Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

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 15 February and 29 March 2019.

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:

These changes were considered to have had no impact on the outcome of the study as the overall conduct of the study remains the same.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: PG-RAW-90-032
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 at 0 hours and at 48 hours for quantitative analysis. All samples were taken for immediate analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

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 indicated that a dissolved test item concentration of approximately 9.78 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.
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 (the first approximate 2 liters used to pre-condition the filter was discarded) 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 Experiment
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.9, 4.1, 9.1, 20, 45 and 100% v/v saturated solution.

Definitive Test
As some loss of test item due to the volatile nature of the test item occurred during the range-finding test, completely filled vessels sealed with ground glass stoppers were used for the 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 (the first approximate 2 liters used to pre-condition the filter was discarded) to give a 100% v/v saturated solution. The 100% v/v saturated solution was used to produce a 50% v/v saturated solution which was further diluted to give a series of test concentrations of 1.9, 4.1, 9.1, 20 and 45% v/v saturated solution.
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 was verified by chemical analysis at 0 and 48 hours.

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 and GM 300 fish 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

Test temperature:

20 °C to 21 °C

pH:

pH 7.9-8.2

Dissolved oxygen:

8.7 - 9.5 mg O2/L

Nominal and measured concentrations:

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.

Definitive Test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.9, 4.1, 9.1, 20, 45 and 100% v/v saturated solution
Measured Test Concentration (mg/L): 0.19, 0.43, 0.98, 2.3, 4.9 and 9.6 mg/L

Details on test conditions:

Test Water
Reconstituted water (Elendt M7 medium) used for the range-finding test, initial experiment and definitive test.

Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ±0.3 and stored at approximately 21 ºC

Range-finding Test
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 and 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.

Exposure Conditions
Nominal 100 mL glass vessels were completely filled with approximately 150 mL of test preparation. 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 using ground glass stoppers 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 and a photoperiod of 16 hours light (between 1215 and 1289 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 contents of 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 48-Hour 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 daily using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.92 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 0.37 - 2.1 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.5 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 0.1 and 1.0% v/v saturated solution, however; 10% immobilization was observed at 10% v/v saturated solution and 100% immobilization was observed at 100% v/v saturated solution.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information test concentrations of 1.9, 4.1, 9.1, 20, 45 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0048 to 9.0 mg/L. There was some reduction in the measured concentrations at 48 hours. To reduce the loss of test item, completely filled vessels sealed with ground glass stoppers were used for the definitive test.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.19 to 9.6 mg/L. There was no significant change in the measured concentrations at 48 hours (ranging from 0.19 to 9.9 mg/L) and so the results were based on the 0-Hour measured test concentrations of 0.19, 0.43, 0.98, 2.3, 4.9 and 9.6 mg/L.

Immobilization Data
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression and 0-Hour measured test concentrations gave the following results:
Time (hours): 24, EC50: 1.5 mg/L
Time (hours): 48, EC50: 0.92 mg/L

Sub-Lethal Effects
Reduced mobility was observed in nominal test concentrations of 1.9, 4.1, 9.1 and 20% v/v saturated solution

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 20 °C to 21 °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 1215 to 1289 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 (study number KC41PP) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

In the positive control test at 0 and 48 hours, the light intensity measured was in excess of the range specified in the study plan (500 to 1000 Lux), the maximum light intensity recorded was 1037 Lux. Given that the recorded light intensity was only marginally in excess of the specified range and as the daphnids in the control group did not show any mortalities or sub-lethal effects, this deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 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 1.1 0.95 - 1.3 0.56 1.0
48 0.8 0.12 - 1.5 0.56 1.0

A level of 20% immobilization was recorded in the 0.32 mg/L test concentration; however, as the immobilization recorded in the 0.56 mg/L test concentration was only 10% immobilization it was considered unlikely that the effect observed was as a result of exposure to the reference item and therefore the NOEC was 0.56 mg/L.
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 (% v/v Saturated Solution)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative ImmobilizedDaphnia		Observations		Cumulative ImmobilizedDaphnia		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	1	5 N	4 N
100	5	5	AI	AI	5	5	AI	AI

