Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

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:

Experimental start date: 15 May 2017 Experimental completion date: 13 July 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: FRET 11-0078
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 ºC in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for immediate quantitative analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.


Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 40 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
A nominal amount of test item (1100 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 filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) 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, 1.0 and 0.10% 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 (1100 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 filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give the required test concentrations of 10, 5.6, 3.2, 1.8 and 1.0% 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:

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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Temperature was maintained at approximately 22 ºC throughout the test.

pH:

7.6 - 8.0

Dissolved oxygen:

7.7 - 8.7 mg O2/L

Nominal and measured concentrations:

Nominal concentrations:
Range-finding test - 0.10, 1.0, 10 and 100% v/v saturated solution
Initial experiment - 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution.
Definitive test - 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution.
The geometric mean measured test concentrations in the definitive test were determined to be 0.080, 0.13, 0.29, 1.2 and 4.4 mg/L.

Chemical analysis of the freshly prepared test solutions at 0 and 24 hours showed measured test concentrations to range from 0.078 to 7.0 mg/L. A decline in measured test concentrations was observed in the old or expired test solutions at 24 and 48 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.064 mg/L, to 4.4 mg/L.
Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the mean measured test concentration only in order to give a “worst case” analysis of the data.

Details on test conditions:

Range-finding Test
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.

Definitive Test
As in the range-finding test 150 mL ground glass conical flasks each 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

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 4.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0% v/v saturated solution, however, immobilization was observed at 10 and 100% v/v saturated solution.
A sub-lethal effect of exposure was observed in the 10% v/v saturated solution test concentration at 24 Hours. This response was reduced mobility.
Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 1.0, 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.64 to 70 mg/L. There was a significant decline in the measured concentrations at 48 hours in the range of less than the limit of quantification (LOQ), determined to be 0.064 mg/L, to 61 mg/L indicating that the test item was unstable under the conditions of the test.
The measured concentration obtained from the 100% v/v saturated solution preparation at 0 hours (70 mg/L) was significantly higher than that obtained from the media preparation trial. This was considered to be due to a media effect with the test item being more readily soluble in the test media than in deionized reverse osmosis water.

Definitive Test
Visual inspection of the data at 24 hours and analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 48 hours gave the following results:
Time (h) EC50 (mg/L) 95% Confidence limits (mg/L)
24 >4.4 Not determined due to nature of data
48 1.3 Not determined due to nature of data

The No Observed Effect Concentrations after 24 and 48 hours exposure were 4.4 and 0.29 mg/L respectively. The Lowest Observed Effect Concentration after 48 hours exposure was 1.2 mg/L.
The slope and standard error of the response curve at 48 hours were 11.6 (SE = 4.7). Due to the nature of the data it was not possible to calculate the slope and error of response curve at 24 hours.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 10% v/v saturated solution test concentration at 24 hours. This response was reduced mobility.

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.

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 definitive test 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:
Time Point
(Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) 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.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	10 N	0	10 N
0.10	0	10 N	0	10 N
1.0	0	10 N	0	10 N
10	0	10 R	10	A/I
100	10	A/I	10	A/I

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Geometric Mean Measured Test 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	5 N	5 N	5 N	5 N
0.080	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.13	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.29	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.2	0	0	0	0	0	0	5 N	5 N	5 N	5 N
4.4	1	0	1	1	3	15	4 R	1 N 4 R	1 N 3 R	4 R

 

Geometric Mean Measured Test 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
0.080	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.13	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.29	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.2	0	0	4	3	7	35	5 N	5 N	1 R	2 R
4.4	5	5	5	5	20	100	A/I	A/I	A/I	A/I

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced mobility

A/I = All daphnia immobilized

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 (immobilisation) value with Daphnia magna is 1.3 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. Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to FRET 11-0078 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:

Time (h)	EC50(mg/L)	95% Confidence limits (mg/L)
24	>4.4	Not determined due to nature of data
48	1.3	Not determined due to nature of data

Chemical analysis of the freshly prepared test solutions at 0 and 24 hours showed measured test concentrations to range from 0.078 to 7.0 mg/L. A decline in measured test concentrations was observed in the old or expired test solutions at 24 and 48 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.064 mg/L, to 4.4 mg/L.

Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Description of key information

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. Groups of twenty, 1st instar Daphnia (less than 24 hours old) were exposed for 48 hours to FRET 11-0078 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:

 

Time (h)	EC50(mg/L)	95% Confidence limits (mg/L)
24	>4.4	Not determined due to nature of data
48	1.3	Not determined due to nature of data

 

Chemical analysis of the freshly prepared test solutions at 0 and 24 hours showed measured test concentrations to range from 0.078 to 7.0 mg/L. A decline in measured test concentrations was observed in the old or expired test solutions at 24 and 48 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.064 mg/L, to 4.4 mg/L.

Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.3 mg/L