2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 09 February 2018 and 11 April 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 15-0735
Physical state/Appearance: clear colorless liquid
Storage Conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

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.

Vehicle:

no

Details on test solutions:

Range-finding Tests:
A nominal amount of test item (50 mg) was dissolved in test water with the aid of ultrasonication and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.

Definitive Test
A nominal amount of test item (200 mg) was dissolved in test water with the aid of ultrasonication for 10 minutes and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg/L.

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 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation 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 within 15 seconds after gentle agitation.

Test temperature:

18 to 22 °C with a maximum deviation of ±1 °C

pH:

pH 7.9

Dissolved oxygen:

8.7-8.8 mg O2/L

Nominal and measured concentrations:

Range-finding Tests
Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L.

Definitive Test
Nominal concentrations: 10, 18, 32, 56 and 100 mg/L

Chemical analysis of the 10 and 100 mg/L test preparations at 0 and 48 hours showed measured test concentrations to be near nominal, indicating that the test item was stable under test conditions.

Details on test conditions:

Experimental Design and Study Conduct
Range-finding Tests
An initial range finding test was conducted using the method described below; however, low analytical recoveries were observed, therefore the test was repeated.
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
In the range finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water with the aid of ultrasonication and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range finding test five 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.

Definitive Test
Based on the results of the range finding tests the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.
A nominal amount of test item (200 mg) was dissolved in test water with the aid of ultrasonication for 10 minutes and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Exposure Conditions
As in the range finding tests 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, containing 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.

Assessments
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 within 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. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparations at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. A set of duplicate samples was taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

41 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: Not determined due to the nature of the data

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 12-150 mg/L

Details on results:

Range finding Tests
No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L.
A sub lethal effect of exposure was observed in the test concentration of 100 mg/L. This response was trapping at the media surface.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Chemical analysis of the 10 and 100 mg/L test preparations at 0 and 48 hours showed measured test concentrations to be near nominal, indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 7.0 to 87 mg/L (between 70% and 105% of the nominal value). Analysis of the test preparations at 48 hours showed similar measured test concentrations, ranging from 6.8 to 82 mg/L (between 68% and 114% of nominal) and so it was considered justifiable to calculate the EC50 values in terms of the 0 hour measured test concentrations.

Immobilization Data
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression method at 24 and 48 hours based on the 0 Hour measured test concentrations gave the following results:
Time (Hour) EC50 (mg/L) 95% Confidence limits(mg/L)
24 41 Not determined due to the nature of the data
48 28 12 - 150
The No Observed Effect Concentrations after 24 and 48 hours exposure were 24 and 7.0 mg/L, respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 59 and 14 mg/L, respectively.
The slopes and their standard errors of the response curves at 24 and 48 hours were 5.5 (standard error = 0.040) and 5.4 (standard error = 0.040) respectively.

Sub-Lethal Effects
A sub lethal effect of exposure was observed in the test concentrations of 18 and 32 mg/L. This response was trapping at the media surface.

Validation Criteria
The test was considered to be valid given that none 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.

Water Quality Criteria
Temperature was maintained at approximately 20 °C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 680 to 721 Lux.

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 LK67NP) 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 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:
Time Point(Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
24 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0

The NOEC 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*.

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2
Control	0	0	5N	5 N	0	0	5 N	5 N
0.10	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	0	0	5 N	5 N	0	0	5 N	5 N
100	3	2	2T	3 T	5	5	AI	AI

Rep  = Replicate

N      = Normal

T       = Trapped at surface

AI     = All daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (mg/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	5N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	1	0	1	5	5 N	5 N	4 N	5 N
56	5	5	5	5	20	100	AI	AI	AI	AI
100	5	4	3	5	17	85	AI	1 N	2 N	AI
Nominal Concentration (mg/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	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	1	2	3	15	5 N	5 N	4 N	3 T
32	0	0	3	0	3	15	5T	5 T	2 T	5 T
56	5	5	5	5	20	100	AI	AI	AI	AI
100	5	5	5	5	20	100	AI	AI	AI	AI

R      =Replicate

N     = Normal

T      = Trapped at surface

AI    = All daphnia immobilized

Water Quality Measurements

Nominal Concentration (mg/L)		0 Hours			24 Hours	48 Hours
		pH	mg O2/L	Temperature °C	Temperature °C	pH	mg O2/L	Temperature °C
Control	R1	7.9	8.7	21	20	8.1	8.9	21
10	R1	7.9	8.8	21	20	8.1	8.8	21
18	R1	7.9	8.7	21	20	8.1	8.8	21
32	R1	7.9	8.7	21	20	8.1	8.8	21
56	R1	7.9	8.7	22	20	8.1	8.8	21
100	R1	7.9	8.7	22	20	8.1	8.9	21

R  = Replicate

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 (immobilisation) value with Daphnia magna is 28 mg/I.

Executive summary:

A study was performed to assess the acute toxicity of FRET 15-0735 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of approximately 20 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: EC50 of 41 and 28 mg/L, respectively. All results are expressed in terms of measured test concentration . Analysis of the test preparations at 0 hours showed measured test concentrations to range from 7.0 to 87 mg/L (between 70% and 105% of nominal). Analysis of the test preparations at 48 hours showed measured test concentrations to be similar, ranging from 6.8 to 82 mg/L (68% to 114% of nominal) and so it was considered justifiable to calculate the EC50 values in terms of the 0 hour measured test concentrations.

Description of key information

A study was performed to assess the acute toxicity of FRET 15-0735 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of approximately 20 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: EC50 of 41 and 28 mg/L, respectively. All results are expressed in terms of measured test concentration . Analysis of the test preparations at 0 hours showed measured test concentrations to range from 7.0 to 87 mg/L (between 70% and 105% of nominal). Analysis of the test preparations at 48 hours showed measured test concentrations to be similar, ranging from 6.8 to 82 mg/L (68% to 114% of nominal) and so it was considered justifiable to calculate the EC50 values in terms of the 0 hour measured test concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

28 mg/L