[No public or meaningful name is available]

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:

06 SEP 2021 - 08 NOV 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

published in the Official Journal of the European Union L 142 of 31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

October 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted 18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-UV method

Details on sampling:

- Concentrations: 1000 mg/L
- Sampling method: The test item is poorly water soluble material, wherefore the OECD Guidance document no. 23 was taken into account for the preparation of the test solutions. In order to achieve the maximum solubility level in the test medium the test solution was prepared as follows (for each water renewal period): a supersaturated solution (1000 mg/L nominal loading) was prepared by dispersing/dissolving the appropriate test item amount into the test medium (ISO medium). This solution was agitated by magnetic stirrer for at least two days and then the non-dissolved test material was separated by filtration through a membrane filter (pore size: 0.22 μm) in order to obtain the saturated test solution. Since the test item has a high absorptive potential (log Pow = 8.22) also, the vessels used for the preparation of the test solution as well as the test aquaria was preconditioned with the respective test solution for at least two days.
- Sample storage conditions before analysis: After the formulation procedure at each water renewal period, the test animals were immediately introduced into the test solutions.

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item is poorly water soluble material, wherefore the OECD Guidance document no. 23 was taken into account for the preparation of the test solutions. In order to achieve the maximum solubility level in the test medium the test solution was prepared as follows (for each water renewal period): a supersaturated solution (1000 mg/L nominal loading) was prepared by dispersing/dissolving the appropriate test item amount into the test medium (ISO medium). This solution was agitated by magnetic stirrer for at least two days and then the non-dissolved test material was separated by filtration through a membrane filter (pore size: 0.22 μm) in order to obtain the saturated test solution. Since the test item has a high absorptive potential (log Pow = 8.22) also, the vessels used for the preparation of the test solution as well as the test aquaria was preconditioned with the respective test solution for at least two days.
- Controls: The dilution water (ISO medium; see section 5.3) without of test item added was used as untreated control solution.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Source: Toma István e.v. Hungary, 2146 Mogyoród, Gödöllői u. 41/B
- Age at study initiation (mean and range, SD): Juveniles were used
- Length at study initiation (length definition, mean, range and SD): The total length of each fish used was within the range of 1-2 cm
- Weight at study initiation (mean and range, SD): The total body weight of the test animals per groups was recorded on the day 0 (at the start of the test)

ACCLIMATION
- Acclimation period: Fish were held for at least 9 days before test initiation in the fish laboratory of TOXI-COOP ZRT.
- Acclimation conditions (same as test or not): under the same conditions as used during the exposure period
- Type and amount of food during acclimation: commercial diet for fish
- Feeding frequency during acclimation: During holding, fish were fed with appropriate, commercial diet for fish at least three times per week until 24-48 hours before the test start.
- Health during acclimation (any mortality observed): 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 and fish appeared in good health, free of apparent malformation; therefore the batch was considered to be acceptable for testing.

FEEDING DURING TEST (as applicable)
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:

The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

The measured temperatures were within a range of 22.2 – 22.9°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 7.66 – 7.97 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 76.1 – 88.5 % of the air saturation value at the temperature used. Aeration of test solution or control was not necessary during the experiment.

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration:

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquariums filled up with 1 litre test solution.
- Renewal rate of test solution (frequency/flow rate): To maintain the test concentration as high as possible throughout the test a semi-static test was performed using 24-h water renewal periods.
- Number of animals: There were 10 animals in the treated and control group respectively (no replicates were used).

At the start of the test (on day 0) 10 fish were introduced into each aquarium, each containing 1 litre testing solution. Before introduction of test animals, the total 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 0.45 g/L in the control and 0.52 g/L in the test item treated group.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium, prepared in the laboratory of TOXI-COOP ZRT.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark; Intensity: 646 lux (measured at the start of the test)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): body weight, body length and analysis of test item concentration

TEST CONCENTRATIONS
- Range finding study
A non-GLP preliminary test was performed at the saturation concentration (limit concentration). Three fish were exposed for 96 hours in the treatment and a control group respectively. No mortality or any sub-lethal effects were observed in the treatment group or in the control during the 96-h exposure period.
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary test, only a single treatment concentration at saturation (i.e. solubility limit of the test item in the test medium) and an untreated control group was tested in a limit test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, body weight, body length of test animals

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, body weight, body length of test animals

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, body weight, body length of test animals

Details on results:

- Observations on body length and weight: Total length of each fish was measured and recorded at the end of the test. The measured values were within the range of 1.65 – 1.95 cm.
- Mortality of control: No mortality or any sub-lethal effects were observed during the 96-h exposure period either in the treatment group (saturated test solution, equivalent to 100 mg/L nominal concentration) or in the control.

