Registration Dossier

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 11, 2017 - September 15, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity 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
- Stability under test conditions: yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 100 mg/L
- Sampling method: The test concentration was analytically determined at the start and at the end of the renewal periods (0, 48, 96 h) by HPLC-UV method.
- Sample storage conditions before analysis: No storage was necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a concentration of 100 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (aquarium water) using ultrasonic bath (approximately 6 minutes). As a limit test was carried out, further dilution of stock solution was not performed.
- Eluate: Aquarium water.
- Controls: The dilution water (circulated and filtered) was used without of addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.
- As the test item was not stable over the 96 h exposure period (based on the analytical method validation, Study Code: 16/415-316AN) the test was performed under semi-static conditions. The frequency of the water renewal periods was 48 hours. Prior to the treatments, at each of the renewal periods test item solution was prepared by the method described above.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish.
- Strain: Brachydanio rerio.
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary.
- Age at study initiation (mean and range, SD): not specified.
- Length at study initiation (length definition, mean, range and SD): 2.6 - 2.9 cm
- Weight at study initiation (mean and range, SD): mean weight: 1.61 - 1.64 g

ACCLIMATION
- Acclimation period: more than 12 days.
- Acclimation conditions (same as test or not): same as test.
- Health during acclimation (any mortality observed): Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

FEEDING DURING TEST
- The fish were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
187 mg/L (as CaCO3)
Test temperature:
20.4 - 22.8 ºC
pH:
7.26 - 7.92
Dissolved oxygen:
88 - 99% of the air saturation value at the temperature used.
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 100 mg/L.
- Measured concentrations: 0 (control), 102 and 100 mg/L at the start and 84.9 mg/l and 69.7 mg/l at the end of the renewal periods. The corresponding measured geometric mean test item concentration was: 88.14 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: one aquarium for the test group and another for the control group.
- Material, size, headspace, fill volume: fill volume: 5 L test solution.
- Aeration: yes.
- Renewal rate of test solution (frequency/flow rate): every 48 h (the test item was not stable over the 96 h exposure period, based on the analytical method validation, Study Code: 16/415-316AN).
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: aquarium water (circulated and filtered).
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 16 hours light and 8 hours darkness.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. The test conditions (pH, temperature, oxygen saturation) were measured daily (twice after 48 hours - at the end of the first as well as at the beginning of the second renewal period) during the test. The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (1.0 g fish/litre testing liquid).

TEST CONCENTRATIONS
- Range finding study: Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions (with 24-hour renewal, in absence of stability data).
- Test concentrations: In the range-finding study, the concentrations tested were 0 (control), 0.1, 1, 10 and 100 mg/L of test item. As the measured concentrations deviated more than 20 percent from the nominal in one case, biological results are based on the measured test item concentration.
- Results used to determine the conditions for the definitive study: Because no toxic response was observed during the preliminary range-finding test, only one test concentration (100 mg/L) and one control group was tested in the definitive study in a semi-static system (limit test).
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
EC0
Effect conc.:
> 88.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 88.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
88.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 88.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 88.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 88.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 88.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: No abnormal behaviour, appearance or any other effect on test animals were detected.
- Observations on body length and weight: There was no considerable difference observed concerning body weights between the groups.
- Mortality of control: 0/7.
- Other adverse effects control: no.
- Abnormal responses: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects. The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Table 3: Cumulative mortality data in the Limit Test

Test Group

Cumulative Mortality (number of dead fish)
[initial population = 7 fish]

3h

6h

24h

48h

72h

96h

Control

0

0

0

0

0

0

100 mg/L (nominal),
88.14 mg/L (measured)

0

0

0

0

0

0

 No sublethal effect was observed.


 Table 4: Measured and calculated data of body weight

Test Group

Measured weight
of 7 fish (g)

Calculated mean
weight of 1 fish (g)

Loading of testing aquarium
(g fish/L testing liquid)

Control

1.64

0.23

0.33

100 mg/L (nominal),
88.14 mg/L (measured)

1.61

0.23

0.32

  There was no considerable difference observed concerning body weights between the groups.

