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 soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Sep 2016 to 25 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

2000

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

NITROGEN TRANSFORMATION TEST
- Since data on the water solubility of the test item were not available, the test item was thoroughly mixed with quartz sand. Subsequently the obtained mixture was added and mixed with the soil by means of a hand stirrer. For an optimum distribution of the test item in the soil, the test item quartz sand mixture was applied at a ratio of about 10 g per kilogram soil dry weight.

CARON TRANSFORMATION TEST
- Since data on the water solubility of the test item were not available, the test item was thoroughly mixed with quartz sand. Subsequently the obtained mixture was added and mixed with the soil in the laboratory mixer. For an optimum distribution of the test item in the soil, the test item quartz sand mixture was applied at a ratio of about 10 g per kilogram soil dry weight.

APPLICATION RATEs (both tests)
- 360 g a.i./ha (equivalent to 371.5 g test item/ha)
- 3600 g a.i./ha (equivalent to 3715 g test item/ha)

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

19.2 - 20.9 °C in a climatic room

Moisture:

- Water content of soil (Nitrogen transformation test): 16.83 - 18.14 g/100 g soil d.w. (equivalent to 46.06 - 49.63 % of WHC).
- Water content of soil (Carbon transformation test): 17.14 - 17.89 g/100 g soil d.w. (equivalent to 46.88 - 48.95 % of WHC)

Organic carbon content (% dry weight):

17.89

Nitrogen content (% dry weight):

18.14

Details on test conditions:

TEST SYSTEM (Nitrogen transformation test)
- Testing vessel: 500 mL wide mouth glass flask
- Amount of soil: 200 g soil dry weight per test vessel
- Lucerne meal: 1.0 g lucerne meal per 200 g soil d.w. and mixed by means of a hand-stirrer
- One additional soil sample (without lucerne meal) was used for determination of the initial NH4-N- and NO3-N-content. The initial NH4-N- and NO3-N-content was < LOQ and 1.82 mg/100 g soil d.w., respectively.
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- The screw caps of the flasks permitted an air exchange.

TEST SYSTEM (Carbon transformation test)
- Testing vessel: 4 L steel test vessels
- Amount of soil: 1000 g soil dry weight per test vessel
- Lucerne meal: 1.0 g lucerne meal per 200 g soil d.w. and mixed by means of a hand-stirrer
- One additional soil sample (without lucerne meal) was used for determination of the initial NH4-N- and NO3-N-content. The initial NH4-N- and NO3-N-content was < LOQ and 1.82 mg/100 g soil d.w., respectively.
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- The lids on the vessels permitted air exchange.

EXPERIMENTAL CONDITIONS (for both tests)
- The incubation of the prepared soil was carried out in wide-mouth glass flasks (500 mL).
- Illumination: darkness
- Water was added to the soil to achieve a water content of approximately 45 % of WHC. The water content of the soil in each test vessel was determined at test start (after application) and adjusted once a week to the required range of 40 - 50 % of WHC.

SOURCE AND PROPERTIES OF SUBSTRATE
- Soil removel: The soil was removed to a depth of 20 cm as mixed samples.
- Soil preparation: The soil was dried and passed through a 2 mm mesh sieve. Before application, the soil was adapted to test conditions.
- Soil storage: At a temperature of approx. 4 °C in containers under aerobic conditions in the dark.
- History and Physico-chemical data of soil see Table 1in "Any other information on materials and methods incl. tables"

