9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

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:

This study was conducted between 29 July 2016 and 22 October 2016

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)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch No.: ADC393100
- Expiration date of the lot/batch: 17 January 2018
- Purity test date: Not reported
- Purity: 81%
- Appearance: Clear lolorless liquid
- Storage condition of test material: Room temperature in the dark

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Culture medium

Details on test solutions:

Range-Finding Test:
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 (4.0 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

Initial Experiment
Based on the result of the range-finding test, a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no effect on algal growth was observed.
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. An aliquot (1000 mL) of the stock solution was inoculated with algal suspension (8.0 mL) to give the required test concentration of 100 mg/L.
The stock solution and prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The experimental preparation and exposure conditions were considered valid. However, the instability of the test item when in solution necessitated chemical analysis of samples on the day of sampling. The sampling procedure was changed for the definitive test, but the experimental preparation and exposure conditions were unchanged from the initial experiment.

Definitive Test
Based on the result of the range-finding test, a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. An aliquot (1000 mL) of the stock solution was inoculated with algal suspension (12.5 mL) to give the required test concentrations of 100 mg/L.
The stock solution and 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 and 72 hours

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 maintained in the laboratory under constant aeration and illumination at 21 ± 2”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 10E3 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 10E4 – 10E5 cells/mL

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24 ±1”C

pH:

7.5

Dissolved oxygen:

Not rpeorted

Salinity:

Not reported

Conductivity:

Note reported

Nominal and measured concentrations:

Chemical analysis of the 100 mg/L test preparation at 0 hours (see Annex 3) showed a measured test concentration of 1.25 mg/L, and at 72 hours was less than the limit of quantification (LOQ) of the analytical method employed were obtained, which was determined to be 0.31 mg/L.
The low measured concentrations in the range-finding test were considered to be due to instability of the test item under test conditions.
Initial Experiment
The initial experiment used frozen samples for chemical analysis. However, due to the instability of the test item under test conditions, as identified in the range-finding test, these samples were not analyzed.
An additional stability test was conducted over a 24-Hour time period (see Annex 3), which showed a decline of test item from 95% of nominal at 0 hours to 56% of nominal at 24 hours.
The definitive test was therefore conducted with analysis of the samples on the day of sampling.
Definitive Test
Verification of Test Concentrations
Analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 113 mg/L. A decline in measured test concentration was observed at 72 hours to 8.0 mg/L (7% of the 0-Hour measured test concentration).
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 concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 30 mg/mL.

Details on test conditions:

Experimental Design and Study Conduct

Range-Finding Test
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 mg/L for a period of 72 hours.
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 (4.0 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.

Initial Experiment
Based on the result of the range-finding test, a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no effect on algal growth was observed.
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. An aliquot (1000 mL) of the stock solution was inoculated with algal suspension (8.0 mL) to give the required test concentration of 100 mg/L.
The stock solution and prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The experimental preparation and exposure conditions were considered valid. However, the instability of the test item when in solution necessitated chemical analysis of samples on the day of sampling. The sampling procedure was changed for the definitive test, but the experimental preparation and exposure conditions were unchanged from the initial experiment.

Definitive Test
Based on the result of the range-finding test, a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. An aliquot (1000 mL) of the stock solution was inoculated with algal suspension (12.5 mL) to give the required test concentrations of 100 mg/L.
The stock solution and 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 and 72 hours (see Annex 3).

Exposure Conditions
As in the range-finding test, 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L 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 4.0 x 105 cells per mL. Inoculation of 1 liter of test medium with 12.5 mL of this algal suspension gave an initial nominal cell density of 5.0 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 – 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:

other: EC10, EC20 and EC50

Effect conc.:

> 30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Inhibiiton

Key result

Duration:

72 h

Dose descriptor:

other: EC10, EC20 and EC50

Effect conc.:

> 30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks:

Inhibition

Key result

Duration:

72 h

Dose descriptor:

other: EC10, EC20 and EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines no effect on growth was observed.
Chemical analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 1.25 mg/L, and at 72 hours was less than the limit of quantification (LOQ) of the analytical method employed were obtained, which was determined to be 0.31 mg/L.
The low measured concentrations in the range-finding test were considered to be due to instability of the test item under test conditions.

