Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

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 27 May 2014 and 21 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliabiility 1 as it has been conducted according to OECD Test Guideline 202 using the Daphnia Immobilisation method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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. The 0-Hour 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 and 24 hours (fresh media) and 24 and 48 hours (old media).

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 approximately 6.6 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.

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. 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

Nominal and measured concentrations:

Range-finding test: Nominal test concentrations of 0.10, 1.0, 10, and 100 % v/v saturated solution.
Definitive test: 10, 18, 32, 56 and 100 % v/v saturated solution.

Details on test conditions:

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 34 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 (first approximate 1 liter discarded in order to pre-condition the filter). 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.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20 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. The 0-Hour 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 (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 1 liter 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.

The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 24 hours (fresh media) and 24 and 48 hours (old media).


Exposure Conditions
As in the range-finding test 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 (464 to 501 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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confience limits of 8.5 - 10 mg/L.

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

6.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

9.3 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, 1.0 and 10% v/v saturated solution. However, immobilization was observed at 100% v/v saturated solution.

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% v/v saturated solution test preparations at 0 and 48 hours showed that measured concentrations of 3.69 and 2.89 mg/L were obtained respectively. Analysis of the 100% v/v saturated solution at 0 and 48 hours showed that measured concentrations of 33.9 and 21.5 mg/L were obtained respectively indicating that the test item was possibly unstable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 3.6 to 30 mg/L. A decline in measured test concentrations was observed in the old or expired test preparations at 24 and 48 hours to between 3.1 and 22 mg/L and hence it was considered appropriate to calculate the 10, 18, 32 and
56% v/v saturated solution results based on the time-weighted mean measured test concentrations, and the 100% v/v saturated solution results on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

Immobilization Data
Analysis of the immobilization data by the probit method (Finney, 1971) at 24 and the trimmed Spearman-Karber method (Hamilton et al, 1977) at 48 hours based on the time-weighted and geometric mean measured concentrations gave the following results:

The 24 h EC50 was 13 mg/L with 95 % confidence limits of 11 - 14 mg/L.
The 48 h EC50 was 9.4 mg/L with 95 % confidence limits of 8.5 - 10 mg/L.

The No Observed Effect Concentrations after 24 and 48 hours exposure were 9.3 and 6.2 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 15 and 9.3 mg/L respectively

The slope and its standard error of the response curve at 24 hours was 12 (SE = 2.7).

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Study Number 41400711) usedpotassiumdichromate 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 thedefinitivetest.
 
Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971) at 24 hours and by the trimmed Spearman-Karber method (Hamiltonet al1977) using the ToxCalc software package (ToxCalc 1999) at 48 hours based on the nominal test concentrations gave the following results:
The 24 h EC50 was 0.87 mg/L with 95 % confidence limits of 0.75 - 1.0. The NOEC was 0.56 and the LOEC was 1.0.
The 48 h EC50 was 0..71 mg/L with 95 % confidence limits of 0.65 - 0.78. The NOEC was 0.56 and the LOEC was 1.0.

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.

The time-weighted mean measured test concentrations were determined to be

Nominal Test Concentration (% v/v Saturated Solution)	Time Weighted Mean Measured Test Concentration (mg/L)
10	3.4
18	6.2
32	9.3
56	15
100	25*

*Result based on the geometric mean measured concentration

Validation Criteria

The test was considered to be valid given that no more than 10% 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

The results of the water quality measurements are given in Table 3. 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, the test preparations were observed to be clear colorless solutions.

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	0
10	0	0
100	10	10

Cumulative Immobilization Data in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	Time-Weighted 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	Control	0	0	0	0	0	0	1	0	0	0	1**	5
10	3.4	0	0	0	0	0	0	0	0	0	0	0	0
18	6.2	0	0	0	0	0	0	1	0	0	0	1**	5
32	9.3	0	0	0	1	1	5	1	4	3	2	10	50
56	15	2	5	5	4	16	80	5	5	5	5	20	100
100	25*	5	5	5	5	20	100	5	5	5	5	20	100

R₁– R₄= Replicates 1 to 4

**Single immobilized daphnid considered not to be significant as it falls within the validation criteria.

*based on geometric mean

Water Quality Measurements

Nominal Concentration (% v/v Saturated Solution)		0 Hours Fresh Media			24 Hours Old Media
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	7.8	9.2	20	7.6	9.0	21
10	R1	7.9	9.2	20	7.9	9.0	21
18	R1	7.9	9.2	20	7.9	8.9	21
32	R1	7.9	9.3	20	7.9	8.9	21
56	R1	7.9	9.3	20	7.9	8.9	21
100	R1	7.9	9.4	20	7.9	8.9	21

R₁= Replicate 1

Nominal Concentration (% v/v Saturated Solution)		24 Hours Fresh Media			48 Hours Old Media
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	7.8	9.1	20	7.7	9.3	21
10	R1	7.7	9.2	20	7.8	9.1	21
18	R1	7.8	9.1	20	7.9	9.0	21
32	R1	7.8	9.2	20	7.9	9.0	21
56	R1	7.8	9.1	20	7.9	9.0	21
100*	R1

