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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:
2017-03 till 2017-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control: 1×100 mL, 6.25 mg/L: 2×100 mL, 12.5 mg/L: 2×100 mL, 25 mg/L: 2×100 mL, 50 mg/L: 2×50 mL, 100 mg/L: 2×50 mL
- Sampling method: samples were taken from each the test concentration at the start and at the end of each water renewal period and from the control at the start of the renewal periods.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: mechanical dispersion (intensive shaking):
For preparation of test solutions (at each renewal period) a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.5 g test item in 5000 mL ISO medium using approx. 2 hours of intensive shaking to obtain clear solution. After the formulation procedure the test animals were immediately introduced into the test solution.

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Brachydanio rerio)
- Source: Akvárium magazin Kft. (Pasaréti Gyula), 1222 Budapest, Dévény u. 36.
- Length at study initiation: 2 ± 1 cm
- Weight at study initiation: mean: 0.25 - 0.27 g

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: as test conditions
- Type and amount of food during acclimation: appropriate, commercial diet for fish
- Feeding frequency during acclimation: at least three times per week until one day before the test start
- Health during acclimation: no mortality 7 days before start of test


FEEDING DURING TEST
The fish were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
249 mg/L (as CaCO3)
Test temperature:
21.7 - 22.6 °C
pH:
7.61 - 8.08
Dissolved oxygen:
69.8 - 94.7 %
Nominal and measured concentrations:
Nominal test concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Measured test concentrations: 4.1, 9.3, 19.2, 37.8 and 79.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquariums
- Fill volume: 2 Liter test liquid
- Renewal rate of test solution: each 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.88 – 0.96 g/L

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: ISO medium, prepared according to recommendation of Annex 2 of the OECD 203 guideline
- Culture medium different from test medium: no


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

EFFECT PARAMETERS MEASURED:
The fish were observed at approximately 3, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality (additional observation period was conducted at approx. 26 h as two fish was found to be dead at this time in the concentration of 50 mg/L nominal).
Measurements of environmental conditions were performed daily (at the start and end of each water renewal period).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes, non-GLP
- Test concentrations: 1, 10, 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.:
39.6 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.:
79.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
19.2 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.:
37.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: In the concentration of 19.2 mg/L (measured) reduced swimming activity and staying mostly at the bottom of aquarium was noticed after approximately 3 and 6 hours of treatment. However, by the 24-h observation period all fish recovered and showed no sign of intoxication during the test. In the two highest concentrations (37.8 and 79.2 mg/L measured) severe sub-lethal effects were observed during the test.
- 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
Reported statistics and error estimates:
The EC50 value was calculated by Probit analysis using SPSS software. Other endpoints were determined directly from the raw data.
Validity criteria fulfilled:
yes
Conclusions:
In a semi-static short term toxicity to fish test the 96-hour LC50 of the test substance to Zebrafish (Brachydanio rerio) was determined to be 39.6 mg/L mean measured concentration.
Executive summary:

The purpose of this study was to evaluate the influence of the test item zebrafish (Brachydanio rerio). Young fish were exposed in an acute toxicity test to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (daily renewal periods). The test method of application and the test species Zebrafish (Brachydanio rerio) are recommended by the test guidelines. Based on the results of the preliminary experiment, nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were investigated in the main study. The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values of each renewal periods) were: 4.1, 9.3, 19.2, 37.8 and 79.2 mg/L. The measured concentrations of the test item were in the range of 73 – 94 % of the nominal at the start and 44 – 74 % at the end of the renewal periods.

All biological results are based on the measured geometric mean concentrations. Seven fish were exposed to each concentration and a control for 96 hours. The fish were observed at least on each day of the test for signs of intoxication and mortality. Body weights of fish were determined prior to test initiation; body length measurement was performed at the end of the test (or when any mortality occurred). Measurements of environmental conditions were performed daily (at the start and end of each water renewal period). The quantification of the test item was performed by a previously validated analytical method. Samples were taken from the test concentrations at the start and at the end of each water renewal period and from the control at the start of the water renewal periods and analysed by a GC – FID method. For determination of the LC50 value Probit analysis was performed using SPSS software. Other endpoints were determined directly from the raw data. All validity criteria were met and therefore the study can be considered as valid. In this 96-hour semi-static acute toxicity test on Zebrafish (Brachydanio rerio) the test item had significant toxic effects on fish. The 96-h LC50 value was determined to be 39.6 mg/L. The highest test concentration causing no mortality within the test period (96-h LC0) was determined to be 19.2 mg/L and the lowest test concentration causing 100 mortality within the test period (96-h LC100) was determined to be 79.2 mg/L.

Description of key information

The acute toxicity to fish was assessed according to OECD guideline 203 and EU-method C.1. In this 96-hour acute toxicity test with zebrafish, the EC50 value was 39.6 mg/L (measured concentration, geometric mean). The no-observed-effect concentration (NOEC) was 19.2 mg/L (nominal concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
39.6 mg/L

Additional information

The purpose of this study was to evaluate the influence of the test item to zebrafish (Brachydanio rerio). Young fish were exposed in an acute toxicity test to aqueous test media containing the test item for 96 hours at a range of concentration in a semi-static test system (daily renewal periods). The test method of application and the test species Zebrafish (Brachydanio rerio) are recommended by the test guidelines. Based on the results of the preliminary experiment, nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were investigated in the main study. The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values of each renewal periods) were: 4.1, 9.3, 19.2, 37.8 and 79.2 mg/L. The measured concentrations of the test item were in the range of 73 – 94 % of the nominal at the start and 44 – 74 % at the end of the renewal periods.

All biological results are based on the measured geometric mean concentrations. Seven fish were exposed to each concentration and a control for 96 hours. The fish were observed at least on each day of the test for signs of intoxication and mortality. The quantification of the test item was performed by a previously validated analytical method. Samples were taken from the test concentrations at the start and at the end of each water renewal period and from the control at the start of the water renewal periods and analysed by a GC – FID method. For determination of the LC50 value Probit analysis was performed using SPSS software. Other endpoints were determined directly from the raw data. All validity criteria were met and therefore the study can be considered as valid. In this 96-hour semi-static acute toxicity test on Zebrafish (Brachydanio rerio) the test item had significant toxic effects on fish. The 96-h LC50 value was determined to be 39.6 mg/L. The highest test concentration causing no mortality within the test period (96-h LC0) was determined to be 19.2 mg/L and the lowest test concentration causing 100 mortality within the test period (96-h LC100) was determined to be 79.2 mg/L.