2-butyloctanoic acid

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

Study period:

2022-02-06 to 2022-02-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other:

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test concentrations include a control and nominal exposure loading rates of 0.48, 1.52, 4.9, 15.6 and 50 mg/L. The test concentrations were determined from a range finding test. After 24, 48, and 72 hours, samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.
At the end of the test (72-hour), microscopic observations were made on a sample taken from a single test replicate of each concentration.

Details on test solutions:

The primary stock solution was prepared using a Water Accommodated Fraction (WAF).
A nominal loading rate of 100 mg/L was prepared by adding a nominal 0.2 g of 2-butyloctanoic acid (Isocarb 12) to 2000 mL of culture medium and mixed for a period of time sufficient to achieve an equilibrated concentration of dissolved and dispersed or emulsified components in the aqueous phase. For this study, mixing time was 24 hours. Following cessation of mixing and a 1 hour period of settling (to allow phase separation) the aqueous phase, i.e. the WAF, was drawn off for testing. The resulting primary stock solution was then filtered to 0.45μm using a vacuum filter.
Prior to stirring and filtering, the solution appeared clear and colourless with some test substance visible as an oily liquid on the solution surface, following stirring and filtering the solution appeared clear and colourless.
Due to the small quantity of test substance required to prepare the nominal test loading rates via WAF preparation, the primary stock was used to prepare the test solutions by direct addition of the appropriate volume to AAP medium followed by approximately 10 minutes of stirring. After stirring, all test solutions were observed as being clear and colourless. The dilution water control consisted of culture medium only.

Test organisms (species):

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

Details on test organisms:

Scymaris laboratory culture from strain CCAP 278/4. A 3-day old culture of the alga in the exponential growth phase was used as inoculum for the test.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

21.8 -22.2 °C

pH:

At the start of the test the pH ranged from 7.05 to 7.18 and at the end of the test ranged from 7.67 to 7.83

Nominal and measured concentrations:

Nominal loading rate of 2-butyloctanoic acid (mg/L): 0.48, 1.52, 4.9, 15.6, and 50.0
Geometric mean measured loading rate of 2-butyloctanoic acid (Isocarb 12) (mg/L): 0.0506, 0.299, 1.02, 3.22, and 10.6. When the measured value was

Details on test conditions:

This study was run with a control and nominal exposure loading rates of 0.48, 1.52, 4.9, 15.6 and 50 mg/L. The test concentrations were determined based on a range finding test. The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 21.8 - 22.2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.
Six replicates of the control and triplicates of each concentration of the test substance were employed. The position of each test replicate vessel in the incubator was randomised daily. One blank vessel (without algal inoculum) was incubated concurrently for each control and test concentration. Blanks were not randomised daily.
The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 μm respectively. Each replicate test vessel was inoculated with 1.298 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.554 × 104 cells/mL. This value was used for growth calculations.
After 24, 48, and 72 hours, samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.
At the end of the test, microscopic observations were made on a sample taken from a single test replicate of each concentration.

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

5.46 mg/L

95% CI:

4.66 - 6.2

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

8.33 mg/L

95% CI:

7.81 - 8.83

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 10.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.299 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, the ErL50, ErL20 and ErL10 values for 2-butyloctanoic acid (Isocarb 12) obtained from the 0-72 hour growth rate analyses, based on geometric mean measured loading rates, were >10.6, 8.33, and 5.46 mg/L, respectively.

Executive summary:

2-butyloctanoic acid: Determination of toxicity to the green alga Pseudokirchneriella subcapitata

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

 

This study was run with a control and nominal exposure loading rates of 0.48, 1.52, 4.9, 15.6 and 50 mg/L, which were determined from a range finding test.

The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 21.8 -22.2 °C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.

Six replicates of the control and triplicates of each concentration of the test substance were employed. The position of each test replicate vessel in the incubator was randomised daily. One blank vessel (without algal inoculum) was incubated concurrently for each control and test concentration. Blanks were not randomised daily.

The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 μm respectively. Each replicate test vessel was inoculated with 1.298 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.554 × 104 cells/mL. This value was used for growth calculations.

The primary stock solution was prepared using a Water Accommodated Fraction (WAF). These are defined as an aqueous fraction containing the dissolved and/or suspended and/or emulsified fraction of a multi-component substances or a mixture.