EFFECT PARAMETERS MEASURED (Nitrogen transformation test)
- Before test start, the following parameters were determined, based on the respective ISO guidelines: pH, water content, WHC and carbon content of microbial biomass. pH was measured at the final sampling on day 28 as well.
- Soil samples (10 g soil d.w. per replicate) were taken at intervals of 3 hours, 7, 14, and 28 days after application and the NH4-N-, NO3-N- and NO2-Ncontents were determined. Soil was extracted by adding 50 mL 1 M KCl solution to the equivalent of 10 g soil d.w. and mixing on a rotator at 150 rpm for 60 minutes. The mixtures were centrifuged and stored deep-frozen prior to analysis at -20 ± 5 °C. The analysis was performed within one week after day 28. For the quantitative determination of the mineralized part of nitrogen a Autoanalyzer was used. The autoanalyzer is a continuous flow analysis system. The autoanalyzer was calibrated before each measurement series by establishing a calibration curve. For each batch of 30 samples a standard was measured for recalibration and adjusting the calibration curve. The calibration curve was calculated with linear regression. The Limits of Quantification (LOQ) for NO3-N, NH4-N and NO2-N were 0.31 mg/100 g soil d.w., 0.12 mg/100 g soil d.w. and 0.14 mg/100 g soil d.w., respectively.
- Ammonium reacts with salicylate and dichloroisocyanuric acid to form an indophenoleblue compound. The intensity of the formed compound is colourimetrically measured at a wavelength of 625 nm. Nitrate is reduced to nitrite by hydrazinesulphate. The nitrite reacts with sulphanilamide in an acidic solution to form a diazocompound. The diazotized product is then coupled with naphthylamine. The intensity of the formed azodye, which is proportional to the sum of the nitrate and nitrite originally present in the sample, is colourimetrically measured at a wavelength of 525 nm. The differences between the nitrate/nitrite sum and the nitrite contents are the nitrate contents. The nitrite contents are
determined without nitrate reduction. The chemicals for the calibration solutions were NaNO2, (NH4)2SO4 and KNO3.

EFFECT PARAMETERS MEASURED (Cabon transformation test)
- Before test start, the following parameters were determined, based on the respective ISO guidelines: pH, water content, WHC and carbon content of microbial biomass. pH was measured at the final sampling on day 28 as well.
- The method is based on the initial respiratory response of microbial populations to which glucose as carbon and energy source has been added (substrate-induced respiration, SIR). Before test start, the optimal glucose concentration was determined as 0.4 %. The carbon transformation was determined over a measurement period of 12 hours on sampling days 0 (3 hours after application), 7, 14 and 28 days after application. On each sampling occasion, 100 g samples of soil (d.w.) were taken, mixed with glucose by means of a hand-stirrer and placed into glass reaction flasks (500 mL). Then small glass vessels containing 18 mL of 1 M NaOH solution were placed in the reaction flasks, which were tightly closed and then connected with a respirometer. The respiration of micro-organisms leads to O2-consumption and formation of CO2 that is absorbed in NaOH solution. The absorption of CO2 decreased the pressure in the reaction flask, which is compensated with O2 delivered by the respirometer. The respirometer determines the cumulative O2-production (corresponding to the O2-consumption by micro-organisms) over a 12-hour measurement period.

Nominal and measured concentrations:

Nominal concentrations: 0.50 mg test item/kg soil dry weight and 4.95 mg test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Dinoterb was tested as reference item in separate studies to verify the sensitivity of the test system.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4.95 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: nitrogen and carbon transformation

Details on results:

No differences greater than 25 % in the nitrogen and carbon transformation were found for the tested concentrations of the test item at the end of the 28-day incubation period in comparison to the respective controls. An overview of the results is provided in Table 2 to Table 7.

Results with reference substance (positive control):

In a separate study the reference item Dinoterb caused a stimulation of nitrogen transformation of + 28.6% and + 40.9% at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application. In a separate study the reference item Dinoterb caused an inhibition of carbon transformation of -34.4% and -39.2% at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application.

Table 2. Effects on Nitrogen Transformation in Soil after Treatment with the test substance

Time Interval (days)	Control		0.50 mg test item/kg soil dry weight equivalent to 371.5 g test item/ha			4.95 mg test item/kg soil dry weight equivalent to 3715 g test item/ha
	NO3-N [mg/kg soil d.w.]	NO3-N [mg/kg soil d.w./day]	NO3-N [mg/kg soil d.w.]	NO3-N [mg/kg soil d.w./day]	Deviation from control [%]^1	NO3-N [mg/kg soil d.w.]	NO3-N [mg/kg soil d.w./day]	Deviation from control [%]^1
0 - 7	50.5	4.52	50.2	4.45	-1.5	50.6	4.43	-1.9
0 - 14	61.3	3.03	60.0	2.93	-3.5	58.5	2.78*	-8.2
0 - 28	77.0	2.08	78.9	2.14	+2.9	73.8	1.94	-6.6

The calculations were performed with non-rounded values

1 based on NO3-nitrogen-production; - = inhibition; + = stimulation

* = statistically significantly different to control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05)

