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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: October 10, 2019; Experimental start date: October 14, 2019; Experimental completion date: October 18, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

Commission Regulation (EU) No. 2016/266, C.11. (2015)

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:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), the test item group (with ascending concentrations) and finally again two blank control vessels. At the start of the test, 16 mL synthetic sewage feed were made up to 250 mL with deionized water and then 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to this first control vessel. Thereafter, in time intervals of about fifteen minutes (an arbitrary but convenient interval) the procedure was repeated, except that the 16 mL synthetic sewage feed were added to the appropriate amounts of reference item or test item in deionized water to obtain a volume of 250 mL, followed by the addition of 250 mL inoculum.
Test item preparation: Based on the results of a solubility/dispersibility pre-experiment, no appropriate stock solution (e.g. of 5 g/L) could be prepared due to the low water solubility of the test item. Therefore, test item amounts of 5.04, 50.01, 500.1, 500.1 and 500.0 mg were weighed by means of an analytical balance and transferred to the designated test vessels with 234 mL deionized water. The test item was mixed into the deionized water by intense stirring for 10 minutes at room temperature to dissolve and finely disperse the maximum practical amount of the test item. No emulsifiers or solvents were used. After stirring, the appearance of the test media was evaluated and the pH measured. All concentrations (loading rates) were clearly above the water solubility limit of the test item under the test conditions. Small undissolved test item particles, homogeneously distributed, were observed in the dispersions. The pH of all dispersions was in the range of 7.3 to 7.4 and therefore, not adjusted. Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added.
Reference item preparation: The reference item 3,5-dichlorophenol (3,5-DCP) was tested in parallel under identical test conditions, and functioned as a positive control. The nominal concentrations tested were 3.2, 10 and 32 mg/L.
Based on the test guidelines, a stock solution of 3,5-DCP was prepared under IES Laboratories Study 20190241 as follows: 0.50 g 3,5-dichlorophenol was directly dissolved in about 950 mL ultrapure water with the aid of ultrasonication and intense stirring. The 3,5-DCP solution was adjusted with NaOH to pH 7.4 and made up to one liter. The final concentration of 3,5-DCP amounted to 500 mg/L. Aliquots of this 3,5-DCP stock solution were mixed with deionized water, synthetic sewage feed and inoculum in the respective test vessels to obtain the desired test concentrations.
- Controls: See above (Method)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Yes. All concentrations (loading rates) were clearly above the water solubility limit of the test item under the test conditions. Small undissolved test item particles, homogeneously distributed, were observed in the dispersions.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: ARA Birs (Birsfelden / Switzerland)
- Method of cultivation: Not applicable
- Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in chlorine free tap water. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
- Pretreatment: A calculated amount of wet sludge was suspended in chlorine free tap water to obtain a concentration equivalent to 3 g dry material per litre. During the holding period of one day prior to use, the sludge was fed once with 50 mL synthetic sewage feed per litre and was kept at room temperature under continuous aeration until use. Before use, on the next day, the pH of the activated sludge inoculum was adjusted from pH 6.4 to pH 7.3 with a 1M NaOH solution.
- Initial biomass concentration: At the start of the test, 16 mL synthetic sewage feed were made up to 250 mL with deionized water and then 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to this first control vessel. Due to this 1:2 dilution an initial biomass concentration of 1.5 g/L (dry weight) was achieved.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

The exposure duration was chosen as recommended by the test guideline.

Post exposure observation period:

Not performed.

Test conditions

Hardness:

Not determined.

Test temperature:

The test was conducted at a temperature of 20 to 21ºC. The temperature was recorded in one control vessel (vessel no. 1) at the start and one (vessel no. 12) at the end of the incubation period.

pH:

The pH of the complete incubation mixture was measured in each vessel at the start and end of the incubation period. At the start, the pH was in the range of 7.2 to 7.4 and at the end in the range of 7.4 to 7.9.
For the test item incubation mixtures the pH was measured also before the addition of synthetic sewage feed and activated sludge. The pH was in the range of 7.3 to 7.4.

