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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 23, 2018 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 203 (Fish, Acute Toxicity 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: 0.0% and 100.0%
- Sampling method: 20 mL of test samples from each group was drawn at 0 and before & after every renewal. Active ingredient concentration in water was determined using the validated analytical method.
- Sample storage conditions before analysis: The representative samples were divided into two equal portions (10 mL). One portion (10 mL) was sent for test concentration analysis and the second portion (10 mL) was stored at -20 ± 5 ºC temperature till the study completion.
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.

An amount of 400 mg VL3 was mixed with RO water and made up to 4 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 2 L 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 fish at the concentration of 100% WAF prepared at 100 mg/L. For control group, only 2 L RO water was used.

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. Same procedure as described above was followed for test solution preparation. The test medium in the aquaria was changed every 24 h and replaced with the freshly prepared test solution. At each renewal, 100% of volume of the tank was renewed. During renewal, fish was transferred by netting to new test vessels.

- Controls: yes, negative control (0% test item)
- Chemical name of vehicle: RO water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0% and 100% WAF prepared at 100 mg/L
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio
- Source: from the Fish-O-Fish Aquarium, Mumbai
- Length at study initiation (length definition, mean, range and SD): between the length 1.4 to 1.9 cm

ACCLIMATION
- Acclimation period: 7 days prior to testing
- Acclimation conditions (same as test or not): in a glass aquarium containing water identical to that to be used in the main study. The water was maintained between 21.3 and 21.7 °C temperature with 93.4 and 97.8% dissolved oxygen content as air saturation value, pH was maintained between 7.20 and 7.70 and total hardness of 78.4 mg/L as CaCO3.
- Type and amount of food during acclimation: The fish were fed with Gemma Micro 500 ZF, France.
- Feeding frequency during acclimation: Daily
- Health during acclimation (any mortality observed): o signs of physical or behavioural abnormalities or mortalities of fish were observed during the acclimatisation period.

FEEDING DURING TEST
Feed was withheld 24 h before the exposure to VL3 and throughout the exposure period.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
78.4 and 89.6 mg/L as CaCO3
Test temperature:
21.2 to 22.0 °C
pH:
7.29 to 7.92
Dissolved oxygen:
85.6 to 98.1%
Nominal and measured concentrations:
Nominal: 0.0 (control) and 100% WAF prepared at 100 mg/L
Measured: VL3 was not detected (hydrolyses), so the result is expressed based on nominal concentration only.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass tank
- Material, size, headspace, fill volume: glass tank of 8 L capacity, filed with 2L
- Aeration: The test solutions were not aerated during the exposure.
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed every 24 h with freshly prepared solutions.
- No. of organisms per vessel/tank: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis water

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light and 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were observed at 3, 6, 24, 48, 72 and 96 h of exposure. Recorded parameters included behavioural responses, clinical signs and mortality.
Temperature, pH and dissolved oxygen content of the test media were measured daily before and after each renewal of the media with freshly prepared test solution. Total hardness was analysed daily for each renewal of the diluent water during the study period.

TEST CONCENTRATIONS : limit test (0.0 (control) and 100% WAF prepared at 100 mg/L)
- Range finding study
- Test concentrations: 0.0 (control), 1.0, 10.0, 25.0, 50.0 and 100.0% WAF prepared at 100 mg VL3/L (n = 10/group)
- Results used to determine the conditions for the definitive study: No mortality and behavioural symptoms were observed in any of the treated and in control group.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 other: % WAF prepared at 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: Fish exposed to 0.0 (control) and 100% WAF prepared at 100 mg/L did not exhibit any treatment related behavioral response
- Mortality of control: No mortality was observed over a period of 96 h at the test concentration of 100% WAF prepared at 100 mg/L as well as in the control group.
Reported statistics and error estimates:
As no mortality was observed at the test concentration of 100% WAF prepared at 100 mg/L, probit analysis was not carried out.

TABLE 1: Mortalities

Group

Test Concentration

(% WAF prepared at 100 mg/L)

Percentage Mortality of Fish at

Cumulative Mortality

(%) at 96 h

3 h

6 h

24 h

48 h

72 h

96 h

G1

0.0 (Control)

0

0

0

0

0

0

0

G2

100.0

0

0

0

0

0

0

0

Key:   h = Hour

TABLE 2: Environmental Parameters of Test Media and Fish Size during Acclimatisation (Main Study)

 

Tank N°

Temperature (˚C)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Mean

SD

T1

21.7

21.6

21.5

21.4

21.3

21.6

21.7

21.5

0.2

T2

21.7

21.5

21.5

21.5

21.5

21.7

21.7

21.6

0.1

 

Tank N°

Dissolved Oxygen (%)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Mean

SD

T1

96.5

97.0

97.8

95.8

93.4

95.1

96.5

96.0

1.4

T2

96.0

97.1

97.5

96.3

94.1

95.2

96.0

96.0

1.1

 

