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

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2017 to 21 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3100 (Aerobic Aquatic Biodegradation)

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of activated sludge: Fresh samples of activated sludge were withdrawn on the 24th May 2017 (day of test start) from the sewage treatment plant in Schmallenberg, Germany, which is mainly fed with municipal wastewater.
- Preparation of inoculum for exposure: Since the sludge had not been taken from a high rate treatment plant and was not thought to contain inhibitors, the samples were not washed with mineral medium. After the arrival in the laboratory, they were kept aerobic until use. The sludge was left for settlement for ca. one hour. Subsequently the supernatant was discarded and the concentration of suspended solids was measured in the remaining sludge.
- Concentration of sludge: The concentration was adjusted to 4.0 g/L and verified by dry mass measurement.
- Initial biomass concentration: The concentration used in the test was 29.6 mg dry mass/litre (7.40 mg dry mass/250 mL)

Duration of test (contact time):

28 d

Initial conc.:

40 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Biochemical oxygen demand (BOD)

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Theoretical oxygen demand (ThOD)

Parameter followed for biodegradation estimation:

other:

Remarks:

ThODNH3 (considering that nitrification is absent)

Parameter followed for biodegradation estimation:

other:

Remarks:

ThODNO3 (considering that nitrification is complete)

Details on study design:

TEST CONDITIONS
- Apparatus: The test flasks (500 mL reaction vessels) were incubated under continuous stirring in respirometer.
- Composition of medium: For the preparation of the stock solutions for mineral medium the following reagents (analytical grade) were used: (a) KH2PO4 8.53 g/L, K2HPO4 21.79 g/L, Na2HPO4 26.66 g/L, NH4Cl 0.52 g/L, pH was adjusted to pH 7.4 ± 0.2 using NaOH (40 g/L); (b) CaCl2 x 2 H2O 36.42 g/L; (c) MgSO4 x 7 H2O 22.53 g/L and (d) FeCl3 x 6 H2O 0.26 g/L. The mineral medium prepared for the test contained 10 mL/L of the mineral stock solution (a) and 1 mL/L of the mineral stock solutions (b) – (d), diluted with deionized water free from inhibitory concentrations of toxic substances (e.g. Cu2+ ions). The organic carbon content was checked at regular intervals (monthly) by dissolved organic carbon (DOC) analysis. The maximum DOC content was verified in the last sampling before and the first sampling after medium preparation and was found to be 0.16 mg/L, corresponding to 0.8 % of the organic carbon content introduced by the test material (20.15 mg C per liter).
- Light conditions: Darkness
- Test temperature: 22 °C, maintained with a built-in thermostat and checked twice per week.
- pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum and it was determinded to be 7.4 ± 0.2. At the end of incubation, the pH was measured again in each test flask and pH 7.4 – 8.6 were found.

PREPARATION OF TEST CONCENTRATIONS
- Reference item: The solution of the reference substance in the mineral medium equivalent to a concentration of 100 mg/L was prepared using an aqueous stock solution (10 g/L). The aqueous stock solution was prepared by dissolving 500 mg of the reference substance in 50 mL deionised water. An aliquot (2.5 mL) of the aqueous stock solution was then diluted to 250 mL with inoculated mineral medium.
- Test item: This amount (40 mg) test item corresponds to 65.6 mg theoretical oxygen demand (ThOD) considering total nitrification and 52.0 mg ThOD considering the total lack of nitrification. No emulsifiers or solvents were used. The required amount to ensure a final concentration of 40 mg/L (10 mg per 250 mL) was added directly on a weight basis. Subsequently, the mineral medium was added to the vessels.
- Toxicity control: A further solution containing both test and reference item at the same concentrations as in the individual solutions (test item 40 mg/L, reference item 100 mg/L) was prepared to determine the possible toxicity of the test item against the inoculum. For this, the required amount of test item was added directly on a weight basis. Subsequently, the required volume of aqueous reference stock solution and mineral medium were added to the vessels.
- Inoculum control: Further flasks with mineral medium only were prepared for inoculum controls. Abiotic controls were also prepared to measure any possible abiotic degradation; a solution of the test item at about 40 mg/L was sterilized by the addition of 10 mL/L NaN3 (100 g/L). In all test assays except of the abiotic controls, 1.85 mL of the inoculum stock solution were transferred for inoculation and the pH was measured. Before test start the pH values of all solutions were determined. At the end of the test the pH values of all solutions were determined again.