Dissolved oxygen:

The concentration of dissolved oxygen did not drop below 60-70 % saturation and the sludge flocs were maintained in suspension during the incubation period. The dissolved oxygen concentration in the incubation mixture measured in each vessel at the start of the incubation period (i.e. after the addition of activated sludge and a few minutes of stirring) was in the range of 8.2 to 8.4 mg/L. Just before measurement of the respiration rates, at the end of the incubation period, the dissolved oxygen concentrations were at least 7.0 mg/L
For the measurement of the dissolved oxygen (DO) an oxygen meter (Oxi 7310P from WTW GmbH, Weilheim, Germany) with a selfstirring DO sensor (StirrOx® G from WTW GmbH) and a 250 mL BOD bottle were used.

Salinity:

Not determined.

Conductivity:

Not determined.

Nominal and measured concentrations:

All test results were based on nominal concentrations of the test and reference item.
Test item: 10, 100 and 1000 mg/L
Reference item (positive control): 3.2, 10 and 32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L beakers
- Type: Open
- Fill volume: 500 mL of incubation mixture
- Aeration: Magnetic stirrers and followers, covered with inert material, were used to assure the aeration and mixing during incubation.
- No. of vessels per concentration (replicates): 1 to 3; Single test vessel replicates were tested for the two lower concentrations, whereas for the highest test item concentration three replicates were prepared.
- No. of vessels per control (replicates): 4; Four blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the range-finding test.
As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates).
- No. of vessels per abiotic control (replicates): No abiotic control was performed.
- Sludge concentration (weight of dry solids per volume): Nominally 1.5 g/L (dry weight)
- Nutrients provided for bacteria: Synthetic Sewage Feed was prepared to contain the following constituents per litre: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 × 2H2O, 0.2 g MgSO4 × 7H2O, 2.8 g K2HPO4 filled up to the final volume with deionized water. The pH of this solution was pH 7.2.
- Biomass loading rate: Not determined

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water, containing less than 1 mg/L DOC (dissolved organic carbon)
- Particulate matter: Not determined

OTHER TEST CONDITIONS
- Adjustment of pH: No pH adjustment was necessary before the addition of synthetic sewage feed and activated sludge since the pH was in the range of 7.3 to 7.4.
- Photoperiod: Throughout the whole exposure period of 3 h
- Light intensity: The test vessels was incubated under normal laboratory lighting.
- Details on termination of incubation: After the incubation period the respiration rate was measured.

EFFECT PARAMETERS MEASURED:
- Measurement of the respiration rates: For measurement of the respiration rate, a well-mixed sample of each incubation mixture was poured into a 250 mL glass BOD bottle after three hours incubation time, and was not further aerated. Subsequently, the dissolved oxygen (DO) concentration was measured with a self-stirring DO sensor attached to an oxygen meter and was continuously recorded for a period of up to 10 minutes or until the oxygen concentration fell below 2 mg/L. The oxygen uptake rate of each sample was determined from the linear part of the respiration curve normally in the range of 2-7 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using fewer concentrations than requested by guideline: Iin line with the test guideline recommendations a range finding study only was required.

RANGE FINDING STUDY
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No definite study was required in agreement with the test guideline.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Sigma-Aldrich) CAS 591-35-5; Batch MKBS0425V; Purity 99.7 % (GC); Expiration Date: 2020-12-29 (i.e. 5 years after opening); Storage Conditions: Cool, dry, tightly closed container in a well ventilated place.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

NOEC

Remarks:

(actually a Loading Rate, NOELR)

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

Arithmethic mean inhibition at this level was 8.4 %.

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Remarks:

(actually an Effective Loading, EL50)

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

Arithmethic mean inhibition at this level was 8.4 %.

