Ethyl 3,5,5-trimethylhexanoate

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:

From 26 September 2016 to 25 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Ethyl 3,5,5-trimethylhexanoate

EC Number:

266-959-4

EC Name:

Ethyl 3,5,5-trimethylhexanoate

Cas Number:

67707-75-9

Molecular formula:

C11H22O2

IUPAC Name:

ethyl 3,5,5-trimethylhexanoate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

-Verification of test concentrations:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

-Culture Medium:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The media used in both the range-finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).

-Experimental preparation:
A nominal amount of test item (25 mg) was dispersed in 500 mL of culture medium (6 replicates) with the aid of orbital shaking at approximately 300 rpm for 24 hours. After 24 hours the shaking was stopped, the replicates pooled, and any undissolved test item removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution.

An aliquot (1500 mL) of each of the stock solutions was separately inoculated with 11.5 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

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

Details on test organisms:

-Test system and supporting information:
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. The master cultures were maintained in the laboratory under constant aeration and constant 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 10^3 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 10^4 - 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

7.8 to 8.4 at test initiation
8.4 to 10.0 at test termination

This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Nominal and measured concentrations:

-Nominal test concentration (% v/v saturated solution):
6.25, 12.5, 25, 50, 100 (%)
-0-hour measured test concentration:
1.18, 2.27, 4.62, 9.62, 19.0 (mg/L)
-72-hour measured test concentration:
0.906, 1.77, 3.66, 7.43, 13.5 (mg/L)
-Geometric mean measured test concentration:
1.0, 2.0, 4.1, 8.5, 16 (mg/L)

Details on test conditions:

-Exposure condition:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each completely filled were used for the control and three flasks each completely filled 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 6.55 x 10^5 cells per mL. Inoculation of 1500 mL of test medium with 11.5 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were sealed with ground glass stoppers 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.

-Test organism observations:
Samples were taken at 24, 48 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 10^3 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. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Reference substance (positive control):

yes

Remarks:

A positive control (Envigo Study Number MG09PL) used potassium dichromate as the reference item. The positive control was conducted between 07 December 2015 and 10 December 2015.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

-Growth data:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2.

Percentage inhibition values are plotted against test concentration in Figure 2.

-Validation criteria:
The following data show that the cell concentration of the control cultures increased by a factor of 102 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 5.09 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 24% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

A positive control (Envigo Study Number MG09PL) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
EyC50 (0 – 72 h) : 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 1.0 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

See attached Table 2 and Figure 2 for details on results

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

ErC10 (0 - 72 h): 3.8 mg/L
ErC50 (0 - 72 h): 9.2 mg/L; 95% confidence limits 8.1 - 11 mg/L

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Information provided by the Sponsor indicated the test item was insoluble in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 17 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by dispersing an excess (50 mg/L) of test item in culture medium with the aid of orbital shaking at approximately 300 rpm for 24 hours. After the shaking period any undissolved test item was removed by filtration (0.2 μm Gelman Acrocap filter, first approximate 100 mL discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.2 to 19 mg/L. A slight decline in measured test concentration was observed at 72 hours to between 0.91 and 14 mg/L (71% to 79% of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the the data.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

ErC10 (0 - 72 h): 3.8 mg/L

ErC50 (0 - 72 h): 9.2 mg/L; 95% confidence limits 8.1 - 11 mg/L