tri{[(2S)-1-ethoxy-1-oxopropan-2-yl]oxy}(vinyl)silane

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:

From December 22, 2017 to April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

exposure-related information

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, avoid humidity
- Stability under test conditions: The test item hydrolyses

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 25, 35, 50, 70 and 100% WAF preparation at 100 mg/l
- Sampling method: The test solutions were subjected for active ingredient analysis using the validated analytical method. For main study, six replicates for control and three replicates for treatment groups were prepared. 10 mL of test samples from each replicates were drawn and mixed together for each group. Collected samples were centrifuged at 2000 rpm for 10 minutes in order to remove algal cell.
- Sample storage conditions before analysis: The representative samples were divided into two equal portions. One portion was sent for test concentration analysis at 0 h and the second portion was stored at -20 ± 5 ºC. Active ingredient concentration in test media was determined using the validated analytical method

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
VL3 hydrolyses very fast (half-lives at pH 1.2 = 3.46 min; pH 4.0 = 4.62 min; pH 7.0 = 19.8 min; pH 9.0 = 9.5 min) to ethyl lactate and the corresponding silanetriols. In the one hand, ethyl lactate is expected to hydrolyze into lactic acid and ethanol (but not completely) and, in the other hand, silanetriols undergo continuous condensation reactions to produce higher molecular weight siloxanes. Test procedure was adopted from the WAF methodology since when preparing the 100 mg/L test item solution, oil droplets were observed at the surface of the media. The concentrations of 100% WAF at 100 mg/L, were prepared by stirring for 10 min at room temperature. After stirring, test solution was kept for re-equilibrium for 10 min. After phase separation (soluble fraction) was collected from lower portion by “L” shaped glass tube without disturbing the phase. The stirring and the re-equilibrium times were kept as short as possible in order to maximize the exposure to VL3, non-condensed silanetriols and the small molecular weight condensation products (the higher molecular weight siloxanes are supposed to be biologically non-available). The separation method of “L” shaped glass was determined to be the best option since the test item forms oily droplets and a filtration method would be not suitable.

Prior to adding the volume of test solution used for exposure to the test vessel, it was pre-conditioned (rinsed) with respective test concentration to saturate the surface of the respective vessel to prevent loss of test concentration due to absorption to the walls of the test vessels. An amount of 100 mg VL3 was mixed with algal culture medium and made up to 1 L. It was kept for stirring using magnetic stirrer for 10 min. at room temperature. After stirring, test solution was kept for re-equilibrium for 10 min. After phase separation, approximate 500 mL test solution was collected from lower portion by “L” shaped glass tube without disturbing the phase. This collected test solution was used for pre-conditioning of test vessels.

An amount of 200 mg VL3 was mixed with algal culture medium and made up to 2 L. It was kept for stirring using magnetic stirrer for 10 min. at room temperature. After stirring, test solution was kept for re-equilibrium for 10 min. After phase separation, approximate 1300 mL test solution was collected from lower portion by “L” shaped glass tube without disturbing the phase. This collected test solution was used for exposure of algal at the concentrations of 25.0, 35.0, 50.0, 70.0 and 100.0% water accommodated fraction (WAF) prepared at 100 mg VL3/L. For control group, only algal culture medium was used. Six replicates for the control group and three replicates for the treatment groups were used. The experiment was conducted in 250 mL conical flasks.

- Controls: yes, negative control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): algae media
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 25.0, 35.0, 50.0, 70.0 and 100.0% water accommodated fraction (WAF) prepared at 100 mg VL3/L

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA
- Age of inoculum (at test initiation): 2 days in pre-culture
- Method of cultivation: Mean initial cell concentration was derived from 3 samples taken from the pre-culture. The cell concentration of the algal stock culture was determined as 1225000 cells/mL by manual counting using a haemocytometer and a microscope. Each replicate was inoculated with 0.5 mL of algal culture to obtain the required cell concentration (approximately 5 x 103- 104 cells/mL).

ACCLIMATION
- Acclimation period: The pre-culture was prepared 2 days prior to the commencement of the study by transferring 1 mL from the latest sub culture into a new culture vessel. The pre-culture was incubated under the same conditions as those required for the test and were used when growing exponentially (5 x 103 - 104 cells/mL). The temperature for pre-culture ranged between 21 - 23 °C.
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 23 °C

pH:

7.49 - 8.04

Nominal and measured concentrations:

Nominal: 0.0 (control), 25.0, 35.0, 50.0, 70.0 and 100.0% WAF prepared at 100 mg VL3/L
Measured: VL3 was not detected (hydrolises), so the result is expressed based on nominal concentration only.