 

Table 3. Effects on Carbon Transformation in Soil after Treatment with the test substance 

Days after application	Control		0.50 mg test item/kg soil dry weight equivalent to 371.5 g test item/ha			4.95 mg test item/kg soil dry weight equivalent to 3715 g test item/ha
	O2-consumption [mg/kg soil d.w./h]	CV   [%]	O2-consumption [mg/kg soil d.w./h]	CV   [%]	Deviation from control [%]^1	O2-consumption [mg/kg soil d.w./h]	CV   [%]	Deviation from control [%]^1
0	14.82	3.5	14.73	1.2	-0.6	14.28	0.8	-3.7
7	14.21	3.4	14.22	1.7	+0.1	13.63	2.7	-4.1
14	13.64	4.3	13.62	1.3	-0.1	13.56	1.7	-0.6
28	10.99	1.0	10.75	2.1	-2.1	10.83	2.3	-1.4

The calculations were performed with non-rounded values.

CV [%] = Coefficient of Variation

1 based on O2-consumption; - = inhibition; + = stimulation

No statistically significantly different to control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05)

Table 4. Analytical Results and Calculations – NO3-N-Content

Days after appli- cation	Treatment group	Repl.	Measured values	Mean value	SD	CV     [%]	mg NO3-N/ kg soil d.w	Mean Values mg NO3-N/ kg soil d.w.	mg NO3-N/ kg soil d.w./ day	Mean values mg NO3- N/ kg soil d.w./ day	Deviation from control [%]
			[mg NO3-N/100 g soil d.w.]
0		1	1.89				18.9
	Control	2	1.88				18.8
		3	1.89	1.89	0.01	0.3	18.9	18.9	-	-	-
	test item 0.50 mg/kg	1 2 3	1.89 1.92 1.89	1.90	0.02	0.9	18.9 19.2 18.9	19.0	-	-	-
	test item 4.95 mg/kg	1 2 3	1.96 1.94 1.96	1.95	0.01	0.6	19.6 19.4 19.6	19.5	-	-	-
7		1	5.10				51.0		4.59
	Control	2	4.88				48.8		4.29
		3	5.17	5.05	0.15	3.0	51.7	50.5	4.69	4.52	-
	test item 0.50 mg/kg	1 2 3	4.92 4.89 5.24	5.02	0.19	3.9	49.2 48.9 52.4	50.2	4.33 4.24 4.79	4.45	-1.5
	test item 4.95 mg/kg	1 2 3	5.24 5.12 4.81	5.06	0.22	4.4	52.4 51.2 48.1	50.6	4.69 4.54 4.07	4.43	-1.9
14		1	6.12				61.2		3.02
	Control	2	6.03				60.3		2.96
		3	6.25	6.13	0.11	1.8	62.5	61.3	3.11	3.03	-
	test item 0.50 mg/kg	1 2 3	5.96 5.91 6.13	6.00	0.12	1.9	59.6 59.1 61.3	60.0	2.91 2.85 3.03	2.93	-3.5
	test item 4.95 mg/kg	1 2 3	5.75 5.99 5.81	5.85	0.12	2.1	57.5 59.9 58.1	58.5	2.71 2.89 2.75	2.78	-8.2
28		1	7.94				79.4		2.16
	Control	2	7.64				76.4		2.06
		3	7.53	7.70	0.21	2.8	75.3	77.0	2.01	2.08	-
	test item 0.50 mg/kg	1 2 3	7.88 7.63 8.15	7.89	0.26	3.3	78.8 76.3 81.5	78.9	2.14 2.04 2.24	2.14	+2.9
	test item 4.95 mg/kg	1 2 3	7.46 7.46 7.23	7.38	0.13	1.8	74.6 74.6 72.3	73.8	1.96 1.97 1.88	1.94	-6.6

Limit of quantification (= LOQ): 0.31 mg/100 g soil d.w.

SD = Standard Deviation

CV [%] = Coefficient of Variation

Repl. = Replicate

The calculations were performed with non-rounded values.

