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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:
2016-04-25 to 2016-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all
- Sampling method: Samples from freshly prepared media after 0, 24, 48 and 72 h and from corresponding aged test media after 24, 48, 72 and 96 h were taken.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until preparation. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate volumes of the test item were pipetted directly into the test vessels filled with appropriate volumes of the dilution water. The test media were mixed with an ultraturrax (1 minute, 12000 rpm). Thereafter, the test vessels were covered with a floating lid.
- Controls: Dilution water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, the test item was clearly dissolved throughout exposure.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Length at study initiation (length definition, mean, range and SD): Fish (average body lenght): 2.3 cm
- Weight at study initiation (mean and range, SD): Fish (average body weight): 0.13 g
- Maintenance of the brood fish: Holding was performed at the test facility at 23 ± 2 °C and diffuse light (0.1 -10 µmol photons x m-2 x s-1, natural photoperiod) in tap water of local origin changed at least once per week. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine. The dissolved oxygen concentration was more than 80% of the air saturation value.

ACCLIMATION
- Acclimation period: At least 12 days
- Acclimation conditions (same as test or not): During test period, the vessels were not aerated. For all other conditions acclimation coditions were the same as in the test.
- Type and amount of food during acclimation: The amount of food (Sera Vipan from Sera GmbH, 52518 Heinsberg, Germany) was 4% of the fish body weight per feeding day.
- Feeding frequency during acclimation: Food was provided 3 times per week. The test fish were not be fed 24h before test star and during the test period.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Day 0: 57 mg/L as CaCO3
Test temperature:
Day 0 - Day 4: 22.9 - 24.0°C (Mean temperature: 23.5°C)
pH:
Day 0 - Day 4: 7.30 - 7.81
Dissolved oxygen:
Day 0 - Day 4: 82 - 100%
Nominal and measured concentrations:
Nominal concentrations: 5, 10, 20, 40, 80 mg/L
Measured concentrations:
Mean value fresh medium (72 h): 2.81, 6.00, 11.9, 27.1, 63.2 mg/L
Mean value old medium (72 h): 2.51, 5.23, 10.3, 23.9, 59.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria of 13 L (dimensions: 25.5/18/28 cm)
- Type (delete if not applicable): Closed with a floating lid (e.g. with aluminium foil)
- Material, size, headspace, fill volume: Glass vessel filled with approximately 10 L of dilution water.
- Aeration: No aeration during the test period.
- Renewal rate of test solution (frequency/flow rate): The test item was renewaled every 24h (3 times).
- No. of organisms per vessel: 7 fish
- No. of vessels per concentration (replicates): One replicate per concentration
- No. of vessels per control (replicates): One replicate
- Biomass loading rate: 0.091 g fish per litre test solution (calculated: 0.91 g fish per vessel)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin filtered on activated charcoal and aerated for at least 24 hours (Hardness: 10 - 250 mg CaCO3/L; pH-value: 6.0 - 8.5).
- Culture medium different from test medium: No.
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all test vessels at the beginning and at the end of the exposure and every 24 hours from fresh and old test media. Total hardness of the water was determined at the beginning out of the control. During the test the water temperature was recorded once per hour with a data logger. The light intensity on the surface of the test aquaria was measured at test start.

OTHER TEST CONDITIONS
- Photoperiod: Daily 15 hours photoperiod (correspond to natural daylight hours)
- Light intensity: 7 - 750 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality, Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 2, 24, 48, 72 and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 (5, 10, 20, 40, 80 mg/L)
- Range finding study: Yes
- Test concentrations: 0 (control) and 10 mg/L (number of fish: control n = 7, 10.0 mg/L n = 3)
- Results used to determine the conditions for the definitive study: Yes (The range finding study was done in a semi-static procedure with daily renewal of the test media, without aeration and without head space, covered with a floating lid)
Reference substance (positive control):
not required
Remarks:
According to the guideline, no reference substance is required.
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
8.45 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
LC50 is calculated as geometric mean of LC100/LC0 based on mean measured initial test item concentration.
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
11.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Reported statistics and error estimates:
The concentrations leading to 0 and 100 % mortality (LCo and LC100) after 96 h were determined directly from the test results. The LC50-values were calculated by sigmoidal regression. The concentrations enclosed to the LC50-value after 24 and 72 hours were chosen as lower and upper confidence limits. The chosen methods for determination of LCx-values were in accordance with the OECD series on testing and assessment No. 54 (2006). Excel, Microsoft Corporation and GraphPad Prism, GraphPad Software, Inc. was used for calculations.