Details on test conditions:

TEST SYSTEM
- Test vessel: flask
- Material, size, headspace, fill volume: 250 mL sterile conical flask
- Aeration: The cells in the cultured flasks were maintained in suspension by agitating the test flasks continuously at 100  2 rpm, using an orbital shaker during the test period.
- Renewal rate of test solution (frequency/flow rate):no renewals
- Initial cells density: mean 5104 cells/mL
- Control end cells density: 603750 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture medium was prepared by adding stock solution I (10 mL), stock solution II (1 mL), stock solution III (1 mL) and stock solution IV (1 mL) and equated up to 1 L in sterilized deionised water.

a) Stock Solution Preparation for Culture Media
- Stock Solution I : Macro Nutrients
Ammonium Chloride Pure (NH4Cl): 1.5 g/L
Magnesium Chloride Hexahydrate (MgCl2.6H2O): 1.2 g/L
Calcium Chloride Dihydrate (CaCl2.2H2O): 1.8 g/L
Magnesium Sulphate Heptahydrate (MgSO4.7H2O): 1.5 g/L
Potassium Dihydrogen Phosphate Purified (KH2PO4): 0.16 g/L
- Stock Solution II : Iron
Ferric Chloride Hexahydrate (FeCl3.6H2O): 64 mg/L
EDTA Disodium Salt (C10H14N2O8Na2·2H2O): 100 mg/L
- Stock Solution III : Trace elements
Boric Acid (H3BO3): 185 mg/L
Manganese (II) Chloride tetrahydrate (MnCl2.4H2O): 415 mg/L
Zinc Chloride (ZnCl2): 3 mg/L
Cobalt Chloride Hexahydrate (CoCl2.6H2O): 1.5 mg/L
Copper (II) Chloride dihydrate (CuCl2.2H2O): 0.01 mg/L
Sodium Molybdate, dihydrate (Na2MoO4.2H2O): 7 mg/L
- Stock Solution IV : Bicarbonate
Sodium Hydrogen Carbonate (NaHCO3) : 50 g/L

Stock solution I and III were sterilized by autoclaving (120 °C for 15 min), Stock solution II and IV were filtered through membrane (0.2 µm) and refrigerated.

- Intervals of water quality measurement: Parameters were evaluated at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 6647 - 6743 (± 15% of the mean value)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : alga cell count (at a regular interval of 24 h), comparison of area under the curve, percent inhibition of cell growth (biomass), growth rate, inhibition of yield.
- Determination of cell concentrations: counting chamber haemocytometer and a microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor 1.42
- Range finding study: Two replicates were taken for each concentration of the test item and three replicates for control and vehicle control.
- Test concentrations: 0.0 (control), 1.0, 10.0, 25.0, 50.0 and 100.0% WAF prepared at 100 mg VL3/L
- Results used to determine the conditions for the definitive study: The percent inhibition of biomass was 0.09, 0.95, 1.64, 48.89 and 59.36 at the test concentrations of 1.0, 10.0, 25.0, 50.0 and 100.0% WAF prepared at 100 mg/L, respectively.
The percent inhibition of growth rate was 0.14, 0.14, 0.14, 14.23 and 18.56 at the test concentrations of 1.0, 10.0, 25.0, 50.0 and 100.0% WAF prepared at 100 mg/L, respectively.

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

38.9 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

64.1 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

39.8 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

48.6 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

49.1 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

74.4 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

73.6 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

35 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

35 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

35 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

50 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

50 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

50 other: % WAF prepared at 100 mg VL3/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Biomass: In the treated groups, it was decreased with increased concentration of VL3 during the 72 h exposure period.
Growth rate: the coefficient of variation of average specific growth rate during the whole test period (0-3) in replicate control culture 0.63%. The mean coefficient of variation for day 0-1, 1-2, and 2-3 in control culture was 6.17%.
Inhibition of biomass: the percent inhibition of biomass was 0.79, 5.25, 44.75, 48.94 and 58.70 for the test concentrations of 25.0, 35.0, 50.0, 70.0 and 100.0% WAF prepared at 100 mg VL3/L, respectively .
Inhibition of growth: the percent inhibition of growth rate was 0.15, 0.90, 12.82, 14.18 and 18.85 for the test concentrations of 25.0, 35.0, 50.0, 70.0 and 100.0% WAF prepared at 100 mg VL3/L, respectively.
Inhibition of yield: the percent inhibition of yield was 0.63, 4.52, 46.15, 49.63 and 59.79 for the test concentrations of 25.0, 35.0, 50.0, 70.0 and 100.0% WAF prepared at 100 mg VL3/L, respectively.

