1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

30 Aug 2013 to 30 Sep 2013

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:

SAMPLING
An agricultural soil (loamy sand) was selected, supplied and analysed. The soil was removed to a depth of 20 cm as mixed samples. Afterwards, the soil was dried and passed through a 2 mm mesh sieve and stored at a temperature of approx. 4 °C in containers under aerobic conditions in the dark. Before application, the soil was adapted to test conditions.
- Control: The control was left untreated, i.e. was prepared with deionised water only.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

19.5 - 21.1 °C

Moisture:

- Nitrogen transformation test: 14.75 - 16.27 g/100 g soil d.w. (equivalent to 40.19 - 44.32 % of WHC)
- Carbon transformation test 15.65 - 16.32 g/100 g soil d.w. (equivalent to 42.62 - 44.47 % of WHC)

Organic carbon content (% dry weight):

1.47

Nitrogen content (% dry weight):

0.14

Details on test conditions:

NITROGEN TRANSFORMATION TEST SYSTEM
Three replicate soil samples were prepared for each treatment rate and the control for the nitrogen transformation test. Sub-samples of 200 g soil dry weight were weighed into each test vessel (500 mL wide mouth glass flask) and then placed into a mixing vessel. Lucerne meal (5 g/kg soil dry weight) was then added to each 200 g soil sample to provide 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
CARBON TRANSFORMATION TEST SYSTEM
Three replicate soil samples were prepared for each treatment rate and the control for the carbon transformation test. For each replicate a sub-sample of 1000 g soil dry weight was weighed into each test vessel and then placed into a mixing vessel of a laboratory mixer. The test item was mixed with deionised water and the test solution was subsequently mixed with the soil in the laboratory mixer. 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. The prepared soil was transferred to steel test vessels (4 L). The lids on the vessels permitted air exchange.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): 12.694435960 degrees East, 51.403774567 degrees North
- Treatments with pesticides or fertilizers: No orgnaic or inorganic fertilisers used since 2003. Last application of plant protection products was in 1990.
- Soil texture
- % sand: 59.5
- % silt: 32.3
- % clay: 8.2
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- pH (in water): 6.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 1.27 mg/100 g soil d.w.
- Total nitrogen (%): 0.14
- Maximum water holding capacity (in % dry weigth): 36.71 g/100 g soil d.w.
- Water content: 8.33 g/100 g soil d.w.
- Cation exchange capacity (mmol/kg): 95
- Organic carbon (%): 1.47
- Initial microbial biomass as % of total organic C: 35.51 = 2.42 % of Corg
- Humus content (%): 2.53

SAMPLING OF NITROGEN TRANSFORMATION TEST
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-N contents 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 the 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 0.05 mg/100 g soil d.w., 0.06 mg/100 g soil d.w. and 0.1 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 colorimetrically 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 colorimetrically 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 from Merck (p.a. quality).

SAMPLING PROCEDURE OF CARBON TRANSFORMATION TEST
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.

PH MEASUREMNETS
The pH-values of the soil used in the tests were measured using a pH-meter at test start (after application) and at the final sampling on day 28.

Nominal and measured concentrations:

- Application rates: 1 L test item/ha (nominally equivalent to 250 g a.s./ha) and 5 L test item/ha (nominally equivalent to 1250 g a.s./ha)
- Test concentrations: 1.30 mg test item/kg soil dry weight and 6.52 mg test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Dinoterb

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.66 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Remarks on result:

other: corresponding to 6.52 mg/kg soil dw test material

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.66 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

respiration rate

Remarks on result:

other: corresponding to 6.52 mg/kg soil dw test material

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 control. An overviwe of all the results is presented in 'Any other information on results incl. tables'.

Results with reference substance (positive control):

- Nitrogen transformation test: In the most recent test, the toxic standard dinoterb caused effects of +33.7 % and +42.6 % (required ≥ 25 %) on the nitrogen transformation at the tested concentrations of 16.00 mg and 27.00 mg/kg soil dry weight, respectively, on day 28 and thus demonstrates the sensitivity of the test system.
- Carbon transformation test: In the most recent test, the toxic standard dinoterb caused effects of -39.3 % and -45.2 % (required ≥ 25 %) on the carbon transformation in a field soil at the tested concentrations of 16.00 mg and 27.00 mg dinoterb per kg soil dry weight, respectively, 28 days after application and thus demonstrates the sensitivity of the test system.

Reported statistics and error estimates:

Calculation of the cumulative O2-consumption after 12 hours using regression analysis was conducted. The goodness of fit (R2) was > 0.99 in all replicates and on all days of measurement. Calculation of standard deviation and coefficient of variation were conducted. Statistical evaluation of the test results (2 sided Student-t-test at 5 % significance level and 2 sided Welch-t-test at 5 % significance level) was performed.

