Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 01 October 2014 and 13 November 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Isobutyl Phenyl Acetate
Chemical nature: 2-methylpropyl 2-phenylacetate
EC No.: 203-007-9
CAS No.: 102-13-6
Molecular weight: 192.2 g/mol
Appearance/physical description: clear, colourless liquid
Empirical formula: C12H16O2
Batch No: SC00010162
Purity: 100%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 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.

Test solutions

Vehicle:
no
Details on test solutions:
Range-Finding Test
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.

Due to the possible light sensitive nature of the test item all test item preparation was performed under laboratory safety lighting/shielded from the light.

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.

A nominal amount of test item (1100mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 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 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 (2.8 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.


Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 2.6, 6.4, 16, 40 and 100% v/v saturated solution.

Experimental Preparation
Due to the possible light sensitive nature of the test item all test item preparation was performed under laboratory safety lighting/shielded from the light.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium 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 stock solutions of 40, 16, 6.4 and 2.6% v/v saturated solution. An aliquot (1500 mL) of each of the stock solutions was separately inoculated with 10.9 mL of algal suspension to give the required test concentrations of 2.6, 6.4, 16, 40 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.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 maintained in the laboratory under constant aeration and constant illumination at 21 ± 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 1E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1E+04 - 1E+05 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

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

Definitive test
Nominal test concentrations of 2.6, 6.4, 16, 40 and 100% v/v saturated solution.
Initial measured concentrations of 2.0, 4.8, 13, 31 and 79 mg/L.
Details on test conditions:
Culture Medium
The culture medium used for the definitive test, range-finding test and for maintenance of the stock cultures was ASTM. However, as the definitive test was conducted in completely filled and sealed vessels, the culture medium was modified by addition of 500 mg/L of sodium bicarbonate to provide a sufficient supply of CO2 and to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

Range-Finding Test
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 mg/L for a period of 72 hours.

Due to the potentially light sensitive nature of the test item all test item preparation was performed under laboratory safety lighting/shielded from the light.

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 test item was dissolved directly in culture medium.

A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. 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 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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 Coulter® Multisizer Particle Counter. 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 – 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 Coulter® Multisizer Particle Counter.

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.


Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 2.6, 6.4, 16, 40 and 100 % v/v saturated solution.

Experimental Preparation
Due to the potentially light sensitive nature of the test item all test item preparation was performed under laboratory safety lighting/shielded from the light.

A nominal amount of test item (500 mg) was dissolved in culture medium with the aid of vigorous magnetic stirring for 1-Hour and the volume adjusted to 5 liters to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 40, 16, 6.4 and 2.6 % v/v saturated solution. An aliquot (1500 mL) of each of the stock solutions was separately inoculated with algal suspension (7.9 mL) to give the required test concentrations of 2.6, 6.4, 16, 40 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 concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.


Exposure Conditions
In the definitive test 250 mL glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and three flasks each completely filled 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 9.48E+05 cells per mL. Inoculation of 1500 mL of test medium with 7.9 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Test Organism Observations
Samples were taken at 0, 25, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.


Water Quality Criteria
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.


Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation at 0 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.

Data Analysis
Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (1nNn – 1nN1) / (tn – t1)

Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = ((µc - µt) / µc) x 100

Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = ((Yc – Yt) / Yc) x 100

Where:

Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group


Determination of ECx Values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits of 7.5 - 10 mg/L.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Effects based on yield were also reported (see attached full study report and summary below). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Range-finding Test
The results showed no significant effect on growth rate at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.

Based on this information test concentrations of 2.6, 6.4, 16, 40 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 (see Appendix 5) showed measured test concentrations of 1.0, 8.1 and 79 mg/L respectively were obtained. A decline in measured test concentrations was observed at 72 hours to 0.23, 0.23 and 20 mg/L for the 1.0, 10 and 100% v/v saturated solution test preparations respectively. Analysis of a 100% v/v saturated solution test sample prepared with the omission of algal cells and stored in a sealed vessel showed a measured test concentration of 62 mg/L was obtained indicating that the decline in measured concentrations observed was likely to be due to a combination of test item volatility and adsorption of the test item to the algal cells.


Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 2.0 to 79 mg/L. A concentration dependant decline in measured concentrations was observed at 72 hours in the range of 0.65 to 77 mg/L (23% to 98% of the initial measured test concentrations). Analysis of samples at 72 hours prepared with the omission of algal cells and incubated alongside the test showed measured test concentrations to range from 2.0 to 77 mg/L (98% to 104% of the initial measured test concentrations) indicating that the test item was adsorbing to the algal cells present rather than being unstable. Given this it was considered appropriate to calculate the results based on the initial measured test concentrations only.


