2-Propenoic acid, 2-methyl-, tris(1-methylethyl)silyl ester

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:

25 May to 28 May 2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

Not possible to verify the concentration was at least 80% of the nominal during the test as the saturated solution was below the level of analytical detection. This does not affect the validity of the study as there was no toxicity at a saturated solution

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tripropan-2-ylsilyl 2-methylprop-2-enoate

EC Number:

700-182-8

Cas Number:

134652-60-1

Molecular formula:

C13 H26 O2 Si

IUPAC Name:

Tripropan-2-ylsilyl 2-methylprop-2-enoate

Details on test material:

- Name of test material (as cited in study report): TIS-M
- Lot/batch No.: 9925
- Expiration date of the lot/batch: 2 December 2010
- Storage condition of test material: Under nitrogen at room temperature (15-25°C)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

not specified

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (Printz-Starr) - (formerly known as Selenastrum capricornutum)
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, GERMANY
- Justification for use: The species is a fast-growing species and is therefore convenient for culturing and testing and is a recommended species by relevant guidelines.
- Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal GrowthMedium to 107 cells/mL.

Study design

Test type:

other: continuously shaken

Water media type:

not specified

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

0 hours - all observations were during the 72 hours exposure period.

Test conditions

Hardness:

No data.

Test temperature:

The cultures were maintained at a temperature in the range of 21 – 24°C. The chosen temperature was maintained within a range of ± 2 °C. Culture temperature was checked at the beginning of the study and every 24 hours in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) in a vessel established for this purpose only within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the study, in the control and each concentration. The pH was not adjusted throughout the test period and the deviation was less than 1.5 units in any one test. The range of the pH was 7.09 – 7.98 at the start and 8.32 – 9.36 at the end of the study.

Dissolved oxygen:

No data.

Salinity:

No data.

Nominal and measured concentrations:

100 % v/v saturated solution and 0.5 mg/L (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Test vessel volume: 250 mL
- Volume of the test liquid in the vessels: 100 mL
- Aeration: The flasks were covered with air-permeable stoppers.
- Initial cells density: approximately 10^4 algal cells per mL test medium
- Control end cells density: No data.
- No. of organisms per vessel: 10^4 cell/mL initially and between 70-82x10^4 after 72hrs.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes - Reconstituted algal growth medium (OECD medium, according to OECD 201). It was also used as dilution water for both the range finding and definitive tests.

OTHER TEST CONDITIONS
- Sterile test conditions: no data.
- Adjustment of pH: pH was not adjusted throughout the test.
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: . The light intensity at the position occupied by algal culture flasks during the test was about 110 μE/m2/s, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm) and it is checked periodically.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: microscope with counting chamber - every 24hrs in the 72hr duration of the study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Range finding study: A preliminary range finding study was undertaken with nominal concentrations (% v/v saturated solution) of 0.01, 0.1, 1, 10 and 100.
- Test concentrations: 100 % v/v saturated solution and 0.5 mg/L (nominal).
- Results used to determine the conditions for the definitive study: No significant toxic response was observed during the preliminary concentration range-finding test, it was decided that only one test concentration (at the solubility level of the test item in the test medium) and one control group would be tested in a limit test. A test concentration (nominally 0.5 mg/L) using acetone as solvent and one additional control contained the solvent substance at the same concentration as that used in the test group at 0.5 mg/L would be tested parallel with the limit test concentration.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Batch Number: 829998)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

- Observation of abnormalities: The shape of the algal cells growing was obviously not affected.

Results with reference substance (positive control):

- Results with reference substance valid? Yes - Potassium dichromate (Batch Number: 829998) is tested at least twice a year to demonstrate satisfactory test conditions.
- The ErC 50 : 0.92 mg/L, (95 % confidence limits: 0.83 – 1.02 mg/L).
- The EbC 50 : 0.55 mg/L, (95 % confidence limits: 0.50 – 0.61 mg/L).
- The EyC 50 : 0.47 mg/L, (95 % confidence limits: 0.43 – 0.52 mg/L).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.

The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The EC50 values of the test item and their confidence limits were not calculated, due to the lack of toxic effects.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

Any other information on results incl. tables

Table 1: Method validation results (Study code: 09/285-316AN)

Selectivity	No interfering component was observed
Reinjection repeatability (7 injections)	Coefficient of Variation < 1.5 %
Linear range	0.2-10ng/ml
Limit of Detection	0.1 |4,g/ml
Limit of Quantification	0.2 jo,g/ml
Recovery from aqueous test media	51-62% (1 ng/ml)
Stability of the samples	At least 21 hours in the autosampler
Stock solution stability	At least seven days at 5 ± 3 °C
Stability of the test item in Water	Less than 30 minutes

Table 2: Data of the regression lines

Date of measurement	Analytical occasion	Constant	X Coefficient	R. Squared
25 May 2010	Start of the study	-1332	35806	0.998
28 May 2010	End of the study	-45	36933	0.995

Table 3: Measured concentrations:

Analytical measurements were performed at the controls (untreated and solvent) and at the applied test concentration levels at the beginning and end of the test. The measured concentrations were below the limit of quantification in the case of the saturated solution as well as the additionally tested concentration of 0.5 mg/L (nominally). Six replicate samples were taken from both test solutions at the start and at the end of the study. Measured concentrations were below the quantification limit of the HPLC method.

