4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2017 to 01 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

2007

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test item was directly dosed into each test flask and pure water was added.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

SPECIES/ORIGIN
Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by a municipal sewage treatment plant (Bensheim, Germany).

CONDITIONING
The activated sludge was used as collected, but coarse particles were removed by settling for 15 minutes and then the upper layer decanted. During holding prior to use overnight, the sludge was fed for both tests daily with 50 mL synthetic sewage per litre and kept aerated at room temperature until use. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.0

Dissolved oxygen:

1.7 – 9.2 mg O2/L

Nominal and measured concentrations:

- Nominal concentration: 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
- Aeration: With compressed air (1.017 L/minute)
- No. of vessels per concentration: 5
- No. of vessels per blank control: 6
- No. of vessels per positive control: 5 replicates per concentration
- Preparation of test flasks: For each replicate a test solution with a final volume of 500 mL was tested per treatment in a glass flask. 16 mL synthetic sewage and an adequate amount of the test item and an adequate volume of the stock solution of the reference item were filled up with pure water to 250 mL before the start of the test. At the start of the test 250 mL activated sludge inoculum with a sludge concentration of 3.0 g/L suspended solids was added, first to two controls, then to the test solutions of the reference item in increasing concentrations, to further two controls, then to the test item in increasing concentrations and finally to additional two controls. During 3 hour aeration period the flasks were stirred on a magnetic stirrer to maintain sludge flocs in suspension. The amount of water was reduced by the amount of the nitrification inhibitor ATU.
- Synthetic swage: 80 g peptone, 55 g meat extract, 15 g urea, 3.5 g NACL, 2.0 g CaCl2· 2H2O, 1.0 g MgSO4 · 7H2O and 14 g K2HPO4 filled up to 5 L deionised water.

WATER PARAMETERS
- Temperature: The room temperature was continuously recorded by a software controlled temperature recording system. In addition, the water temperature was measured in one control medium at the start and the end of the incubation period.
- pH: The pHvalue was determined at the start and at the end in at least one replicate of the test concentrations and controls.

EFFECT PARAMETERS MEASURED
- Measurement of respiration rate: For the measurement of the respiration rate a well-mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2/L/minute) was determined over periods up to 10 minutes in the range between approximately 9.2 to 1.7 mg O2/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of respiration due to nitrification

Remarks on result:

other:

Remarks:

95% C.L.: 23.1 - >1000 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

TOXICITY OF TEST ITEM
- Inhibition of Respiration Rate (Total Respiration)
The test item showed slightly dose-related inhibiting effects on the total respiration rate of activated sludge bacteria. In comparison to the inoculum controls the respiration rates of the activated sludge were slightly inhibited at test concentrations of 10 and 32 mg/L and moderately inhibited at a test item concentration of 100 mg/L and 320 mg/L. A test item concentration of 1000 mg/L resulted in the strongest inhibiting effect on activated sludge bacteria. The inhibition was 16.4%, 16.4%, 20.6%, 24.3% and 34.4% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. Concentrations exceeding 1000 mg/L nominal were not tested.
The R^2 of the 3-parametric normal CDF was 0.656, the analysis of variance and test for lack of fit showed that a significant amount of variances is explained by the regression model. There was no significant lack of fit. The 3-hour EC10 and EC20 for total respiration were established to be < 10 mg/L and 56.0 mg/L, respectively; the EC50 could not be determined.

- Inhibition of Respiration Rate (Heterotrophic Respiration)
The test item showed no clear dose-related inhibiting effects on the heterotrophic respiration rate of activated sludge bacteria. In comparison to the inoculum controls the respiration rates of the activated sludge were moderately inhibited at all test concentrations from 10 mg/L to 1000 mg/L. The inhibition was 29.5%, 17.3%, 26.0%, 26.7% and 31.6% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. Concentrations exceeding 1000 mg/L nominal were not tested.
The R^2 of the 3-parametric normal CDF was 0.771, the analysis of variance and test for lack of fit showed that a significant amount of variances is explained by the regression model. The lack of fit was significant. As a consequence, no 3-hour EC10, EC20 and EC50 values for heterotrophic respiration could be determined.