Initial Experiment
The initial experiment used frozen samples for chemical analysis. However, due to the instability of the test item under test conditions, as identified in the range-finding test, these samples were not analyzed.
An additional stability test was conducted over a 24-Hour time period, which showed a decline of test item from 95% of nominal at 0 hours to 56% of nominal at 24 hours.
The definitive test was therefore conducted with analysis of the samples on the day of sampling.

Definitive Test
Verification of Test Concentrations
Analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 113 mg/L. A decline in measured test concentration was observed at 72 hours to 8.0 mg/L (7% of the 0-Hour measured test concentration).
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 concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 30 mg/mL

rowth Data
Cell density values wer determined at each sampling time and pH values at 0 and 72 hours.
From the data given, 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 nominal test concentration of 100 mg/L over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h) : > 30 mg/L
ErC20 (0 - 72 h) : > 30 mg/L
ErC50 (0 - 72 h) : > 30 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 100 mg/L test group using a Student’s t-test incorporating Levene’s test for homogeneity of variance.
There was a statistically significant difference (P0.05) between the control and 100 mg/L test group. Visual inspection of the data indicates little growth inhibition in the 100 mg/L test group. The control was calculated to have a CV of 0% for average specific growth rate, which was considered to impact the statistical analysis in this case. Based on visual inspection of the data the "No Observed Effect Concentration" (NOEC) based on growth rate was considered to be 30 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h) : > 30 mg/L
EyC20 (0 - 72 h) : > 30 mg/L
EyC50 (0 - 72 h) : > 30 mg/L
Where: EyCx is the test concentration that reduced yield by x%.
There was a statistically significant difference (P0.05) between the control and 100 mg/L test group. Visual inspection of the data indicates low inhibition of yield in the 100 mg/L test group. The control was calculated to have a CV of 0% for average specific growth rate, which is considered to impact the statistical analysis in this case. Based on visual inspection of the data the "No Observed Effect Concentration" (NOEC) based on yield was considered to be 30 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 103 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 : 5.00 x 10E3 cells per mL
Mean cell density of control at 72 hours : 5.14 x 10E5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 4% 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 0% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Water Quality Criteria
The pH values of the control and each test preparation were 81 - 8.2.
Temperature was maintained at 24 ± 1 ºC throughout the test.

Observations on Test Item Solubility
At the start of the test, the control and 100 mg/L test cultures were observed to be clear colorless solutions.
At 72 hours, the control and 100 mg/L test cultures were observed to be pale green dispersions

Results with reference substance (positive control):

Positive Control
A positive control (Envigo Study Number FP48BQ) 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 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) 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 based on the geometric mean measured test concentrations gave EC50 values of greater than 30 mg/L. The No Observed Effect Concentration (NOEC) is considered to be 30 mg/L.

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.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

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 mg/L test preparation at 0 hours showed a measured test concentration of 113 mg/L.  A decline in measured test concentration was observed at 72 hours to 8.0 mg/L (7% of the 0-Hour measured test concentrations) 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.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 30 mg/L. The No Observed Effect Concentration was considered to be 30 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L (30 mg/L geometric mean measured concentration).

Description of key information

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.

Analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 113 mg/L.  A decline in measured test concentration was observed at 72 hours to 8.0 mg/L (7% of the 0-Hour measured test concentrations) 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.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 30 mg/L. The No Observed Effect Concentration was considered to be 30 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L (30 mg/L geometric mean measured concentration).

Key value for chemical safety assessment

EC50 for freshwater algae:

30 mg/L

EC10 or NOEC for freshwater algae:

30 mg/L