Tank N°

pH

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Mean

SD

T1

7.20

7.33

7.60

7.69

7.68

7.69

7.35

7.51

0.21

T2

7.25

7.30

7.62

7.61

7.62

7.70

7.40

7.50

0.18

 

Total Hardness (as CaCO3) mg/L

Water

78.4

Tank N°

pH

1

2

3

4

5

6

7

8

9

10

Mean

SD

T1

1.8

1.5

1.6

1.4

1.8

1.7

1.8

1.5

1.6

1.5

1.6

0.1

T2

1.9

1.6

1.5

1.6

1.7

1.5

1.6

1.4

1.8

1.7

1.6

0.1

Key: SD = Standard Deviation, - = Not applicable

TABLE 3: Mean of Physico-Chemical Parameters of Test Media (Main test)

Group

Test Concentration

(% WAF prepared at 100 mg/L)

pH

Temperature (°C)

Dissolved Oxygen (%)

Initial

Final

Initial

Final

Initial

Final

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

G1

0.0 (Control)

7.60

0.10

7.58

0.22

21.4

0.1

21.6

0.3

96.0

2.4

89.0

2.5

G2

100

7.62

0.10

7.55

0.29

21.6

0.3

21.5

0.1

96.1

2.3

87.4

1.2

 

Total Hardness (mg/L as CaCO3)

Total Hardness (mg/L)

Mean

SD

84.0

4.6

Keys: Initial = 0 h, Final = 24 h, SD= Standard deviation

TABLE 4: Physico-Chemical Parameters of Test Media

A. Temperature (°C)

Group

Test Concentration

(% WAF prepared at 100 mg/L)

0 - 24 h

24 - 48 h

48 - 72 h

72 - 96 h

Initial

0 h

Final

24 h

Initial

24 h

Final

48 h

Initial

48 h

Final

72 h

Initial

72 h

Final

96 h

G1

0.0 (Control)

21.5

21.8

21.4

21.3

21.3

21.8

21.2

21.3

G2

100

22.0

21.6

21.6

21.5

21.4

21.6

21.3

21.4

B. Dissolve Oxygen (%)

Group

Test Concentration

(% WAF prepared at 100 mg/L)

0 - 24 h

24 - 48 h

48 - 72 h

72 - 96 h

Initial

0 h

Final

24 h

Initial

24 h

Final

48 h

Initial

48 h

Final

72 h

Initial

72 h

Final

96 h

G1

0.0 (Control)

97.6

89.4

92.8

89.3

95.3

91.5

98.1

85.6

G2

100

97.6

88.3

98.1

88.3

95.8

85.8

93.0

87.2

C. pH

Group

Test Concentration

(% WAF prepared at 100 mg/L)

0 - 24 h

24 - 48 h

48 - 72 h

72 - 96 h

Initial

0 h

Final

24 h

Initial

24 h

Final

48 h

Initial

48 h

Final

72 h

Initial

72 h

Final

96 h

G1

0.0 (Control)

7.50

7.79

7.57

7.50

7.58

7.71

7.74

7.31

G2

100

7.55

7.92

7.71

7.63

7.69

7.29

7.51

7.35

D. Total Hardness (mg/L as CaCO3)

Hour

0

24

48

72

Total Hardness (mg/L)

84.0

89.6

84.0

78.4

TABLE 5: Behavioral Response

Group

Test Concentration

(% WAF prepared at 100 mg/L)

Noof

Fish Used

Behavioral Response observed at

3 h

6 h

24 h

48 h

72 h

96 h

G1

0.0 (Control)

10

1(10)

1(10)

1(10)

1(10)

1(10)

1(10)

G2

100.0

10

1(10)

1(10)

1(10)

1(10)

1(10)

1(10)

Behavioral Response: 1 = Normal

Note: Figures presented outside parentheses refer the clinical symptom and inside parentheses refer the total number of fish. 

Active Ingredient Concentration and Stability of Ethyl Lactate in Test Media (For 0 day)

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

0.00

ND

-

-

G2 (100)

I

92.0

26778

66.86277

72.68

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

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

0.00

ND

-

-

G2 (100)

I

92.0

9649

23.80864

25.88

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

Purity (% w/w)

92.0

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

Note: (1) Dilution factor for G1 and G2 were 2.5 and 2.5, respectively. (2) For stability of RO water for 0 day, 0 h samples were prepared and injected on to HPLC, no peak response of VL3 was observed. Test item having hydrolysing properties in aqueous phase. The VL3 was hydrolysed and gave degradant in form of ethyl lactate.

Active Ingredient Concentration and Stability of Ethyl Lactate in Test Media (For 1 day)

Groups and Conc. of sample

(%WAF preparation

at 100mg/L)

Injection

Theoretical

Conc. in

Test Media

(mg/L)

1 D, 0 h

Peak

Area of Sample

(Y)

Analysed Conc.

