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Short-term toxicity to fish

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:

June from the 16th to the 20th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted July 17, 1992

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)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations, samples (4 replicates; 5 ml per replicate) were taken from the test solution (100 mg/l) and the control at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent. After the formulation procedure the test animals were immediately introduced into the test solution.
For preparation of test solution an amount of 0.3 g test item was dissolved in 3000 ml dilution water (OECD medium) using approx. 25 min ultrasonic dispersion resulting the nominal concentration of 100 mg/l.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium magazin Kft. (Pasaréti Gyula), Budapest.
- Age at study initiation: juveniles, age not specified.
- Length at study initiation: within a range of 2.0 ± 1 cm.
- Sex: both female and male (were not separated).
- Animal health: apparently healthy animals were used in the test.

ACCLIMATION
- Acclimation period: at least 12 days before test initiation.
- Acclimation conditions: same conditions as used during the exposure period.
- Type of food during acclimation: fish were fed with 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: the health of the breeding was continuously monitored and any mortality or abnormal behaviour recorded. No significant mortality (less than 5 % of population) occurred in seven days before the start of the experiment, therefore the batch was considered to be acceptable for testing.
- Light: 16 h light – 8 h darkness photoperiod.
- Temperature: 21 – 25 °C, constant within ± 1 °C.
- Oxygen saturation: at least 80 % of air saturation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

22.2 – 22.6 °C

pH:

7.09 – 7.76

Dissolved oxygen:

79.1 – 99.5 %

Nominal and measured concentrations:

100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Aeration: test solutions were not aerated during the test.
- No. of organisms per vessel: 10 animals
- No. of vessels per concentration: one replicates.
- No. of vessels per control: one replicates.
- Biomass loading rate: the loading of the test-aquariums was calculated and was 0.92 g/l in the control and 0.83 g/l in the test item treated group

TEST MEDIUM / WATER PARAMETERS
- Choice of dilution water: based on the results of preliminary solubility experiments the test item could not be completely dissolved at a concentration level of 100 mg/l in ISO medium, therefore OECD medium, reconstituted water, according to OECD 201, was used as dilution water in the experiment (in which the test item can be diluted completely at 100 mg/l).
- Source/preparation of dilution water: OECD medium, reconstituted water, prepared in deionised water.
- Stock solution 1 (macro nutrients): NH4Cl 15.0 mg/l, MgCl2 5.6 mg/l, CaCl2 × 2 H2O 18.0 mg/l, MgSO4 × 7 H2O 15.0 mg/l, KH2PO4 1.6 mg/l.
- Stock solution 2 (iron): FeCl3 × 6 H2O 64.0 µg/l, Na2EDTA × 2H2O 100.0 µg/l.
- Stock solution 3 (trace elements): H3BO3 185.0 µg/l, MnCl2 × 4 H2O 415.0 µg/l, ZnCl2 3.0 µg/l, CoCl2 × 6 H2O 1.5 µg/l, CuCl2 × 2 H2O 0.01 µg/l, Na2MoO4 × 2 H2O 7.0 µg/l.
- Stock solution 4 (bicarbonate): NaHCO3 50.0 mg/l.
- Hardness: the total hardness of OECD medium was determined as 53.4 mg/l (as CaCO3).

OTHER TEST CONDITIONS
- Photoperiod: the light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness.

OBSERVATIONS
The fish were observed at approximately 4, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen and temperature were carried out daily.
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).

TEST CONCENTRATIONS
No toxicity was expected up to the limit test concentration (i.e. 100 mg/l); therefore, a single concentration of 100 mg/l (limit concentration) was tested in a limit test.

VALIDITY OF THE STUDY
- The mortality of the fish in the control should not exceed one fish at the end of the test.
- The dissolved oxygen concentration in the test solutions should not fall below 60 % of air saturation value during the study.
- Constant conditions should be maintained as far as possible throughout the test
- There must be evidence that the concentration of the substance being tested has been satisfactorily maintained and it should be at least 80 per cent of the nominal one throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results and conclusions should be based on the measured concentration.

Duration:

96 h

Dose descriptor:

LC50

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 (nominal).

BODY LENGHT
The measured values were within the range of 2.15-2.70 cm.

MEASURED CONCENTRATIONS
In the untreated control group the test item was not detected. The measured concentration values of test item were 111 mg/l at the start and 108 mg/l at the end of the test.

Sublethal observations / clinical signs:

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	2.77	0.28	0.92
100 mg/l	2.5	0.25	0.83

Body length of test animals

Test Group	Body length [cm]
Control	2.4	2.5	2.3	2.4	2.3	2.35	2.65	2.3	2.7	2.3
100 mg/l	2.6	2.25	2.55	2.25	2.35	2.15	2.25	2.2	2.6	2.2

pH-values measured during the experiment

Test Group	pH
	0h	24h	48h	72h	96h
Control	7.76	7.71	7.73	7.66	7.67
100 mg/l	7.09	7.16	7.27	7.44	7.56

Dissolved Oxygen Concentrations measured during the experiment

Test Group	Dissolved Oxygen Concentration [%]
	0h	24h	48h	72h	96h
Control	96	88.2	84.4	81	81.3
100 mg/l	99.5	86.1	84	79.1	80.8

Temperatures measured during the experiment

Test Group	Temperature [°C]
	0h	24h	48h	72h	96h
Control	22.5	22.3	22.4	22.5	22.3
100 mg/l	22.6	22.3	22.2	22.4	22.2

Validity criteria fulfilled:

yes

Remarks:

no mortality was observed in control, dissolved oxygen did not fall below 60 % of air saturation value, constant conditions were maintained throughout the test (e.g. temperature, pH, testi item concentrations ± 20 %)

Conclusions:

LC50 (96h) > 100 mg/l (nominal)

Executive summary:

The short-term toxicity to fish of test item was investigated in Zebrafish (Brachydanio rerio), aqccording to the OECD testing guideline 203. A limit test was chosen, using a single concentration of 100 mg/l. Fish were exposed to aqueous test media containing the test item for 96 hours in a static test.

Fish were observed at approximately 4, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen and temperature were carried out daily. Concentrations of the test item were determined using reverse phase HPLC method with VIS detection: in the untreated control group the test item was not detected; in the test vessel the measured concentration values were 111 mg/l at the start and 108 mg/l at the end of the test.

Mortality and any sub-lethal effects were not observed during the 96-h exposure period either in the treated or in the control group, therefore the LC50 was indicated as higher than 100 mg/l.

Conclusion

LC50 (96h) > 100 mg/l (nominal)

Description of key information

Not harmful/toxic for short-term exposure to fish.

LC50 (96h) for freshwater fish > 100 mg/l test mat. (nominal) based on mortality

Additional information

The short-term toxicity to fish of test item was investigated in Zebrafish (Danio rerio), according to the OECD testing guideline 203. Concentrations of the test item were determined using reverse phase HPLC method with VIS detection: in the untreated control group the test item was not detected; in the test vessel the measured concentration values were 111 mg/l at the start and 108 mg/l at the end of the test. Mortality and any sub-lethal effects were not observed during the 96-h exposure period either in the treated or in the control group, therefore the LC50 was indicated as higher than 100 mg/l.