- Other: Analysis of Test Concentration
VL3 is degraded in ethyl lactate and lactic acid. As per sponsor’s requirement, the test media was analysed for VL3 and ethyl lactate concentrations. The stability of VL3 and ethyl lactate in test media was performed during method validation (JRF Study N° 228-2-13-17733) in test media. Test media was analysed for VL3 and ethyl lactate active ingredient to monitor the concentration and stability of test solution at 0, 24, 48 and 72 h during main study. No peak response of VL3 was observed, while degraded product ethyl lactate was observed at the concentration of 25.0, 35.0, 50.0, 70.0 and 100.0% water accommodated fraction (WAF) prepared at 100 mg VL3/L. Since, VL3 was not detected, the result is expressed based on nominal concentration only

Reported statistics and error estimates:

Probit analysis method for Percent Inhibition of Growth Rate. Calculation of NOELR, LOELR, EL10, EL20, and EL50 use of ANOVA and Probit analysis method.

TABLE 1: Mean Values of Algal Biomass, Yield and Specific Growth Rate and Validity Criteria

A. Mean Values of Algal Biomass, Yield, Specific Growth Rate

Group	Test Concentration (% WAF Prepared at 100 mg/L)	Mean Number of Cells Count/mL at					Specific Growth Rate (0 – 3)
		0 h	24 h	48 h	72 h	Yield
G1	0.0 (Control)	5104	27917	131250	603750	598646	1.59
G2	25.0		27500	130000	600000	594896	1.59
G3	35.0		28333	120833	576667	571563	1.57
G4	50.0		27500	69167	327500*	322396*	1.39*
G5	70.0		25833	62500	306667*	301563*	1.37*
G6	100.0		20000	55000	245833*	240729*	1.29*

B. Validity Criteria

Group	Test Concentration (% WAF Prepared at 100 mg/L)	Replicate	Specific Growth Rate				Mean % Coefficient of Variation for Days 0-1, 1-2, and 2-3
			0-1	1-2	2-3	0-3	Mean	SD	CV
G1	0.0 (Control)	R1	1.68	1.59	1.49	1.59	1.59	0.10	6.29
		R2	1.77	1.58	1.48	1.61	1.61	0.15	9.32
		R3	1.68	1.51	1.57	1.59	1.59	0.09	5.66
		R4	1.59	1.57	1.57	1.57	1.58	0.01	0.63
		R5	1.77	1.52	1.49	1.60	1.59	0.15	9.43
		R6	1.68	1.51	1.56	1.59	1.58	0.09	5.70
		Mean	-			1.59	Mean CV: 6.17
		SD				0.01
		CV				0.63

Key: h = Hour,*=Significantly lower than control at 1% level (p£0.01), SD = Standard deviation, - = Not applicable

TABLE 2: Percentage (%) Inhibition of Biomass, Growth Rate, and Yield

Group	Test Concentration (% WAF prepared at mg/L)	Percentage (%) Inhibition of
		Biomass (EbL) 0 - 72 h	Growth Rate (ErL) 0 - 72 h	Yield (EyL)0 - 72 h
G1	0.0 (Control)	-	-	-
G2	25.0	0.79	0.15	0.63
G3	35.0	5.25	0.90	4.52
G4	50.0	44.75	12.82	46.15
G5	70.0	48.94	14.18	49.63
G6	100.0	58.70	18.85	59.79

Key:  - = Not applicable, h = Hour

Note:  Growth rate is a logarithmic term. Therefore, E_bLand E_rL values are not numerically comparable. All values mentioned above are based on the nominal concentrations selected for the study