- Inhibition of Respiration Rate (Nitrification Respiration)
In comparison to the inoculum controls the respiration rates of the activated sludge were not inhibited at a test concentration of 10 mg/L and moderately inhibited at a test item concentrations of 32 mg/L and 100 mg/L. Test item concentrations of 320 and 1000 mg/L resulted in increasing inhibiting effects on activated sludge bacteria. The inhibition was -0.9%, 14.9%, 13.4%, 21.0% and
37.9% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. Concentrations exceeding 1000 mg/L nominal were not tested.
The R^2 of the 3-parametric normal CDF was 0.349, the analysis of variance and test for lack of fit showed that a significant amount of variances is explained by the regression model. There was no significant lack of fit. The 3-hour EC10, EC20 and EC50 for nitrification respiration were established to be 45.3 mg/L, 194.5 mg/L and >1000 mg/L, respectively; the NOEC was determined to be at a test item concentration of 100 mg/L.

Results with reference substance (positive control):

TOXICITY OF REFERENCE ITEM (3,5-Dichlorophenol)
- Inhibition of Respiration Rate (Total Respiration)
In comparison to the controls the total respiration rate of the activated sludge was inhibited by 26.0% at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 42.4% and 82.4%, respectively.
The R^2 of the 3-parametric normal CDF was 0.854, the analysis of variance and test for lack of fit showed that a significant amount of variances is explained by the regression model. There was no significant lack of fit.

- Inhibition of Respiration Rate (Heterotrophic Respiration)
In comparison to the controls the heterotrophic respiration rate of the activated sludge was inhibited by 17.3% at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 22.4% and 72.9%, respectively.
The R^2 of the 3-parametric normal CDF was 0.929, the analysis of variance and test for lack of fit showed that a significant amount of variances is explained by the regression model. A significant lack of fit was found.

- Inhibition of Respiration Rate (Nitrification Respiration)
In comparison to the controls the nitrification respiration rate of the activated sludge was inhibited by 37.2% at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 68.3% and 94.7%, respectively.
The R^2 of the 3-parametric normal CDF was 0.716, the analysis of variance and test for lack of fit showed that a significant amount of variances is explained by the regression model. There was no significant lack of fit.

Reported statistics and error estimates:

For ECx value determination, a non-linear, 3-parametric normal Cumulative Distribution Function (CDF) with Levenberg-Marquardt optimization was chosen, followed by an analysis of variance and test for lack of fit.
For NOEC determination, the data were checked for normal distribution by the Shapiro-Wilk's Test (α=0.01), the following variance homogeneity was tested by the Levene's Test (α=0.01).
For poorly normal distributed data where variance homogeneity requirements may be seen as fulfilled (total respiration and nitrification respiration of test item), a non-parametric trend analysis by contrasts followed by the step-down Jonckheere-Terpstra test procedure (onesided smaller, (α=0.05)) was performed. For data passing the variance homogeneity check and revealing a linear trend (heterotrophic respiration of test item; total and nitrification respiration of reference item), the Williams Multiple Sequential t-test Procedure (one-sided smaller, (α=0.05)) was used. The results demonstrated, that the data for heterotrophic respiration of the reference item failed the variance homogeneity test, but the normal distribution requirements were fulfilled. Therefore the multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm (one-sided smaller, (α=0.05)) was used to determine the LOEC and NOEC values, if possible.

Table 1. Influence of the test substance on total respiration of activated sludge in a 3-hour respiration inhibition test

Nominal concentration (mg/L)	Oxygen consumption rate (mg O2/L/hour)	Inhibition (%)	pH values		Oxygen concentration (mg O2/L)
			Start	End	Start	End
Control	41.7	-	7	7	5.0	7.7
10	34.9*	16.4	7	7	5.1	7.2
32	34.9*	16.4	7	7	5.7	8.4
100	33.1*	20.6	7	7	5.0	7.8
320	31.6*	24.3	7	7	5.2	8.1
1000	27.4*	34.4	7	7	4.9	7.8
3,5-dichlorophenol 1	30.8*	26.0	7	8	5.4	8.6
3,5-dichlorophenol 4	24.0*	42.4	7	8	4.8	8.0
3,5-dichlorophenol 16	7.3*	82.4	7	7	5.2	8.6

*: statistically significant difference from control (3,5-dichlorophenol: Williams Multiple Sequential t-test (α= 0.05 %, one sided smaller); SYN547407: step-down Jonckheere-Terpstra Test (α= 0.05 %, one sided smaller)).

