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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 the test substance concentration in WAF or evaluation about equilibrium between aqueous phase and solid were not carried out, because like reported in OECD Guidance No. 23 (2000(6)) and AST D6081-98 (2004) the "loading rate" has therefore been advocated for expressing exp[osures 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 prepartion of a WAF. WAFs may be thus considered analogous to the term "nominal concentration" used for typical test substances, with all the minitations inherent to that term.

Vehicle:

no

Details on test solutions:

- STANDARD WATER:
Standard water used for tests and WAF was prepared from the following stock solutions:
A. NaHCO3              2.59       g/l
B. CaCL2.2H20       11.76       g/l
C. MgSO4.7H2O       4.93       g/l
D. KCl                     0.236       g/l
25 ml of each of the four stock solutions were mixed and made up to 1 litre with deionised water (conductivity < 5 uScm-1)
The obtained solution was aerated until the diossolved oxygen concentration equalled the air saturation value. The pH was 7.8 ±0.2; the ratio of Ca:Mg ions was 4:1 and of Na:K ions was 10:1. The total alkalinity of the solution was 0.8 mmol per litre.

- TEST SUBSTANCE TREATMENT:
Preparation and treatment of the test substrate were 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 substanmces and mixtures.

- Limit test for direct addition:
A limt test was performed at 100 mg(active ingredient)/l, a concentration of test substance higher than 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. A 48 hour period of stirring was performed to achieve maximum solubility.

- WAF limit test (water accommodated fraction):
WAF was not prepared by serial dilution of a single stock WAF according to ASTM D6081 -98 (2004).
3,000 mg of test substance (final concentration of 100 mg/l) was added directly to 30 litres of water and mixed for a period of 96 hours. A 24 period of settling followed cessation of mixing and the aqueous phase was then drawn off for testing.
Part of the aqueous phase was removed and transferred into a separator funnel for a number of hours (unspecified). The definitive aqueous phase obtained was aerated by stirring for 24 hours and it was considered WAF at 100 mg/l of Cosmacol EOI.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Organism: Brachydanio rerio
- Strain: Teleostei, Cyrinidae (Hamilton Buchanan)
- Length at study initiation (length definition, mean, range and SD): 2.1 ± 1.0 cm

ACCLIMATION
All fishes were held in the laboratory for at least 12 days before they are used for testing. They were held in water of the quality to be used in the test for at least seven days immediately before testing and under the following conditions:
- Light: 12 to 16 hours photoperiod daily
- Temperature: 22 ±1 °C
- Oxygen concentration: at least 80 per cent of air saturation value
- Feeding: daily until 24 hours before the test is started

FEEDING DURING TEST
- Frequency: None

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

CaCl2 x 2H2O 11.76 g/l

Test temperature:

22 °C ±1 °C

pH:

7.8 ± 0.2

Dissolved oxygen:

Not less than 60 % of the air-saturation value at the selected temperature

Salinity:

Not applicable.

Conductivity:

<5 uScm-1

Nominal and measured concentrations:

100 mg/l direct addition (nominal)
100 mg/l loading rate (nominal)

Details on test conditions:

TEST METHOD
Apparatus
For the kind of toxicity test performed, in which no flow of the test solution occurs (solutions remain unchanged throughout the duration of the test), glass tanks (10 litres) were used. The tanks were equipped with adequate apparatus for temperature and illumination control
Prior to the test, the animals were stabilised in coagulation baths with water recycle through carbon filters. An adequate level of oxygenation was provided by bubbling air into the tanks which were equipped with programmable illumination and temperature control.
The pH meter was Thermo no int. 2047.
The conductivity and oxygen meter was YSI no. int. 2046.

ASSAY
Direct addition limit test
10 fish were exposed in static conditions to the substance, directly added (100 mg/l) to 10 litres of standard water as described below:
Duration: 96 hours
Illumination: 12 to 16 hours illumination daily
Temperature: 22 ±1°C
Dissolved oxygen concentration: not less than 60 % of the air-saturation value at the selected temperature
Feeding: none.
The fish were inspected after the first 2 to 4 hours and at least at 24 hour intervals. The fish were considered dead if touching the caudal peduncle produced no reaction and no breathing movements were visible. Records were kept of visible abnormalities (e.g. loss of equilibrium, changes in swimming behaviour, respiratory function, pigmentation etc.).
Measurements of pH, dissolved oxygen and temperature were carried out daily.

WAF limit test (Water Accommodated Fraction)
10 fish were exposed in a static condition in 10 litres of WAF (100 mg/l) obtained from the test substance as described below:
Duration: 96 hours
Illumination: 12 to 16 hours illumination daily
Temperature: 22 ±1°C
Dissolved oxygen concentration: not less than 60 % of the air-saturation value at the selected temperature
Feeding: none.
The fish were inspected after the first 2 to 4 hours and at least at 24 hour intervals. The fish were considered dead if touching the caudal peduncle produced no reaction and no breathing movements were visible. Records were kept of visible abnormalities (e.g. loss of equilibrium, changes in swimming behaviour, respiratory function, pigmentation etc.).
Measurements of pH, dissolved oxygen and temperature were carried out daily.
One control (without the test substance) was run.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

Direct addition of test material

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

Loading rate

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 ng/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

Direct addition of test material

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

Loading rate

Basis for effect:

mortality (fish)

Details on results:

Direct addition limit test
The fish were inspected after the first 2 to 4 hours and at least at 24 intervals. All fish in the first 4 hours did not exhibit visible abnormalities. After 24 - 78 and 72 hours, all fish were alive and did not show visible abnormalities. At the end of the test (after 96 hours) all fish were alive and in good health.

WAF limit test
The fish were inspected after the first 2 to 4 hours and at least at 24 intervals. All fish in the first 4 hours did not exhibit visible abnormalities. After 24 - 78 and 72 hours, all fish were alive and did not show visible abnormalities. At the end of the test (after 96 hours) all fish were alive and in good health.

Test validity
For a test to be valid, the following conditions should be fulfilled at the end of the assay:
The mortality in the control(s) should not exceed 10 % (or one fish if less than ten are used) at the end of the test;
The dissolved oxygen concentration must have been at least 60 % of the air saturation value throughout the test.
In this study, checks of test substance concentration in WAF or evaluations about equilibrium between aquesous phase and solid phase were not carried out. Based on the lack of test substance solubility and OECD Guidance No. 23 2000(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 (Girling et al., 1992). The loading rate is the mass to volume ratio of the mixture to medium used in the preparation of a WAF. WAFs may thus be considered analogous to the term nominal concentration used for typical test substances, with all the limitations inherent to that term.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Not applicable.

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 of the test material was determined to be >100 mg/l and the 96 hour NOEC of the test material was determined to be 100 mg/l (the highest concentration tested), under the conditions of the test.

Executive summary:

In this guideline (OECD 203, EU C.1) study, conducted with GLP certification, the test material (EC 291-443-0) was determined to have the following effect thresholds:

LC50: >100 mg/l

NOEC: 100 mg/l

Testing was performed as a limit test and in duplicate, using both the direct addition method and the Water Accomodated Fraction (WAF) methods of test solution preparation. Testing was conducted as a static test in both cases. No effects were observed on the test species (Danio rerio) during the conditions of the test.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Additional information

96 hour LC50: >100 mg/l

96 hour NOEC: 100 mg/l