A nominal loading rate of 100 mg/L was prepared by adding a nominal 0.2 g of 2-butyloctanoic acid to 2000 mL of culture medium and mixed for a period of time sufficient to achieve an equilibrated concentration of dissolved and dispersed or emulsified components in the aqueous phase. For this study, mixing time was 24 hours. Following cessation of mixing and a 1 hour period of settling (to allow phase separation) the aqueous phase, i.e. the WAF, was drawn off for testing. The resulting primary stock solution was then filtered to 0.45μm using a vacuum filter.

Prior to stirring and filtering, the solution appeared clear and colourless with some test substance visible as an oily liquid on the solution surface, following stirring and filtering the solution appeared clear and colourless.

Due to the small quantity of test substance required to prepare the nominal test loading rates via WAF preparation, the primary stock was used to prepare the test solutions by direct addition of the appropriate volume to AAP medium followed by approximately 10 minutes of stirring. After stirring, all test solutions were observed as being clear and colourless. The dilution water control consisted of culture medium only.

After 24, 48, and 72 hours, samples were removed from each test and blank vessel for analyses. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density. At the end of the test, microscopic observations were made on a sample taken from a single test replicate of each concentration. Concentrations of the test substance were measured at 0 and 72 hours using the validated LC-MS. 

The data for cell counts obtained at 24, 48 and 72 hours was entered into electronic data files and the 0-72 hour data statistically analysed using CETIS. A statistical test was performed to check for significant differences (p <0.05) between the control and test concentrations for each response variable. Further statistical procedures were applied to determine EL50, EL20 and EL10 values along with the associated 95% confidence intervals, where appropriate, for yield and average specific growth rate.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Effect parameters based on growth rate:

NOELR: 0.299 mg/L

ErL50: >10.6 mg/L 

ErL20: 8.33 mg/L (95% confidence interval 7.81 - 8.83 mg/L)

ErL10: 5.46 mg/L (95% confidence interval 4.66 - 6.20 mg/L)

 

Effect parameters based on yield:

NOERL: 0.299 mg/L

ErL50: 6.73 mg/L (95% confidence interval 5.87 - 7.65 mg/L)

ErL20: 3.32 mg/L (95% confidence interval 2.46 - 4.12 mg/L)

ErL10: 2.08 mg/L (95% confidence interval 1.28 - 2.75 mg/L)

 

Description of key information

An algae test for 2-butyl octanoic acid was conducted according to OECD 201 guidelines with Pseudokirchneriella subcapitata. The test was GLP compliant. The results of the GLP test showed that under the test conditions with Pseudokirchneriella subcapitata, the ErL50, ErL20 and ErL10 values for 2-butyloctanoic acid obtained from the 0-72 hour growth rate analyses, based on geometric mean measured loading rates, were >10.6, 8.33, and 5.46 mg/L, respectively. In addition, the toxicity of the similar substance Reaction mass of n-undecanoic-acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid was assessed in a fresh water algal groth inhibition test with Pseudokirchnerella subcapitata according to OECD guideline 201. The ErC10 of 2.7 mg/L (95% CL 2.0 – 3.5 mg/L), NOErC of 1.3 mg/L and the  ErC50 of 13 mg/L (95% CL 11 – 15 mg/L) after 72 hours of exposure can be considered reliable without restrictions. Moreover, the ECOSAR calculation of the test substance 2-butyl octanoic acid confirms that range of acute toxicity with a value of 16.975 mg/L and chronic toxicity with a calculated NOEC of 5.076 mg/L..

Key value for chemical safety assessment

EC50 for freshwater algae:

10.6 mg/L

EC10 or NOEC for freshwater algae:

5.46 mg/L

Additional information

An algae test with 2-butyl octanoic acid was conducted according to OECD 201 guidelines with Pseudokirchneriella subcapitata. The test was GLP compliant. The results of the GLP test showed that under the test conditions with Pseudokirchneriella subcapitata, the ErL50, ErL20 and ErL10 values for 2-butyloctanoic acid obtained from the 0-72 hour growth rate analyses, based on geometric mean measured loading rates, were >10.6, 8.33, and 5.46 mg/L, respectively. An algae test with Reaction mass of 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid is not available. Toxicity in freshwater algae Pseudokirchneriella subcapitata with the similar substance Reaction mass of n-undecanoic-acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid was tested according to OECD guideline No. 201. The study met the acceptability criteria and was conducted GLP compliant, therefore considered reliable without restrictions. The ECOSAR calculation of the test substance 2-butyl octanoic acid also predicts that range of toxicity with a value of 16.975 mg/L.