Isotridecyl isononanoate

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:

08 and 12 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isotridecyl isononanoate

EC Number:

255-673-5

EC Name:

Isotridecyl isononanoate

Cas Number:

42131-27-1

Molecular formula:

C22H44O2

IUPAC Name:

Isotridecyl isononanoate

Specific details on test material used for the study:

Test substance name: Isotridecyl isononanoate
Scymaris reference number: 1014TS074
Physical state: Liquid
Lot/batch number: 0001067681
Certificate of Analysis Re-test date: 19 April 2018
Sample storage: Room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

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.
Samples taken at the start of the study (0 hours) were taken from excess solutions. 100 mL sample was added to a 200 mL volumetric flask and 2 mL 0.5 mg/L D10-Phenanthrene in hexane is added. The contents were mixed for 2 minutes, made up to 200 mL with water and an aliquot taken from the hexane layer for analysis. This constituted a 50:1 extraction ratio.
Samples taken at 72 hours were prepared from test vessels, where 100 mL (whole contents of test vessel) was poured into a 200 mL volumetric flask, and 2 mL 0.5 mg/L D10-Phenanthrene in hexane was added. The contents were mixed for 2 minutes, made up to 200 mL with water and an aliquot taken from the hexane layer for analysis. This constituted a 50:1 extraction ratio.

Test solutions

Vehicle:

yes

Details on test solutions:

The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium.
This study was run with a culture medium control, solvent control and nominal exposure concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L.
Primary stock concentrates of Isotridecyl isononanoate with nominal concentrations of 10 and 20 g/L were prepared by weighing a nominal 0.1 and 0.2 g of test substance (actual weights: 0.09987 and 0.19990 g respectively) directly into 10 mL volumetric flasks and made up to volume with tetrahydrofuran (THF). The stocks were both observed to be clear and colourless solutions.
Test solutions were prepared by the direct addition of the appropriate amount of concentrate to dilution water via a microliter syringe into a stirring solution in a volumetric flask. The solvent control was prepared in the same way using solvent only. The control consisted of culture medium only.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3-day old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.7-22.1

pH:

7.16-8.63

Nominal and measured concentrations:

Nominal concentration 0.125 mg/L : Mean measured concentration 0.055 mg/l
Nominal concentration 0.25 mgl/L : Mean measured concentration 0.10 mg/l
Nominal concentration 0.5 mgl/L : Mean measured concentration 0.25 mg/l
Nominal concentration 1.0 mgl/L : Mean measured concentration 0.48 mg/l
Nominal concentration 2.0 mgl/L : Mean measured concentration 1.0 mg/l

Details on test conditions:

Test procedure and apparatus
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 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 culture medium control and solvent 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 sampling occasion.
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 0.462 mL of the inoculum culture to give a nominal cell density of 0.5 × 10^4 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.410 × 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 samples taken from a single replicate of the control and each test substance concentration.

Control : 0µL stock, 0 µL additional THF, made up to 1000mL with dilution water
Solvent Control : 0µL stock, 100 µL additional THF, made up to 1000mL with dilution water
0.125 mg/L : 12.5 µL stock (10 g/L), 88 µL additional THF, made up to 1000 mL with dilution water
0.25 mgl/L : 25 µL stock (10 g/L), 75 µL additional THF, made up to 1000 mL with dilution water
0.5 mgl/L : 50 µL stock (10 g/L), 50 µL additional THF, made up to 1000 mL with dilution water
1.0 mgl/L : 100 µL stock (10 g/L), 0 µL additional THF, made up to 1000 mL with dilution water
2.0 mgl/L : 100 µL stock (20 g/L), 0 µL additional THF, made up to 1000 mL with dilution water

All test solutions were observed as clear and colourless. Due to the low solubility of the test substance observed in the solubility trial and range finding test, the test solutions were dispensed to test replicates from vigorously stirring solutions with re-stirring or vigorous shaking between each pouring to ensure homogeneity of the dispensed solutions.
A solvent was used in this study to assist in dosing the test compound due to its apparent low solubility in test media, the concentration of solvent used in all exposure solutions with the exception of the control was 100 μL/L.
The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.

To demonstrate that nominal exposure concentrations were being achieved, the concentrations of Isotridecyl isononanoate in the test solutions were measured using gas chromatography / mass spectrometry.

At the start of the test, samples were taken from excess solutions. At the end of the test, one test vessel from each concentration was taken for whole sample analysis (all three test vessels taken for nominal 0.25 mg/L test concentration). The method of whole sample analysis was used due to the low solubility of the compound.

The pH of the control and test solutions was measured at the start of the test with a calibrated pH meter, using the excess remaining after filling the test vessels. At the end of the test the pH of one replicate vessel (containing algae) from each test concentration was determined.
The temperature of the incubator was measured daily. The light intensity was measured once during the study, in each of four representative positions, using a photometer reading in lux (cosine).

The data for cell counts obtained at 24, 48 and 72 hours was entered into electronic data files and statistically analysed using CETIS, where appropriate procedures were applied to test for significant differences (p <0.05) between the controls and test concentrations and determine EC50, EC20 and EC10 values and the associated 95% confidence intervals where appropriate. The individual statistical procedures applied to each set of data are detailed within the results.
Lowest Observed Effect Concentration (LOEC) is defined as the lowest tested concentration at which the substance is observed to have a statistically significant reducing effect on growth (at p <0.05) when compared with the controls, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. The No Observed Effect Concentration (NOEC) is defined as the test concentration immediately below the LOEC.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical data
All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the study were 81-104% of nominal and at the end were 18-34% of nominal.
Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ was less than 30% and less than 20% at levels greater than the LOQ.
On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

Biological data
Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass.

Growth rates
The growth rate (0 to 72 hours) was calculated for each replicate culture, according to the formula:

Growthrate = ( Ln ( N2 / N1 ) ) / t

where:
N1 = Cell density at start of test
N2 = Cell density at end of test
t = Time interval (days)

The growth rates were examined by one-way analysis of variance and a two-sample t test to identify significant differences (p<0.05) between the control and solvent control. There was a significant effect (p=0.0021) between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). ECx was calculated using the linear interpolation (ICPIN) method.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:
NOEC = 1.0 mg/L
LOEC > 1.0 mg/L
ErC50 > 1.0 mg/L
ErC20 > 1.0 mg/L
ErC10 > 1.0 mg/L

Based on the growth rate, there was a significant difference (p <0.05) between the mean measured 0.25 mg/L concentration and the solvent control. However, as the growth rate was not significantly different (p <0.05) from the solvent control in any of the higher concentrations, the LOEC was not affected.

Additional biological data
The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the solvent control and all test concentrations appeared normal.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on growth rate at the end of the test, the results obtained after 72 hours were:
NOEC = 1.0 mg/L
LOEC > 1.0 mg/L
ErC50 > 1.0 mg/L
ErC20 > 1.0 mg/L
ErC10 > 1.0 mg/L