2-Propanamine, compd. with boron trifluoride, reaction products with Bu glycidyl ether

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 hours and stored frozen for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Vehicle:

no

Details on test solutions:

The culture medium used for the range-finding tests, initial experiments and definitive test was the same as that used to maintain the stock culture.
The culture medium is

NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

See test solution details

Test temperature:

24 ± 1 °C

pH:

7.5

Dissolved oxygen:

> 3 mg/l

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 81% to 116% of nominal and so the results are based on nominal test concentrations only.

Details on test conditions:

Initial Range-Finding Test
The test concentrations to be used in the initial experiments were determined by a preliminary range-finding test. The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (6.3 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.
Initial Experiments
Based on the results of the initial range-finding test, three initial experiments were conducted covering a test concentration range of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. On all three occasions significant inhibition of growth was observed to have occurred at the lowest test concentration employed. Given this it was considered likely that the results obtained from the initial range-finding test were erroneous. As such a second range-finding test was conducted in a similar manner.
Definitive Test
Based on the results of the second range-finding test the following test concentrations were assigned to the definitive test: 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
Experimental Preparation
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 8 minutes and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot (900 mL) of each of the 0.10, 0.32, 1.0, 3.2 and 10 mg/L stock solutions was separately inoculated with algal suspension (6.0 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
Exposure Conditions
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.51 x 105 cells per mL. Inoculation of 900 mL of test medium with 6.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Assessments
Test Organism Observations
Samples were taken at 19, 43 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. Theshape and size of the algal cells was inspected microscopically and any abnormalities recorded.
3.5.2 Water Quality Criteria
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.
Verification of Test Concentrations
Samples were taken from the control and each group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 hours and stored frozen for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.3 - < 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.38 - < 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the initial range-finding test were evaluated.
The results of the initial range-finding test showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 29% to 76% of nominal. Examination of the data could find no cause for the lower than expected results as a sufficient quantity of test item had been shown to be used. Whilst the results were not as expected, they did demonstrate that the test item was stable over the test duration.

Initial Experiments
Based on the results of the initial range-finding test, three initial experiments were conducted covering a test concentration range of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. On all three occasions significant inhibition of growth was observed to have occurred at the lowest test concentration employed. Given this it was considered likely that the results obtained from the initial range-finding test were erroneous. As such a second range-finding test was conducted.

Second Range-finding Test
The results of the second range-finding test showed no effect on growth at the test concentration of 0.10 mg/L. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L.
Based on these results a test concentration range of 0.10, 0.32, 1.0, 3.2 and 10 mg/L was selected for the definitive test.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 81% to 116% of nominal and so the results are based on nominal test concentrations only.
Accordingly the following results were determined from the data:


The following results were determined from the data:

Inhibition of growth rate
ErC10 (0 - 72 h) : 0.36 mg/L
ErC20 (0 - 72 h) : 0.62 mg/L
ErC50 (0 - 72 h) : 1.6 mg/L; 95% confidence limits 1.3 – 2.0 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control and 0.10 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.10 mg/L. The "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.32 mg/L. Correspondingly, the EC10 based on growth rate was 0.36 mg/l.


Inhibition of Yield
EyC10 (0 - 72 h) : 0.17 mg/L
EyC20 (0 - 72 h) : 0.24 mg/L
EyC50 (0 - 72 h) : 0.44 mg/L; 95% confidence limits 0.38 – 0.50 mg/L
Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out. There were no statistically significant decreases in yield (P≥0.05), between the control and 0.10 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.10 mg/L.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The effect of the Boron (2-propanamine) trifluoro-, rxn prod with butyl glycidyl ether on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:

NOEC (0 - 72 h) : 0.1 mg/L
ErC10 (0 - 72 h) : 0.36 mg/L
ErC20 (0 - 72 h) : 0.62 mg/L
ErC50 (0 - 72 h) : 1.6 mg/L; 95% confidence limits 1.3 – 2.0 mg/L

NOEC (0 - 72 h) : 0.1 mg/L
EyC10 (0 - 72 h) : 0.17 mg/L
EyC20 (0 - 72 h) : 0.24 mg/L
EyC50 (0 - 72 h) : 0.44 mg/L; 95% confidence limits 0.38 – 0.50 mg/L

Executive summary:

The effect of the Boron (2-propanamine) trifluoro-, rxn prod with butyl glycidyl ether on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:

NOEC (0 - 72 h) : 0.1 mg/L
ErC10 (0 - 72 h) : 0.36 mg/L
ErC20 (0 - 72 h) : 0.62 mg/L
ErC50 (0 - 72 h) : 1.6 mg/L; 95% confidence limits 1.3 – 2.0 mg/L

 

NOEC (0 - 72 h) : 0.1 mg/L
EyC10 (0 - 72 h) : 0.17 mg/L
EyC20 (0 - 72 h) : 0.24 mg/L
EyC50 (0 - 72 h) : 0.44 mg/L; 95% confidence limits 0.38 – 0.50 mg/L

Description of key information

The effect of the Boron (2-propanamine) trifluoro-, rxn prod with butyl glycidyl ether  on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results - EC50=1.6 mg/l (based on growth rate) and 0.44 mg/l (based on yield).

Key value for chemical safety assessment

EC50 for freshwater algae:

1.6 mg/L