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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 25 March 2018 Experimental completion date 19 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 1,3-bis(tert-butylperoxyisopropyl)benzene CAS 2212-81-9
Batch: 1403454131
Purity: 91.1%
Physical state/Appearance: white flakes
Expiry Date: 01 January 2024
Storage Conditions: room temperature 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 the concentration to be used for the definitive test was analyzed.

Definitive test
Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours, from additional media incubated alongside 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.

A further sample of the 100% v/v saturated solution test preparation containing no algal cells was incubated alongside the test to provide a sample for uninoculated analysis at 72 hours.
Vehicle:
no
Details on test solutions:
Range-Finding Test
Information provided by the Sponsor indicated that the test item had a low water solubility and high log Kow and therefore fell into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD, 2000), the method of preparation of the test media was based on the recommendations of this document.

The test concentration to be used in the definitive test was 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 (20 mg) was dispensed onto the surface of 2 liters of culture medium prior to stirring via magnetic stirrer for 23 hours at a rate such that a dimple was formed at the media surface. After 23 hours the stirring was stopped and the mixture allowed to stand for 1-Hour, prior to removal of the aqueous phase by mid-depth siphoning 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 (1.6 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.

Experimental Preparation
A nominal amount of test item (25 mg) was dispensed onto the surface of 2.5 liters of culture medium prior to stirring via magnetic stirrer for 23 hours at a rate such that a dimple was formed at the media surface. After 23 hours the stirring was stopped and the mixture allowed to stand for 1-Hour, prior to removal of the aqueous phase by mid-depth siphoning to give a 100% v/v saturated solution.

An aliquot (1 liter) of this saturated solution was inoculated with algal suspension (8.7 mL) to give the required test concentration of 100% v/v saturated solution.

The stock solution and the 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
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 kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
All test and control cultures were inspected microscopically at 72 hours.
Test temperature:
24 +/- 1°C
pH:
The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 9.5 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in Test Guidelines.
Nominal and measured concentrations:
Range-finding test
0.10, 1.0, 10 and 100% v/v

Definitive test
100% v/v
Details on test conditions:
Range-finding test
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 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 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 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. Only the concentration to be used for the definitive test was analyzed.

Experimental preparation
A nominal amount of test item (25 mg) was dispensed onto the surface of 2.5 liters of culture medium prior to stirring via magnetic stirrer for 23 hours at a rate such that a dimple was formed at the media surface. After 23 hours the stirring was stopped and the mixture allowed to stand for 1-Hour, prior to removal of the aqueous phase by mid-depth siphoning to give a 100% v/v saturated solution.

An aliquot (1 liter) of this saturated solution was inoculated with algal suspension (8.7 mL) to give the required test concentration of 100% v/v saturated solution.

The stock solution and the 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 100% v/v saturated solution 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 5.77 x 105 cells per mL. Inoculation of 1 liter of test medium with 8.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 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
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Range-finding test
The results showed no effect on growth rate at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 72 hours showed measured test concentrations of 0.0054 and less than the LOQ, determined to be 0.00031 mg/L respectively indicating that the test item was either unstable and/or was adsorbing to the algal cells present.

Definitive Test
Verification of Test Concentrations
Analysis of the 100% v/v saturated solution test preparation at 0 hours (see Annex 4) showed a measured test concentration of 0.0078 mg/L was obtained. A decline in measured test concentration was observed after each 24-Hour period in the range of 0.0045 mg/l at 24 hours, 0.0021 mg/L at 48 hours and less than the LOQ of the analytical method employed, determined to be 0.00031 mg/L at 72 hours.
Analysis of an additional 100% v/v saturated solution test preparation containing no algal cells which was incubated alongside the test showed a measured test concentration of 0.0059 mg/L was obtained at 72 hours (75% of the 0-Hour measured test concentration). This result confirms that the majority of losses observed over the test period were due to adsorption of the test item to the algal cells present rather than instability of the test item. It was therefore considered appropriate to calculate the results based on the geometric mean of the 0-Hour measured test concentration and the 72-Hour measured concentration obtained from the preparation containing no algal cells as this gave a more representative measurement of the concentration of test item to which the algae were exposed.

Growth Data
It is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.0068 mg/L over the 72-Hour exposure period.