*No measurements taken as 100% immobilization occurred therefore media not renewed

Results of analytical investigations

Preparation of Test Samples

Nominal Concentra­tion of Test Item	Sample Volume	Final Volume	Sample Preparation Factor
[% v/v Saturated Solution]	[mL]	[mL]	F
Control	200	10	0.05
10	200	10	0.05
18	200	10	0.05
32	200	10	0.05
56	200	10	0.05
100	200	10	0.05

 

Linearity Data

Concentration of Test Item	Mean Area
[mg/L]	[counts]
0	2.219 x 10^4
1.01	3.955 x 10^5
5.03	1.804 x 10^6
10.1	3.744 x 10^6
50.3	1.847 x 10^7
101	3.714 x 10^7
99.6	3.653 x 10^7
604	2.144 x 10^8
805	2.850 x 10^8
1006	3.526 x 10^8

 

Results for Range-Finding Samples

Time Point	Nominal Concentration of Test Item in Range-Finding Sample cnom	Measured Concentration  of Test Item in Sample Vial x	Sample Preparation Factor F	Determined Concentration of Test Item in Range-Finding Sample c
[hours]	[% v/v Saturated Solution]	[mg/L]		[mg/L]
0	10	73.8	0.05	3.69
	100	678	0.05	33.9
48	10	57.8	0.05	2.89
	100	430	0.05	21.5

 

Results for Spiked Recovery Samples

Nominal Concentra­tion of Test Item cnom		Fortified Concentra­tion of Test Item in the Spiked Sample cfort		Measured Concentra­tion of Test Item in the Sample Vial x		Sample Preparation Factor F		Determined Concentra­tion of Test Item in the Spiked Sample c		Mean Analytical Recovery Rate R		Precision (Relative Standard Deviation of Recovery)
[mg/L]		[mg/L]		[mg/L]				[mg/L]		[%]		[%]
2		2.00		38.2		0.05		1.91		99		1.8
		2.00		39.8		0.05		1.99
		2.00		40.2		0.05		2.01
		2.00		39.6		0.05		1.98
		2.00		39.6		0.05		1.98
30		29.9		580		0.05		29.0		98		1.3
		29.9		576		0.05		28.8
		29.9		580		0.05		29.0
		29.9		588		0.05		29.4
		29.9		594		0.05		29.7
Acceptance Target									80-120		<10

For reporting purposes, x has been calculated from c retrospectively.

 

Results for Test Samples

Time Point		Nominal Concentration of Test Item in Test Sample cnom		Measured Concentration  of Test Item in Sample Vial x		Sample Preparation Factor F		Determined Concentration of Test Item in Test Sample   c
[hours]		[% v/v Saturated Solution]		[mg/L]				[mg/L]
0		Control		<LOQ		0.05		<LOQ
(Fresh)		10		75.4		0.05		3.77
		18		144		0.05		7.22
		32		224		0.05		11.2
		56		388		0.05		19.4
		100		592		0.05		29.6
24		Control		<LOQ		0.05		<LOQ
(Old)		10		61.8		0.05		3.09
		18		116		0.05		5.82
		32		183		0.05		9.16
		56		228		0.05		11.4
		100		436		0.05		21.8
24		Control		<LOQ		0.05		<LOQ
(Fresh)		10		72.0		0.05		3.60
		18		131		0.05		6.54
		32		200		0.05		10.0
		56		332		0.05		16.6
48		Control		<LOQ		0.05		<LOQ
(Old)		10		62.8		0.05		3.14
		18		106		0.05		5.30
		32		143		0.05		7.17
		56		296		0.05		14.8

LOQ =Limit of Quantification

- = not applicable

For reporting purposes, x has been calculated from c retrospectively.

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 and geometric mean measured test concentrations:
The 48 h EC50 was 9.4 mg/L with 95 % confidence limits of 8.5 - 10 mg/L The NOEC was 6.2 mg/L and the LOEC was 9.3 mg/L.

Executive summary:

The acute toxicity to invertebrates of the test substance, IFF 215 (Floriane), was assessed according to OECD Test Guideline 202 using the Daphnia Immobilisation method. The 48 h EC50 was 9.4 mg/L with 95 % confidence limits of 8.5 - 10 mg/L The NOEC was 6.2 mg/L and the LOEC was 9.3 mg/L.

Description of key information

The acute toxicity to invertebrates of the test substance, IFF 215 (Floriane), was assessed according to OECD Test Guideline 202 using the Daphnia Immobilisation method. The 48 h EC50 was 9.4 mg/L with 95 % confidence limits of 8.5 - 10 mg/L The NOEC was 6.2 mg/L and the LOEC was 9.3 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

9.4 mg/L