Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}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:

Experimental start date 30 May 2017. Experimental completion date 20 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: Lyrame
Physical state/Appearance: Yellow viscous liquid
Storage Conditions: Under nitrogen at approximately 4 ºC in the dark

Analytical monitoring:

yes

Details on sampling:

Range-finding Test
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
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours

Chemical Analysis of Test Loading Rates
Samples were taken from the control and the 100 mg/L loading rate WAF test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates (replicates R1 – R4) at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

Validation of Mixing Period
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item 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. It is evident from this work that increasing the stirring period did not increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours.

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.
After heating to 60 ºC in order to aid handling, 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.

Definitive Test
Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L to confirm that no immobilization or adverse reactions to exposure were observed.
After heating to 60 ºC in order to aid handling, a nominal amount of test item (200 mg) was added to the surface of 2 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

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 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:

yes

Total exposure duration:

48 h

Test temperature:

Temperature was maintained at 22 ºC throughout the test

pH:

7.7 - 7.8

Dissolved oxygen:

8.4 - 8.6 mg O2/L

Nominal and measured concentrations:

Nominal test concentrations: 1.0, 10 and 100 mg/L (range finding). 100 mg/L (definitive)

Measured test concentrations
Range finding: Chemical analysis of the 100 mg/L loading rate test preparation at 0 hours showed a measured test concentration of 0.29 mg/L. There was a significant decline in the measured concentration at 48 hours indicating that the test item was not stable under test conditions.
Definitive test: Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to be 1.0 and 0.22 mg/L, respectively. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to be 0.29 mg/L and less than the limit of quantification of the analytical method employed, which was determined to be 0.14 mg/L, respectively.
The dissolved test item may have been one or several components of the test item. 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

Details on test conditions:

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. 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.

Definitive Test
As in the range-finding test, 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, 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.
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. Concentrations at which 100% immobilization was observed after 24 hours exposure were not renewed.

Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. 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.

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:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at 1.0, 10 and 100 mg/L loading rate WAF.
A sub lethal effect of exposure was observed in the 10 mg/L loading rate. This response was trapping at the surface.
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.

Definitive Test
Immobilization Data
There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours.
Exposure of Daphnia magna to the test item gave EL50 values 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.

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

Water Quality Criteria
Temperature was maintained at 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 505 to 598 lux.

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 on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.
At the start of the 0-Hour test preparation mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with yellow oily globules of test item on the 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 and oily layer on the surface and yellow globules of test item settled on the bottom.
At the start of the 24-Hour test preparation mixing period the 100 mg/L loading rate was observed to be a colorless water column with a yellow oily layer of test item on the 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 a globule of test item and a yellow oily layer on the surface.
Microscopic inspection of the WAFs in both preparations showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the control and 100 mg/L loading rate were observed to be clear, colorless solutions.

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, 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.

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.

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Loading Rate (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
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

Nominal Loading Rate (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
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 EL50 (immobilisation) value with Daphnia magna is >100 mg/L.

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 Daphnia (less than 24 hours old) were exposed for 48 hours to Water Accommodated Fraction (WAF) of the test item at a nominal loading rate 100 mg/L 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: EL50 (immobilisation) value with Daphnia magna is >100 mg/L.

All results are expressed in terms of nominal loading rates. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to be 0.29 mg/L and less than the limit of quantification of the analytical method employed, which was determined to be 0.14 mg/L, respectively.

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. Four replicates of five Daphnia (less than 24 hours old) were exposed for 48 hours to Water Accommodated Fraction (WAF) of the test item at a nominal loading rate 100 mg/L 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: EL50 (immobilisation) value with Daphnia magna is >100 mg/L.

All results are expressed in terms of nominal loading rates. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to be 0.29 mg/L and less than the limit of quantification of the analytical method employed, which was determined to be 0.14 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L