Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

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: 06 May 2014; Experimental Completion Date: 06 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

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. In cases where the test item is a complex mixture and is poorly soluble in water, 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. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a 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.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

EC Number:

940-953-8

Molecular formula:

not applicable UVCB

IUPAC Name:

Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

Test material form:

other: solid block

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L loading rate

- Sampling method: 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.

- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

TEST WATER:
Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined below:

Ingredient Final Concentration (mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 64.75
KCl 5.75

VALIDATION OF MIXING PERIOD:
Preliminary 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 and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. 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 samples taken for chemical analysis.

There was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

RANGE-FINDING TEST:
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.

The test item was melted at approximately 80°C for 60 minutes prior to use to ensure homogeneity. Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test 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 (the first approximate 75-100 mL discarded) 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 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 frozen 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:
The test item was melted at approximately 80°C for 120 minutes prior to use to ensure homogeneity. A nominal amount of test item (500 mg) was added to the surface of 5 liters of test water to give the 100 mg/L loading rate. After the addition of the test item, the test 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 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 and 48 hours

Test organisms

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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

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

Test temperature:

Temperature was maintained at approximately 21 C throughout the test.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Nominal and measured concentrations:

Definitive Test:
Nominal: 100 mg/L loading rate WAF
Measured: 0.17 mg/L (0 hours) and 0.13 mg/L (48 hours)

Details on test conditions:

DEFINITIVE TEST:
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 approximately 21°C with a photoperiod of 16 hours light (463 to 488 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.

The test preparations were not renewed during the exposure period.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test Organism Observations:
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.

Water Quality Criteria:
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

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:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Details on results:

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. A single immobilized daphnid was observed in the control group during the range-finding test. However, as this only represented 10% immobilization and no further immobilization was observed during the test, this was considered not to impact the test.

Based on this information, a single loading rate of four replicates, of 100 mg/L, 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 test preparation at 0 and 48 hours showed measured concentrations of 0.17 and 0.13 mg/L respectively indicating that the test item was stable under test conditions.


DEFINITIVE TEST:
Chemical Analysis of Test Loading Rates:
Analysis of the 100 mg/L loading rate WAF test preparation at 0 and 48 hours (see Appendix 'Analytical Investigations') showed measured concentrations of 0.17 and 0.14 mg/L respectively.

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.

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 significant 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:
24 and 48 h EL50: >100 mg/L loading rate WAF

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF. A single immobilized daphnid was observed in the control and 100 mg/L loading rate WAF test group. However, as this only represents 5% immobilization and no further immobilization was observed during the test this was considered not to impact the outcome of the test.

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

Validation Criteria:
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Water Quality Criteria:
The results of the water quality measurements are given in Table 3. Temperature was maintained at approximately 21°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

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.

Observations of 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 colorless water column with globules of test item at the water surface. After 23 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was observed to be a clear colorless water column with solidified test item floating at the water 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.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971*) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al 1977 *) using the ToxCalc software package (ToxCalc 1999) at 48 hours based on the nominal test concentrations gave the following results:

24 h EC50: 0.87 mg/L
24 h NOEC: 0.56 mg/L
24 h LOEC: 1.0 mg/L

48 h EC50: 0.71 mg/L
48 h NOEC: 0.56 mg/L
48 h LOEC: 1.0 mg/L

The No Observed Effect Concentration is based upon less than 10% immobilization at this concentration.

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

Any other information on results incl. 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	1[1]	1^*
1.0	0	0
10	0	0
100	0	0

*Single immobilized daphnid considered not to be significant as represents only 10% immobilization and no further immobilization was observed during the range-finding test.

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	1*	5
	R2	0			0
	R3	0			0
	R4	0			1
100	R1	0	1*	5	0	1*	5
	R2	1			1
	R3	0			0
	R4	0			0

 

R₁– R₄= Replicates 1 to 4

*Single immobilized daphnid observed in control and 100 mg/L loading rate WAF test group. Considered not to impact test as only represents 5% immobilization and no further immobilization was observed during the test.

Table 3     Water Quality Measurements

Nominal Loading Rate (mg/L)		0 Hours			24 Hours	48 Hours
		pH	mg O2/L	T°C	T°C	pH	mg O2/L	T°C
Control	R1	7.8	9.4	21	21	8.1	8.8	20
100	R1	7.9	9.1	21	21	8.1	8.6	21

R₁= Replicate 1

Applicant's summary and conclusion

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 EL50 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 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 medium 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 single nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 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…….

Analysis of the 100 mg/L loading rate WAF test preparation at 0 and 48 hours showed measured concentrations of 0.17 and 0.14 mg/L respectively.

 

Given that the 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.

 

Exposure of Daphnia magna to the test item gave EL₅₀values of 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.