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.

Accordingly the following results were determined from the data:


Inhibition of Growth Rate
ErC10 (0 - 72 h): 4.3 mg/L
ErC20 (0 - 72 h): 5.6 mg/L
ErC50 (0 - 72 h): 8.6 mg/L; 95% confidence limits 7.5 - 10 mg/L

where ErCx is the test concentration that reduced growth rate by x%.

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, 2.0 and 4.8 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 4.8 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 13 mg/L.


Inhibition of Yield
EyC10 (0 - 72 h): 4.2 mg/L
EyC20 (0 - 72 h): 4.4 mg/L
EyC50 (0 - 72 h): 4.9 mg/L; 95% confidence limits 4.7 - 5.0 mg/L

Where:

EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences between the control and 2.0 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 2.0 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 4.8 mg/L.
Results with reference substance (positive control):
Positive control studies using potassium dichromate as the reference item were conducted in a conventional test system (Harlan Study Number 41403074) and in a modified test system using completely filled and sealed vessels (Harlan Study Number 41403365).

Exposure conditions for the modified positive control were similar to those in the definitive test (i.e. completely filled and sealed vessels). The data evaluation for both positive control tests was similar to those in the definitive test. Both tests were conducted using ASTM culture medium, however for the modified test the culture medium contained 500 mg/L of sodium bicarbonate to provide a sufficient supply of CO2 and to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.2 mg/L; 95% confidence limits 1.1 – 1.4 mg/L (41403074) and 0.97 - 1.4 mg/L (41403365)
EyC50 (0 – 72 h): 0.63 mg/L; 95% confidence limits 0.57 – 0.70 mg/L (4140374) and 0.52 mg/L; 95% confidence limits 0.44 - 0.60 mg/L (41403365)

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L (41403074) and 0.32 mg/L (41403365)
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L (41403074) and 0.32 mg/L (41403365)
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.00 mg/L (41403074) and 0.80 mg/L (41403365)
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L (41403074) and 0.80 mg/L (41403365)

The results from the positive control test with potassium dichromate in a conventional test system were within the normal ranges for this reference item. Additionally, the results of the positive control test with potassium dichromate in a modified test system were comparable with the conventional test system.

It is evident from these results that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system. As such it can be considered that the results obtained from the definitive test give a reliable result for the test item.
Reported statistics and error estimates:
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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Any other information on results incl. tables

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 94 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.

 

Mean cell density of control at 0 hours: 5.57E+03 cells per mL

Mean cell density of control at 72 hours: 5.22E+05 cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 24% 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 – 72 h) was 5% 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.

 

Water Quality Criteria

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

 

The pH value of the control cultures was observed to increase from pH 8.5 at 0 hours to pH 10.0 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.

 

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, 2.0 and 4.8 mg/L test cultures were observed to be green dispersions whilst the 13, 31 and 79 mg/L test cultures were observed to be clear colorless solutions.

Applicant's summary and conclusion

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 gave the following results:
The growth rate EC50 was 8.6 mg/L with 95 % confidence limits of 7.5 - 10 mg/L. The corresponding NOEC was 4.8mg/L and the EC10 was 4.3 mg/L.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be 8.6 mg/L and 4.3 mg/L respectively.
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 was designed to be compatible with 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 poorly 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.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 79 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,Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 2.6, 6.4, 16, 40 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 (100 mg/L) of test item in culture medium 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 Gelman Acrocap filter, first approximate 500 mL 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 2.0 to 79 mg/L. A concentration dependant decline in measured concentrations was observed at 72 hours in the range of 0.65 to 77 mg/L (23% to 98% of the initial measured test concentrations. Analysis of samples at 72 hours prepared with the omission of algal cells and incubated alongside the test showed measured test concentrations to range from 2.0 to 77 mg/L (98% to 104% of the initial measured test concentrations) indicating that the test item was adsorbing to the algal cells present rather than being unstable. Given this it was considered appropriate to calculate the results based on the initial measured test concentrations only.

Conclusion

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results based on the initial measured test concentrations:

 

Response Variable

EC50(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

Growth Rate

8.6

7.5

-

10

4.8

13

Yield

4.9

4.7

-

5.0

2.0

4.8