[No public or meaningful name is available]

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 start date 26 June 2020

Experimental completion date 31 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Molecular formula:

C20H36O2

IUPAC Name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Test material form:

liquid: viscous

Details on test material:

Chemical name: 1‐[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan‐1‐yl)oxy]pentan‐2‐ol and 1‐[(1,4,4‐trimethyltricyclo[6.3.1.02,5]dodecan‐8‐yl)oxy]pentan‐2‐ol and isomers

Specific details on test material used for the study:

Identification: FRET 18-0091
Chemical Name: 1-[(4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-yl)oxy]pentan-2-ol and 1-[(1,4,4-trimethyltricyclo[6.3.1.02,5]dodecan-8-yl)oxy]pentan-2-ol and isomers
Batch: RDEA571-42
Purity: 92%
Physical State/Appearance: Light yellow viscous liquid
Expiry Date: 31 December 2021
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding Test
A sample of each test concentration was taken for immediate chemical analysis at 0 hours from the bulk test preparations, and at 48 hours from the pooled replicates in order to determine the stability of the test item under test conditions. A duplicate set of samples was taken on each occasion and stored frozen for further analysis if required.

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

Verification of Test Concentrations
Samples were taken from the control and each of the test groups from the bulk test preparation at 0 and 24 hours and from the pooled replicates at 24 and 48 hours for quantitative analysis. The 0 and 24 (fresh and old/expired) hour samples were stored frozen prior to being analyzed with the 48 hour samples which were taken for immediate analysis.

Duplicate sets of samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Experimental Design and Study Conduct
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 Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). 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 dissolved test item concentration of approximately 0.89 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 Gelman Acrocap filter (the first approximate 500 mL 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.

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 120 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
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 Gelman Acrocap filter (the first approximate 500 mL 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 the remaining 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 first instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 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 (Raphidocelis subcapitata) and GEMMA Micro 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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

20 degrees C to 21 degrees C

pH:

pH recorded daily using a physical meter
Throughout the test, while there were no treatment related differences for pH.
Range 7.8-8.0.

Dissolved oxygen:

The dissolved oxygen concentration were measured using a digital meter.
Range 8.8-8.9mg O2/L

Salinity:

Not applicable as fresh water study.

Nominal and measured concentrations:

Range finding test 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:

Preliminary Study
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 120 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.

In the definitive test 150 mL glass vessels 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 and 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 a 2.0 mL wide bore glass pipette from the 24 Hour old test media into the fresh test media.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.87 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range finding test are given in Table 1.
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 100% v/v saturated solution. These responses were reduced mobility and trapping at the surface.
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 fresh test preparations at 0 hours (see Annex 6) showed measured test concentrations to range from 0.019 to 0.97 mg/L. Chemical analysis of the old/expired test preparations at 48 hours showed measured test concentrations to range from 0.012 to 0.79 mg/L. There was decline in the measured concentrations indicating that the test item was not stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.13 to 1.1 mg/L.
A decline in the measured old/expired test concentrations was observed at 24 and 48 hours to between 0.087 and 1.0 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

Results with reference substance (positive control):

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

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)
10	0.12
18	0.19
32	0.31
56	0.59
100	1.0

 Sub-Lethal Effects

Sub‑lethal effects of exposure were observed in the 1.0 mg/L test concentration.  These responses were reduced mobility and trapping at the surface, please refer to the attached table "Cumulative Immobilization Data and Observations in the Definitive Test".

 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.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

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 geometric mean measured test concentrations:

At the time of 48 hours -

EC50 = 0.87 mg/L
NOEC = 0.59 mg/L
LOEC = 1.0 mg/L

95% Confidence Limits 0.74 - 0.99 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.

Methods

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 approximately 0.89 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item 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 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 21 °C under semi‑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 Gelman Acrocap filter (the first approximate 500 mL 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 remaining test concentrations.  Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

  Results

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.13 to 1.1 mg/L.  Analysis of the old/expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.087 and 1.0 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.  The geometric mean measured test concentrations were determined to be 0.12, 0.19, 0.31, 0.59 and 1.0 mg/L.

 

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

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			NOEC (mg/L)	LOEC (mg/L)
48	0.87	0.74	-	0.99	0.59	1.0

 

NOEC  = No observed effect concentration

LOEC  = Lowest observed effect concentration