4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: November 05, 2019; Experimental start date: November 11, 2019; Experimental completion date: January 24, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of decision 2018-01-10; Swiss Ordinance relating to GLP, adopted May 18, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of GLP, as revised in 1997 and adopted November 26, 1997 by decision of the OECD Council [C(97)186/Final].

Test material

Test material information

Constituent 1

Reference substance name:

4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Molecular formula:

C23H18Cl2F4N2O4

IUPAC Name:

4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid

Details on test material:

Off-white

Specific details on test material used for the study:

- Expiration date of the lot/batch: End of November 2022
- Storage condition of test material: < 30 °C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and a loading rate of 100 mg/L treatment
- Sampling method: For the determination of the test item concentrations, duplicate samples were taken from the test media of the test concentration and the control at the start and at the end of each renewal period.
- Sample storage conditions before analysis: All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis.
- Analysed samples: The concentrations of the test item were measured in one of the duplicates for all taken samples.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test (Day 0) and at each test medium renewal (Day 1, Day 2 and Day 3) the test concentration (undiluted filtrate) was prepared, using 3 separate dispersions of test medium for convenience, by dispersing 500 mg of the test item (dosing range: 500.04 and 500.30 mg) in 5000 mL of test water twice and 300 mg of the test item (dosing range: 300.00 and 300.24 mg) in 3000 mL of test water once, (13 liters total volume). These preparations were supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer for three hours at room temperature in the dark. The stirring time was based on the stirring pre-experiment, which showed, that the maximum amount of dissolved test item was reached after this stirring time. After stirring, the suspensions of the test item were filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 μm) and combined. As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The test medium was freshly prepared just before introduction of the fish (at the start of the exposure) and at the beginning of each renewal period i.e., 24 hours. For the treatment and the control 12 liters of test medium were used for exposure of the fish.
- Controls: A control (test water without test item) was tested in parallel.
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the entire test duration.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

•TEST ORGANISM
- Common name: Rainbow trout
- Source: The fish were obtained from a commercial breeder at least 9 days before start of the test (fish breeding farm Störk, 88348 Bad Saulgau, Germany).
- Age at study initiation: The study was performed with juvenile fish. The fish were obtained from a commercial breeder at least 9 days before start of the test.
- Length at study initiation: From the acclimatized test fish batch, a representative sub-batch of 10 fish was measured at the start of the test. The mean total body length (measured from the mouth to the end of the tail fin) of the fish was 4.52 ± 0.14 cm (Mean ± SD) and the mean body wet weight was 1.17 ± 0.26 g (Mean ± SD). The measured fish were not used for the test.
- Weight at study initiation: From the acclimatized test fish batch, a representative sub-batch of 10 fish was measured at the start of the test. The mean body wet weight was 1.1 ± 0.30 g (Mean ± SD). The measured fish were not used for the test.

•ACCLIMATION
- Acclimation period: The fish were acclimatized to the test water and test temperature for seven days prior to test start.
- Acclimation conditions: Same as test
- Type and amount of food during acclimation: During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Hokovit, Hofmann Nutrition AG, CH-4922, Bützberg, Switzerland).
- Health during acclimation (any mortality observed): During the 12 days prior to the main test, 0.5 % (1 of 194 fish) mortality was observed in the test fish batch and all surviving fish were healthy. No medication was applied during holding and acclimatization of the fish.

Study design

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted test water (EPA Medium) was used in the study.

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Test conditions

Hardness:

The total hardness of the reconstituted test water was measured to be 125 mg/L (as CaCO3), i.e. in the recommended range of 40-250 (preferably <180) mg/L as CaCO3 (in the reconstituted test water before the initiation of the test).

Test temperature:

The water temperature in the test vessels was maintained between 13 and 14 °C during the test period. For this, the aquaria were placed in a temperature regulated water bath.

pH:

The pH throughout the test ranged from 7.0 to 7.3

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 9.2 to 10.2 mg/L at the start of the medium renewal periods. At the end of the medium renewal periods the dissolved oxygen concentration was in the range of 7.4 to 9.3 mg/L (i.e. 74-94 % saturation)

Conductivity:

The conductivity was 352 μS/cm (measured in the reconstituted test water before the initiation of the test).

