test vessels were set on an automatic shaker at a shaking rate of approximately 100 rpm. Temperatures during the exposure period averaged ± standard deviation (range) 24.1 ± 0.1°C (22.8°C-24.2°C), light intensity averaged 7766 ± 435 lux (6830 lux-8590 lux), and pH values ranged from 7.2 to 7.8 at test initiation, from 7.5 to 9.6 with algae at test termination, and from 7.2 to 7.4 without algae at test termination.

 

The effect criteria were as follows: inhibition of cell density relative to the control, as denoted by EC25 and EC50 values for 25% and 50% inhibition, respectively, at 72 and 96 hours; inhibition of growth rate (day-1) relative to the control, as denoted by ErC50 value for 50% inhibition at 72 and 96 hours; and inhibition of biomass (difference between the area under the control growth curve and the area under the growth curve at each mean measured concentration), as denoted by EbC50 value for 50% inhibition at 72 and 96 hours. No-observed-effect concentrations or NOECs (P < 0.05) were also calculated for each of the three criteria.

 

The 72-hour results, based on mean measured butyl propionate concentrations, were as follows:

The 72-hour EC25, EC50, and NOEC values for cell density were 109, 175, and 82.2 mg/l, respectively.

The 72-hour ErC50 and NOEC values for growth rate (day-1) were 204 and 82.2 mg/l, respectively.

The 72-hour EbC50 and NOEC values for biomass (area under the growth curve) were 166 and 82.2 mg/l, respectively.

 

The 96-hour results, based on mean measured butyl propionate concentrations, were as follows:

The 96-hour EC25, EC50, and NOEC values for cell density were 134, 239, and 82.2 mg/l, respectively.

The 96-hour ErC50 and NOEC values for growth rate (day-1) were 261 and 152 mg/l, respectively.

The 96-hour EbC50 and NOEC values for biomass (area under the growth curve) were 182 and 82.2 mg/l, respectively.