TABLE 3: EL₁₀, EL₂₀and EL₅₀Values

Exposure Time (72 h)	EbLxValue for Biomass Inhibition	Concentration (% WAF Prepared at 100 mg/L)	95% Confidence Limits (% WAF Prepared at 100 mg/L)		Regression Equation (y = a + bx)
			Lower Limit	Upper Limit
	EbL10	38.9	28.5	53.2	y = -3.5 + 4.6x
	EbL20	48.6	37.8	62.6
	EbL50	74.4	56.3	98.3
Exposure Time (72 h)	ErLxValue for Growth Rate Inhibition	Concentration (% WAF Prepared at 100 mg/L)	95% Confidence Limits (% WAF Prepared at 100 mg/L)		Regression Equation (y = a + bx)
			Lower Limit	Upper Limit
	ErL10	64.1	50.1	82.1	y = -2.6 + 3.5x
	ErL20	-	-	-
	ErL50	-	-	-
Exposure Time (72 h)	EyLxValue for YieldInhibition	Concentration (% WAF Prepared at 100 mg/L)	95% Confidence Limits (% WAF Prepared at 100 mg/L)		Regression Equation (y = a + bx)
			Lower Limit	Upper Limit
	EyL10	39.8	28.6	55.3	y = -3.9 + 4.8x
	EyL20	49.1	37.6	64.2
	EyL50	73.6	55.2	98.1

TABLE 4: pH Values recorded at 0 and 72 h

Group	Test Concentration(% WAF prepared at mg/L)	0 h [Initial]						72 h [Final]
		R1	R2	R3	R4	R5	R6	R1	R2	R3	R4	R5	R6
G1	0.0 (Control)	8.01	8.00	8.02	8.04	8.04	8.03	7.80	7.79	7.68	7.78	7.49	7.59
G2	25.0	7.91	7.96	7.98	-	-	-	7.54	7.60	7.72	-	-	-
G3	35.0	7.89	7.91	7.96	-	-	-	7.57	7.60	7.59	-	-	-
G4	50.0	7.85	7.86	7.81	-	-	-	7.49	7.63	7.54	-	-	-
G5	70.0	7.79	7.89	7.81	-	-	-	7.57	7.49	7.70	-	-	-
G6	100.0	7.81	7.88	7.84	-	-	-	7.56	7.57	7.52	-	-	-

Key: R = Replicate, - = Not applicable

TABLE 5: Temperature and Illumination Record

For pre-culture

Observation at Hour	Temperature (°C)		Mean Illumination (Lux)
	Maximum	Minimum
0 h	23	21	6470
24 h	23	21	6530
48 h	23	21	6490

For test flasks

Observation at Hour	Temperature (°C)		Mean Illumination (Lux)
	Maximum	Minimum
0 h	22	21	6647
24 h	23	22	6730
48 h	23	21	6653
72 h	23	22	6743

Key: h = Hour

TABLE 6: Algal Cell Count, Yield and Specific Growth Rates

Group	Test Concentration (% WAF prepared at 100 mg/L)	Replicate	Cell Count at			Yield	Specific Growth Rate
			24 h	48 h	72 h	Cells/mL	0-1	1-2	2-3	0-3
G1	0.0 (Control)	R1	27500	135000	600000	594896	1.68	1.59	1.49	1.59
		R2	30000	145000	637500	632396	1.77	1.58	1.48	1.61
		R3	27500	125000	600000	594896	1.68	1.51	1.57	1.59
		R4	25000	120000	575000	569896	1.59	1.57	1.57	1.57
		R5	30000	137500	612500	607396	1.77	1.52	1.49	1.60
		R6	27500	125000	597500	592396	1.68	1.51	1.56	1.59
G2	25.0	R1	32500	132500	600000	594896	1.85	1.41	1.51	1.59
		R2	25000	130000	597500	592396	1.59	1.65	1.53	1.59
		R3	25000	127500	602500	597396	1.59	1.63	1.55	1.59
G3	35.0	R1	30000	120000	575000	569896	1.77	1.39	1.57	1.57
		R2	25000	120000	585000	579896	1.59	1.57	1.58	1.58
		R3	30000	122500	570000	564896	1.77	1.41	1.54	1.57
G4	50.0	R1	27500	70000	312500	307396	1.68	0.93	1.50	1.37
		R2	32500	62500	347500	342396	1.85	0.65	1.72	1.41
		R3	22500	75000	322500	317396	1.48	1.20	1.46	1.38
G5	70.0	R1	25000	57500	312500	307396	1.59	0.83	1.69	1.37
		R2	27500	60000	297500	292396	1.68	0.78	1.60	1.36
		R3	25000	70000	310000	304896	1.59	1.03	1.49	1.37
G6	100.0	R1	20000	52500	245000	239896	1.37	0.97	1.54	1.29
		R2	17500	50000	240000	234896	1.23	1.05	1.57	1.28
		R3	22500	62500	252500	247396	1.48	1.02	1.40	1.30

