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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation 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: 10 mL of test samples from each replicates were drawn at 0 and before & after every renewal. The representative samples were divided into two equal portions.
- Sample storage conditions before analysis: One portion was sent for test concentration analysis and the second portion was stored at -20 ± 5 ºC temperature till the study completion. Active ingredient concentration in water 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.

An amount of 100 mg VL3 was mixed with reconstituted water and made up to 1000 mL. It was kept for stirring using magnetic stirrer for 10 minutes at room temperature. After stirring, test solution was kept for re-equilibrium for 10 minutes. 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 exposure of daphnia at the concentration of 100% WAF prepared at 100 mg/L. For control group, only reconstituted 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 test vessel was changed at 24 h and replaced with the freshly prepared test solution. At renewal, 100% of volume of the test vessel was renewed. During renewal, daphnia was transferred by micropipette to new test vessels.

- Controls: yes, negative control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): reconstituted water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0% and 100%
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: MicroBio Test Inc, Kleimoer 15, 9030 Mariakerke (Gent), Belgium
- Age of parental stock (mean and range, SD): less than 24 h old (first instar nymph)
- Feeding during test: Feed was not provided during the exposure period
They showing no signs of stress such as presence of males and ephippia, delay in the production of the first brood, discolouration, etc. and having normal behavior.

ACCLIMATION
- Acclimation period: Gravid females were acclimatised to the test conditions for a minimum period of 48 h.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: live algal cells
- Health during acclimation (any mortality observed): no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Freshly hatched daphnids belonging to the same stock (less than 24 h-old) were collected with the help of a micropipette and was used for the study.
First instar daphnids (less than 24 h old neonates) were separated from the adults in a culture of daphnia derived from a single mother. This culture had previously been acclimatised to the study conditions and was immediately transferred to labelled glass beakers (five for each replicate). Transfer was performed using a micropipette having a capacity of 100 - 1000 μL. The mobility of the test organisms was verified immediately after their introduction by gentle swirling of the test container with visual inspection.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
184.8 and 190.4 mg/L as CaCO3
Test temperature:
19.7 and 21.2 °C
pH:
7.14 and 7.79
Dissolved oxygen:
8.20 and 8.87 mg/L
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: Beaker
- Material, size, headspace, fill volume: Glass beaker of 600 mL capacity
- Aeration: The diluent water was aerated, prior to use, for the test so that dissolved oxygen concentration has reached saturation
- Renewal rate of test solution (frequency/flow rate): test medium was renewed at 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates):4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Daphnia culture water with a total hardness of 184.8 and 190.4 mg/L as CaCO3.
(1) Calcium chloride solution: A quantity of 11.76 g CaCl2.2H2O was dissolved in distilled water and the volume was made up to 1 litre.
(2) Magnesium sulphate solution: A quantity of 4.93 g MgSO4.7H2O was dissolved in distilled water and the volume was made up to 1 litre.
(3) Sodium bicarbonate solution: A quantity of 2.59 g NaHCO3 was dissolved in distilled water and the volume was made up to 1 litre.
(4) Potassium chloride solution: A quantity of 0.23 g KCl was dissolved in distilled water and the volume was made up to 1 litre.
From the above stock solutions a volume of 25 mL each of solution (a) to (d) were taken in a volumetric flask and the total volume was made up to 1 litre by adding distilled water. This media was aerated thoroughly for two days and used for conducting the study.
- Culture medium different from test medium: no
- Intervals of water quality measurement: before and after each renewal

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light and 8 h dark cycle
- Light intensity: 1310 and 1330 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility and behavioural symptoms at 0, 24, and 48 h of exposure. Temperature, pH, and dissolved oxygen content before and after each renewal. Total hardness of reconstituted water was measured once during acclimatisation and test period using the titrimetric method. Light intensity measured daily.

VEHICLE CONTROL PERFORMED: yes

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):
yes
Remarks:
The validity and reliability of the test system Daphnia magna was confirmed by conducting a positive control study (JRF Study N° 502-3-07-19120) using potassium dichromate.
Duration:
24 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:
mobility
Key result
Duration:
48 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:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF prepared at 100 mg VL3/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF prepared at 100 mg VL3/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Daphnids exposed to 100% WAF prepared at 100 mg VL3/L and control (without VL3) did not exhibit any abnormal behavioural symptoms at 24 and 48 h.
- Other biological observations: No immobilisation was observed at the test concentration of 100% WAF prepared at 100 mg VL3/L as well as in the control (without VL3) at 24 and 48 h.
- Mortality of control: no mortality.
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: Immobility Data at 0, 24 and 48 h Exposure Period

Group

Test Concentration

(%WAF prepared at 100 mg/L)

N° of Replicates

N° of Daphnia/group

Immobility N° and % at

0 h

%

24 h

%

48 h

%

G1

0.0 (Control)

4

20

0

0

0

0

0

0

G2

100.0

4

20

0

0

0

0

0

0

Key: h = Hour

TABLE 2: Mean Values of Water Parameters of Test Solution

Groups

Nominal Concentration (% WAF Prepared at 100 mg/L)

pH

Temperature (°C)

