Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 24 March 2013 Experimental Completion Date: 5 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method
for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere
(ECETOC 1996,
OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing
the test item with water for a prolonged period. Pre-study work showed that a mixing period of 23 hours was sufficient to ensure equilibration between the
test item and water phase. At the completion of mixing and following a 1-Hour standing period, the test item phase is separated by siphon and the test
organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in
terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in
the WAF.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Isostearamide DEA

IUPAC Name:

Isostearamide DEA

Constituent 2

Reference substance name:

CAS No. 52794-79-3

IUPAC Name:

CAS No. 52794-79-3

Test material form:

other: complex mixture

Details on test material:

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is given in the attached Appendix 1.

Identification: Isostearamide DEA CAS No. 52794-79-3
Batch: OE11124 (6/202593/00)
Purity: not applicable – complex mixture
Expiry Date: not supplied
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical Analysis of Test Loading Rates
Water samples were taken from the control and the 100 mg/L loading rate WAF test vessel at 0 (fresh media), 24 (old media), 72 (fresh media) and
96 (old media) hours for quantitative analysis. The samples were stored at approximately -20 °C prior to analysis.

Duplicate samples, and samples at 24 (fresh media), 48 (old and fresh media), and 72 (old media) hours were taken and stored at approximately -20 °C for
further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in the attached Appendix 4.

Test solutions

Vehicle:

no

Details on test solutions:

Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared, in duplicate, in deionized reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and the concentration of the test item in the WAFs was verified by chemical analysis (see attached Appendix 3).


Definitive Test
Based on the results of Acute Toxicity to Daphnia Magna Test (Harlan Study Number: 41202251) and the Algal Growth Inhibition Test (Harlan Study Number: 41205522) 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.


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.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours (see attached
Appendix 4).

Test organisms

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 07 March 2013. Fish were maintained in a glass fiber tank with a "single pass" water
renewal system. Fish were acclimatized to test conditions from 15 March 2013 to 25 March 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 10.2 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 0 mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.8 cm
(sd = 0.2) and a mean weight of 0.88 g (sd = 0.13) at the end of the definitive test. Based on the mean weight value this gave a loading rate of
0.31 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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

Total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

The test vessels were sealed and maintained at approximately 14ºC to 16°C

pH:

The pH was measured using a Hach HQ30d Flexi handheld meter.
pH range of 8.0 - 8.3.
Please see Physico-Chemical Measurements in the attached Appendix 5.

Dissolved oxygen:

The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Please see Physico-Chemical Measurements in the attached Appendix 5.

Nominal and measured concentrations:

Based on the results of Acute Toxicity to Daphnia Magna Test (Harlan Study Number: 41202251) and the Algal Growth Inhibition Test
(Harlan Study Number: 41205522) 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.

The No Observed Effect Loading rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Details on test conditions:

Exposure Conditions
In the definitive 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.

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.

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.


Physico-Chemical Measurements
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after
each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to
each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH and
dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna
Instruments HI 93510 digital thermometer.


Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Validation of Mixing Period
Pre-study work (see attached Appendix 3) indicated that there was no significant increase in the measured test concentration present in the WAF by extending the preparation period for longer than 24 hours.
`
Definitive Test
Mortality Data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 1.

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

The No Observed Effect Loading rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.


Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.


Physico-Chemical Measurements
The results of the physico-chemical measurements are given in the attached Appendix 5. Temperature was maintained at 14 °C to 16 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

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 in the control group.


Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion
(see Table 2).


Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start and end of each mixing period, and after the 1-Hour settlement period the 100 mg/L loading rate was observed to be a clear colorless
water column with oily globules of test item on the surface. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test
item to be present. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colorless solution.


Chemical Analysis of Test Loading Rates
Chemical analysis of the freshly prepared test media at 0 and 72 hours (see Appendix 4) showed measured test concentrations of 0.0305 and
0.0635 mg/L respectively were obtained. Measured test concentrations in the old or expired test media at 24 and 96 hours of 0.0418 and
0.0652 mg/L respectively were obtained.

Given that toxicity 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.


Validation Criteria
All of the validation criteria were met, there were no mortalities or signs of stress in the control group and the ASV remained ≥94% throughout the
test.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

An estimate of the LL50*values was given by inspection of the mortality data.

*LL = Lethal Loading rate

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1     Cumulative Mortality Data in the DefinitiveTest

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0

 

Table 2     Vortex Depth Measurements at the Start and End of Each Mixing Period

FIRST MIXING PERIOD                                                                                                                                              

	Nominal Loading Rate (mg/L)
	Control		100
	*	+	*	+
Height of Water Column (cm)	35.5	35.5	35.5	35.5
Depth of Vortex (cm)	~ 0.2	~ 0.2	~ 0.2	~ 0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

SECOND MIXING PERIOD

	Nominal Loading Rate (mg/L)
	Control		100
	*	+	*	+
Height of Water Column (cm)	35.5	35.5	35.5	35.5
Depth of Vortex (cm)	~ 0.2	~ 0.2	~ 0.2	~ 0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

 

* = Start of mixing period

+ = End of mixing period

THIRD MIXING PERIOD

	Nominal Loading Rate (mg/L)
	Control		100
	*	+	*	+
Height of Water Column (cm)	35.5	35.5	35.5	35.5
Depth of Vortex (cm)	~ 0.2	~ 0.2	~ 0.2	~ 0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

FOURTH MIXING PERIOD

	Nominal Loading Rate (mg/L)
	Control		100
	*	+	*	+
Height of Water Column (cm)	35.5	35.5	35.5	35.5
Depth of Vortex (cm)	~ 0.2	~ 0.2	~ 0.2	~ 0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

 

* = Start of mixing period

+ = End of mixing period

Applicant's summary and conclusion

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.


* LL = Lethal Loading Rate

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…….

In the definitive 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 16 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.

 

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

 

Given that toxicity 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.

*LL = Lethal Loading Rate