Rep  = Replicate

N      = Normal

AI     = All Daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	24 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5N	5 N	5 N	5 N
1.9 (0.19)	0	0	0	0	0	0	5 N	5 N	5 N	5 N
4.1(0.43)	0	0	0	0	0	0	5 N	5 N	5 N	5 N
9.1(0.98)	0	3	2	2	7	35	5R	2 R	3 R	3 R
20(2.3)	4	3	2	4	13	65	1 R	2 R	3 R	1 R
45(4.9)	5	5	5	5	20	100	AI	AI	AI	AI
100(9.6)	5	5	5	5	20	100	AI	AI	AI	AI
Nominal Concentration (% v/v Saturated Solution)	48 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.9 (0.19)	0	1	1	0	2	5	5 N	4 N	1 R 3 N	5 N
4.1(0.43)	0	0	0	0	0	0	1 R 4 N	3 R 2 N	2 R 3 N	1 R 4 N
9.1(0.98)	3	3	3	2	11	55	2R	2 R	2 R	3 R
20(2.3)	5	5	3	5	18	90	AI	AI	2 R	AI
45(4.9)	5	5	5	5	20	100	AI	AI	AI	AI
100(9.6)	5	5	5	5	20	100	AI	AI	AI	AI

Rep  = Replicate

N      = Normal

R      = Reduced mobility

AI     = All Daphnia immobilized

( )    = Measured 0 hour concentration mg/L

Water Quality Measurements

Nominal Concentration (% v/v Saturated Solution)		0 Hours			24 Hours	48 Hours
		pH	mg O2/L	Temperature °C	Temperature ºC	pH	mg O2/L	Temperature °C
Control	Rep1	8.0	9.5	20	21	8.0	9.4	21
1.9 (0.19)	Rep1	8.0	9.2	20	21	7.9	8.8	21
4.1(0.43)	Rep1	8.0	9.2	21	21	7.9	8.8	21
9.1(0.98)	Rep1	8.0	9.2	21	21	7.9	8.8	21
20(2.3)	Rep1	8.0	9.1	21	21	8.2	8.7	21
45(4.9)	Rep1	8.0	9.1	21	21	8.1	8.9	20
100(9.6)	Rep1	8.0	9.1	21	21	8.0	8.9	21

Rep  = Replicate

( )    = Measured 0 hour concentration mg/L

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 (immobilisation) value with Daphnia magna is 0.92 mg/I.

Executive summary:

A study was performed to assess the acute toxicity of PG-RAW-90 -032 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of 9.78 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of aqueous solubility of the test item in Elendt M7 media under test conditions.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.9, 4.1, 9.1, 20, 45 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 21 °C under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (the first approximate 2 liters used to pre-condition the filter was discarded) to give a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the required remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. As loss of test item occurred during the range-finding test whilst using glass vessels covered to reduce evaporation, completely filled glass vessels sealed using ground glass stoppers were used during the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.19 to 9.6 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on the 0-Hour measured test concentrations of 0.19, 0.43, 0.98, 2.3, 4.9 and 9.6 mg/L. Exposure of Daphnia magna to the test item gave the following results based on the 0-Hour measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			NOEC (mg/L)	LOEC (mg/L)
48	0.92	0.37	-	2.1	0.43	0.98

Description of key information

A study was performed to assess the acute toxicity of PG-RAW-90 -032 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of 9.78 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of aqueous solubility of the test item in Elendt M7 media under test conditions.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.9, 4.1, 9.1, 20, 45 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 21 °C under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (the first approximate 2 liters used to pre-condition the filter was discarded) to give a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the required remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. As loss of test item occurred during the range-finding test whilst using glass vessels covered to reduce evaporation, completely filled glass vessels sealed using ground glass stoppers were used during the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.19 to 9.6 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on the 0-Hour measured test concentrations of 0.19, 0.43, 0.98, 2.3, 4.9 and 9.6 mg/L. Exposure of Daphnia magna to the test item gave the following results based on the 0-Hour measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			NOEC (mg/L)	LOEC (mg/L)
48	0.92	0.37	-	2.1	0.43	0.98

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.92 mg/L