Reported statistics and error estimates:

A limit test was carried out and no toxic effects were observed, therefore statistical analysis was not necessary.

Validity criteria fulfilled:

yes

Remarks:

- No mortality was observed in the control during the test. - The dissolved oxygen concentration did not fall below 60 % of air saturation value in any of the test vessels during the test. - Test concentration was analytically measured during the test.

Conclusions:

In this 96-hour semi-static acute toxicity test on Zebrafish (Danio rerio) the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the LC50 and the LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.

Executive summary:

This short-term toxicity to fish test was performed according to OECD Guideline 203 under GLP compliance. The purpose of this study was to evaluate the acute toxicity of the test item on Zebrafish (Danio rerio). Based on the results of a non-GLP preliminary test, the study was conducted as a limit test using a nominal concentration of 100 mg/L (i.e. 100% v/v saturated solution). Fish were exposed to aqueous test media containing the test item for 96 hours in a semi-static test (24-h water renewal periods). Ten fish were exposed to the test concentration and control (no replicates were used) and were observed twice a day (in the morning and afternoon) during the exposure at approx. 2-3, 5-6, 24, 30, 48, 54, 72, 78 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen concentration and water temperature were carried out daily (at the start and end of each water renewal period). For determination of the test item concentrations, samples were taken from the treatment and the control group at the start and at the end of the first and the last water renewal periods. Concentrations were determined using HPLC-UV method.

The measured concentrations of the test item treated group were below the LOQ (i.e. < 0.05 mg/L) at each analytical occasion. Test item was not detected in the control samples. No mortality or any sub-lethal effects were observed during the 96-h exposure period either in the treatment group (saturated solution, equivalent to 100 mg/L nominal concentration) or in the control. Accordingly, the 96-h NOEC was determined to be at least the saturation concentration (i.e. 100% v/v saturated solution, equivalent to 100 mg/L nominal concentration) and the LOEC and the EC50 values were determined to be higher than saturation. 

In conclusion, in this 96-hour semi-static acute toxicity test on Zebrafish (Danio rerio) the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the LC50 and the LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.

Description of key information

Key, short-term toxicity to fish, OECD 203, semi-static, limit-test, GLP: LC₅₀ (96h) > 100 mg/L nominal, analytical concentration <LOQ (0.05 mg/L) for zebra fish (Danio rerio)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L

Additional information

This short-term toxicity to fish test was performed according to OECD Guideline 203 under GLP compliance. The purpose of this study was to evaluate the acute toxicity of the test item on Zebrafish (Danio rerio). Based on the results of a non-GLP preliminary test, the study was conducted as a limit test using a nominal concentration of 100 mg/L (i.e. 100% v/v saturated solution). Fish were exposed to aqueous test media containing the test item for 96 hours in a semi-static test (24-h water renewal periods). Ten fish were exposed to the test concentration and control (no replicates were used) and were observed twice a day (in the morning and afternoon) during the exposure at approx. 2-3, 5-6, 24, 30, 48, 54, 72, 78 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen concentration and water temperature were carried out daily (at the start and end of each water renewal period). For determination of the test item concentrations, samples were taken from the treatment and the control group at the start and at the end of the first and the last water renewal periods. Concentrations were determined using HPLC-UV method.

The measured concentrations of the test item treated group were below the LOQ (i.e. < 0.05 mg/L) at each analytical occasion. Test item was not detected in the control samples. No mortality or any sub-lethal effects were observed during the 96-h exposure period either in the treatment group (saturated solution, equivalent to 100 mg/L nominal concentration) or in the control. Accordingly, the 96-h NOEC was determined to be at least the saturation concentration (i.e. 100% v/v saturated solution, equivalent to 100 mg/L nominal concentration) and the LOEC and the EC50 values were determined to be higher than saturation. 

In conclusion, in this 96-hour semi-static acute toxicity test on Zebrafish (Danio rerio) the test item had no toxic effect at the saturation concentration following nominal loading of 100 mg test item/L; the LC50 and the LOEC are higher than the solubility limit of the test item in the test medium, corresponding to a nominal concentration of > 100 mg/L.