 

Table 2.Influence of the test substance on heterotrophic respiration of activated sludge in a 3-hour respiration inhibition test

Nominal concentration (mg/L)	Oxygen consumption rate (mg O2/L/hour)	Inhibition (%)	pH values		Oxygen concentration (mg O2/L)
			Start	End	Start	End
Control	23.5	-	7	8	5.1	8.1
10	16.6*	29.5	7	8	5.5	8.4
32	19.4*	17.3	7	8	5.1	7.6
100	17.4*	26.0	7	7	5.3	8.3
320	17.2*	26.7	7	8	5.0	8.6
1000	16.1*	31.6	7	8	5.1	8.5
3,5-dichlorophenol 1	19.4*	17.3	7	8	5.4	8.4
3,5-dichlorophenol 4	18.2*	22.4	7	8	5.6	8.2
3,5-dichlorophenol 16	6.4*	72.9	7	7	4.9	9.2

* : statistically significant difference from control (3,5-dichlorophenol: Williams Multiple Sequential t-test (α= 0.05 %, one sided smaller); SYN547407: step-down Jonckheere-Terpstra Test (α= 0.05 %, one sided smaller))

Table 3.Influence of the test substance on nitrification respiration of activated sludge in a 3-hour respiration inhibition test

Nominal concentration (mg/L)	Oxygen consumption rate (mg O2/L/hour)	Inhibition (%)
Control	18.2	-
10	18.3	-0.9
32	15.4	14.9
100	15.7	13.4
320	14.3*	21.0
1000	11.3*	37.9
3,5-dichlorophenol 1	11.4	37.2
3,5-dichlorophenol 4	5.8	68.3
3,5-dichlorophenol 16	1.0	94.7

*: statistically significant difference from control (Williams Multiple Sequential t-test (α= 0.05 %, one sided smaller))

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the NOEC for nitrification respiration was determined to be 100 mg/L and the EC50 for nitrification respiration was >1000 mg/L.

Executive summary:

Activated sewage sludge was incubated under GLP for three hours in the presence of the substance according to OECD TG 209 at concentrations of 10, 32, 100, 320 and 1000 mg/L, together with a blank control and reference item control. Total respiration, heterotrophic respiration and nitrification respiration were determined.

The 3-hour EC₁₀, EC₂₀ and EC₅₀ values for nitrification respiration were determined to be 45, 195 and >1000 mg/L, respectively. The 3-hour NOEC was 100 mg/L. The 3-hour EC₁₀, EC₂₀ and EC₅₀ values for heterotrophic respiration could not be determined. The 3-hour EC₁₀ and EC₂₀ values for total respiration were determined to be <10 and 56 mg/L, respectively, and the EC₅₀ could not be determined.

Description of key information

3-h NOEC = 100 mg/L, Activated sludge, inhibition of nitrification respiration, OECD TG 209, Hammesfahr 2017

3-h EC50 >1000 mg/L, Activated sludge, inhibition of nitrification respiration, OECD TG 209, Hammesfahr 2017

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

Activated sewage sludge was incubated under GLP and to OECD TG 209 for three hours in the presence of the substance at concentrations of 10, 32, 100, 320 and 1000 mg/L, together with a blank control and reference item control. Total respiration, heterotrophic respiration and nitrification respiration were determined.
The 3-hour EC10, EC20 and EC50 values for nitrification respiration were determined to be 45, 195 and >1000 mg/L, respectively. The 3-hour NOEC was 100 mg/L. The 3-hour EC10, EC20 and EC50 values for heterotrophic respiration could not be determined. The 3-hour EC10 and EC20 values for total respiration were determined to be <10 and 56 mg/L, respectively, and the EC50 could not be determined.