Key:   h = Hour, R = Replicate Note: Values are total number of cells counted per mL

Yield = Biomass at the end of Exposure period (72 h) – Biomass at the start of the Exposure period

TABLE 7 -10: Active Ingredient Concentration and Stability of Ethyl Lactate in Test Media

Groups and Conc. of sample (%WAF preparation at 100mg/L)	Injection N°	Theoretical Conc. in Test Media (mg/L)	0 D, 0 h
			Peak Area of Sample (Y)		Analysed Conc. (mg/L)	% Recovery
G1 (0.0)	I	0.00000	ND		0.00000	0.00
G2 (25.0)	I	23.00000	5413		19.23191	83.62
G3 (35.0)	I	32.20000	7819		25.00735	77.66
G4 (50.0)	I	46.00000	10536		31.52931	68.54
G5 (70.0)	I	64.40000	13733		39.20349	60.87
G6 (100.0)	I	92.00000	19805		53.77890	58.46
Intercept with y-axis (a)			-2598.86	Correlation of coefficient (r)		0.999
Slope of the line (b)			1041.48	Purity (% w/w)		92.0

Groups and Conc. of sample (%WAF preparation at 100mg/L)	Injection N°	Theoretical Conc. in Test Media (mg/L)	0 D, 24 h
			Peak Area of Sample (Y)		Analysed Conc. (mg/L)	% Recovery
G1 (0.0)	I	0.00000	ND		0.00000	0.00
G2 (25.0)	I	23.00000	4935		18.08450	78.63
G3 (35.0)	I	32.20000	6613		22.11243	68.67
G4 (50.0)	I	46.00000	10750		32.04301	69.66
G5 (70.0)	I	64.40000	17845		49.07406	76.20
G6 (100.0)	I	92.00000	26793		70.55311	76.69
Intercept with y-axis (a)			-2598.86	Correlation of coefficient (r)		0.999
Slope of the line (b)			1041.48	Purity (% w/w)		92.0

Groups and Conc. of sample (%WAF preparation at 100mg/L)	Injection N°	Theoretical Conc. in Test Media (mg/L)	0 D, 48 h
			Peak Area of Sample (Y)		Analysed Conc. (mg/L)	% Recovery
G1 (0.0)	I	0.00000	ND		0.00000	0.00
G2 (25.0)	I	23.00000	1774		10.49675	45.64
G3 (35.0)	I	32.20000	6438		21.69235	67.37
G4 (50.0)	I	46.00000	7985		25.40582	55.23
G5 (70.0)	I	64.40000	15779		44.11477	68.50
G6 (100.0)	I	92.00000	22449		60.12564	65.35
Intercept with y-axis (a)			-2598.86	Correlation of coefficient (r)		0.999
Slope of the line (b)			1041.48	Purity (% w/w)		92.0

Groups and Conc. of sample (%WAF preparation at 100mg/L)	Injection N°	Theoretical Conc. in Test Media (mg/L)	0 D, 72 h
			Peak Area of Sample (Y)		Analysed Conc. (mg/L)	% Recovery
G1 (0.0)	I	0.00000	ND		0.00000	0.00
G2 (25.0)	I	23.00000	1205		9.13090	39.70
G3 (35.0)	I	32.20000	2640		12.57552	39.05
G4 (50.0)	I	46.00000	3828		15.42723	33.54
G5 (70.0)	I	64.40000	10999		32.64071	50.68
G6 (100.0)	I	92.00000	17897		49.19888	53.48
Intercept with y-axis (a)			-2598.86	Correlation of coefficient (r)		0.999
Slope of the line (b)			1041.48	Purity (% w/w)		92.0

Keys: ND = Not Detected and Conc. = Concentration.

Note:(1) Dilution factor for G1, G2, G3, G4, G5 and G6 were 2.5, 2.5, 2.5, 2.5, 2.5 and 2.5, respectively.

