Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

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:

5 June 2008 - 31 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

67/548/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name: COSMACOL EOI
- INCI: C12-13 Alkyl ethylexanoate
- Composition: Hexanoic acid, 2-ethyl-,C12-15-alkyl esters concentrations >=90 %
- CAS No.: 90411-66-8
- Chelab sample ID: 08/36240/01

Analytical monitoring:

yes

Details on sampling:

In this study checks of test substance concentrations in WAF or evaluations about equilibrium between aqueous phase and solid were not carried out, because like reported in OECD Guideance no 23 (2000(6)) and ASTM D6081-98 (2004) the "loading rate" has therefore been advocated for expressing exposures of mixturees that neither wholly dissolve not completely form a stable dispersion or emulsion over the required test range. The loading rate is the mass to volume ratio of the mixture to medium used in the preparation of a WAF. WAFs may be thus considered analygous to the term "nominal concentration" used for typical test substances, with all the limitations inherent to that term.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Test substance preparation:
By mutual consent with the sponsor for the preparation and general treatment of the test substance the following guidance has been followed:
ENV/JM/MONO(2000)6; OECD series on testing and assessment number 23 guidance document on aquatic toxicity testing of difficult substances and mixtures.

- WAF test (Water Accommodated Fraction):
WAF (Water accommodated fraction) was obtained as follows:
The solutions of teh test substance were prepared by adding the chosen quantity to 30 litres of standard water.
Solutions were mixed for 96 hours at ambient temperature. Following cessation of mixing and 24 hours of settling, the aqueous phase is drawn off for testing.
Part of the aqueous phase was removed and transferred into a separator funnel for some hours. The aqueous fraction was used for the assay after 24 hours of agitation at room temperature.
The WAF are considered at the following nominal values: 25 mg/l, 50 mg/l and 100 mg/l.

- Standard water:
Standard water, used for WAF preparation, has been prepared from the solutions reported below (all prepared with deionised water, conductivity < 10 uScm-1):
NaHCO3              2.59 g/l
CaCL2.2H2O       11.76 g/l
MgSO4.7H2O       4.93 g/l
KCl                       0.23 g/l
25 ml of each solution (A-D) are mixed together and this solution is diluted to a final volume of 1 litre with deionised water. Standard water is aerated for ca. 24 h until the pH is stable and the oxygen uptake rate of the solution reaches equilibrium with air.
Standard water used for the test had the following parameters:

pH 7.8 ±0.2;
Total alkalinity of the solution was 110 - 120 mg CaCO3/l;
Ratio of Ca:Mg ions 4:1
Dissolved oxygen concentration > 7 mg/l
The solution can be stored at 20 ±2 °C for 15 days.

- Acetone solubilisation test:
The solvent used for solubilisation of the test substance in dilution water was acetone. 0.5 g of the test substance are dissolved in 10 ml of acetone and diluted to a final concentration of 5 mg/l. The acetone concentration in the final test solution is lower than 100 mg/l and its toxicity at this concentration is checked to evaluate any eventual interference with the IC50 determination in teh test substance.

Test organisms (species):

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

Details on test organisms:

The test is performed using the kit Algaltox, microalgae (species Selenastrum capricornutum ATCC 22662 also classified as Pseudokircneriella subcapitata).

- Pre-culture inoculum:
Exponentially growing algae are pre-cultured; the cellular density was evaluated in order to calculate the volume of inoculum for the test.
The pre-culture was prepared following the instructions of the reference reported below:
Algatoxkit F TM Freshwater Toxicity Test with microalgae_standard operational procedure 7.5.
The algae were in a vial, soaked in a maintenance medium. When the medium was removed, the algae were re-suspended in 5 ml of matrix dissolving medium, vortex and centrifuged at 3,000 rpm for 10 min.
When the supernatant is removed, the algae are re-suspended in 10 ml of deionised water then centrifuged at 3,000 rpm for 10 minutes and the supernatant removed. The pellet (with algae) is re-suspended in 25 ml of standard water.
The cells were counted with a Burker cytometer.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Total alkalinity of the solution was 110 - 120 mg CaCO3/l

Test temperature:

The solution was stored at 20 ±2 °C

pH:

pH 7.8 ±0.2

Dissolved oxygen:

Dissolved oxygen concentration > 7 mg/l

Details on test conditions:

The matrix where they are immobilised allows them to survive for many months without losing any vitality. The algae are de-immobilised and then transferred in a adapted culture medium where they immediately start to grow. Exponentially growing algae are exposed to different concentrations of the test substance in batch cultures over a period of normally 72 hours for various generation (24-48-72 hours). The growth inhibition after 72 hours induced by the test substance compared with the control culture is determined.