(mg/L)

%

Recovery

G1 (0.0)

I

0.00

ND

 

-

G2 (100)

I

92.0

12202

30.22566

32.85

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

Purity (% w/w)

92.0

Groups and Conc. of sample

(%WAF preparation

at 100mg/L)

Injection

Theoretical

Conc. in

Test Media

(mg/L)

1 D, 24 h

Peak

Area of Sample

(Y)

Analysed Conc.

(mg/L)

%

Recovery

G1 (0.0)

I

0.00

ND

 

-

G2 (100)

I

92.0

22605

56.37384

61.28

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

Purity (% w/w)

92.0

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

Note: (1) Dilution factor for G1 and G2 were 2.5 and 2.5, respectively. (2) For stability of RO water for 1 day, 0 h samples were prepared and injected on to HPLC, no peak response of VL3 was observed. Test item having hydrolysing properties in aqueous phase. The VL3 was hydrolysed and gave degradant in form of ethyl lactate.

Active Ingredient Concentration and Stability of Ethyl Lactate in Test Media (For 2 day)

Groups and Conc. of sample

(%WAF preparation

at 100mg/L)

Injection

Theoretical

Conc. in

Test Media

(mg/L)

2 D, 0 h

Peak

Area of Sample

(Y)

Analysed Conc.

(mg/L)

%

Recovery

G1 (0.0)

I

0.00

ND

 

-

G2 (100)

I

92.0

28997

72.44028

78.74

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

Purity (% w/w)

92.0

Groups and Conc. of sample

(%WAF preparation

at 100mg/L)

Injection

Theoretical

Conc. in

Test Media

(mg/L)

2 D, 24 h

Peak

Area of Sample

(Y)

Analysed Conc.

(mg/L)

%

Recovery

G1 (0.0)

I

0.00

ND

 

-

G2 (100)

I

92.0

9731

24.01475

26.10

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

Purity (% w/w)

92.0

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

Note: (1) Dilution factor for G1 and G2 were 2.5 and 2.5, respectively. (2) For stability of RO water for 2 day, 0 h samples were prepared and injected on to HPLC, no peak response of VL3 was observed. Test item having hydrolysing properties in aqueous phase. The VL3 was hydrolysed and gave degradant in form of ethyl lactate.

Active Ingredient Concentration and Stability of Ethyl Lactate in Test Media (For 3 day)

Groups and Conc. of sample

(%WAF preparation

at 100mg/L)

Injection

Theoretical

Conc. in

Test Media

(mg/L)

3 D, 0 h

Peak

Area of Sample

(Y)

Analysed Conc.

(mg/L)

%

Recovery

G1 (0.0)

I

0.00

ND

 

-

G2 (100)

I

92.0

14922

37.06245

40.29

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

Purity (% w/w)

92.0

Groups and Conc. of sample

(%WAF preparation

at 100mg/L)

Injection

Theoretical

Conc. in

Test Media

(mg/L)

3 D, 24 h

Peak

Area of Sample

(Y)

Analysed Conc.

(mg/L)

%

Recovery

G1 (0.0)

I

0.00

ND

 

-

G2 (100)

I

92.0

17266

42.95414

46.69

Intercept with y-axis (a)

176.78

Correlation of coefficient (r)

0.999

Slope of the line (b)

994.62

Purity (% w/w)

92.0

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

Note: (1) Dilution factor for G1 and G2 were 2.5 and 2.5, respectively. (2) For stability of RO water for 3 day, 0 h samples were prepared and injected on to HPLC, no peak response of VL3 was observed. Test item having hydrolysing properties in aqueous phase. The VL3 was hydrolysed and gave degradant in form of ethyl lactate.

Validity criteria fulfilled:
yes
Remarks:
Control: In the control group no mortality observed during the study period. Dissolved Oxygen Concentration: The dissolved oxygen concentration in the test media did not fall below 85.6% of air saturation value during the test.
Conclusions:
The LL50 of VL3 is greater than 100% WAF prepared at 100 mg/L in Zebrafish, Danio rerio.
Executive summary:

The acute toxicity of the test item in fish was evaluated in accordance to the OECD Guideline 203 (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. This study was performed to assess the 96h-LL50 of water accommodated fraction (WAF) of VL3 to the Zebrafish, Danio rerio. Based on the results of the preliminary range finding study in which the doses of 0.0 (control), 1.0, 10.0, 25.0, 50.0 and 100.0% WAF prepared at 100 mg VL3/L were evaluated, twenty fish were divided into two groups of ten fish. The definitive study was conducted as a semi-static limit study, in which one group of fish was exposed to the concentration of 100% WAF prepared at 100 mg VL3/L and one group served as control. 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 100% WAF prepared at 100 mg/L. Since, VL3 was not detected, the result is expressed based on nominal concentration only.  No mortality or other abnormalities were observed and all validity criteria were fulfilled. The 96h-LL50 of VL3 was found greater than 100% WAF prepared at 100 mg/L.

Description of key information

Key study: Accordign to OECD 203. GLP study. The 96h-LL50 of VL3 is greater than 100% WAF prepared at 100 mg/L in Zebrafish, Danio rerio.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information