Table 5. Analytical Results and Calculations – NH4-N-Content

Days after application		0	7	14	28
Treatment group	Repl.	mg NH4-N/ 100 g soil d.w.	mg NH4-N/ 100 g soil d.w.	mg NH4-N/ 100 g soil d.w.	mg NH4-N/ 100 g soil d.w.
	1	0.92	<LOQ	<LOQ	<LOQ
Control	2	0.96	<LOQ	<LOQ	<LOQ
	3	0.97	<LOQ	<LOQ	<LOQ
test item 0.50 mg/kg	1 2 3	0.94 0.94 0.99	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ
test item 4.95 mg/kg	1 2 3	0.98 0.98 0.92	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ

Limit of Quantification (= LOQ): 0.12 mg/100 g soil d.w.

Repl. = Replicate

 

Table 6. Analytical Results and Calculations – NO2-N-Conten

Days after application		0	7	14	28
Treatment group	Repl.	mg NO2-N/ 100 g soil d.w.	mg NO2-N/ 100 g soil d.w.	mg NO2-N/ 100 g soil d.w.	mg NO2-N/ 100 g soil d.w.
	1	<LOQ	<LOQ	<LOQ	<LOQ
Control	2	<LOQ	<LOQ	<LOQ	<LOQ
	3	<LOQ	<LOQ	<LOQ	<LOQ
test item 0.50 mg/kg	1 2 3	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ
test item 4.95 mg/kg	1 2 3	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ

Limit of Quantification (= LOQ): 0.14 mg/100 g soil d.w.

Repl. = Replicate

Table 7. Results and Calculations – Carbon Transformation

Days after appli- cation	Treatment group	Repl.	Measured values O2-consumption [mg/kg] 12 h^1	O2-consumption	Mean²	SD	CV     [%]	Deviation from control [%]
				mg/kg soil d.w./h
0		1	184.92	15.41
	Control	2	172.89	14.41
		3	175.88	14.66	14.82	0.52	3.5	-
	test item 0.50 mg/kg	1 2 3	175.16 175.92 179.29	14.60 14.66 14.94	14.73	0.18	1.2	-0.6
	test item 4.95 mg/kg	1 2 3	171.43 172.76 170.00	14.29 14.40 14.17	14.28	0.12	0.8	-3.7
7		1	171.95	14.33
	Control	2	175.41	14.62
		3	164.02	13.67	14.21	0.49	3.4	-
	test item 0.50 mg/kg	1 2 3	167.76 173.58 170.70	13.98 14.47 14.23	14.22	0.24	1.7	+0.1
	test item 4.95 mg/kg	1 2 3	158.56 166.85 165.13	13.21 13.90 13.76	13.63	0.36	2.7	-4.1
14		1	171.15	14.26
	Control	2	157.36	13.11
		3	162.49	13.54	13.64	0.58	4.3	-
	test item 0.50 mg/kg	1 2 3	162.56 161.94 165.78	13.55 13.50 13.82	13.62	0.17	1.3	-0.1
	test item 4.95 mg/kg	1 2 3	161.46 165.79 160.85	13.46 13.82 13.40	13.56	0.22	1.7	-0.6
		1	132.42	11.04
28	Control	2	132.67	11.06
		3	130.37	10.86	10.99	0.11	1.0	-
	test item 0.50 mg/kg	1 2 3	129.98 125.93 131.22	10.83 10.49 10.94	10.75	0.23	2.1	-2.1
	test item 4.95 mg/kg	1 2 3	129.10 133.24 127.52	10.76 11.10 10.63	10.83	0.25	2.3	-1.4

1 Calculated with regression analysis over 12 hours

2 Mean O2-consumption per hour

SD = Standard Deviation

CV [%] = Coefficient of Variation

Repl. = Replicate

The calculations were performed with non-rounded values.

Validity criteria fulfilled:

yes

Remarks:

See Validity Criteria in "Any other information on materials and methods incl. tables"

Conclusions:

The test item (tested at 0.50 mg/kg dry soil corresponding to 371.5 g test item/ha and 4.95 mg/kg dry soil corresponding to 3715 g test item/ha) caused no adverse effects (deviation from control < 25 %) on soil nitrogen transformation (measured as NO3-N-production) and on soil carbon transformation (measured as O2-consumption) at the end of the 28-day incubation period. Therefore, the NOEC was determined to be 4.95 mg/kg dry soil.