Table 1: Measured concentrations of the percent of the initial concentrations of the test item

Sampling date 0 h (Fresh medium) 24 h (Old medium) 24 h (Fresh medium) 48 h (Old medium) 48 h (Fresh medium) 72 h (Old medium) 72 h (Fresh medium) 96 h (Old medium)  
Nominal concentration of the test item (mg/L) Meas. conc. (mg/L) Meas. conc. (mg/L) % Meas. conc. (mg/L) Meas. conc. (mg/L) % Meas. conc. (mg/L) Meas. conc. (mg/L) % Meas. conc. (mg/L) Meas. conc. (mg/L) % Mean value fresh medium (mg/L) Mean value old medium (mg/L) %
80 63.2 59.0 93 - - - - - - - - - 63.2 59 93
40 28.1 22.9 82 26.1 24.9 95 - - - - - - 27.1 23.9 88
20 11.8 9.66 82 11.8 10.5 89 12.0 10.1 85 11.9 10.7 90 11.9 10.3 86
10 6.65 5.7 86 5.12 4.43 87 6.31 5.66 90 5.91 5.12 87 6 5.23 87
5 2.83 2.49 88 2.99 2.67 89 2.70 2.44 90 2.71 2.43 90 2.81 2.51 89
Control < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ  -

 

Meas. conc.= Measured concentration of the test item, dilution and enrichment factor taken into account

% = Percent of initial concentration of the test item

- = not determined due to 100% mortality

LOQ = Limit of quantification (50 µg/L test item)

Table 2: Environmental conditions including mean measured initial concentration, oxygen saturation, pH-value, temperature and total hardness during the test period.

Exposure Time (h)

Mean measured initial concentration (mg/L)

Oxygen Saturation (%; old and new)

pH-value (old and new)

Temperature (°C; old and new)

Total Hardness (mg/L as CaCO3; old and new)

0

63.2

99

7.81

23.5

57

27.1

99

7.78

23.5

11.9

99

7.75

23.6

6.00

99

7.69

23.7

2.81

99

7.65

23.7

Control

99

7.55

23.7

24

63.2

96

7.72

23.6

-

27.1

90.5

7.69

23,3

11.9

92

7.65

23,4

6.00

91.5

7.61

23,3

2.81

93

7.48

23,3

Control

93.5

7.36

23,8

48

63.2

-

-

-

-

27.1

99

7.6

23.6

11.9

95.5

7.57

23.4

6.00

96.5

7.54

23.2

2.81

96.5

7.48

23.1

Control

94

7.37

23.4

72

63.2

-

-

-

-

27.1

-

-

-

11.9

94.5

7.57

23.4

6.00

94

7.55

23.2

2.81

96.5

7.51

23.3

Control

95

7.37

23.6

96

63.2

-

-

-

-

27.1

-

-

-

11.9

95

7.5

23.9

6.00

89

7.48

24.0

2.81

93

7.47

23.9

Control

90

7.30

24.2

Table 3: Cumulative Mortality [%] in the Definitive Test

Mean measured initial concentration (mg/L)

Cumulative Mortality (%) after different exposure periods (h)

2

24

48

72

96

63.2

100

100

100

100

100

27.1

0

57

100

100

100

11.9

0

0

0

71

100

6.00

0

0

0

0

0

2.81

0

0

0

0

0

Control

0

0

0

0

0

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of Ethyl isovalerate on Danio rerio was determined in a GLP study according to OECD 203. Individuals were exposed to 0, 5, 10, 20, 40, 80 mg/L of the test substance, which were equivalent to 0, 2.83., 6.65, 11.8, 28.1, 63.2 mg/L mean measured initial concentrations for 96 hours under semi-static test conditions. The test concentrations were analytically verified by GS-MS. The LC50 after 96 hours was calulated as 8.45 mg /L (geometric mean of LC100/LC0 based on mean measured concentration).

Description of key information

LC50 (96 h): 8.45 mg /L (Danio rerio)

Key value for chemical safety assessment

LC50 for freshwater fish:
8.45 mg/L

Additional information

The toxicity of the test item on Danio rerio was determined in a GLP study according to OECD 203. Individuals were exposed to 0, 5, 10, 20, 40, 80 mg/L of the test substance, which were equivalent to 0, 2.83., 6.65, 11.8, 28.1, 63.2 mg/L mean measured initial concentrations, for 96 hours under semi-static test conditions. The test concentrations were analytically verified by GS-MS. The LC50 after 96 hours was calulated as 8.45 mg /L (geometric mean of LC100/LC0 based on mean measured concentration).