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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 June 2008 - 31 July 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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:

no

Details on sampling:

In this study, checks of test substance concentrations in WAF or evaluations about equilibrium between aqueous phase and solid phase were not carried out because, in line with OECD Guidance No. 23 200(6) and ASTM D6081 -98 (2004), the loading rate has been advocated for expressing exposures of mixtures that neither wholly dissolve nor 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 this considered analogous to the term 'nominal concentration' used for typical substances, with all the limitations inherent to that term.

Vehicle:

no

Details on test solutions:

- Standard water:
All solutions and dilution series of teh test substance were prepared according to ISO 6341:1996/corr1:1998 in standard water with the following characteristics:
pH 7.8 ±0.2;
Total alkalinity of the solution was 110 - 120 mg CaCO3/l;
Hardness: 250 mg/l ± 25 mg/l (CaCO3);
Ratio of Ca:Mg ions: 4:1
Dissolved oxygen concentration: > 7 mg/l
Composition
NaHCO3: 2.59 g/l
CaCL2.2H2O: 11.76 g/l
MgSO4.7H2O: 4.93 g/l
KCl: 0.23 g/l
Standard water was aerated for 24 hours or until the dissolved oxygen concentration equaled the air saturation value.

- Test substance preparation:
- Test substance treatment:
Preparation and treatment of the test substanmce are carried out according to: ENV/JM/MONO(2000)6; OECD series on testing and assessment number 23 guidance document on aquatic toxicity testing of difficult substances and mixtures.

- Limit test for direct addition:
A limit test was performed at 100 mg (active ingredient)/l, a concentration of test substance higher than the reported substance solubility, by direct addition of the test substance to the water. It should be noted that a high level of uncertainty is associated with solubility estimates for a poorly water-soluble substance like Cosmacol EOI. 100 mg of test substance were directly added to 100 ml of standard water.

- Test with acetone solubilisation:
9 mg of the test substance was dissolved in 10 ml of acetone. This solution was diluted up to 0.99 mg/l. The acetone concentration in the final test was below 100 mg/l. Controls were performed with acetone solutions in standard water without the test substance.

- Test with WAF (Water Accomodated Fraction):
WAF was not prepared by serial dilution of a single stock WAF according to ASTM D6081 -98(2004), but each concentration was prepared by adding the chosen quantity to 30 litres of standard water.
Solutions were mixed for 96 hours. 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 6 hours. The definitive aqueous phases obtained were aerated by stirring for 24 hours and were then considered WAF for Cosmacol EOI at various concentrations: 0.08 mg/l, 0.4 mg/l, 2 mg/l, 10 mg/l and 100 mg/l.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source:
Young daphnids obtained from dormant eggs (ephippia DaphtoxKit F) were used for the test according to the following procedure:
The contents of one vial of ephippia were poured into a microseive and rinsed thoroughly with tap water to eliminate all traces of the storage medium.
The ephippia were transferred into the hatching petri dish (a 10 cm diameter dish) in 50 ml standard freshwater, prepared by air bubbling. The petri dish was covered and incubated for 72 - 90 hours at 21 ±1 °C, under continuous illumination (6,000 to 8,000 lux).
- Feeding during test: Not specified

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/l ± 25 mg/l (CaCO3). Total alkalinty was 110 - 120 mg CaCO3/l

Test temperature:

21 ± 1C

pH:

7.8 ±0.2

Dissolved oxygen:

> 7 mg/l
Standard saturated water was aerated for 24 hours or until the dissolved oxygen concentration equalled the air saturation value.

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Direct test material addition: 100 mg/l (nominal)
Acetone Stabilisation: 0.09 mg/l, 0.009 mg/l, 0.0009 mg/l (nominal)
WAF: 0.08, 0.4, 2, 10, & 100 mg/l (nominal)

Details on test conditions:

- Test principles:
Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test substance at a range of concentrations for a period of 48 hours. Immobilisation is recorded at 48 hours and compared with control values. The results are analysed in order to calculate the EC50 at 48 h.

