Cyclohexanecarboxylic acid, 2,2,6-trimethyl-, 2-propen-1-yl ester, (1R,6S)-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 08 July 2014; Experimental Completion Date: 10 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: US EPA Ecological Effects Test Guidelines OPPTS 850.1000 “Special Considerations for Conducting Aquatic Laboratory Studies”

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/L) of test item in test water for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorious Sartopore, first approximate 1 liter discarded in order to pre-condition the filter) to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl (1R,6S)-2,2,6-trimethylcyclohexane-1-carboxylate

EC Number:

810-519-1

Cas Number:

1648784-10-4

Molecular formula:

C13H22O2

IUPAC Name:

prop-2-en-1-yl (1R,6S)-2,2,6-trimethylcyclohexane-1-carboxylate

Test material form:

other: liquid

Details on test material:

Identification: NACET10419
Chemical Name: (6S)-Allyl 2,2,6-trimethylcyclohexanecarboxylate
CAS No.: 1450841-11-8(planar)
Physical state/Appearance: clear colorless liquid
Batch: KU31213
Purity: 100%
Expiry Date: not supplied
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 18, 32, 56 and 100% v/v saturated solution (definitive test)

- Sampling method: Samples were taken from the control and each 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.

- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

TEST WATER:
Reconstituted water (ISO medium) used for both the range-finding and definitive test is defined below. Prior to use, the water was diluted 50:50 with deionized reverse osmosis water in order to reduce the hardness.

Ingredient (mg/L) Final Concentration
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 64.75
KCl 5.75

PROCEDURE:
PRELIMINARY MEDIA PREPARATION TRIAL:
Information provided indicated the test item was insoluble in water.

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 saturated solution method of preparation was the most appropriate for this test item.

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 (2250 mg) was dispersed in 22.5 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 Gelman Acrocap filter (first approximate 500 mL 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 100 % v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 21°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 250 mL test and control vessel contained 200 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. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

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.

Experimental Preparation:
A nominal amount of test item (2250 mg) was dispersed in 22.5 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 1 liter 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, 18, 32 and 56% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

The reconstituted water was diluted with deionised reverse osmosis water to give an approximate theoretical total hardness of 150 mg/L as CaCO3.

Test temperature:

Temperature was maintained at approximately 20°C throughout the test.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Nominal and measured concentrations:

Definitive Test:
Nominal test concentration (% v/v Saturated Solution): 10, 18, 32, 56, 100
Time Weighted/Geometric Mean Measured Test Concentration (mg/L): 1.1, 1.4, 2.9, 4.1, 7.2

Details on test conditions:

DEFINITIVE TEST:
Exposure conditions:
250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 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 at approximately 20°C with a photoperiod of 16 hours light (610 to 670 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.

EVALUATIONS:
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 for approximately 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 HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

The temperature was also measured every hour in one replicate of the control using a Testo temperature logger.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

time-weighted/geometric mean measured test concentration

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Limits: 1.5 - 1.8 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

time-weighted/geometric mean measured test concentration

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

time-weighted/geometric mean measured test concentration

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

No significant 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.

A single immobilized daphnid was observed in the 1.0% v/v saturated solution test group. However, as no more than 10% immobilization was observed, and no further immobilization was observed at the higher test concentration of 10% v/v saturated solution, this was considered not to have had an impact on the outcome of the test.

Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.

Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations of 1.3 and 11 mg/L respectively were obtained. There was a significant decline in the measured concentrations at 48 hours to 0.66 and 6.2 mg/L indicating that the test item was not stable under test conditions.

DEFINITIVE TEST:
Verification of Test Concentrations:
Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 1.2 to 9.5 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24 and 48 hours to between 0.84 and 5.4 mg/L and hence it was considered appropriate to calculate the results based on the time-weighted/geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean method was used for the highest concentrations as the concentrations were only calculated over one 24 hours period, the time weighted mean method was used for the rest of the concentrations as data for the full 48 hours was available.

The time-weighted/geometric mean measured test concentrations were determined to be:
Nominal test concentration (% v/v Saturated Solution): 10, 18, 32, 56, 100
Time Weighted/Geometric Mean Measured Test Concentration (mg/L): 1.1, 1.4, 2.9, 4.1*, 7.2*
* Geometric mean measured concentration

Initial analysis of the 10% v/v saturated solution at 0 and 24 hours showed that measured concentrations of 0.12 and 0.92 mg/L were obtained respectively. However, as this did not fit with the pattern of a decline in measured concentration that was observed in the other concentrations, analysis of the duplicate samples was conducted. Duplicate analysis showed that measured concentrations of 1.2 and 1.3 mg/L were obtained respectively, which although didn’t show a decline, were considered to be more consistent with the values obtained from the other test concentrations.

Immobilization Data:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

Analysis of the immobilization data by the trimmed Spearman-Karber method (Hamilton et al, 1977) at 24 and 48 hours based on the time-weighted mean measured/geometric mean measured test concentrations gave the following results:

24 h EC50: 2.7 mg/L
48 h EC50: 1.6 mg/L

The No Observed Effect Concentrations after 24 and 48 hours exposure were 1.4 and 1.1 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 2.9 and 1.4 mg/L respectively.

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 ≥3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA:
Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

OBSERVATIONS ON TEST ITEM SOLUBILITY:
Throughout the duration of the test all control and test solutions 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.

Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al 1977 ) at 48 hours based on the nominal test concentrations gave the following results:

24 h EC50: 0.87 mg/L
24 h NOEC: 0.56 mg/L
24 h LOEC: 1.0 mg/L
48 h EC50: 0.71 mg/L
48 h NOEC: 0.56 mg/L
48 h LOEC: 1.0 mg/L

The No Observed Effect Concentration is based upon 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.

Any other information on results incl. tables

Table 1     Cumulative Immobilization Data in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)	Cumulative Immobilized Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	1*
10	0	0
100	10	10

*      Considered to be due to natural causes as no more than 10% immobilization occurred and no further immobilization was observed at the higher concentration of 10% v/v saturated solution

Table 2     Cumulative Immobilization Data in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	Time-Weighted /Geometric Mean Measured Test Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)
		24 Hours						48 Hours
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control		0	0	0	0	0	0	0	0	0	0	0	0
10	1.1	0	0	0	0	0	0	0	0	0	0	0	0
18	1.4	0	0	0	0	0	0	1	1	3	4	9	45
32	2.9	0	4	3	2	9	45	5	5	5	5	20	100
56	4.1	5	5	5	5	20	100	5	5	5	5	20	100
100	7.2	5	5	5	5	20	100	5	5	5	5	20	100

R₁– R₄= Replicates 1 to 4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the time-weighted mean/geometric mean measured test concentrations:
48 h EC50: 1.6 mg/L
The No Observed Effect Concentrations after 48 hours exposure was 1.1 mg/L. The Lowest Observed Effect Concentrations after 48 hours exposure was 1.4 mg/L.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1010 (April 1996).

Methods:

Information provided indicated the test item was insoluble in water.

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 saturated solution method of preparation was the most appropriate method.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of approximately 20°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 (0.2 µm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results:

Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 1.2 to 9.5 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24 and 48 hours to between 0.84 and 5.4 mg/L and hence it was considered appropriate to calculate the results based on the time-weighted mean/geometric mean measured test concentration only in order to give a “worst case” analysis of the data

Exposure of Daphnia magna to the test item gave the following results based on the time-weighted mean/geometric mean measured test concentrations:

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)
48	1.6	1.5	-	1.8	1.1	1.4