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Ecotoxicological information

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:
2020-06-02 to 2020-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
adopted 18 June 2019
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
440/2008/EC, 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
October 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For determination of the test item concentrations, five replicate samples were taken from the test solution (5×3 mL) and five replicate samples from the control solution (5×3 mL) at the start and at the end of the test. The sampling was performed from different part of the aquariums.
- Sample storage conditions before analysis: no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent. An amount of 0.5015 g test item was dissolved in 5015 mL dilution water (ISO medium) in order to obtain the nominal concentration of 100 mg/L.
- Controls: negative control
- Evidence of undissolved material: no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Source: Akvárium Magazin Kft. (Pasaréti Gyula), 1222 Budapest, Dévény u. 36., Hungary
- Age at study initiation (mean and range, SD): juveniles
- Length at study initiation: total length 1-2 cm
- Weight at study initiation: 0.07 g (calculated mean of 1 fish)

ACCLIMATION
- Acclimation period: at least 9 days before test initiation
- Acclimation conditions:
Fish were held for in the fish laboratory under the following conditions:
Water: ISO medium (same as used for the test)
Photoperiod: 12 to 16 hours light daily
Temperature: 21 – 25°C
Oxygen concentration: at least 80 % of air saturation
- Type and amount of food during acclimation:
- Feeding frequency during acclimation: three times per week or daily until 24 – 48 hours before the test is started
- Health during acclimation: fish were apparently healthy and no mortality was observed

FEEDING DURING TEST
- Food type: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
22.4 – 22.8 °C
pH:
7.67 – 9.41
Dissolved oxygen:
81.3 – 90.9 % of air saturation
Salinity:
not applicable
Conductivity:
not specified
Nominal and measured concentrations:
nominal: 100 mg/L
measured (after 96 hours): 98.2 mg/L (arith. mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- Type: open
- Material, fill volume: glass, fill volume = 5 L
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: 0.15 g/L in the control and 0.13 g/L in the test item treated group

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium prepared using deionised water (prepared in TOXI-COOP ZRT. by BWT Christ Ministil water purification system)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 639 lux

EFFECT PARAMETERS MEASURED: Fish were observed at approximately 2, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline: Based on the results of a preliminary test, no toxicity of the test item was expected on Zebrafish up to 100 mg/L (limit concentration). Therefore a limit test was performed using only this concentration level and a control in order to demonstrate that the test item is not toxic to Zebrafish up to at least the limit concentration of 100 mg/L.
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: not observed
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Since a limit test was performed (and mortality did not occur), no statistical analysis was necessary. Endpoints were determined directly from the raw data.

Table 1: Cumulative Mortality Data

Test Group

Cumulative mortality
(initial population = 10 fish / test group)

2h

6h

24h

48h

72h

96h

Control

0

0

0

0

0

0

100 mg/L

0

0

0

0

0

0

 

Table 2: Measured and calculated Data of Body Weight

Test Group

Measured weight
of 10 fish (g)

Calculated mean
weight of 1 fish (g)

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

Control

0.77

0.08

0.15

100 mg/L

0.65

0.07

0.13

 

Table 3: Body Length of Test Animals

Test Group

Body length [cm]

Control

1.80

1.90

2.00

1.85

1.85

1.90

1.75

1.95

1.80

1.90

100 mg/L

1.70

1.85

2.00

1.95

1.90

2.00

1.75

1.80

1.90

1.85

 

Table 4: pH-values measured during the Experiment

Test Group

pH

0h

24h

48h

72h

96h

Control

7.89

7.82

7.67

7.91

7.91

100 mg/L

9.41

8.90

8.58

8.45

8.17

 

Table 5: Dissolved Oxygen Concentrations measured during the Experiment

Test Group

Dissolved Oxygen Concentration [%]

0h

24h

48h

72h

96h

Control

89.5

84.5

83.2

82.9

81.3

100 mg/L

90.9

89.0

88.2

87.4

87.0

 

Table 6: Temperatures measured during the Experiment

Test Group

Temperature[°C]

0h

24h

48h

72h

96h

Control

22.4

22.7

22.8

22.7

22.6

100 mg/L

22.6

22.8

22.8

22.7

22.7

Table 7: Concentration of Test Item measured in the Test Solutions

Nominal concentration mg/L

Measured concentrations

Start(June 02, 2020)

End(June 06, 2020)

Concentration (mg/L) 

% of the nominal

Concentration (mg/L) 

% of the nominal

Control

not detected

-

not detected

-

100

83.41

83

99.49

99

100.80

101

96.80

97

102.58

103

99.35

99

101.09

101

98.05

98

101.26

101

97.62

98

Validity criteria fulfilled:
yes
Conclusions:
In a static acute toxicity test according to OECD TG 203 with Danio rerio the 96-hour LC50 of the test item was determined to be >100 mg/L (nominal).
Executive summary:

To evaluate the acute toxicity of the test item on Zebrafish (Danio rerio) a static 96-hour test according to OECD TG 203, EU Method C.1. and US EPA OCSPP 850.1075 was carried out in compliance with the GLP principles. Based on the results of a preliminary study 10 animals were exposed to a single limit concentration of 100 mg/L (nominal). A negative control with ISO Medium but without the test item was performed in parallel. The test item concentration was analytically verfied by using HPLC-UV and found to be 83 –103 % of the nominal at the start, and 97 – 99 % of the nominal at the end of the test. Therefore, all biological results are based on nominal concentrations. Fish were observed at approximately 2, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. All validity criteria of the test guidelines were fulfilled. As a result, no toxic effect on fish was observed  at the limit concentration of 100 mg/L. Accordingly, the 96-hour LC50 value was determined to be > 100 mg/L. The 96-hour NOEC was determined to be 100 mg/L.

Description of key information

In a static acute toxicity test according to OECD TG 203 with Danio rerio the 96-hour LC50 of the test item was determined to be >100 mg/L (nominal).

Key value for chemical safety assessment

Additional information

To evaluate the acute toxicity of the test item on Zebrafish (Danio rerio) a static 96-hour test according to OECD TG 203, EU Method C.1. and US EPA OCSPP 850.1075 was carried out in compliance with the GLP principles. Based on the results of a preliminary study 10 animals were exposed to a single limit concentration of 100 mg/L (nominal). A negative control with ISO Medium but without the test item was performed in parallel. The test item concentration was analytically verfied by using HPLC-UV and found to be 83 –103 % of the nominal at the start, and 97 – 99 % of the nominal at the end of the test. Therefore, all biological results are based on nominal concentrations. Fish were observed at approximately 2, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. All validity criteria of the test guidelines were fulfilled. As a result, no toxic effect on fish was observed  at the limit concentration of 100 mg/L. Accordingly, the 96-hour LC50 value was determined to be > 100 mg/L. The 96-hour NOEC was determined to be 100 mg/L.