- Test Method:
- Apparatus:
DaphTox kit (Ecotox MicroBioTests lot DM110208 exp. date 31/10/2008)
Microplate test containers constructed of biologically inert materials
Other apparatus that will come into contact with the test solutions to be made entirely of chemically inert material
Incubator (ECOTOX No. int 2064)
pH meter (THERMO No. int. 2047)
Conductivity and oxygen meter (YSI No. int. 2046)
Refrigerator (LIEBHERR No. int. 2055)

- Assay:
- Test setup with direct addition:
The bioassays are conducted in disposable multiwell test plates with 30 test wells. Each plate is provided with 4 wells fo rthe controls and 4 wells (A, B, C, D) for each test substanceconcentration. Additionally, the plates are provided on the left side with a column of rinsing wells to prevent dilution of the test substance during the transfer of the neonates from teh hatching petridish to the test wells. The wells are labelled vertically as rows X for the controls and 1 to 5 for the test substance dilution.
Each well of the test plates was filled with 10 ml test substance solution (or standard freshwater in the control column).
Assay was performed in quintuplicate for each dilution with 20 animals divided into four groups of five animals each.
A parafin strip and the cover were put on the multiwell plate and incubated at 21 ±1 °C for 48 hours with an illumination cycle of 16 hours light and 8 hours dark. The test wells were not aerated during the test. The test was carried out without adjustment of pH. The daphnids were not fed during the test.
After the incubation period the test plates were put on the transparent stage of the light table with a dark light strip and the number of dead and immobilised test organisms was determined.
The daphnids which were not able to swim after gentle agitation of the liquid for 15 seconds were considered to be immobilised, even if they could still move their antennae.
Multiwell Plate:

- Test with acetone solution:
The bioassays are conducted in disposable multiwell test plates with 30 test wells. Each plate is provided with 4 wells fo rthe controls and 4 wells (A, B, C, D) for each test substanceconcentration. Additionally, the plates are provided on the left side with a column of rinsing wells to prevent dilution of the test substance during the transfer of the neonates from teh hatching petridish to the test wells. The wells are labelled vertically as rows X for the controls and 1 to 5 for the test substance dilution.
Each well of the test plates was filled with 10 ml test substance solution (or standard freshwater in the control column).
Assay was carried out with test substance concentrations of 0.09 mg/l, 0.009 mg/l, 0.0009 mg/l.
9 mg of the test substance was dissolved in 10 ml of acetone and then diluted to the test concentrations reported above. .
Assay was performed in triplicate for each dilution with 20 animals divided into four groups of five animals each.
A parafin strip and the cover were put on the multiwell plate and incubated at 21 ±1 °C for 48 hours with an illumination cycle of 16 hours light and 8 hours dark. The test wells were not aerated during the test. The test was carried out without adjustment of pH. The daphnids were not fed during the test.
After the incubation period the test plates were put on the transparent stage of the light table with a dark light strip and the number of dead and immobilised test organisms was determined.
The daphnids which were not able to swim after gentle agitation of the liquid for 15 seconds were considered to be immobilised, even if they could still move their antennae.

- WAF Test (Water Accommodated Fraction):
The bioassays are conducted in disposable multiwell test plates with 30 test wells. Each plate is provided with 4 wells fo rthe controls and 4 wells (A, B, C, D) for each test substanceconcentration. Additionally, the plates are provided on the left side with a column of rinsing wells to prevent dilution of the test substance during the transfer of the neonates from the hatching petridish to the test wells. The wells are labelled vertically as rows X for the controls and 1 to 5 for the test substance dilution.
The test was performed with five WAFs: 5 mg/l, 10 mg/l, 25 mg/l, 50 mg/l and 100 mg/l of product.
Assay was performed in triplicate for each dilution with 20 animals divided into four groups of five animals each.
A parafin strip and the cover were put on the multiwell plate and incubated at 21 ±1 °C for 48 hours with an illumination cycle of 16 hours light and 8 hours dark. The test wells were not aerated during the test. The test was carried out without adjustment of pH. The daphnids were not fed during the test.
After the incubation period the test plates were put on the transparent stage of the light table with a dark light strip and the number of dead and immobilised test organisms was determined.
The daphnids which were not able to swim after gentle agitation of the liquid for 15 seconds were considered to be immobilised, even if they could still move their antennae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Lot: 7G062107G from Carlo Erba)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