(2) For stability of algal media for 0 h and 24 h samples were prepared and injected on to HPLC, no peak response of VL3 was observed.

Validity criteria fulfilled:

yes

Remarks:

Coefficient of variation of average specific growth rates in the replicate control cultures = 0.63%. Cell concentration in the control cultures increased exponentially (factor of 118.3). The mean coefficients of variation in control culture was 6.17%.

Conclusions:

The EL50 of the test item in green algae is greater than 100% WAF prepared at 100 mg/L based on growth rate. The NOELR was determined to be 35.0% WAF prepared at 100 mg VL3/L.

Executive summary:

A growth inbition test on Pseudokirchneriella subcapitata was performed according to the OECD Guideline 201 (GLP study). VL3 hydrolyses very fast (half-lives at pH 1.2 = 3.46 min; pH 4.0 = 4.62 min; pH 7.0 = 19.8 min; pH 9.0 = 9.5 min) to ethyl lactate and the corresponding silanetriols. In the one hand, ethyl lactate is expected to hydrolyze into lactic acid and ethanol (but not completely) and, in the other hand, silanetriols undergo continuous condensation reactions to produce higher molecular weight siloxanes. Test procedure was adopted from the WAF methodology since when preparing the 100 mg/L test item solution, oil droplets were observed at the surface of the media. The concentrations of 100% WAF at 100 mg/L, were prepared by stirring for 10 min at room temperature. After stirring, test solution was kept for re-equilibrium for 10 min. After phase separation (soluble fraction) was collected from lower portion by “L” shaped glass tube without disturbing the phase. The stirring and the re-equilibrium times were kept as short as possible in order to maximize the exposure to VL3, non-condensed silanetriols and the small molecular weight condensation products (the higher molecular weight siloxanes are supposed to be biologically non-available). The separation method of “L” shaped glass was determined to be the best option since the test item forms oily droplets and a filtration method would be not suitable. Exponentially growing cultures of P.subcapitata were exposed to nominal test concentrations of 0.0 (control), 25.0, 35.0, 50.0, 70.0 and 100.0% water accommodated fraction (WAF) prepared at 100 mg VL3/L. The algal cultures were assessed for the growth by visual cell count at 24, 48 and 72 h. The test media was analysed for VL3 and ethyl lactate concentrations. No peak response of VL3 was observed, while degraded product ethyl lactate was observed at the concentration of 25.0, 35.0, 50.0, 70.0 and 100.0% water accommodated fraction (WAF) prepared at 100 mg VL3/L. Since, VL3 was not detected, the result is expressed based on nominal concentration only. A concentration-effect relationship was observed and statistically analysed to obtain effect concentrations. The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErL10 = 64.1 (50.1 - 82.1)% WAF prepared at 100 mg/L. ErL20 and ErL50 could not be calculated because 18.85% growth rate inhibition was observed at the highest concentration 100% WAF prepared at 100 mg/L. Based on the result it can be concluded that ErL20 and ErL50 are greater than 100% WAF prepared at 100 mg/L. The biomass inhibition effect concentrations with their respective 95% lower and upper confidence limits for the biomass inhibition were EbL10 = 38.9 (28.5 - 53.2)% WAF prepared at 100 mg/L, EbL20 = 48.6 (37.8 - 62.6)% WAF prepared at 100 mg/L and EbL50 = 74.4 (56.3 - 98.3)% WAF prepared at 100 mg/L. The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyL10 = 39.8 (28.6 - 55.3)% WAF prepared at 100 mg/L, EyL20 = 49.1 (37.6 - 64.2)% WAF Prepared at 100 mg/L and EyL50 = 73.6 (55.2 - 98.1)% WAF prepared at 100 mg/L. Based on the results of the statistical analysis, the NOELR (No Observable Effect Loading Rate) and LOELR (The Lowest Observable Effect Loading Rate) values for biomass (cell growth), growth rate and yield were 35.0 and 50.0% WAF prepared at 100 mg VL3/L, respectively.

Description of key information

Key study: According to OECD 201. GLP study. The 72h-EL50 of the test item in green algae is grater than 100% WAF prepared at 100 mg/L based on growth rate. The 72h-NOELR was determined to be 35.0% WAF prepared at 100 mg VL3/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

35 mg/L