Methyl 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoate

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 conducted between 13 February 2017 and 12 May 2017

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 (Trade Name): Lilial ME Anthranilate (Lilianth BHT):
Identification (IUPAC Name): Reaction mass of methyl 2-{[(1E)-3-(4-tert-butylphenyl)-2-methylprop-1-en-1-yl]amino}benzoate and methyl 2-{[(1Z)-3-(4-tert-butylphenyl)-2-
methylprop-1-en-1-yl]amino}benzoate
Physical state/Appearance: Yellow liquid – observed to have formed a yellow, wet looking crystalline solid when opened for use

Analytical monitoring:

yes

Details on sampling:

Range-finding test:
A sample of each test concentration was taken for chemical analysis at 0 and 24 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 initial test were analyzed.

Definitive test:
Samples were taken from the control and the 100% v/v saturated solution test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the pooled replicates at 24 and 48 hours for quantitative analysis. Samples were analyzed on the day of sampling. Duplicate samples were taken and stored frozen for further analysis if necessary.
.

Vehicle:

no

Details on test solutions:

Range-finding Tests
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 10000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Initial Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 10000 g for
30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 10000 g for 30 minutes to give the required test concentration of 100% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
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 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Temperature was maintained at 19 ºC to 22 ºC throughout the test

pH:

7.6 -8.0

Dissolved oxygen:

7.1 - 9.0 mg O2/L

Nominal and measured concentrations:

Range-finding test:
Nominal test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution.
Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to be less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0053 mg/L, and 0.21 mg/L, respectively. There was no significant change at 24 hours indicating that the test item was stable under test conditions.

Initial test:
Nominal test concentrations: 10, 18, 32, 56 and 100% v/v saturated solution
Due to a technical issue with the instrument used for the chemical analysis the results of the test and duplicate samples were inconclusive.

Definitive test:
Nominal test concentrations: 100% v/v saturated solution
Chemical analysis of the freshly prepared 100% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to be 0.012 and 0.013 mg/L, respectively. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0053 mg/L. Hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.
The geometric mean measured test concentration was determined to be 0.0058 mg/L.

Details on test conditions:

Range-finding Tests
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.43 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the initial test were determined by preliminary range-finding tests.
An initial range finding test showed extensive immobilization between 24 and 48 hours, due to this a second range finder was conducted to determine if the initial test immobilization was a contamination issue or a test item effect.
In the second range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
In the range-finding test 10 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. Each 150 mL test and control vessel was completely filled with test media and sealed 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.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
A sample of each test concentration was taken for chemical analysis at 0 and 24 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 initial test were analyzed.

Initial Test
Based on the results of the range-finding test, the following test concentrations were assigned to the initial test: 10, 18, 32, 56 and 100% v/v saturated solution.
A technical issue with the instrument used for the chemical analysis meant that the results of the test and duplicate samples were inconclusive. Due to this and given that no effects were seen in the initial test, the definitive test was conducted at a nominal test concentration of 100% v/v saturated solution.

Definitive Test
As in the range-finding and initial tests 150 mL glass stoppered conical flasks completely filled with test preparation were used. At the start of the test five 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 sealed 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.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Tests
In the initial range finding test there was extensive immobilization at the 1.0, 10 and 100% v/v saturated solution test concentrations, between 24 and 48 hours, which was considered to be due to contamination. Hence it was considered appropriate to perform a second range finding test to confirm this.

No significant immobilization was observed at any of the test concentrations, however, significant sub-lethal effects were observed at the 100% v/v saturated solution.
Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the initial test.
Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to be less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0053 mg/L, and 0.21 mg/L, respectively. There was no significant change at 24 hours indicating that the test item was stable under test conditions.

Initial Test
No immobilization was observed at any of the test concentrations, however, due to a technical issue with the instrument used for the chemical analysis the results of the test and duplicate samples were inconclusive.
Based on this information a test concentration of 100% v/v saturated solution was selected for the definitive test.

Definitive Test
Verification of Test Concentrations
The geometric mean measured test concentration was determined to be 0.0058 mg/L.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a geometric mean measured test concentration of 0.0058 mg/L for a period of 48 hours.
Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 0.0058mg/L. The No Observed Effect Concentration was 0.0058 mg/L.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the test.

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 19 ºC to 22 ºC 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 431 to 573 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 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.
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) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
24 1.3 1.0 - 1.8 1.0 1.8
48 1.2 1.1 - 1.3 0.56 1.0
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*.

* The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give an EC50-24 hour for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L.

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	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	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

 

Nominal Concentration (% v/v Saturated Solution)	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
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

Validity criteria fulfilled:

yes

Remarks:

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.

Conclusions:

The 48 h EC50 (immobilisation) value with Daphnia magna is > 0.0058 mg/I.

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. Four replicates of five 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to Lilianth BHT concentrations of the substance dispersed in test water.

The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: EC50value of greater than 0.0058 mg/L. 

All results are expressed in terms of the geometric mean measured test concentration. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0053 mg/L.

Description of key information

A study was performed to access the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. Four replicates of five 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to Lilianth BHT concentrations of the substance dispersed in test water.

The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: EC50value of greater than 0.0058 mg/L. The No Observed Effect Concentration was 0.0058 mg/L.

All results are expressed in terms of the geometric mean measured test concentration. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0053 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.006 mg/L