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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 19 February 2013 Experimental Completion Date: 12 March 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 the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

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: amber colored liquid

Details on test material:

The integrity of the supplied data relating to the identity, purity and stability of the test item is the responsibility of 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
Description: amber colored liquid
Batch: OE11124 (6/202593/00)
Purity: not applicable – complex mixture
Expiry date: not supplied
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

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 group (replicates R1 – R4 pooled) at 0 and 48 hours for
quantitative analysis. Samples were stored at approximately -20 °C prior to analysis.

Duplicate samples 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 6.

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 5).


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

The loading rate to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.

Amounts of test item (10, 40 and 200 mg) were each separately added to the surface of 10, 4 and 2 liters of reconstituted water to give the
1.0, 10 and 100 mg/L loading rates respectively. 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 mixtures 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 WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was
removed by mid-depth siphoning to give the 1.0, 10 and 100 mg/L loading rate WAFs.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21 °C to 22 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized
Daphnia magna were recorded.

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

A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test
conditions. All samples were stored at approximately -20 °C prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
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.


Experimental Preparation
An amount of test item (200 mg) was added to the surface of 2 liters of reconstituted water to give the 100 mg/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 to give the 100 mg/L loading rate WAF.

The concentration and stability of the test item in the test preparation was verified by chemical analysis at 0 and 48 hours (see attached Appendix 6).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target
organism in freshwater ecosystems.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see attached Appendix 2) in a temperature controlled room at
approximately 20 °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.

A positive control (Harlan Study Number: 41203341) used potassium dichromate as the reference item. Details of the positive control are given in
the attached Appendix 3.


Test Water
Reconstituted water used for both the range-finding and definitive tests is defined in the attached Appendix 4.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

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

Test temperature:

Temperature was maintained at 20 °C to 21 °C throughout the test.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

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 pH was measured using a measured using a Hach HQ30d Flexi handheld meter.
There were no treatment related differences for pH.
See attached Appendix 7 for results.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a Hach HQ30d Flexi handheld meter.
See attached Appendix 7 for results.

Salinity:

Freshwater used.

Nominal and measured concentrations:

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.

Details on test conditions:

Exposure Conditions
As in the range-finding 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 21 °C with a photoperiod of 16 hours
light and 8 hours darkness with 20 minute dawn and dusk transition periods with a light intensity ranging from 628 to 688 lux. 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.

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.

Physico-Chemical Measurements
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of
the test. 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 the mixing period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Test item prepared as a Water Accommodated Fraction (WAF)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Test item prepared as a Water Accommodated Fraction (WAF)

Details on results:

RESULTS
Validation of Mixing Period
Pre-study work (see attached Appendix 5) 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.


Range-finding Test
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

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

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 definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or
adverse reactions to exposure were observed.

Chemical analysis of the 100 mg/L loading rate WAF at 0 and 48 hours (see attached Appendix 6) showed that measured test concentrations of
0.0991 mg/L and less than the Limit of Quantitation, determined to be 0.070 mg/L, were obtained respectively, indicating that the test item was
possibly unstable over the test duration.

Definitive Test
Immobilization Data
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EL*50 (mg/L Loading rate WAF)
24 >100
48 >100

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF.

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


Physico-Chemical Measurements
The results of the physico-chemical measurements are given in the attached Appendix 7.

Temperature was maintained at 20 °C to 21 ºC 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 the mixing period and was observed to be a dimple at the water surface on each occasion
(see Table 3).


Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.

At the start of the mixing period the 100 mg/L loading rate was observed to be a clear coloulress water column with an oily globule of test item
floating on the surface. After 23 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was observed to remain as a clear colorless water column with an oily globule of test item floating 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 test preparations at 0 and 48 hours (see attached Appendix 6) showed measured test concentrations of less than the limit of
quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.070 mg/L. This does not infer that no test item
was in solution, just that which was so, was at a concentration of less than the LOQ.

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 and the dissolved test item was below the quantifiable limit of the analytical method, the
results were based on nominal loading rates only.


Validation Criteria
Both validation criteria were met during the test, there were no immobilized daphnids on the control group and the oxygen concentration was
≥8.1 mg/L throughout the test.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal range for this reference item.
See the attached Appendix 3
Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)
24 h EC50 0.75 mg/L nominal other: Positive control mortality 95% Confidence limits (mg/L) 0.56 - 1.0
48 h EC50 0.45 mg/L nominal other: Positive control mortality 95% Confidence limits (mg/L) 0.42 - 0.48

Reported statistics and error estimates:

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977 ) at 48 hours based on the nominal test concentrations.

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 preparation 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 settlement 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.

Any other information on results incl. tables

TABLES

Table 1     Cumulative Immobilization Data in the Range-finding Test

Nominal Loading Rate (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	0	0
100	0	0

 

  Table 2     Cumulative Immobilization Data in the Definitive Test

Nominal Loading Rate (mg/L)		Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
100	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

 

R₁– R₄= Replicates 1 to 4

 

Table 3     Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/L)
	Control		100
	*	+	^*	^+
Height of Water Column (cm)	12.5	12.5	12.0	12.0
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:

CONCLUSION
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
100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

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, "Daphniasp., 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, 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 100 mg/L as a ‘Limit’ Test for 48 hours at a temperature of 20 °C to 21°C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

 

Results…….

The 48-Hour EL*₅₀ for the test item to Daphnia magna 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 loading rate WAF.

 

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.070 mg/L. This does not infer that no test item was in solution, just that which was so was at a concentration of less than the LOQ.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

 

*EL = Effective Loading Rate