Reference substance:

benzoic acid, sodium salt

Remarks:

- Purity : 99% - ThOD: 1.665 mg O2/mg - initial concentration: 100 mg/L

Test performance:

The test was considered as valid, because it fulfilles the validity criteria of the guideline: With a maximum of 4 %, the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20 %. The percentage degradation of the reference item has exceeded the pass level of 60 % by day 14. The oxygen uptake of the inoculum blank is < 60 mg/L in 28 days and the pH value was inside the range of 6.0 - 8.5 except of one replicate of the toxicity control. This replicate had a pH of 8.6 and showed plausible results.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: No biodegradation of the test item in the test media could be calculated since less oxygen was consumed in the test media than in the blank inoculum.

Details on results:

An overview of the result is provided in Table 1 and 2 in 'Any other information on results incl. tables'.

BIODEGRADATION OF THE TEST ITEM
The percent biodegradation of the test item was calculated based on the theoretical oxygen demand of 1.30 mg O2/mg test item (ThODNH3) and of 1.64 mg O2/mg test item (ThODNO3). Less oxygen was consumed in the test media than in the blank inoculum. No biodegradation within the 10-day-window could be calculated since the start criterium of the window (10 % degradation rate) was not reached within the 28 days of incubation.

BIODEGRADATION IN THE TOXCITY CONTROL
Within 14 days of exposure, biodegradation amounted to 69 % based on the ThODNH3 and to 65 % based on the ThODNO3. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 40 mg/L because biodegradation in the toxicity control was > 25 % within 14 days.

Results with reference substance:

The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg. In the procedural controls, the reference item was degraded by an average of 89 % by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 93 %.

Table 1. An overview of the results

Time (days)	Percentage Biodegradation1
	Test item based on				Procedure control based on		Toxicity control based on
	ThODNH3		ThODNO3		ThOD		ThODNH3		ThODNO3
	Flask No.		Flask No.		Flask No.		Flask No.		Flask No.
	1	2	1	2	1	2	1	2	1	2
0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
1	-1.9	1.9	-1.5	1.5	16.8	26.4	22.9	24.7	21.5	23.3
2	-3.8	0.0	-3.0	0.0	42.6	45.0	33.9	34.3	31.9	32.3
3	-3.8	0.0	-3.0	0.0	53.4	55.8	40.7	44.8	38.3	42.2
4	-3.8	-1.9	-3.0	-1.5	60.6	63.0	44.4	62.7	41.8	59.0
5	-3.8	-1.9	-3.0	-1.5	67.9	69.7	54.9	67.7	51.7	63.8
6	-3.8	-1.9	-3.0	-1.5	74.5	76.3	57.6	70.0	54.3	65.9
7	-4.8	-1.0	-3.8	-0.8	77.8	80.2	60.2	71.1	56.6	67.0
8	-4.8	-1.0	-3.8	-0.8	81.4	82.6	61.5	72.5	57.9	68.3
9	-6.7	-1.0	-5.3	-0.8	82.6	83.2	62.0	73.0	58.4	68.7
10	-6.7	-1.0	-5.3	-0.8	85.0	84.4	62.5	73.4	58.8	69.1
11	-3.8	-1.9	-3.0	-1.5	85.9	85.3	62.7	73.7	59.0	69.4
12	-3.8	0.0	-3.0	0.0	87.1	86.5	63.1	74.6	59.4	70.2
13	-5.8	-1.9	-4.6	-1.5	87.7	87.1	62.7	75.0	59.0	70.6
14	-5.8	-1.9	-4.6	-1.5	88.9	88.3	63.1	75.5	59.4	71.1
15	-6.7	-1.0	-5.3	-0.8	89.8	88.0	62.9	75.7	59.2	71.3
16	-6.7	-1.0	-5.3	-0.8	89.8	89.2	62.9	76.2	59.2	71.7
17	-4.8	-2.9	-3.8	-2.3	90.4	88.6	62.9	75.7	59.2	71.3
18	-4.8	-1.0	-3.8	-0.8	90.4	89.8	62.9	76.2	59.2	71.7
19	-4.8	-1.0	-3.8	-0.8	91.6	89.8	62.9	76.6	59.2	72.2
20	-6.7	-2.9	-5.3	-2.3	91.0	90.4	62.9	76.6	59.2	72.2
21	-6.7	-1.0	-5.3	-0.8	92.2	90.4	62.9	77.1	59.2	72.6
22	-3.8	-1.9	-3.0	-1.5	91.9	90.1	62.7	77.3	59.0	72.8
23	-3.8	-1.9	-3.0	-1.5	91.9	91.3	62.7	77.3	59.0	72.8
24	-3.8	-1.9	-3.0	-1.5	93.1	91.3	62.7	77.8	59.0	73.2
25	-4.8	-2.9	-3.8	-2.3	92.8	92.2	62.5	78.0	58.8	73.4
26	-5.8	-1.9	-4.6	-1.5	93.7	91.9	62.2	77.8	58.6	73.2
27	-5.8	-1.9	-4.6	-1.5	93.7	91.9	62.7	78.2	59.0	73.7
28	-5.8	-1.9	-4.6	-1.5	93.7	93.1	62.7	78.2	59.0	73.7
Mean (Day 28)	-4		-3		93		70		66