The test concentration of 0.0068 mg/L was the highest attainable test concentration that could been prepared due to the limited solubility of the test item in water.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 to 72 hour): >0.0068 mg/L
ErC20 (0 to 72 hour): >0.0068 mg/L
ErC50 (0 to 72 hour): >0.0068 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 0.0068 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981).

There were no statistically significant decreases in growth rate (P≥0.05), between the control and 0.0068 mg/L test group and therefore the NOEC based on growth rate was 0.0068 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): >0.0068 mg/L
EyC20 (0 to 72 hour): >0.0068 mg/L
EyC50 (0 to 72 hour): >0.0068 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 decreases in yield (P≥0.05), between the control and 0.0068 mg/L test group and therefore the NOEC based on yield was 0.0068 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 251 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 103 cells per mL
Mean cell density of control at 72 hours : 1.25 x 106 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 17% 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 control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be 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.
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 0.0068 mg/L based on the geometric mean measured test concentration. The No Observed Effect Concentration was 0.0068 mg/L.

This study showed that there were no toxic effects at the limit of water solubility (100% v/v saturated solution).
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

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solution was prepared by stirring an excess (10 mg/L) of test item in culture medium using a magnetic stirrer for 23 hours. After 23 hours the stirring was stopped and the mixture was allowed to stand for 1-Hour, prior to removal of the aqueous phase by mid-depth siphoning, to give a 100% v/v saturated solution of the test item.

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 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 0.0078 mg/L was obtained. A decline in measured test concentration was observed after each 24-Hour period in the range of 0.0045 mg/L at 24 hours, 0.0021 mg/L at 48 hours and less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.00031 mg/L at 72 hours.

Analysis of an additional 100% v/v saturated solution test preparation containing no algal cells which was incubated alongside the test showed a measured test concentration of 0.0059 mg/L was obtained at 72 hours (75% of the 0-Hour measured test concentration). This result confirms that the majority of losses observed over the test period were due to adsorption of the test item to the algal cells present rather than instability of the test item. It was therefore considered appropriate to calculate the results based on the geometric mean of the 0-Hour measured test concentration and the 72-Hour measured concentration obtained from the preparation containing no algal cells as this gave a more representative measurement of the concentration of test item to which the algae were exposed.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50values based on the geometric mean measured test concentration of greater than 0.0068 mg/L. The No Observed Effect Concentration (NOEC) was determined to be 0.0068 mg/L.

This study showed that there were no toxic effects at the limit of water solubility (100% v/v saturated solution).

Description of key information

The analgous test substance CAS 25155-25-3 [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was not found to be toxic to Pseudokirchneriella subcapitata at a nominal loading rate of 1.0 mg/L, equivalent to an initial mean measured concentration of 0.0471 mg/L.

A study with the target substance (CAS 2212 -81 -9) is was performed to strengthen the Read Across.

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 0.0068 mg/L based on the geometric mean measured test concentration.  The No Observed Effect Concentration was 0.0068 mg/L.

This study showed that there were no toxic effects at the limit of water solubility (100% v/v saturated solution).

Both studies show that the test substances did not give an effect at the limit of water solubility.

Key value for chemical safety assessment

Additional information

The toxicity of analogous CAS 25155 -25 -3 [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was evaluated with Pseudokirschneriella subcapitata in a study performed in accordance with OECD testing guideline 201 and GLP requirements.

The algae were exposed to nominal test concentrations of 1 mg/L under static conditions during 72 hours. The mean measured concentrations were not maintained between 80 and 120 % of nominal.

pH range and dissolved oxygen concentration were reported and in agreement with the guideline recommendations. Quality criteria were fulfilled.

The test substance was not found to be toxic to Pseudokirchneriella subcapitata at a nominal loading rate of 1.0 mg/L, equivalent to an initial mean measured concentration of 0.0471 mg/L.

A study with the target substance (CAS 2212 -81 -9) is was performed to strengthen the Read Across.

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.

Exposure to the test item gave EC50 values of greater than 0.0068 mg/L based on the geometric mean measured test concentration.  The No Observed Effect Concentration was 0.0068 mg/L.

This study showed that there were no toxic effects at the limit of water solubility (100% v/v saturated solution).