Details on results:

At all tested concentrations up to and including 1000 mg/L, the inhibition of the respiration rate for individual replicates after the incubation period of three hours was in the range of 6.3 to 11.7 % compared to the control. For the three replicates of the highest test concentration of 1000 mg/L the mean inhibition of the respiration rate was calculated to be 8.4 %, compared to the control. Although (due to the very low variability of results) this low reduction in the respiration rate was mathematically found to be statistically significantly different from the control (results of a Welch t-test, one-sided smaller, α = 0.05), the inhibition is considered as not biologically relevant.
- Effect concentrations exceeding solubility of substance in test medium: Yes. All concentrations (loading rates) were clearly above the water solubility limit of the test item under the test conditions. Small undissolved test item particles, homogeneously distributed, were observed in the dispersions.
- Adsorption (e.g. of test material to the walls of the test container): Not observed
- Blank controls oxygen uptake rate: The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 38 and 35 mg O2 per dry weight of activated sludge per hour and for two blank controls prepared at the end of the test were 34 and 39 mg O2/gh. All four values are higher than the 20 mg O2/gh stipulated in the guidelines.
- Coefficient of variation of oxygen uptake rate in control replicates: The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 6 % and is therefore, as stipulated in the guidelines, not more than 30 %.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 6.0 mg/L with 95 % confidence limits from 3.0 to 10 mg/L. The 3-hour EC50 is within the guidelines-recommended range of 2 to 25 mg/L, confirming the suitability of the activated sludge used.
- Other: As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates).

Reported statistics and error estimates:

The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration and statistically analysed using a suitable software (ToxRat® Professional, (Version 3.3.0), ToxRat® Solutions GmbH).
The three replicates of the highest test item concentration were compared with the four blank control replicates using the Welch t-test with one-tailed (smaller) hypothesis and α = 0.05. Although (due to the very low variability of results) the low reduction in the respiration rate was mathematically found to be statistically significantly different from the control, the inhibition (8.4 %) is considered as not biologically relevant. The NOEC was considered to be the highest test concentration.
The 3-hour EC10, EC20, EC50 and EC80 values of the test item and their 95 %-confidence limits could not be calculated because of the absence of a toxic effect of the test item on the respiration rate of activated sludge at the tested concentrations.
The 3-hour EC50 of the reference item 3,5-dichlorophenol was derived by Probit Analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item had no inhibitory effect on the respiration rate of aerobic wastewater micro-organisms of activated sludge after the incubation period of three hours at the highest tested concentration of 1000 mg/L.

Executive summary:

The inhibitory effect of the test item on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010 and to the Commission Regulation (EU) No. 2016/266, C.11. (2015). The validity criteria were met.

A range-finding test using the three test item concentrations 10, 100 and 1000 mg/L was performed. Single test vessel replicates were tested for the two lower concentrations, whereas for the highest test item concentration three replicates were prepared. All concentrations (loading rates) were clearly above the water solubility limit of the test item under the test conditions. Small undissolved test item particles, homogeneously distributed, were observed in the dispersions. As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates). Four blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the range-finding test. The vessels were prepared in the following order: two blank controls, the reference item group (with ascending concentrations), the test item group (with ascending concentrations) and finally again two blank control vessels.

The results of the blank and positive control treatments confirmed the suitability of the activated sludge and the method used. All the validity criteria were fulfilled. In conformity with the guidelines, a definitive test was not necessary since in the range finding test the test item showed no inhibition of the respiration rate at the highest tested concentration (performed as triplicate). The test item had no biologically relevant inhibitory effect on the respiration rate of activated sludge at all tested concentrations up to and including 1000 mg/L after the incubation period of three hours. For the three replicates of the highest test concentration of 1000 mg/L the mean inhibition of the respiration rate was calculated to be 8.4 %, compared to the mean control. Although this low reduction in the respiration rate was mathematically found to be statistically significantly different from the control, the inhibition is considered as not biologically relevant. Thus, the 3-hour NOEC of the test item to activated sludge micro-organisms was ≥1000 mg/L. Concentrations exceeding 1000 mg/L were not tested. The 3-hour EC10, EC20, EC50 and EC80 could not be calculated, due to the absence of a relevant toxic effect at the tested concentrations. The NOEC (actually a Loading Rate, NOELR) is therefore ≥1000 mg/L and the EC50 (actually an Effective Loading, EL50) >1000 mg/L.

In conclusion the test item had no inhibitory effect on the respiration rate of aerobic wastewater micro-organisms of activated sludge after the incubation period of three hours at the highest tested concentration of 1000 mg/L.