(carboxylatomethyl)dimethyltetradecylammonium

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 start date 15 September 2017. Experimental completion date 24 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

please see any other information on materials and methods

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

please see any other information on materials and methods

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(carboxylatomethyl)dimethyltetradecylammonium

EC Number:

220-006-9

EC Name:

(carboxylatomethyl)dimethyltetradecylammonium

Cas Number:

2601-33-4

Molecular formula:

C18H37NO2

IUPAC Name:

(carboxylatomethyl)dimethyltetradecylammonium

Test material form:

other: aqueous solution

Details on test material:

Identification: FSM-005W
CAS Number: 2601-33-4
Batch: 170321
Purity: 19.9% aqueous solution of FSM-005W (solid)
Physical state/Appearance: clear colorless liquid
Expiry Date: 20 November 2018

Specific details on test material used for the study:

Identification: FSM-005W
Batch: 170321
Purity: 19.9%
Physical state/Appearance: Clear colourless liquid
Expiry Date: 20 November 2018
Storage Conditions: Refrigerated at approximately 4 °C in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding Test
A sample of each test concentration 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
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding Test
A nominal amount of test item (251 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg ai/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg ai/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (502.5 mg) was dissolved in test water and the volume adjusted to 1 liter to give a 100 mg ai/L stock solution from which a series of dilutions was made to give the required test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg ai/L.
The stock solution and each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 to 22 °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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

Temperature was maintained at 21 °C to 22 °C throughout the test

pH:

7.9-8.0 at 0 hours to 7.8 at 48 hours
there were no treatment related differences for oxygen concentration or pH

Dissolved oxygen:

8.9-9.2 at 0 hours to 8.4-8.6 mg O2/L at 48 hours
there were no treatment related differences for oxygen concentration or pH

Nominal and measured concentrations:

Range-finding Test
nominal test concentrations of 0.10, 1.0, 10 and 100 mg ai/L.

Definitive Test
nominal 1.0, 1.8, 3.2, 5.6 and 10 mg ai/L.
Measured 0.866, 1.88, 3.2, 5.2, 9.74 mg ai/L.

Details on test conditions:

Test Water
Reconstituted water (Elendt M7 medium) used for both the range-finding and definitive tests

Range-finding Test
In the range-finding test 5 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °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. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration 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 the following test concentrations were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg ai/L.

Exposure Conditions
As in the range-finding test, 150 mL glass jars containing approximately 100 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0 mg ai/L, however, immobilization was observed at 10 and 100 mg ai/L.
Sub-lethal effects of exposure were observed in the 10 mg ai/L test concentration. This response was reduced mobility.
Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg ai/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Immobilization Data
The No Observed Effect Concentrations after 24 and 48 hours exposure were 5.6 and 3.2 mg ai/L, respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 10 and 5.6 mg ai/L, respectively.
The slopes and their standard errors of the response curves at 24 and 48 hours were 2.6 (SE = 0.13) and 4.3 (SE = 0.041) respectively.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 3.2, 5.6 and 10 mg ai/L test concentrations and the control. These responses were reduced mobility, covered with debris and trapping at the surface

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 equal to or greater than 3 mg/L in the control and test vessels.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control (Envigo study number XT01HS) 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, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

24 hours
EC50 1.3 mg/L; 1.0 -1.8 (95% confidence limits)
NOEC 1.0 mg/L
LEOC 1.8 mg/L

48 hours
EC50 1.2 mg/L; 1.1 -1.3 (95% confidence limits)
NOEC 0.56 mg/L
LEOC 1.0 mg/L

The No Observed Effect Concentration is based upon equal to or 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

Immobilization Data

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal concentrations gave the following results:

Time (Hour)	EC50 (mg ai/L)	95% Confidence limits (mg ai/L)
24	>10	Not determined
48	4.8	Not determined

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal  Concentration (mg ai/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5N	5N	0	0	5N	5N
0.1	0	0	5N	5N	0	0	5N	5N
1.0	0	0	5N	5N	0	0	5N	5N
10	0	2	5R	3R	5	5	A.I	A.I
100	5	5	A.I	A.I	5	5	A.I	A.I

R1-R2 = Replicates 1 and 2

A.I = All immobilized

R = Reduced mobility

N = Normal

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal  Concentration (mg ai/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)							Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	4N, 1T	5N	5N	5N
1.0	0	0	1	0	1	5	5N	5N	4N	5N
1.8	0	0	0	0	0	0	5N	5N	5N	5N
3.2	0	0	0	0	0	0	5N	5N	5N	5N
5.6	0	0	0	0	0	0	5N	5N	5N	5N
10	4	1	2	4	11	55	1R	3R, 1N	3R	1R

Nominal  Concentration (mg ai/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)							Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	4N, 1T	5N	5N	5N
1.0	0	0	1	0	1	5	5N	5N	4N	5N
1.8	0	0	0	0	0	0	5N	5N	5N	5N
3.2	0	0	3	0	3	15	3N, 2R	4N, 1R	2R	5N
5.6	0	2	4	5	11	55	5N	1N, 2C	1R	A.I
10	5	5	5	5	20	100	A.I	A.I	A.I	A.I

R1-R4 = Replicates 1 to 4

A.I = All immobilized

R = Reduced mobility

T= Trapped at surface

N= Normal

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:
At 48 hours
EC50: 4.8 mg ai/L
NOEC: 3.2 mg ai/L
LEOC: 5.6 mg ai/L

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 Regula-ion (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg active ingredient (ai)/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal concentration and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

Time (Hour)	EC50 (mg ai/L)	95% Confidence limits (mg ai/L)	NOEC (mg ai/L)	LOEC  (mg ai/L)
48	4.8	Not determined	3.2	5.6