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 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:

Experimental Starting Date: 29 April 2013 Experimental Completion Date: 24 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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and the 100 mg/L loading rate WAF at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media). Duplicate samples, and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media), were taken and stored at approximately -20 ºC for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 10 April 2013. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 9 May 2013 to 21 May 2013. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.5 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.4 cm (sd = 0.3) and a mean weight of 1.09 g (sd = 0.28) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.38 g bodyweight/liter.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Hardness:

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3.

Test temperature:

The test was conducted ata temperature of between 14 and 15 deg C.

pH:

The pH was measured to be between 7.9 and 8.3 during the cours of the study, in both the control and test vessels.

Dissolved oxygen:

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Salinity:

Not applicable as a fresh water study was conducted

Nominal and measured concentrations:

A limit test was conducted with the test substance being tested at a nominal concentration of 100 mg/L, please note WAFs were used throughout the study.

Samples of the control and the 100 mg/L loading rate WAF were taken at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Analysis of the fresh media for the 100 mg/L loading rate WAF showed measured concentrations of between less than the control and 0.19 mg C/L. Analysis of the old or expired media showed measured concentrations of between less than the control and 0.21 mg C/L. The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Details on test conditions:

Experimental preparation:
An amount of test item (2100 mg) was added to the surface of 21 liters of dechlorinated tap water to give the 100 mg/L loading rate. After the addition of the test item, the dechlorinated tap 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 75-100 mL discarded) to give the 100 mg/L loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 and 72 hours (fresh media) and 24 and 96 hours (old media).

TEST SYSTEM
As in the range-finding test, 20 liter glass exposure vessels were used for each test concentration. At the start of the test 7 fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

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

Data from the control group was shared with similar concurrent studies.

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build-up of nitrogenous waste products.

TEST MEDIUM / WATER PARAMETERS
Information on water quality can be found within appendix 2 which has been attached in the attached background

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: In the range-finding test fish were exposed to a single nominal loading rate of 100 mg/L as the results of the Acute Toxicity to Daphnia (Harlan Study Number 41301081) indicated that toxicity was not expected at this level.

Range finding study
- Test concentrations: 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 mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

yes

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range finding test:
Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given below:

cumulative mortality
Nominal Loading Rate (mg/L) 3 hours 6 hours 24 hours 48 hours 72 hours 96 hours
Control 0 0 1 1 2 2
100 0 0 0 0 0 0

The results showed no mortalities at 100 mg/L loading rate WAF. Whilst mortalities were observed in the control, this was not considered to affect the integrity of the result obtained as no mortalities were observed in the 100 mg/L loading rate WAF.
Based on this information, a single loading rate of 100 mg/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 mortalities or sub-lethal effects of exposure were observed.

Sublethal observations / clinical signs:

Definitive Test:

Mortality Data:

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in the table below:

Nominal Loading Rate (mg/L)	Cumulative Mortality (initial population = 7)						% mortality
	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours	96 h0urs
Control	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0

There were no mortalities in 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)	LL*50 (mg/L Loading Rate (WAF)
3	>100
6	>100
24	>100
48	>100
72	>100
96	>100

There were no sub-lethal effects of exposure observed in the test.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL*50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 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, seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14 °C to 15 ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results:

The 96-Hour LL*50 based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Samples of the control and the 100 mg/L loading rate WAF were taken at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Analysis of the fresh media for the 100 mg/L loading rate WAF showed measured concentrations of between less than the control and 0.19 mg C/L. Analysis of the old or expired media showed measured concentrations of between less than the control and 0.21 mg C/L.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or 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 fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL*50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L