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 aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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 medium and the control at the start and at the end of each test medium renewal period. For sampling at the end of the renewal periods, the contents of the replicate test vessels were combined prior to sampling.
- 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 duplicate samples from all sampling dates.

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 the test medium renewal (Day 1) the single test concentration (undiluted filtrate) was prepared by suspending 100 mg of the test item (100.14 and 100.25 mg, respectively) in 1000 mL of test water. This preparation was 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 suspension of the test item was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 μm). 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 undiluted filtrate with a loading rate of 100 mg/L was used as test medium.
- Evidence of undissolved material: 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 test medium was prepared just before the introduction of the daphnids (i.e., the start of the exposure) and at the test medium renewal (Day 1).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

•TEST ORGANISM
- Common name: Water flea
- Strain/clone: A clone of the species Daphnia magna (originally from the Daphnia Collection of the University of Basel/Switzerland in 2015) is successfully bred in IES Ltd Laboratories.
- Source: Daphnia Collection of the University of Basel/Switzerland in 2015
- Age at study initiation: At the start of the test, the test organisms were ≤ 24 hours old and were not first brood progeny.
- Feeding during test: The daphnids were not fed during the test.

•ACCLIMATION
- Acclimation period: No acclimatisation was necessary, since the bred of the daphnia was under identical conditions to those of the test.
- Type and amount of food during the breeding: An algal suspension of the green algae Desmodesmus subspicatus CHODAT, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH, 49324 Melle/Germany).
- Feeding frequency: During breeding, daphnids were generally fed three times a week.

•CULTURING CONDITIONS
The cultivation of the parental daphnids is performed in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, temperature of 21°C, main ions, and total hardness). Each stock animal was maintained separately in a 100-mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy. The culture contains no ephippia, the first offspring were produced before day 12, the cultured daphnids did not shown any signs of stress during the 48 hours prior to test start (e.g., abnormal behavior or >5 % mortality). Additionally, the cultured daphnids produced an average of at least 3 offspring per female per day over a 7-day period prior to test initiation.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted test water (ISO Test water) according to the OECD Guideline No. 202 was used in the study.

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 mmol/L = 250 mg/L as CaCO3

Test temperature:

The test was performed under temperature-controlled conditions (climate room with continuous monitoring of the room temperature and the water temperature in a separate vessel placed in the test area). The test water temperature was maintained between 21 and 22 °C.

pH:

At the beginning and end of the test medium renewal periods, the pH value of the test medium and the control was maintained at 7.8

Dissolved oxygen:

The dissolved oxygen concentration in the test medium and control was at least 8.0 mg/L

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 medium of the undiluted filtrate with a loading rate of 100 mg/L were 51.1 μg/L at the start of the test and 64.4 μg/L at the start of the medium renewal period. At the end of the medium renewal periods, 57.8 μg/L ( =113 % of the initially measured concentration) and 45.0 μg/L ( = 70 % of the initially measured concentration) were found, respectively. The mean concentrations of the test item for each of the two renewal periods of 24 hours were calculated as the geometric means of the concentrations measured at the start and the end of the test medium renewal periods. From the two geometric mean values obtained, the mean measured test item concentration during the test period of 48 hours was calculated as an arithmetic mean. The mean measured test item concentration of the undiluted filtrate with the loading rate of 100 mg/L during the test period of 48 hours was calculated to be 54 μg/L.

Details on test conditions:

•TEST SYSTEM
- Test vessel: Beakers
- Type: Open. The test vessels were loosely covered with glass sheets to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: Glas, 100 mL, open - thus unlimited headspace, 50 mL
- Aeration: No. The test vessels were not aerated during the test.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4
- Biomass loading rate: 1 Daphnid / 10 mL test media

•TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium
- Ingredients / [mmol/L] / Concentration [mg/L]
• CaCl2 × 2H2O / 2.0 / 294
• MgSO4 × 7H2O / 0.5 / 123
• NaHCO3 / 0.75 / 65
• KCl / 0.075 / 5.8
- Alkalinity: 0.8 mmol/L
- Ca/Mg ratio (molar): 2.0÷0.5 = 4

•OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod:16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: Light intensity during the light period was 16 to 17 µmol/(m²·s).

•EFFECT PARAMETERS MEASURED
- Immobility of the daphnids and other non-lethal effects (e.g. abnormal behavior or appearance) after 24 and 48 hours of exposure.