Sample Code	Measured Concentrations
	At the start	At the end
	mg/L	mg/L
Control	Not detected.	Not detected.
Control with acetone	Not detected.	Not detected.
0.5 mg/L	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2
Saturated solution	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2
	<0.2	<0.2

 

Table 5: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period:

Areas under the Growth Curves (A) and % inhibition of A
Test group		0-24 h	0-48 h		0-72 h
	A	%	A	%	A
Control	36	0.0	314	0.0	1458	0.0
Solvent Control	36	0.0	306	2.5	1430	1.9
100 % v/v saturated solution	36	0.0	300	4.5	1422	2.5
0.5 mg/L (nominal)	34	5.6	292	7.0	1410	3.3

 

Table 6: Yield (Y) and Percentage Inhibition of Y during the Test Period:                                      

Yield (Y) and % inhibition of Y (0-72 h)
Test group	Y	%
Control	75.2	0.0
Solvent Control	74.2	1.3
100 % v/v saturated solution	74.5	0.9
0.5 mg/L (nominal)	74.5	0.9

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield values were not statistically significantly different from the untreated control value at the used test groups (for results in tables 5 and 6).

Table 7: Influence of TIS-M on the Growth of Pseudokirchneriella subcapitata:

Parameter (0-72h)	Biomass (b)	Growth rate (r)	Yield (y)
The results below were determined directly from the raw data.
EC50	> 100% v/v saturated solution	> 100% v/v saturated solution	> 100% v/v saturated solution
95 % conf. limits	Not calculated.	Not calculated.	Not calculated.
NOEC	100% v/v saturated solution	100% v/v saturated solution	100% v/v saturated solution
LOEC	> 100% v/v saturated solution	> 100% v/v saturated solution	> 100% v/v saturated solution

Remarks:

Under the conditions of this algal growth inhibition test, no toxic effect was observed at both tested concentration levels. As the concentration of 0.5 mg/L (nominally) represented a likely higher level than the limit of solubility, it can be stated that the test item TIS-M had no toxic effect at saturation; the EC50 results (for biomass, growth rate and yield) and the Lowest Observed Effect Concentration are higher than the solubility level of the test item in the test medium (100% v/v saturated solution). Correspondingly the No Observed Effect Concentration was determined as 100% v/v saturated solution.

Definitions:

-Cell Density: the number of cells per mL

-Growth: the increase of cell density over the test period

-Biomass (b): the actual number of cells per volume of medium (cells/mL) calculated as the area under the growth curve (A)

-Yield (y): the cell density at the end of the test minus the starting cell density

-Average Specific Growth Rate (μ): the increase in cell density per time unit

-EbC50: the calculated concentration of test item which results in a 50% reduction of biomass (b) relative to the control

-ErC50: the calculated concentration of test item which results in a 50% reduction of growth rate (μ) relative to the control

-EyC50: the calculated concentration of test item which results in a 50% reduction of yield (y) relative to the control

-NOEC: (No Observed Effect Concentration) the highest test concentration at which no significant inhibition of growth is observed relative to the control

-LOEC: (Lowest Observed Effect Concentration) the lowest test concentration at which a significant inhibition of growth is observed relative to the control

Validity:

- The cell density in the control cultures, increased by a factor of more than 16 (76.17) within 72 hours.

- The mean coefficient of variation (CV) for section-by-section specific growth rates in the control cultures did not exceed 35 % during the course of the test (days 0-1; 1-2; 2-3).

- The section-by-section growth rates remained nearly constant indicating the exponential growth for the entire study period.

- The coefficient of variation of average specific growth rates during the whole test period in the control cultures did not exceed 7 %.

- The pH of the controls did not vary by more than 1.5 units.

Therefore the study can be considered as valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

It was determined that the registered substance had no toxic effect at saturation, as the EC50 and LOEC results are higher than the solubility level of the test item in the test medium.

Executive summary:

The toxicity of the registered substance to aquatic algae was assessed according to the EU C.3 Method which was a growth inhinition test. A positive control substance Potassium dichromate was also used. A solvent control was also used. The 72-hour EC50 (biomass, yield and growth rate) and LOEC values was > 100% v/v saturated solution, so it was determined that the test item TIS-M had no toxic effect at saturation.