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Ecotoxicological information

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:
From December 22, 2017 to April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
exposure-related information
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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, EbLand ErL 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

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

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

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

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

Additional information