Dissolved Oxygen (%)

Total Hardness

(as CaCO3)

mg/L water

Initial

Final

Initial

Final

Initial

Final

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

187.6 ± 4.0

G1

0.0 (Control)

7.56

0.19

7.41

0.16

20.0

0.1

21.1

0.1

8.53

0.19

8.51

0.19

G2

100.0

7.60

0.18

7.52

0.14

19.9

0.1

21.0

0.1

8.75

0.07

8.72

0.06

Key: SD = Standard Deviation, h = Hour

TABLE 3: Behavioral Symptoms (Main study)

Group

Test

 Concentration

(% WAF Prepared at 100 mg/L)

Behavioral Symptoms Observed at

0 h

24 h

48 h

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

G1

0.0 (Control)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

G2

100.0

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

Key:  h = Hour, R = Replicate

Behavioral symptom: 1 = Normal

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

TABLE 4: Record of Physico-chemical Parameters of Test Solution

Parameter: pH

Group

Test

Concentra-tion

 (% WAF Prepared at 100 mg/L)

Values at

0 Day

1 Day

Initial

Final

Initial

Final

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

G1

0.0 (Control)

7.38

7.37

7.39

7.40

7.35

7.38

7.27

7.14

7.67

7.73

7.74

7.76

7.38

7.53

7.59

7.62

G2

100.0

7.42

7.44

7.44

7.45

7.37

7.41

7.39

7.41

7.77

7.74

7.78

7.79

7.63

7.64

7.66

7.67

Parameter: Temperature (°C)

Group

Test

Concentra-tion

 (% WAF Prepared at 100 mg/L)

Values at

0 Day

1 Day

Initial

Final

Initial

Final

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

G1

0.0 (Control)

20.6

20.3

20.1

20.0

20.9

20.7

20.5

21.1

19.9

19.7

19.8

19.9

21.2

21.2

21.1

21.0

G2

100.0

20.0

20.1

20.1

20.1

20.8

20.9

20.6

20.7

20.0

20.0

19.9

19.8

21.0

21.0

20.9

20.9

Parameter: Dissolved Oxygen (mg/L)

Group

Test

Concentra-tion

 (% WAF Prepared at 100 mg/L)

Values at

0 Day

1 Day

Initial

Final

Initial

Final

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

G1

0.0 (Control)

8.23

8.33

8.51

8.40

8.20

8.31

8.50

8.38

8.63

8.73

8.72

8.69

8.60

8.71

8.66

8.68

G2

100.0

8.73

8.79

8.81

8.87

8.71

8.75

8.80

8.81

8.74

8.68

8.66

8.73

8.70

8.66

8.65

8.67

Key: R = Replicate, h = Hour

Parameter: Light Intensity

Hours

0 h

24 h

48 h

Light Intensity (Lux)

1330

1310

1320

TABLE 5: 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

92.00

24801

60.5029

65.76

Intercept with y-axis (a)

490.22

Correlation of coefficient (r)

0.999

Slope of the line (b)

1004.53

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

92.00

24211

59.0345

64.17

Intercept with y-axis (a)

490.22

Correlation of coefficient (r)

0.999

Slope of the line (b)

1004.53

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 reconstituted 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 degradanting form of ethyl lactate.

TABLE 6: 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

92.00

15631

37.6813

40.96

Intercept with y-axis (a)

490.22

Correlation of coefficient (r)

0.999

Slope of the line (b)

1004.53

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

92.00

14464

34.7769

37.80

Intercept with y-axis (a)

490.22

Correlation of coefficient (r)

0.999

Slope of the line (b)

1004.53

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 reconstituted 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 degradanting form of ethyl lactate.

Validity criteria fulfilled:
yes
Remarks:
Control immobilisation 0% (<10%) at the termination. Dissolved oxygen concentration was ≥ 8.20 mg/L in the control and test vessels at the end of the test. Daphnia in the control groups did not show immobilisation or other signs or disease or stress.
Conclusions:
The EL550 (48 h), NOELR and LOELR of the test item in Daphnia magna is greater than the test concentration of 100% WAF prepared at 100 mg VL3/L.
Executive summary:

An acute Daphnia magna immobilization test was performed according to the OECD Guideline 202 (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. 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 used, the main study was restricted to limit study and it was performed at the nominal test concentration of 100% WAF prepared at 100 mg VL3/L. Experiment was conducted in semi-static condition i.e., the test media was changed at 24 h interval. There were two groups, each one consisted of four replicates with 5 daphnids per replicate. 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. As the study was conducted as a limit test of 100% WAF prepared at 100 mg VL3/L and no immobility observed up to 48 h, the 24 and 48 h EL50 could not be calculated and considered to be greater than test concentration of 100% WAF prepared at 100 mg VL3/L. The NOELR and LOELR of the test item is also considered greater than test concentration of 100% WAF prepared at 100 mg VL3/L.

Description of key information

Key study: Test according to OECD 202. GLP study. The 48h-EL50, NOELR and LOELR of the test item in Daphnia magna is greater than the test concentration of 100% WAF prepared at 100 mg VL3/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information