Validity criteria fulfilled:

yes

Remarks:

See Validity of the test in 'Test performance'

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance was not readily biodegradable under the conditions of the test.

Executive summary:

The ready biodegradability of the test item was investigated at a concentration of 40 mg/L in a manometric respirometry test using domestic aerobic activated sewage sludge incubated at 22 °C in the dark over 28 days under continuous stirring. The manometric respirometry test was carried out according to the OECD TG 301 F (1992) and EPA OPPTS 835.3110. The study was in compliance with GLP criteria. The biochemical oxygen demand (BOD) was measured daily and the percentage degradation was calculated from the theoretical oxygen demand (ThOD) after correcting for the BOD of inoculum controls. No biodegradation of the test item in the test media could be calculated since less oxygen was consumed in the test media than in the blank inoculum. No biodegradation within the 10-day-window could be calculated since the start criterium of the window (10 % degradation rate) was not reached within the 28 days of incubation. In the toxicity control, containing both the test item and the reference item sodium benzoate, biodegradation was greater than 25 %. According to the guidelines this indicates that the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration. In the procedural controls, the reference substance sodium benzoate was degraded by an average of 89 % by exposure day 14, and reached an average biodegradation of 93 % by the end of the test (day 28), thus confirming suitability of the activated sludge. The difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20 %. Therefore, the test can be considered as valid. Based on the findings, it is concluded that the test item was not readily biodegradable under the conditions of the test according to the guidelines listed above.

Description of key information

The test substance was not readily biodegradable under the conditions of the test.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test item was investigated at a concentration of 40 mg/L in a manometric respirometry test using domestic aerobic activated sewage sludge incubated at 22 °C in the dark over 28 days under continuous stirring. The manometric respirometry test was carried out according to the OECD TG 301 F (1992). The biochemical oxygen demand (BOD) was measured daily and the percentage degradation was calculated from the theoretical oxygen demand (ThOD) after correcting for the BOD of inoculum controls.

No biodegradation of the test item in the test media could be calculated since less oxygen was consumed in the test media than in the blank inoculum. No biodegradation within the 10-day-window could be calculated since the start criterium of the window (10% degradation rate) was not reached within the 28 days of incubation. In the toxicity control, containing both the test item and the reference item sodium benzoate, biodegradation was greater than 25%. According to the guidelines, this indicates that the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration. In the procedural controls, the reference substance sodium benzoate was degraded by an average of 89% by exposure day 14, and reached an average biodegradation of 93% by the end of the test (day 28), thus confirming suitability of the activated sludge. The difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20%. Therefore, the test can be considered as valid. Based on the findings, it is concluded that the test item was not readily biodegradable under the conditions of the test according to the guidelines.