83.45 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (total fraction)

Remarks:

WAF

Basis for effect:

mobility

Details on results:

Data were analysed by statistical software http://www.epa.gov/eerd/stat2.htm#probit.

Results for limit test (direct addition)
100 daphnids (in quintuplicate, 20 animals divided into four groups of five animals each)
Total number of dead and % effect
immobilised test organsims/100
Control 0/100 0%
Cosmacol EOI (100 mg/l) 100/100 100%
100/100 100%
100/100 100%
100/100 100%
100/100 100%

It is important to note that in this test the substance made micelles in the water that trapped daphnids and kept the correct oxygenation.

Results for acetone solubilisation
A) Test with test substance and 60 daphnids (in triplicate, 20 animals divided into four groups of five animals each).

Total number of dead and % effect
immobilised test organsims/100
Control 0/60 0%
Cosmacol EOI 0.09 mg/l 0/60 0%
Cosmacol EOI 0.009 mg/l 0/60 0%
Cosmacol EOI 0.0009 mg/l 0/60 0%

B) Control test acetone diluted in standard water without test substance, and 60 daphnids (in triplicate, 20 animals divided into four groups of five animals each).

Total number of dead and % effect
immobilised test organsims/100
Control 0/60 0%
Acetone 1 % 30/60 50%
Acetone 0.5 % 5/60 8.3%
Acetone 0.25 % 0/60 0%
Acetone 0.1 % 0/60 0%
Acetone 0.01 % 0/60 0%

WAFs Test

Total number of dead and
immobilised test organsims/100 % effect
Control 0/60 0%
Cosmacol EOI (100 mg/l) 45/60 75%
Cosmacol EOI (75 mg/l) 18/60 30%
Cosmacol EOI (50 mg/l) 5/60 8%
Cosmacol EOI (25 mg/l) 0/60 0%
Cosmacol EOI (10 mg/l) 0/60 0%
Cosmacol EOI (5 mg/l) 0/60 0%

EC50 48 hours = 83.45 mg/l 95 % confidence limits 77.84 mg/l; 90.45 mg/l
X² tabulated value (0.05) 9.488 X² tabulated value (0.05) 2.456

WAFs Test

	Total number of dead and immobilised test organsims/100	% effect
Control	0/60	0%
Cosmacol EOI (100 mg/l)	45/60	75%
Cosmacol EOI (75 mg/l)	18/60	30%
Cosmacol EOI (50 mg/l)	5/60	8%
Cosmacol EOI (25 mg/l)	0/60	0%
Cosmacol EOI (10 mg/l)	0/60	0%
Cosmacol EOI (5 mg/l)	0/60	0%

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EC50 of the test material to Daphnia Magna was determined to be 83.45 mg/l (95% confidence limits 77.84 mg/l - 90.45 mg/l) under the conditions of the test.

Executive summary:

In this guideline (OECD 202, EU C.2) study, conducted with GLP certification, the 48 hour EC50 of the test material (EC 291 -443 -0) to Daphnia Magna was determined to be 83.45 mg/l (95% confidence limits 77.84 mg/l - 90.45 mg/l) under the conditions of the test. The test was conducted under static conditions, using three test solution preparation methods (direct addition, acetone stabilisation, and WAF), only the results of the WAF test were considered to be reliable.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

83.45 mg/L