•RANGE-FINDING STUDY:
- Test concentrations: 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study: 48-hour immobilization: 1.0 mg/L / 0%; 10 mg/L / 0%; 100 mg/L / 0%

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (CAS 7778-50-9), Batch No. MKBR3876V) obtained from Sigma-Aldrich

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

No adverse effects were observed in the test animals.

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable

Remarks:

No adverse effects were observed in the test animals.

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

No adverse effects were observed in the test animals.

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Remarks:

LOEC

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

not determinable

Remarks:

No adverse effects were observed in the test animals.

Details on results:

- Behavioural abnormalities: No adverse effects were observed in the test animals.
- Mortality of control: None
- Other adverse effects control: None
- Any observations: None
- Effect concentrations exceeding solubility of substance in test medium: Yes.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The 24-hour EC50 of 1.8 mg/L showed that the sensitivity of the test organisms was within the range given by the guideline (OECD 202: 24-hour EC50: 0.60-2.1 mg/L).
- Dose-response test: The reference test was carried out to check the sensitivity of the test system as used by IES Ltd. Daphnia were exposed for a period of 48 hours to potassium dichromate concentrations of 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L and a control (test water without test item). Twenty daphnia were exposed per concentration.
- ECx: The 24-hour EC50 was 1.8 mg/L with a 95 % confidence interval between 1.6 and 2.0 mg/L. The 48-hour EC50 was 1.4 mg/L with 95 % confidence limits of 1.2 and 1.6 mg/L.
- Other:
•Potassium Dichromate - Acute Toxicity to Daphnia magna in a 48-Hour Immobilization Test (IES Ltd Study 20190394).
•Experimental Starting Date: October 28, 2019
•Experimental Completion Date: October 31, 2019
•The reference test was based on the OECD Guideline No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.
•The reference test was carried out to check the sensitivity of the test system as used by IES Ltd.
•Effect of Potassium Dichromate on the Mobility of Daphnia magna:
• Nominal Test Item Concentration [mg/L] / No. of Daphnids Tested / Immobilized Daphnids after 24 Hours / Immobilized Daphnids after 48 hours
• Control / 20 / 0 / 0
• 0.19 / 20 /0 / 0
• 0.38 / 20 / 0 / 0
• 0.75 / 20 / 0 / 0
• 1.5 / 20 / 5 (25 %) / 12 (60 %)
• 3.0 / 20 / 20 (100 %) / 20 (100 %)

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 acutely toxic to aquatic invertebrates (Daphnia magna) in a 48-hour semi-static limit test up to the level of its water solubility and a nominal loading rate of 100 mg/L.

Executive summary:

The acute toxicity of the test item to young (≤ 24 hours old) water flea (Daphnia magna) was determined in a 48 hour semi-static test according to the OECD Guideline for the Testing of Chemicals, No. 202, adopted 2004. 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. On the basis of the pre-test results, a limit test was performed in accordance with the test guideline 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 with test medium renewal after 24 hours was performed to keep the concentration of the test item in the test medium as constant as possible during the test period of 48 hours. For each treatment, 20 daphnids were used, distributed over four replicates (five individuals per vessel). The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Observations were also performed for other non-lethal effects (e.g. abnormal behaviour or appearance).

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. At the start of the two medium renewal periods (0 and 24 hours), the analytically determined concentrations of the test item in the freshly prepared test medium (undiluted filtrate with a loading rate of 100 mg/L) were 51 and 64 μg/L. At the end of the two 24-hour renewal periods (24 and 48 hours) concentrations of the test item were 58 and 45 μg/L, respectively, i.e. 113 and 70 % of the initially measured concentrations. The mean concentrations of the test item for each of the two renewal periods of 24 hours were calculated as the geometric means of the concentrations measured at the start and the end of the test medium renewal periods. From the two geometric mean values obtained, the mean measured test item concentration during the test period of 48 hours was calculated as an arithmetic mean. The mean measured test item concentration of the undiluted filtrate with the loading rate of 100 mg/L during the test period of 48 hours was calculated to be 54 μg/L.

The test item showed no toxicity under the conditions of the test. Accordingly no 48-hour EC50 and NOEC or could be determined. The 48-hour EL50 was derived to be >100 mg/L and the NOELR ≥100 mg/L.

In conclusion, the test item was found not acute toxic to daphnids (Daphnia magna) in a 48-hour semi-static limit test up to the level of its water solubility and a nominal loading rate of 100 mg/L.