4-(1,1-dimethylethyl)cyclohexyl methacrylate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 18 January 2018 Experimental completion date 08 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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 Test
A sample of each test concentration was taken for chemical analysis at 0 and 72 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 from the control and each test group from the bulk test preparation at 0 hours, from additional samples incubated alongside the test at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
Information provided by the Sponsor indicated the test item was not readily soluble 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.
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.

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).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ±1 ºC throughout the test.

pH:

The pH value of the test cultures was observed to increase from pH 7.6 at 0 hours to pH 9.2 at 72 hours
The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 8.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

Range-Finding Test.
Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.

Definitive Test
Nominal test concentrations 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
Geometric Mean Measured Test Concentration (mg/L): 0.019, 0.038, 0.091, 0.31, 1.2

Details on test conditions:

Range-Finding Test
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.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
A nominal amount of test item (125 mg) was dispensed on to the surface of 2.5 liters of culture medium prior to stirring via magnetic stirrer at a rate such that a dimple was formed at the media surface 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 stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.1 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a haemocytometer and light microscope. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a haemocytometer and light microscope.
A sample of each test concentration was taken for chemical analysis at 0 and 72 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: 1.0, 3.2, 10, 32 and 100% v/v saturated solution. Prior to use the test item was heated to 35 ºC in order to liquefy.
Experimental Preparation
A nominal amount of test item (125 mg) was dispensed on to the surface of 2.5 liters of culture medium prior to stirring via magnetic stirrer at a rate such that a dimple was formed at the media surface 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 stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 6.2 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and 3 flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.21 x 10^5 cells per mL. Inoculation of 900 mL of test medium with 6.2 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Range-finding Test
The results showed no significant effect on growth rate at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth rate was observed to be reduced at 100% v/v saturated solution.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 1.0, 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.024 to 1.5 mg/L. A decline in measured test concentrations was observed at 72 hours to between less than the LOQ, determined to be 0.011 mg/L, and 0.41 mg/L. This decline in measured test concentrations indicated that the test item was unstable under the conditions of the test.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.028 to 1.8 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.024 to 1.4 mg/L at 24 hours, 0.020 to 1.1 mg/L at 48 hours, and from less than the LOQ, determined to be 0.011 mg/L, to 0.34 mg/L at 72 hours.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0055 mg/L) was used to enable calculation of the geometric mean measured concentration.

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
The effects on growth observed in the definitive test were not as significant as those seen in the range-finding test. As similar measured test concentrations were obtained from the chemical analyses it was considered that the test systems had been dosed correctly in both instances. As such the significant inhibitory effects observed at 100% v/v saturated solution in the range-finding test were attributable to contamination from an unknown source.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 to 72 hour): 1.03 mg/L
ErC20 (0 to 72 hour): >1.2 mg/L
ErC50 (0 to 72 hour): >1.2 mg/L
Where ErCx is the test concentration that reduced growth rate by x%.
It was not possible to calculate ErC20 or ErC50 values as only 13% inhibition of growth rate was observed at the maximum attainable test concentration of 1.2 mg/L.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control, 0.019, 0.038, 0.091 and 0.31 mg/L test concentrations (P≥0.05), however the 1.2 mg/L test concentration was significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.31 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 1.2 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 0.29 mg/L
EyC20 (0 to 72 hour): 0.49 mg/L
EyC50 (0 to 72 hour): 1.2 mg/L
Where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences between the control, 0.019, 0.038, 0.091 and 0.31 mg/L test concentrations (P≥0.05), however the 1.2 mg/L test concentration was significantly different (P<0.05) and, therefore the NOEC based on yield was 0.31 mg/L. Correspondingly the LOEC based on yield was 1.2 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 282 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.41 x 10^6 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hours) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.019, 0.038, 0.091 and 0.31 mg/L test cultures were observed to be green dispersions whilst the 1.2 mg/L test cultures were observed to be pale green dispersions.

Results with reference substance (positive control):

A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Geometric mean measured test concentrations

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a Percentage of the 0-Hour Measured Test Concentration
1.0	0.019	69
3.2	0.038	58
10	0.091	46
32	0.31	55
100	1.2	67

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 hour period and based on the geometric mean measured test concentrations gave the following results:
Inhibition of Growth Rate
ErC10 (0 to 72 hour): 1.03 mg/L
ErC20 (0 to 72 hour): >1.2 mg/L
ErC50 (0 to 72 hour): >1.2 mg/L
NOEC: 0.31 mg/L
LOEC: 1.2 mg/L

The 72h-ErC10 = 1.03 mg/L (geomean) and the 72h-ErC50 > 1.2 mg/L (geomean), which is equal to the 100% v/v saturated solution. The 72h-ErC50 is greater than the maximum water solubility of the test substance, therefore.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Information provided by the Sponsor indicated the test item was not readily soluble 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 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, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium with the aid of magnetic stirring at a rate such that a dimple was formed at the media surface 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 groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.028 to 1.8 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.024 to 1.4 mg/L at 24 hours, 0.020 to 1.1 mg/L at 48 hours, and from less than the limit of quantification (LOQ), determined to be 0.011 mg/L, to 0.34 mg/L at 72 hours.

Given this decline in measured test concentrations 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. These were determined to be 0.019, 0.038, 0.091, 0.31 and 1.2 mg/L.

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

Response Variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	1.03	>1.2*	>1.2	0.31	1.2
Yield	0.29	0.49	1.2	0.31	1.2

*It was not possible to calculate E_rC₂₀or E_rC₅₀values as only 13% inhibition of growth rate was observed at the maximum attainable test concentration of 1.2 mg/L.

Description of key information

A study according to OECD guideline 201 under GLP, was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata.

Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution for 72 hours. The geometric mean measured test concentration were determined to be 0.019, 0.038, 0.091, 0.31 and 1.2 mg/L.

The 72h-ErC10 = 1.03 mg/L (geomean) and the 72h-ErC50 > 1.2 mg/L (geomean), which is equal to the 100% v/v saturated solution. The 72h-ErC50 is greater than the maximum water solubility of the test substance, therefore.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.2 mg/L

EC10 or NOEC for freshwater algae:

1.03 mg/L