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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 05 February 2018 Experimental completion date 29 March 2018
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:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: 4-(1,1-dimethylethyl)cyclohexyl methacrylate (CAS 46729-07-1)
Batch: DAR-16030
Purity: 89.2%
Physical state/Appearance: white solid prior to heating
clear colorless liquid after heating
Expiry Date: 01 August 2018
Storage Conditions: approximately 4 ºC in the dark
Analytical monitoring:
yes
Details on sampling:
Range-finding Tests
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
Samples were taken for quantitative analysis from the control and each test group, from the freshly prepared bulk test preparation at 0 and 24 hours and from old or expired pooled replicates at 24 and 48 hours. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary. Only samples from the NOEC were analyzed.
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Preparation
A nominal amount of test item (550 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
Visual inspection of the centrifuged test samples showed that undissolved test item remained. As such the use of centrifugation as a means to remove dispersed test item was considered inappropriate for this test item. From the filtered test samples it was evident that there was no increase in the dissolved test item concentration when the preparation period was extended beyond 24 hours, furthermore, there were no differences between the pre-conditioning volumes discarded.
Following discussions with the Sponsor, their preferred method of preparation was a slow stir method as the values obtained from the high energy input system were in excess of the predicted water solubility value of approximately 1 mg/L. Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred via magnetic stirrer at a speed such that a dimple was formed at the water surface 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 1 liter discarded).

Range-finding Tests
A nominal amount of test item (250 mg) was dispersed in 5 liters of test water with the aid of magnetic stirring at a speed such that a dimple is formed at the water surface for a period of 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 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
Amounts of test item (599.8 and 549.8 mg for each 24 hour period respectively) were 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) 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.
Due to a technical oversight, the method of test item preparation differed from that requested by the sponsor. This, however, was considered not to have adversely affected the results of the test as a greater measured test concentration was achieved in the definitive test when compared to the range finding test and no immobilization was observed.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
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 (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 20 °C to 21 °C throughout the test
pH:
Recorded at start and termination of the test:
8.1 - 8.2 at start, 8.6 - 8.9 at termination
Dissolved oxygen:
Recorded at start and termination of the test.
9.1 - 9.4 mg O2/L at start, 8.6 -8.9 mg O2/L at termination
Salinity:
Not applicable
Nominal and measured concentrations:
Range-finding Tests: 0.10, 1.0, 10 and 100% v/v saturated solution (nominal)

Definitive Test: 10, 18, 32, 56 and 100% v/v saturated solution (nominal)
Details on test conditions:
Range-finding Tests
In the initial 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. Prior to use the test item was heated to 35 °C in order to liquefy.
In the initial 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 with a photoperiod of 16 hours light and 8 hours darkness for a period of 24 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 hours the number of immobilized daphnids were recorded.
As an inconsistent pattern of immobilization was observed, testing was terminated after 24 hours exposure and the test repeated as detailed above. The number of immobilized daphnids in the second test were recorded after 24 and 48 hours. The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
Exposure Conditions
As in the range-finding test 150 mL glass beakers 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 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.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Tests
No immobilization was observed at the test concentration of 10% v/v saturated solution, however, immobilization was observed at 0.10, 1.0 and 100% v/v saturated solution.
No sub-lethal effects of exposure were observed throughout 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 to range from 0.14 and 1.4 mg/L. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was not stable under test conditions.

Definitive Test
Analysis of the freshly prepared test media at 0 and 24 hours showed the measured test concentration to range from 1.9 to 2.0 mg/L. Analysis of the old or expired test media at 24 and 48 hours showed the measured test concentration had declined, to range from 0.73 and 0.94 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

Immobilization Data
There was no immobilization in 20 daphnids exposed to a test concentration of 1.3 mg/L for a period of 48 hours.
The NOEC after 24 and 48 hours exposure was 1.3 mg/L.
The differences observed in immobilization between the second range finding test and definitive test was considered to be due to a potential contamination in the second range finding test. A higher measured test concentration was achieved in the 100% v/v saturated solution in the definitive test and no daphnids were observed to be immobilized.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the test.

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.

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 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 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: 24 hours. EC50 (mg/L):0.79. 95% Confidence Limits (mg/L): 0.73 - 0.86. NOEC (mg/L): 0.56. LOEC (mg/L): 1.0
Time Point: 48 hours. EC50 (mg/L):0.75. 95% Confidence Limits (mg/L): 0.56 - 1.0. NOEC (mg/L): 0.56. LOEC (mg/L): 1.0

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.

The geometric mean measured test concentration was determined to be:

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

100

1.3

Inspection of the immobilization data gave the following results:

Time
(hour)

EC50 (mg/L)

95% Confidence limits (mg/L)

24

>1.3

Not determined

48

>1.3

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the geometric mean measured test concentration gave a 48 hour EC50 value of greater than 1.3 mg/L. The NOEC was greater than or equal to 1.3 mg/L.
This study showed that there were no toxic effects at saturation.
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 2.0 mg/L was obtained from a saturated solution method of preparation employing a high energy input system (propeller stirring at approximately 1500 rpm). At the request of the Sponsor a slow stir method of preparation was selected for testing.

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 (54.5 and 50 mg/L for each 24 hour period respectively) 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, first approximate 1 liter discarded in order to pre-condition the filter) 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.

Due to a technical oversight, the method of test item preparation differed from that requested by the sponsor. This, however, was considered not to have adversely affected the results of the test as a greater measured test concentration was achieved in the definitive test when compared to the range finding test.

Results

Chemical analysis of the freshly prepared 100% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 1.9 to 2.0 mg/L. Analysis of the old or expired 100% v/v saturated solution test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.73 and 0.94 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 1.3 mg/L. The No Observed Effect Concentration (NOEC) was 1.3 mg/L. This study showed that there were no toxic effects at saturation.

Description of key information

A study according to OECD guideline 202 under GLP, was performed to assess the effect of the test item.

Daphnia magna was exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours. The geometric mean measured test concentration was determined to be 1.3 mg/L for 100% v/v saturated solution.

The 48h-EC50 > 1.3 mg/L (geomean), which is equal to the 100% v/v saturated solution.The 48h-EC50 is greater than the maximum water solubility of the test substance, therefore.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.3 mg/L

Additional information