Executive summary:

The purpose of this study was to determine the effects of the test item on the activity of soil microflora with regard to nitrogen transformation (mineralization) and carbon transformation (respiration) in a laboratory test over a period of 28 days of exposure. The test was performed in accordance with OECD TG 216 and 217 (2000) by measuring the nitrogen turnover and the short-term substrate induced respiration and it was incompliance with GLP criteria. The test concentrations are control, 0.50 mg test item/kg dry soil and 4.95 mg test item/kg dry soil. Test concentrations related to a soil depth of 5 cm and a soil density of 1.5 g/cm3. The application rates are 360 g a.i./ha (equivalent to 371.5 g test item/ha) and 3600 g a.i./ha (equivalent to 3715 g test item/ha). Determination of the nitrogen transformation (NO3-nitrogenproduction) in soil enriched with lucerne meal (concentration in soil 0.5 %) was made for soil treated with two rates of the test substance. Comparison of test item treated soil was made with a non-treated control soil. Three replicates were used per treatment and concentration. NH4-nitrogen, NO3- and NO2- nitrogen were determined by using the Autoanalyzer.The soils were analysed at 0, 7, 14 and 28 days after treatment. Determination of carbon transformation in soil after addition of glucose was made for soil treated with two rates of the test substance. Comparison of test item treated soil was made with a non-treated control soil. Three replicates were used per treatment and concentration. A respirometer system was used to determine the O2- consumption over a period of 12 hours at different sampling intervals. The soils were analysed at 0, 7, 14 and 28 days after treatment.

No adverse effects of the test item on nitrogen and carbon transformation in soil were observed at either tested concentration (0.50 mg test item/kg dry soil and 4.95 mg test item/kg dry soil) after 28 days. In a separate study the reference item Dinoterb caused a stimulation of nitrogen transformation of +28.6 % and +40.9 % at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application. In a separate study the reference item Dinoterb caused an inhibition of carbon transformation of -34.4 % and -39.2 % at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application. Therefore, the NOEC of the test substance was determined to be 4.95 mg/kg dry soil.

Description of key information

28-d NOEC = 4.95 mg a.i./kg dry soil (loamy sand soil), Nitrification and carbon transformation, OECD TG 216 & 217, Schulz 2016

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

4.95 mg/kg soil dw

Additional information

The purpose of this study was to determine the effects of the test item on the activity of soil microflora with regard to nitrogen transformation (mineralization) and carbon transformation (respiration) in a laboratory test over a period of 28 days of exposure. The test was performed in accordance with OECD TG 216 and 217 (2000) by measuring the nitrogen turnover and the short-term substrate induced respiration and it was in compliance with GLP criteria. The test concentrations are control, 0.50 mg test item/kg dry soil and 4.95 mg test item/kg dry soil. Test concentrations related to a soil depth of 5 cm and a soil density of 1.5 g/cm3. The application rates are 360 g a.i./ha (equivalent to 371.5 g test item/ha) and 3600 g a.i./ha (equivalent to 3715 g test item/ha). Determination of the nitrogen transformation (NO3-nitrogenproduction) in soil enriched with lucerne meal (concentration in soil 0.5 %) was made for soil treated with two rates of the test substance. Comparison of test item treated soil was made with a non-treated control soil. Three replicates were used per treatment and concentration. NH4-nitrogen, NO3- and NO2- nitrogen were determined by using the Autoanalyzer. The soils were analysed at 0, 7, 14 and 28 days after treatment. Determination of carbon transformation in soil after addition of glucose was made for soil treated with two rates of the test substance. Comparison of test item treated soil was made with a non-treated control soil. Three replicates were used per treatment and concentration. A respirometer system was used to determine the O2- consumption over a period of 12 hours at different sampling intervals. The soils were analysed at 0, 7, 14 and 28 days after treatment.

No adverse effects of the test item on nitrogen and carbon transformation in soil were observed at either tested concentration (0.50 mg test item/kg dry soil and 4.95 mg test item/kg dry soil) after 28 days. In a separate study the reference item Dinoterb caused a stimulation of nitrogen transformation of +28.6 % and +40.9 % at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application. In a separate study the reference item Dinoterb caused an inhibition of carbon transformation of -34.4 % and -39.2 % at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 days after application. Therefore, the NOEC of the test substance was determined to be 4.95 mg/kg dry soil.