- Apparatus:
AlgalTox kit (Ecotox MicroBioTests batch DM110208 exp. date 31/10/2008).
Glassware and laboratory plastics without adsorption occurence or release of substances that could interfere with the assay
Illuminated thermostatic refrigerator (ECOTOX No. int. 2064)
Conductivity and oxygen meter (YSI No. int. 2046)
pH meter (THERMO No. int. 2047)
Refrigerator (LIEBHERR No. int. 2055)
Optic microscope
Burker cytometer

- Test substance assay:
For each concentration of the test substance and of the control, conical culture flasks were used.
In order to obtain an acceptable statistic of trhe algal growth inhibition al the tests were carried out in triplicate (501 ml for each sample). The algal inoculum is calculated in order to get a starting concentration of 104cell/ml and in order to leave the concentration of the test substance unaltered (i.e. considering dilution factors). pH is measured at the beginning of the test.

- Incubation:
All the flasks were covered to avoid:
• Air contaminants;
• Excessive evaporation of water;
• To allow the passage of CO2
The flasks were then transferred to a thermostatic refrigerator (23 ±2 °C) under continuous illumination conditions (intensity range 6,000 to 10,000 lux) with agitation.

- Assay:
- Pre-culture inoculum:
Exponentially growing algae are pre-cultured; the cellular density was evaluated in order to calculate the volume of inoculum for the test.
The pre-culture was prepared following the instructions of the reference reported below:
Algatoxkit F TM Freshwater Toxicity Test with microalgae_standard operational procedure 7.5.
The algae were in a vial, soaked in a maintenance medium. When the medium was removed, the algae were re-suspended in 5 ml of matrix dissolving medium, vortex and centrifuged at 3,000 rpm for 10 min.
When the supernatant is removed, the algae are re-suspended in 10 ml of deionised water then centrifuged at 3,000 rpm for 10 minutes and the supernatant removed. The pellet (with algae) is re-suspended in 25 ml of standard water.
The cells were counted with a Burker cytometer.
 
- Test substance assay:
For each concentration of the test substance and of the control, conical culture flasks were used.
In order to obtain an acceptable statistic of trhe algal growth inhibition al the tests were carried out in triplicate (501 ml for each sample). The algal inoculum is calculated in order to get a starting concentration of 104cell/ml and in order to leave the concentration of the test substance unaltered (i.e. considering dilution factors). pH is measured at the beginning of the test.
Incubation
All the flasks were covered to avoid:
• Air contaminants;
• Excessive evaporation of water;
• To allow the passage of CO2
The flasks were then transferred to a thermostatic refrigerator (23 ±2 °C) under continuous illumination conditions (intensity range 6,000 to 10,000 lux) with agitation.

- Counting:
Every 24 h (up to 72 h total), algae are counted with a Burker cytometer to determine the algal density.
At the end of the test, the pH is measured for each concentration of the test substance and of the control.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Lot: 7G062107G from Carlo Erba)

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

< 0.001 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (total fraction)

Remarks:

Acetone solubilisation

Basis for effect:

growth rate

Remarks on result:

other: Result disregarded due to testing method.

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

42.39 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (total fraction)

Remarks:

WAF

Basis for effect:

growth rate

Details on results:

See tables in relevant section.

Validity of the test
For a test to be valid the follo9wing conditions should be fullfilled:
1) At least a 16 fold growth in control cultures at the end of the test (72 hours) must be shown;
2) pH variation can be at least 1.5 units
3) Potassium dichromate test result must give an ErC50 range between 0.6 and 1.3 mg/l.
All three validation criteria were met.

When possible, data were analysed by statistical software http://www.epa.gov/eerd/stat2.htm#icp.

Acetone solubilisation test (72 h)

Start inoculum = 5.33 x 10⁴

Number of replicates	Concentration (mg/l)	Average response	CV%
3	0	5533333.333	4.28
3	0.00025	5433333.333	4.63
3	0.00045	4733333.333	4.4
3	0.0009	196666.667	7.77

IrC50 (72 h) <0.0009 mg/l

Test start pH 7.14

Test finish pH 7.12

WAF test (72 h)

Start inoculum = 6.0 x 10⁴

Number of replicates	Concentration (mg/l)	Average response	CV%
3	0	5100000	5.88
3	25	4700000	4.85
3	50	1600000	6.25
3	100	626666.667	4.88

IrC50 (72 h) 42.3898 mg/l (40.0524 - 44.8498)

SD of 0.6613

Test start pH 7.01

Test finish pH 7.16

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour IrC50 of the test material to Pseudokirchneriella subcapitata was determined to be 42.3898 mg/l (± 0.6613) under the conditions of the test.

Executive summary:

In this guideline (OECD 201, EC C.3) study, conducted with GLP certification. The test material (EC 291 -443 -0) was determined to have a 48 hour IrC50 of 42.3898 mg/l to Pseudokirchneriella subcapitata. The study was conducted as a static test using the Water Accomodated Fraction (WAF) test solution preparation method.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

42.39 mg/L

Additional information

The result of the key study is reported as an IC50 (growth rate), which for the purposes of classification and labelling is interpred as an EC50 (growth rate).