hydrogenation products of (esterification products of 2-ethylhexan-1-ol with (Estolide formation products of oleic acid and Fatty acids, C8-18 and C18-unsatd. (branched or linear)).

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:

Experimental Starting Date: 22 April 2013 Experimental Completion Date: 21 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 10 g/L loading rate WAFs
- Sampling method: Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and the 10 g/L loading rate WAF test group
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).

Reconstituted water used for both the range-finding and definitive tests. The reconstituted water was made up as follows:

Stock Solutions
a) CaCl2.2H2O 11.76 g/L
b) MgSO4.7H2O 4.93 g/L
c) NaHCO3 2.59 g/L
d) KCl 0.23 g/L


Preparation:
An aliquot (25 mL) of each of solutions a-d was added to each liter (final volume) of deionized water with a conductivity of <5 μS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 21 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

Temperature was maintained at 21 °C to 24 ºC throughout the test.

pH:

There were no treatment related differences for pH.

Dissolved oxygen:

There were no treatment related differences for dissolved oxygen concentration.

Salinity:

Not applicable as the study is a freshw ater study

Nominal and measured concentrations:

Range finding test: nominal concentrations of 1, 10 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
In the initial test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20 °C to 22 °C with a photoperiod of 16 hours light (603 to 612 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/L to confirm that no immobilization or adverse reactions to exposure were observed.
- Range finding study:
- Test concentrations:1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/L to confirm that no immobilization or adverse reactions to exposure were observed.

Additional test (required for other world wide notifications):
At the request of the Sponsor, an additional "Limit test" was conducted at a single loading rate of 10 g/L to confirm that no immobilization or adverse reactions to exposure were observed at this higher nominal loading rate.

An amount of test item (20 g) was added to the surface of 2 liters of reconstituted water to give the 10 g/L loading rate. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 10 g/L loading rate WAF.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 10 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Range finding test:
No immobilization was observed at 1.0, 10 and 100 mg/L loading rate WAF.

Initial test:
No immobilization was observed at 100 mg/L loading rate WAF.

At the request of the Sponsor, a single loading rate of four replicates, of 10 g/L, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

Definitive test:
There was no immobilization in 20 daphnids exposed to a 10 g/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EL 50 (g/L loading rate WAF)
24 > 10
48 > 10

Results with reference substance (positive control):

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

Based on this information, a single loading rate of four replicates, of 100 mg/L, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the initial test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

Positive control:

Analysis of the immobilization data by the trimmed Spearman-Karber method (Hamilton et al 19771) at 24 and 48 hours based on the nominal test concentrations gave the following results. The results from the positive control with potassium dichromate were within the normal range for this reference item , as can be seen below:

Time point (hours)	EC50 (mg/L)	95% confidence limits	NOEC	LOEC
24	1	0.91 - 1.2	0.56	1.0
48	0.71	0.65 - 0.76	0.32	0.56

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 10 g/L loading rate WAF. The No Observed Effect Loading rate was 10 g/L loading rate WAF.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test and an initial test, at the request of the Sponsor, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a single nominal loading rate of 10 g/L for 48 hours at a temperature of 21 °C to 24 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results:

Exposure of Daphnia magna to the test item gave EL*50 values of greater than 10 g/L loading rate WAF. The No Observed Effect Loading Rate was 10 g/L loading rate WAF.

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed a measured concentration of 1.69 mg C/L. Analysis of the old media at 48 hours showed a measured concentration of less than the control.

The dissolved test item may have been one or several components of the test item. Given that the toxicity of the test item cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 10 g/L loading rate WAF. The No Observed Effect Loading rate was 10 g/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10 g/L