Table 1: Effects on Nitrogen Transformation in Soil after Treatment with the Test Item

Days after application	Control		1.30 mg test item/kg soil dry weight equivalent to 1 L test item/ha			6.52 mg test item/kg soil dry weight equivalent to 5 L test item/ha
	NO3-N [mg/kg soil d.w.]	CV [%]	NO3-N [mg/kg soil d.w.]	CV [%]	Deviation from control [%] (1)	NO3-N [mg/kg soil d w.]	CV [%]	Deviation from control [%]^1)
0	18.3	1.1	17.8	2.2	-2.6	18.3	0.8	+0.4
7	46.7	2.6	48.9	2.3	+4.6	47.6	4.8	+2.0
14	55.3	2.3	56.3	1.5	+1.7	54.2	2.9	-2.1
28	66.6	4.8	67.1	6.9	+0.9	65.4	3.3	-1.8

The calculations were performed with non-rounded values
CV [%] = Coefficient of Variation

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

No statistically significant differences between the control and the test item treatments were calculated.

Table 2: Effects on Carbon Transformation in Soil after Treatment with the Test Item

Days after application	Control		1.30 mg test item/kg soil dry weight equivalent to 1 L test item/ha			6.52 mg test item/kg soil dry weight equivalent to 5 L 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	12.29	1.2	12.37	0.6	+0.6	12.12	1.3	-1.4
7	11.86	0.2	11.82	2.0	-0.4	11.43	1.6	-3.7
14	11.19	1.8	10.98	1.5	-1.9	10.64	4.7	-4.9
28	9.88	1.0	9.92	2.1	+0.4	10.00	1.2	+1.3

The calculations were performed with non-rounded values.

CV [%] = Coefficient of Variation

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

No statistically significant differences between the control and the test item treatments were calculated.

 

Table 3: Analytical Results and Calculations – NO3-N-Content

Days after appli- cation	Treatment group	Repl.	Measured values	Mean value	SD	CV     [%]	mgNO3-N/kg soil d.w.	mg NO3-N/ kg soil d.w./ day	Deviation from control [%]
			[mg NO3-N/100 g soil d.w.]
		1	1.85
0	Control	2	1.82
		3	1.81	1.83	0.02	1.1	18.3	-	-
	test item 1.30 mg/kg	1 2 3	1.74 1.78 1.82	1.78	0.04	2.2	17.8	-	-2.6
	test item 6.52 mg/kg	1 2 3	1.82 1.85 1.83	1.83	0.02	0.8	18.3	-	+0.4
		1	4.53
7	Control	2	4.74
		3	4.74	4.67	0.12	2.6	46.7	6.7	-
	test item 1.30 mg/kg	1 2 3	5.00 4.78 4.88	4.89	0.11	2.3	48.9	7.0	+4.6
	test item 6.52 mg/kg	1 2 3	4.91 4.88 4.50	4.76	0.23	4.8	47.6	6.8	+2.0
		1	5.62
14	Control	2	5.59
		3	5.39	5.53	0.13	2.3	55.3	4.0	-
	test item 1.30 mg/kg	1 2 3	5.62 5.55 5.72	5.63	0.09	1.5	56.3	4.0	+1.7
	test item 6.52 mg/kg	1 2 3	5.60 5.31 5.34	5.42	0.16	2.9	54.2	3.9	-2.1
		1	6.70
28	Control	2	6.95
		3	6.32	6.66	0.32	4.8	66.6	2.4	-
	test item 1.30 mg/kg	1 2 3	7.18 6.70 6.26	6.71	0.46	6.9	67.1	2.4	+0.9
	test item 6.52 mg/kg	1 2 3	6.32 6.75 6.54	6.54	0.22	3.3	65.4	2.3	-1.8

Limit of quantification (=LOQ): 0.05 mg/100 g soil d.w.

SD = Standard Deviation

CV [%] = Coefficient of Variation

Repl. = Replicate

The calculations were performed with non-rounded values.

Table 4: 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.
Control	1 2 3	1.02 1.01 1.03	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ
test item 1.30 mg/kg	1 2 3	1.02 1.05 0.98	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ
test item 6.52 mg/kg	1 2 3	0.95 0.99 1.01	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ

Limit of Quantification (=LOQ): 0.06 mg/100 g soil d.w.

Repl. = Replicate

 

Table 5: Analytical Results and Calculations – NO2-N-Content

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.
Control	1 2 3	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ
test item 1.30 mg/kg	1 2 3	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ
test item 6.52 mg/kg	1 2 3	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ

Limit of Quantification (=LOQ): 0.1 mg/100 g soil d.w.

