Trimethoxy(3,3,3-trifluoropropyl)silane

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:

Experimental start date: 29 April 2020, Experimental completion date: 28 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxy(3,3,3-trifluoropropyl)silane

EC Number:

207-059-3

EC Name:

Trimethoxy(3,3,3-trifluoropropyl)silane

Cas Number:

429-60-7

Molecular formula:

C6H13F3O3Si

IUPAC Name:

trimethoxy(3,3,3-trifluoropropyl)silane

Test material form:

liquid

Specific details on test material used for the study:

Identification: ST2092NM
Batch: 802496
Purity: 100%
Physical state/Appearance: Clear colorless liquid
Expiry Date: 31 October 2020
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis.
All samples were stabilized by the addition of hexane and frozen prior to providing for analysis.
An additional sample of each test concentration was incubated alongside to provide samples at 24 and 48 hours and stored frozen for further analysis if necessary.
A set of 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:

PREPARATION AND APPLICATION OF TEST SOLUTION
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 Raphidocelis 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.
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 from which a series of dilutions was made to give further stock solutions of 0.10, 1.0 and 10 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (4.7 mL) to give an initial nominal cell density of 5.00 x 10E3 cells/mL.
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.

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/EC Test Guidelines, no effect on algal growth was observed.
A nominal amount of test item (500 mg) was dissolved in culture medium and the volume adjusted to 5 liters to give a 100 mg/L stock solution. An aliquot (5 liters) of the stock solution was inoculated with algal suspension (36.6 mL) to give an initial nominal cell density of 5.00 x 10E3 cells/mL.
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

Test organisms (species):

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

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at 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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10E5 to 10E6 cells/mL.

Culture Medium:
The culture medium used for both the range-finding and definitive tests was modified by the addition of 250 mg of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with conducting the test in an enclosed system (Herman et al 1990).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

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

pH:

7.7 - 9.8

Nominal and measured concentrations:

Definitve test:
Nominal: 100 mg/L
Measured (geometic mean): 5.8 mg/L

Details on test conditions:

TEST SYSTEM
Completely filled 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 groups.
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 6.83 x 10E5 cells per mL. Inoculation of 5 liters of test medium with 36.6 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10E3 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 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.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM PARAMETERS
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.
The appearance of the test media was recorded daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10E3 cells/mL) was taken as the starting cell density.
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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.8 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 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/EC Test Guidelines, no effect on growth was observed.
Chemical analysis of the test preparations at 0 and 72 hours showed a decrease in measured test concentrations indicating that the test item was unstable under test conditions.

DEFINITIVE TEST:
Verification of Test Concentrations:
Analysis of the test preparation at 0 hours showed a measured test concentration of 72 mg/L, and of less than the limit of detection (LOD), determined to be 5 mg/L for the method of analysis utilized in this study, at 24, 48 and 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 LOD of the analytical method following current regulatory advice a value of half the LOD (i.e. 2.5 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentration was determined to be 5.8 mg/L.

Growth Rate:
The growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72 Hour exposure period.
Accordingly the following results were determined from the data:
ErC10 (0 to 72 hour): >5.8 mg/L
ErC20 (0 to 72 hour): >5.8 mg/L
ErC50 (0 to 72 hour): >5.8 mg/L
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 Shapiro-Wilk’s Test on Normal Distribution and Levene’s Test on Variance Homogeneity. There were no statistically significant differences (P0.05), between the control and 5.8 mg/L test group and therefore the No Observed Effect Concentration (NOEC) based on growth rate was 5.8 mg/L.

Inhibition of Yield:
EyC10 (0 to 72 hour): >5.8 mg/L
EyC20 (0 to 72 hour): >5.8 mg/L
EyC50 (0 to 72 hour): >5.8 mg/L
There were no statistically significant differences (P0.05), between the control and 5.8 mg/L test group and therefore the NOEC based on yield was 5.8 mg/L.

Validation Criteria:
The following data show that the cell concentration of the control cultures increased by a factor of 66 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 10E3 cells per mL
Mean cell density of control at 72 hours : 3.29 x 10E5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 22% 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 hour) was 2% 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 at 72 hours.

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 7.7 at 0 hours to pH 9.8 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 the Test Guidelines.

Observations on Test Item Solubility:
At the start of the test period, the control and test cultures were observed to be clear colorless solutions. After the 72 Hour test period the control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

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

ErC50 (0 to 72 hour): 1.2 mg/L; 95% confidence limits 1.0 to 1.3 mg/L
EyC50 (0 to 72 hour) : 0.51 mg/L; 95% confidence limits 0.45 to 0.58 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

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

Reported statistics and error estimates:

A Student’s t test incorporating Shapiro-Wilk’s Test on Normal Distribution and Levene’s Test on Variance Homogeneity was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using ToxRatPro software package (TOXRAT).

Any other information on results incl. tables

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.059	-	3.33E+05	-
	R2	0.060		3.77E+05
	R3	0.058		3.19E+05
	R4	0.059		3.51E+05
	R5	0.056		2.67E+05
	R6	0.057		2.96E+05
	Mean	0.058		3.24E+05
	SD	0.001		3.92E+04
5.8	R1	0.064	[10]	4.81E+05
	R2	0.061	[5]	4.11E+05
	R3	0.061	[5]	4.07E+05
	R4	0.063	[9]	4.46E+05
	R5	0.062	[7]	4.25E+05
	R6	0.062	[7]	4.42E+05
	Mean	0.062	[7]	4.35E+05	[34]
	SD	0.001		2.72E+04

*        In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R      =Replicate

SD   = Standard Deviation

[ ]    = Increase in growth compared to controls

-       = Not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on hte growth of Raphidocelis subcapitata has been investigated and based on the geometric mean measured test concentrations gave an EC50 value of greater than 5.8 mg/L. The No Observed Effect Concentration (NOEC) was 5.8 mg/L.

Executive summary:

Introduction:

A study was perford to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as 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" and Method C.3 of Commission Regulation (EC) No 761/2009.

Methods:

Following a preliminary range-finding test, Raphidocelis 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 test preparation at 0 hours showed a measured test concentration of 72 mg/L, and of less than the limit of detection (LOD), determined to be 5 mg/L for the method of analysis utilized in this study, at 24, 48 and 72 hours.

Given this decline in measured test concentrations it was 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 calculated to be 5.8 mg/L.

Exposure of Raphidocelis subcapitata to the test item gave an EC₅₀ value based on the geometric mean measured test concentration of greater than 5.8 mg/L. The No Observed Effect Concentration (NOEC) was 5.8 mg/L.