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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentrations, samples (5 mL per replicate) were taken from the test solution (100 mg/L) and from the control at the start and at the end of the test. From the treatment group five replicate samples from the control two replicate samples were taken.
Vehicle:
no
Details on test solutions:
The test solution used in the test was prepared by mechanical dispersion. An amount of 0.5 g test item was dissolved in 5000 mL dilution water (ISO medium) in order to obtain the nominal concentration of 100 mg/L. The test solution was freshly prepared in the testing laboratory just before introduction of the test animals (start of the experiment).
Test organisms (species):
other: Brachydanio rerio; Zebrafish.
Details on test organisms:
Species: Zebrafish (Brachydanio rerio)
Origin: Akvárium magazin Kft. (Pasaréti Gyula) 1222 Budapest, Dévény u. 36.
Number of animals: There were 10 animals at the treated and control group respectively (no parallels were used).
Age and size of animals: Juveniles; size within a range of 2.0 ± 1 cm. The loading of the test aquaria did not exceed 1.0 g / L.
Sex: Both female and male (were not separated).
Animal health: Apparently healthy animals were used in the test.
Acclimatization, holding of fish prior to the study: Fish were held for at least 12 days before test initiation in the fish laboratory of TOXI-COOP ZRT. under the same conditions as used during the exposure period.
During holding, fish were fed with appropriate, commercial diet for fish at least three times per week until one day before the test start. The health of the breeding was continuously monitored and any mortality or abnormal behaviour recorded. No mortality occurred in seven days before the start of the experiment, therefore the batch was considered to be acceptable for testing.
Fish were held prior to the study in reconstituted water (ISO medium, prepared according to Annex 2 of the OECD guideline 203).
Light: 12 to 16 hours photoperiod daily;
Temperature: 21 – 25°C, constant within ± 1°C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
According to the guidelines.
Post exposure observation period:
No.
Hardness:
The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
The measured temperatures were within a range of 22.3 – 22.8°C in the test aquariums (the maximum deviation did not exceed ± 1°C).
pH:
The test was carried out without adjustment of pH. The measured pH values were in the range of 6.77 – 7.37 during the test.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 73.3 – 89.5 % of the air saturation value at the temperature used. Test solutions were not aerated during the test.
Salinity:
No.
Conductivity:
Not reported.
Nominal and measured concentrations:
0 - 100 mg/L.
Details on test conditions:
The water temperature, pH value and dissolved oxygen concentration were determined at the start of the test (i.e. introduction of the fish) and on the subsequent days (once daily) in the test concentration and the control.
Test type: Static test. Based on the results obtained during analytical method validation (Study number: 880-100-1637, see Section 8 Analytical methods, Gaal 2016) the test item is stable for the duration of 96 in ISO Medium, therefore a static test was performed.

Light-Dark Cycle: The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness.
Feeding: The fish were not fed during the test.
A preliminary test was performed at the concentration of 100 mg/L (limit concentration). No test item effects were observed during the preliminary test.

Body Weight Measurement and Introduction of Fish:
At the start of the test (on day 0) 10 fish were introduced into each aquarium, each containing 5 litre testing solution. Before introduction of test animals, the body weight of fish was measured and recorded: first the weight of a vessel filled with water was determined. Then the ten fish were selected randomly and placed into this vessel. The weight of the group of fish was registered and the average body weight calculated. On the basis of the above, the loading of the test-aquariums was calculated and was 0.51 g/L in the control and 0.58 g/L in the test item treated group.
Observations: 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.
Body Length Measurement: The body length of each fish were measured at the end of the test in order to check their compliance with the size range recommended for the species by the test guideline (OECD No. 203). The measured values were within the range of 2.30-2.80 cm.
Reference substance (positive control):
no
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 (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
There were no mortality and any sub-lethal effect observed in 10 fish in the control group or in 10 fish exposed to a test concentration of 100 mg/L. The 96-h No Observed Effect Concentration (NOEC) and the LC0 were determined to be 100 mg/L. The LC50 the LOEC, and LC100 values were determined to be > 100 mg/L.
Sublethal observations / clinical signs:

Validity of the Study

 There was no mortality observed in control or in the treatment group during the study.

 The dissolved oxygen concentration in the test solutions did not fall below 60 % of air saturation value during the study.

 No significant change (more than one unit) in the pH value was observed during the test.

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Analytical Results:

A single concentration of 100 mg/L (limit test concentration) and a concurrent control was tested in the main test. The concentration of the test item was analytically determined at the start and at the end of the test. In the untreated control group the test item was not detected.

The measured mean concentration of the test item was 121 mg/L at the start and 108 mg/L at the end of the test. The concentration of 121 mg/L at the start of experiment was slightly out of the ± 20 % range. However, as no toxicity was observed in this limit test even in the slightly higher concentration the biological results and endpoints can be based on the nominal concentration.

Validity criteria fulfilled:
yes
Conclusions:
The 96-h LC50 is >100 mg/L. The 96-h NOEC is 100 mg/L.
Executive summary:

In this 96-hour static acute toxicity test on Zebrafish (Brachydanio rerio) the test item had no toxic effect on fish up to a concentration of 100 mg/L. Accordingly, the 96-h LC50 value was determined to be > 100 mg/L. The 96-h NOEC was determined to be 100 mg/L.

Results are based on nominal concentrations.

Description of key information

The 96-h LC50 is >100 mg/L. The 96-h NOEC is 100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

The LC50 is not 100 mg/L, as one is forced to enter in the field above, but the LC50 is >100 mg/kg.