Table 5: Temperature

Test Group

Temperature (°C)

Measuring

0h1)

24h

482)

481)

72

962)

Control

21.7

20.9

20.5

21.2

20.4

20.7

100 mg/L (nominal),
88.14 mg/L (measured)

22.8

21.0

20.4

22.8

20.4

20.6

 

 

Table 6: Dissolved oxygen concentration

Test Group

Dissolved oxygen concentration (%)

Measuring

0h1)

24h

482)

481)

72

962)

Control

99

91

92

97

94

92

100 mg/L (nominal),
88.14 mg/L (measured)

99

89

90

96

90

88

 

 

Table 7: pH

Test Group

pH

Measuring

0h1)

24h

482)

481)

72

962)

Control

7.35

7.91

7.39

7.77

7.82

7.45

100 mg/L (nominal),
88.14 mg/L (measured)

7.26

7.92

7.51

7.47

7.76

7.74

1) : measurement at the start of the renewal period in the fresh test medium

2) : measurement at the end of the renewal period in the old test medium

 

 

Table 8: Body length of test fish at the end of the test

Test Group

Body length [cm]

1

2

3

4

5

6

7

Control

2.7

2.9

2.6

2.7

2.7

2.8

2.9

100 mg/L (nominal),
88.14 mg/L (measured)

2.8

2.7

2.8

2.9

2.6

2.7

2.6

 Remark: At the end of the test,body length of test fish were measured in order to check their compliance with the size range recommended for the species by the test guideline [Commission Regulation (EC) No 440/2008;Annex Part C, C.1]


Validity criteria fulfilled:
yes
Remarks:
All validity criteria were fulfilled, see "Overall remarks".
Conclusions:
The 96h-EC50 of the test item in zebrafish was found to be higher than 88.14 mg/L (measured) and the NOEC was 88.14 mg/L (measured), both based on mortality.
Executive summary:

An acute toxicity test on zebrafish (Brachydanio rerio) was performed in accordance with OECD 203 and EU method C.1, following GLP. Based on the results obtained in a preliminary range-finding test where no toxic response was observed, a Limit Test was carried out using only one concentration (100 mg/L) and one control group in a semi-static system. As the measured concentrations deviated more than 20 percent from the nominal in one case, biological results are based on the measured test item concentration, the measured geometric mean test item concentration was 88.14 mg/L. The renewal period of the water was 48 h, due to the test item was not stable over the 96 h exposure period. Seven fish were exposed to the test item or control for 96 h. All validity criteria were met. The 96h-EC50 of the test item in zebrafish was found to be higher than 88.14 mg/L (measured) and the NOEC was 88.14 mg/L (measured), both based on mortality.

Description of key information

Key study: OECD 203 and EU method C.1. GLP study. The 96h-EC50 of the test item in zebrafish was found to be higher than 88.14 mg/L (measured) and the NOEC was 88.14 mg/L (measured), both based on mortality.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
88.14 mg/L

Additional information

Key study: An acute toxicity test on zebrafish (Brachydanio rerio) was performed in accordance with OECD 203 and EU method C.1, following GLP. Based on the results obtained in a preliminary range finding test where no toxic response was observed, a Limit Test was carried out using only one concentration (100 mg/L) and one control group in a semi-static system. As the measured concentrations deviated more than 20 percent from the nominal in one case, biological results are based on the measured test item concentration, the measured geometric mean test item concentration was 88.14 mg/L. The renewal period of the water was 48 h, due to the test item was not stable over the 96 h exposure period. Seven fish were exposed to the test item or control for 96 h. All validity criteria were met. The 96h-EC50 of the test item in zebrafish was found to be higher than 88.14 mg/L (measured) and the NOEC was 88.14 mg/L (measured), both based on mortality.