Nominal and measured concentrations:

The nominal loading rate in the sole treatment was 100 mg/L.
- The analytically determined concentrations of the test item in the test media of the undiluted filtrate with the loading rate of 100 mg/L were between 99 and 153 μg/L at the start of the medium renewal periods (0, 24, 48 and 72 hours). During the test medium renewal period of 24 hours a decrease of the concentration of the test item in the test media were observed. At the end of the four 24-hour renewal periods, measured concentrations between 53 and 97 μg/L were observed, equivalent to 41 to 75 % of their respective initially measured concentrations. Therefore, the biological results were based on the mean measured test item concentration calculated as an arithmetic mean of the four geometric means determined of the test item concentration measured at the start and end of the test medium renewal periods. The test mean measured concentration of was determiend to be 95 μg/L.

Details on test conditions:

•TEST SYSTEM
- Test vessel: Mono block aquarium
- Material, size, headspace, fill volume: Glass, 15 L, 3 L, 12 L
- Aeration: The test water was aerated prior to the preparation of the test media until oxygen saturation was reached. The test medium and the control were not aerated during the test.
- No. of organisms per vessel: At the start of the test, seven fish were introduced into each aquarium in a random order.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The loading rate of the fish was 0.68 g fish wet weight per liter medium.

•TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water was used in the study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations:
- Ingredient / Concentration mmol/L / Concentration mg/L
• CaCl2 × 2H2O / 1.0 / 147
• MgSO4 × 7H2O / 0.25 / 61.5
• NaHCO3 / 0.38 / 32.5
• KCl / 0.038 / 2.9
- Alkalinity: The alkalinity was measured to be 0.6 mmol/L (in the reconstituted test water before the initiation of the test)
- Ca/Mg ratio (molar): 1.0÷0.25 = 4

•OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test water after equilibration was between 7.1 and 7.3. Thus no pH adjustment was required.
- Photoperiod: A 16-hour light to 8-hour dark photoperiod with a 30-minute transition period was used.
- Light intensity: The light intensity during the light period was approximately 15 to 16 microeinstein [μE/(m²·s)]

•EFFECT PARAMETERS MEASURED
The test fish were observed for mortality and visible abnormalities approximately 2 hours after the start of the exposure and further on each exposure day in the morning and the afternoon. The fish were checked for the mandatory clinical signs presented below, including the corresponding sub-categories (as presented in Annex 4 of the test guideline):
•E: Loss of equilibrium
•S: Abnormal swimming behavior
•V: Ventilatory function
•P: Abnormal skin pigmentation
•O: Other visible abnormalities

•TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable in a limit test

•RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No effects observed

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loss of equilibrium, Abnormal swimming behavior, Ventilatory function, Abnormal skin pigmentation and other visible abnormalities

Basis for effect:

behaviour

Remarks on result:

other:

Remarks:

All fish survived until the end of the test and no visible abnormalities were observed.

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EL50

Remarks:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loss of equilibrium, Abnormal swimming behavior, Ventilatory function, Abnormal skin pigmentation and other visible abnormalities

Basis for effect:

behaviour

Remarks on result:

other:

Remarks:

All fish survived until the end of the test and no visible abnormalities were observed.

Effect concentrations 3

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

All fish survived until the end of the test and no visible abnormalities were observed.

Effect concentrations 4

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 95 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

behaviour

Remarks:

Loss of equilibrium, Abnormal swimming behavior, Ventilatory function, Abnormal skin pigmentation and other visible abnormalities

Remarks on result:

not determinable

Remarks:

This was the sole concentration tested and represents the water accomodated fraction solubility limit. All fish survived until the end of the test and no visible abnormalities were observed.

