4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

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 01 August 2017. Experimental completion date 07 September 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:

yes

Remarks:

Please see any other information on materials and method section

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Physical state/Appearance: Very pale yellow liquid
Storage Conditions: Approximately 4 °C in darkness

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
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.
Samples were taken from the control and each surviving test group from the bulk test preparation at 0 and 24 hours and from the pooled replicates at 24 and 48 hours for quantitative analysis. All 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:

Preliminary Media Preparation Trial
Information provided by the Sponsor indicated the water solubility of the test item to be 18 mg/L.
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

Saturated Solution Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)
Discussion
Whilst the centrifuged samples yielded the highest measured test concentrations, visual observations made on the samples indicated that undissolved test item remained and as such the use of centrifugation as a means to remove undissolved test item was considered inappropriate for this test item.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 17 mg/L.

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% v/v saturated solution.
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 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution.
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). 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.

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 3) 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 22 °C throughout the test,

pH:

7.8 - 8.0

Dissolved oxygen:

8.0 - 8.8 mg O2/L

Nominal and measured concentrations:

Range finding test:
0.10, 1.0 and 10% v/v saturated solution.
Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations of 1.7 and 12 mg/L were obtained respectively. After 48 hours exposure, measured concentrations of 0.77 and 7.3 mg/L were obtained respectively indicating that the test item was not stable under test conditions.

Definitive test:
10, 18, 32, 56 and 100% v/v saturated solution.
Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 1.7 to 13 mg/L. There was no significant change in the measured concentrations of the old or expired media at 24 and 48 hours and so the results are based on average measured concentrations of the freshly prepared media only which were determined to be 1.7, 3.0, 4.8, 6.8 and 13 mg/L.

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. 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.

Definitive Test
In the definitive test ground glass stoppered conical flasks 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 sealed as a precautionary measure at the request of the sponsor 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.

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
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.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 4.0 - 5.0 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.1 mg/L

Nominal / measured:

meas. (initial)

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, 1.0 and 10% v/v saturated solution, however, immobilization was observed at 100% v/v saturated solution.
Sub-lethal effects of exposure were observed in the 0.10 and 1.0% v/v saturated solution test groups. These responses were trapping at the surface and a pale color.

Definitive Test
Immobilization Data
The No Observed Effect Concentrations after 24 and 48 hours exposure were 3.0 and 1.7 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 4.8 and 3.0 mg/L respectively.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 1.7, 3.0 and 4.8% v/v saturated solution test concentrations. These responses were reduced movement and trapping at the surface

Water Quality Criteria
Temperature was maintained at 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 503 to 523 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.
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.
Throughout the positive control 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.
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 Definitive Test

Average Freshly Prepared 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
1.7	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.0	0	0	0	1	1	5	3 R 2 N	5 R	5 R	4 R
4.8	2	3	3	3	11	55	3 R	2 R	2 R	2 R
6.8	5	5	5	5	20	100	A/I	A/I	A/I	A/I
13	5	5	5	5	20	100	A/I	A/I	A/I	A/I

Average Freshly Prepared 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
1.7	0	0	0	0	0	0	3 R 2 T	5 R	5 R	1 R 4 N
3.0	4	0	0	1	5	25	1 R	5 R	5 R	4 R
4.8	5	5	5	5	20	100	A/I	A/I	A/I	A/I
6.8	5	5	5	5	20	100	A/I	A/I	A/I	A/I
13	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

T = Trapped at the surface

R = Reduced movement

A/I = All daphnia immobilized

Validity criteria fulfilled:

yes

Remarks:

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and 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 3.1 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 nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 22 °C under semi-static test conditions. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: 48 h EC50 (immobilisation) value with Daphnia magna is 3.1 mg/L. All results are expressed in terms of average measured concentration of the freshly prepared media only, which were determined to be 1.7, 3.0, 4.8, 6.8 and 13 mg/L.

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 nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 22 °C under semi-static test conditions. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following values determined: 48 h EC50 (immobilisation) value with Daphnia magna is 3.1 mg/L. All results are expressed in terms of average measured concentration of the freshly prepared media only, which were determined to be 1.7, 3.0, 4.8, 6.8 and 13 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.1 mg/L