(6R-trans)-7-amino-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

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:

2017

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 were taken from the test concentration and the control at the start and at the end of each water renewal period.

Vehicle:

no

Details on test solutions:

The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent. The test solution was prepared by dissolving an amount of 0.5 g test item in 5000 mL dilution water (ISO medium) resulting a nominal concentration of 100 mg/L. This solution was ultrasonicated for approx. 15 min and then agitated by orbital shaker for about 1 hour to obtain clear solution. The test solution was prepared just before introduction of test animals (start of each renewal period).

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 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: 12 to 16 hours photoperiod daily;
Temperature: 21 – 25°C, constant within ± 1°C.
Fish were held prior to the study in reconstituted water (ISO medium, prepared according to Annex 2 of the OECD guideline 203).

Test type:

semi-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 22.5 – 23.1°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 6.44 – 7.80 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 65.6 – 95.9 % of the air saturation value at the temperature used. Test solutions were slightly aerated from the 2nd day of the test (due to the decline of oxygen concentration observed during the first 48-h period).

Salinity:

No.

Conductivity:

Not reported.

Nominal and measured concentrations:

0 - 100 mg/L.
The analytically measured test item concentrations remained within ± 20 % of the nominal during the test. Therefore, the biological endpoints are based on the nominal test item concentration.

Details on test conditions:

Test type: Semi-static test. Based on the results obtained during analytical method validation the test item is not stable for the duration of 48 or 96 hours in ISO Medium, therefore a semi-static test was performed with daily water renewal periods.

The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent. The test solution was prepared by dissolving an amount of 0.5 g test item in 5000 mL dilution water (ISO medium) resulting a nominal concentration of 100 mg/L. This solution was ultrasonicated for approx. 15 min and then agitated by orbital shaker for about 1 hour to obtain clear solution. The test solution was prepared just before introduction of test animals (start of each renewal period).

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.

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:

LOEC

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)

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 each water renewal period. In the untreated control group the test item was not detected.

The measured concentration of 7-ACT varied between 91.2 – 108 % of the nominal during the test.

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

A 96-hour semi-static acute toxicity test on Zebrafish (Brachydanio rerio) according to OECD 203 was performed. The test item had no toxic effect on fish up to a tested 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.

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/L.