Repl. = Replicate

 

Table 6: 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^-1*h^-1]
		1	146.10	12.18
0	Control	2 3	149.52 146.95	12.46 12.25	12.29	0.15	1.2	-
	test item 1.30 mg/kg	1 2 3	148.89 147.44 148.94	12.41 12.29 12.41	12.37	0.07	0.6	+0.6
	test item 6.52 mg/kg	1 2 3	143.93 144.87 147.66	11.99 12.07 12.31	12.12	0.16	1.3	-1.4
		1	142.40	11.87
7	Control	2	142.68	11.89
		3	142.05	11.84	11.86	0.03	0.2	-
	test item 1.30 mg/kg	1 2 3	138.84 142.08 144.51	11.57 11.84 12.04	11.82	0.24	2.0	-0.4
	test item 6.52 mg/kg	1 2 3	134.83 137.13 139.35	11.24 11.43 11.61	11.43	0.19	1.6	-3.7
		1	133.34	11.11
14	Control	2	132.47	11.04
		3	137.13	11.43	11.19	0.21	1.8	-
	test item 1.30 mg/kg	1 2 3	132.38 133.34 129.60	11.03 11.11 10.80	10.98	0.16	1.5	-1.9
	test item 6.52 mg/kg	1 2 3	132.05 130.11 120.93	11.00 10.84 10.08	10.64	0.49	4.7	-4.9
		1	119.81	9.98
28	Control	2	117.93	9.83
		3	117.77	9.81	9.88	0.09	1.0	-
	test item 1.30 mg/kg	1 2 3	116.12 120.21 120.77	9.68 10.02 10.06	9.92	0.21	2.1	+0.4
	test item 6.52 mg/kg	1 2 3	121.50 118.53 120.09	10.13 9.88 10.01	10.00	0.12	1.2	+1.3

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

Conclusions:

The test item (tested at 1.30 mg/kg dry soil and 6.52 mg test item/kg dry soil) caused no adverse effects (deviation from control <25 %, OECD 216/217) 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 28-d NOEC was determined to be 6.52 mg test item/kg dry soil, which corresponds to 1.66 mg a.i./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 by measuring the nitrogen turnover and the short-term substrate induced respiration. Agricultural soil (loamy sand) was used as substrate, which was spiked applied with the test material in the following rates: Control, 1.30 mg test item/kg dry soil and 6.52 mg test item/kg dry soil. Test concentrations were related to a soil depth of 5 cm and a soil density of 1.5 g/cm3. 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 material. 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 material. Comparison of test item treated soil was made with a nontreated 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. For the nitrogen transformation, no effects were observed when compared with the control (<5% during 28 d) at a conc. up to 6.52 mg/kg dry soil (corresponding to 1.66 mg ai/kg dry soil). Also for the carbon transformation test, no effects were observed when compared with the control (<5% during 28 d) at a conc. up to 6.52 mg/kg dry soil (corresponding to 1.66 mg ai/kg dry soil). Therefore, the 28-d NOEC was determined to be 6.52 mg test item/kg dry soil, which corresponds to 1.66 mg a.i./kg dry soil.

Description of key information

All available data was assessed and the study representing the worst-case effect was included as key study. Other studies are included as supporting information. The effect value from the key study was selected for the CSA. 

28-d NOEC = 6.52 mg test item/kg dry soil, which corresponds to 1.66 mg a.i./kg dry soil, OECD TG 216 and 217, Schulz 2013

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1.66 mg/kg soil dw

Additional information

Three 28-d studies are available for this endpoint. The study following standard guidelines (i.e. OECD TG 216 and 217), GLP, and representing the worst-case effect (i.e. shows the lowest NOEC value) was selected as the key study. In the key study (Schulz 2013; Reliability 1), the effects of the test item on the activity of soil microflora with regard to nitrogen transformation (mineralization) and carbon transformation (respiration) were determined in a laboratory test. Agricultural soil (loamy sand) was used as substrate, which was spiked with the test material in the following rates: Control, 1.30 mg test item/kg dry soil and 6.52 mg test item/kg dry soil. Test concentrations were related to a soil depth of 5 cm and a soil density of 1.5 g/cm3. The nitrogen transformation (NO3-nitrogenproduction) in soil enriched with lucerne meal (concentration in soil 0.5 %) was determined for soil treated with two rates of the test material. Three replicates were used per treatment and concentration. NH4-nitrogen, NO3- and NO2- nitrogen were determined at 0, 7, 14 and 28 days after treatment. Carbon transformation in soil after addition of glucose was determined for soil treated with two rates of the test material. Three replicates were used per treatment and concentration. O2-consumption over a period of 12 hours at different sampling intervals was determined. The soils were analysed at 0, 7, 14 and 28 days after treatment. The 28-d NOEC was determined to be 6.52 mg test item/kg dry soil, which corresponds to 1.66 mg a.i./kg dry soil. The results were supported by the study from Butterfield et al. (1979; Reliability 2), that was not performed in accordance with a standard guideline. This supporting study shows that the effect of the test substance on soil nitrification was an extension of the lag period when applied at a 100 ppm rate and the impact on soil respiration was limited. Another supporting study from Mumma and Bogus (data unknow) has significant methodological deficiencies and incomplete of result tables, and was not performed in accordance with a standard guideline, therefore was assigned reliability 3 (unreliable). No significant effects were reports in 14CO2 production from cellulose and protein. No effect values were determined in the study.