Registration Dossier

Ecotoxicological information

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 201, EEC Directive 92/69/EEC C.3, EPA – TSCA 40 CFR 797.1500 (1992) and in accordance with Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA – TSCA 40 CFR 797.1500 (1992)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): n-propyl propionate
- Molecular formula: C6H12O2
- Physical state: liquid
- Analytical purity: 99.96%
- Lot/batch No.: QC1355V1C1
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: 62.5, 125, 250, 500, 1000, and 2000 mg n-propyl propionate/L

Test solutions

Vehicle:
no
Details on test solutions:
All test solutions were made via direct addition of n-propyl propionate to the (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 (0.881 g/mL). Based on this, 35.5, 70.9, 142, 284, 571, and 1135 mL aliquots of n-propyl propionate were added to approximately 400 mL AAM in 0.5-L volumetric flasks for the 62.5, 125, 250, 500, 1000, and 2000 mg n-propyl propionate/L test solutions, respectively. The 500, 1000, and 2000 mg/L test solutions were sonicated for 10, 15 and 15 minutes, respectively, to aid in dissolution of the test material.
The flasks were then brought to volume with AAM, stoppered, and inverted several times to mix thoroughly. The control test solution was AAM with no test material added.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata (syn: Selenastrum capricornutum)
- Source (laboratory, culture collection): The algae were obtained from University of Toronto Culture Collection at the University of Toronto, Toronto, Ontario, Canada.

ACCLIMATION
- Culturing media and conditions: same as test

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 measured.
Test temperature:
24.5°C–24.6°C
pH:
6.7-9.4
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 62.5, 125, 250, 500, 1000, and 2000 mg n-propyl propionate/L AAM, and a medium control
Mean measured concentrations over days 0 to 4: 30.5, 62.0, 123, 245, 513, and 1004 mg n-propyl propionate/L
Details on test conditions:
The culture medium used was algal assay medium(AAM). The growth and test medium used was that designed for the EPA Algal Assay Bottle Test.
Culture conditions are as follow: Organism used was Pseudokirchneriella subcapitata, temperature maitained at 24 ± 2°C, Light (lux): 4300 ± 650,
Photoperiod: Continuous, pH: approximately 7.0-7.5, Culture Conditions: Axenic, Culture Volume: 200 mL. Test vessels were sterilized 250-mL borosilicate Erlenmeyer flasks with Shimadzu closures, each containing 100 mL test medium. Each flask was labeled with a unique number for identification purposes.
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 (0.940 mL).
The definitive test was conducted using four replicate test vessels were prepared per test concentration and control, each containing 100 mL of test solution. Three replicates at each test concentration and the control group were inoculated with approximately 10,000 cells/mL. Inoculations were made after all the replicate test vessels at each test concentration were poured. The fourth replicate at each test concentration and the control group was not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the interference of the test material and to monitor pH and concentration of the test material without the algal biomass.
The exposure phase was carried out aseptically under static conditions for 4 days (approximately 96 hours). The replicate test flasks were placed in a walk-in environmental chamber (Lab-Line Environmental Chamber, Lab-Line Inc., Melrose, Illinois) according to a computer-generated randomization scheme. The replicate test flasks were randomized daily after sampling for cell counts. The incubator was thermostated at 24 ± 2°C with continuous light at approximately 8000 ± 1600 lux.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1 004 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
245 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Mean cell densities at 72 hours were 149.2, 148.8, 136.6, 135.5, 106.9, 26.14, and 11.12 x 104 cells/mL for the control, 30.5, 62.0, 123, 245, 513, and 1004 mg/L test levels, respectively. The 72-hour calculated EC25 and EC50 values (95% confidence intervals) for cell density were 184 (84.5-403) and 328 (153-703) mg/L, respectively.The 72-hour NOEC value for cell density was determined to be 123 mg/L.

Mean cell densities at 96 hours were 416.1, 399.8, 388.2, 388.7, 303.3, 111.3, and 51.81 x 104 cells/mL for the control, 30.5, 62.0, 123, 245, 513, and 1004 mg/L test levels, respectively. The 96-hour calculated EC25 and EC50 values (95% confidence intervals) for cell density were 199 (101-394) and 365 (187-712) mg/L, respectively. The 96-hour NOEC value for cell density was determined to be 123 mg/L.

Mean specific growth rates at 72 hours were 1.668, 1.665, 1.637, 1.636, 1.548, 1.041, and 0.728 day-1 for the control, 30.5, 62.0, 123, 245, 513, and 1004 mg/L test levels, respectively. The 72-hour calculated ErC50 value (95% confidence intervals) for specific growth rate was 850 (305->1004) mg/L. The 72-hour NOEC value for specific growth rate was determined to be 245 mg/L.

The 72-hour calculated EbC50 value (95% confidence intervals) for biomass was 311 (169-573) mg/L. The 72-hour NOEC value for biomass was
determined to be 123 mg/L. The 96-hour calculated EbC50 value (95% confidence intervals) for biomass was 340 (196-588) mg/L. 96-hour NOEC value for biomass was determined to be 123 mg/L.

Microscopic evaluation of cells at each test concentration and the control revealed no abnormal observations other than clumping at the 513 mg/L test level.
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

Probe study:

The dose levels selected for evaluating the effects of n-propyl propionate on the growth of Pseudokirchneriella subcapitata were based on the results of a probe test. The probe test was conducted between 29 May and 2 June 2003 using four nominal n-propyl propionate concentrations of 1.00, 10.0, 100, and 1000 mg/L, plus a medium control. Percent inhibition compared to controls was 7, 4, 14, and 66% for the 1.00, 10.0, 100, and 1000 mg/L test levels, respectively. The information derived from this test was used to set the range of concentrations for the definitive test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 96-hour (growth rate) EC50 of n-propyl propionate to Pseudokirchneriella subcapitata (syn. Selenastrum
capricornutum) under static conditions was > 1004 mg/l and the NOEC being 245 mg/l.
Executive summary:

The purpose of this study was to determine the effects of n-propyl Propionate on the growth of Pseudokirchneriella subcapitata under static conditions in a closed-bottle system. The results are reported as the 72- and 96-hour No-Observed-Effect Concentration (NOEC) for yield , cell density and growth rate. Additionally 72 and 96 -hour EC50 values for the above endpoints were determined.

The nominal test concentrations were- medium control, 62.5,125, 250, 500, 1000, and 2000 mg 245, 513, and 1004 mg n-propyl propionate/L AAM.

Mean measured concentrations over days 0 to 4 were 30.5, 62.0, 123, 245, 513, and 1004 mg n-propyl propionate/L.

Based on mean measured concentrations the 96-hour (growth rate) EC50 of n-propyl propionate to Pseudokirchneriella subcapitata under static conditions was > 1004 mg/l and the NOEC being 245 mg/l.