Effect concentrations 5

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 95 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

not determinable

Remarks:

This was the sole concentration tested and represents the water accomodated fraction solubility limit. All fish survived until the end of the test and no visible abnormalities were observed.

Effect concentrations 6

Duration:

96 h

Dose descriptor:

EC50

Remarks:

LOEC

Effect conc.:

> 96 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

behaviour

Remarks:

Loss of equilibrium, Abnormal swimming behavior, Ventilatory function, Abnormal skin pigmentation and other visible abnormalities

Remarks on result:

not determinable

Remarks:

This was the sole concentration tested and represents the water accomodated fraction solubility limit. All fish survived until the end of the test and no visible abnormalities were observed.

Details on results:

All fish survived until the end of the test and no visible abnormalities were observed in the test fish in the control and the test mean measured concentration of 95 μg/L.
- Behavioural abnormalities: None.
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: None.
- Any observations that might cause a difference between measured and nominal values: Yes. From the loaded test item amount of 100 mg/L, only 95 μg/L could be found analytically.
- Effect concentrations exceeding solubility of substance in test medium: Yes, the sole treatment was in excess of the test item water solubility.

Reported statistics and error estimates:

No statistics needed due to the absence of toxicity of the test item. The 24 and 48-hour EC50 of the test item could not be calculated.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item was found to be not acuteky toxic to fish (Oncorhynchus mykiss) in a 96-hour semi-static test up to 100 mg/L

Executive summary:

The acute toxicity of the test item to juvenile rainbow trout (Oncorhynchus mykiss) was determined in a 96 hour semi-static test according to the OECD Guideline for the Testing of Chemicals, No. 203, adopted 2019. The validity criteria were met.

Due to the low water solubility of the test item, a suspension of the test item with the loading rate of 100 mg/L was prepared by using ultrasonic treatment for 15 minutes and intensive stirring for 3 hours to reach a maximum concentration of dissolved test item in test water. The stirring time was based on a pre-experiment. After stirring, the suspension was filtered through a 0.45 μm membrane filter. The undiluted filtrate was used as test medium. The preparation of the test medium was based on the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019. A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including the solubility limit of the test item in test water at a loading rate of 100 mg/L. Thus, the only tested concentration was the undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L. Additionally a control was tested in parallel. A semi-static test design, with medium renewal every 24 hours, was applied. Seven fish were used in the treatment and control groups. The fish were observed for visible abnormalities and mortality at 2 hours after test start and twice every 24 hours thereafter (in the morning and the afternoon).

No remarkable observations were made concerning the appearance of the test medium, i.e. the undiluted filtrate. The test medium was a clear solution throughout the entire test duration. The analytically determined concentrations of substance in the test media prepared with undiluted filtrate with the loading rate of 100 mg/L were between 99 and 153 μg/L at the start of the medium renewal periods (0, 24, 48 and 72 hours). During the test medium renewal period of 24 hours a decrease of the concentration of the test item in the test media were observed. At the end of the four 24-hour renewal periods, measured concentrations between 53 and 97 μg/L were observed, equivalent to 41 to 75 % of their respective initially measured concentrations.

The mean concentrations of the test item during the renewal periods of 24 hours were calculated as the geometric means of the concentrations measured at the start and the end of each of the four test medium renewal periods. From the four geometric mean values obtained, the mean measured test item concentrations during the test period of 96 hours were calculated as an arithmetic mean. The mean measured concentration of the undiluted filtrate with the loading rate of 100 mg/L during the test period of 96 hours was calculated to be 95 μg/L. The test item showed no toxicity under the conditions of the test up to the level of its water solubility. Accordingly no 96-hour LC₅₀ and NOEC or LOEC could be determined. The 96-hour LL₅₀ was derived to be >100 mg/L and the NOELR is ≥100 mg/L.

In conclusion, the test item was found to be not acutely toxic to rainbow trout (Oncorhynchus mykiss) in a 96-hour semi-static limit test up to the level of its water solubility and a nominal loading rate of 100 mg/L.