N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

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:

05 Nov 1992 to 18 Feb 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

BBA Part VI, 1-1

Version / remarks:

1990

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

Two agricultural soils, a sandy loam (Soil I) and a loam (Soil II) were used.
Before use, all plants (plant parts, e.g. roots) were removed from the soil and sieved through a 2 mm sieve. For that purpose, the soils were carefully dried at room temperature in order to avoid a moisture content of less than 40 % of their MWC. The soils were stored for about 2 weeks under incubation conditions in closed plastic boxes. The soils were aerated at least every second day by thoroughly mixing until the determination of the microbial biomass which was prior to the start of the study.

Preparation of the application solution:
The test substance was applied as a solution in acetone.
The treatment was based on a field rate of 150 g/ha assuming a penetration depth of 5 cm and a soil density of 1.5 cm3
- 10 x field rate:
The 10x field rate of 1500 g/ha corresponds to 2000 µg per kg dry soil or 200 µg per 100 g dry soil. For this purpose 40 mg of the test substance were dissolved in 200 mL acetone. By applying 1 mL of this solution to 100 g dry soil, the 10x field rate concentration was achieved.
- 1 x field rate:
The low rate corresponds to the recommended field rate (150 g/ha). i.e. 200 µg/kg dry soil or 20 µg per 100 g dry soil. For this purpose, 10 mL of the solution prepared as described above were filled to volume of 100 mL with acetone. For treatment 1.0 mL was applied to 100 g of soil (dry weight basis).

Application:
The soil samples were treated by adding dropwise the respective application solutions to the soils by means of a Hamilton syringe (1 mL per sample). During treatment, the soils were thoroughly mixed. After application, the soil samples of the experiments were amended with 0.5 g lucerne meal per 100 g dry soil, containing approx. 3 % nitrogen. Finally, the soil moisture content of all samples was adjusted to 40 % MWC for Soil I and to 39 % MWC Soil II by adding bidistilled water.
The soil samples of the control were treated with 1 ml acetone/100 9 soil (dry weight)

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

Soil I: 40 % MWC
Soil II: 39 % MWC

Details on test conditions:

TEST SYSTEM
- Amount of soil: 100 g
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: 3

SOURCE AND PROPERTIES OF SUBSTRATE
- History of site: Soil I has not been subjected to any pesticide, organic nor inorganic fertilizer treatment at least one year before use. Soil ll has not been subjected to any pesticide, organic nor inorganic fertilizer treatment
- Soil taxonomic classification: Soil I: sandy loam; Soil II: loam
- Soil classification system: USDA classification
- pH: Soil I: 6.6 - 6.9; Soil II: 7.3
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/100 g dry weight): Soil I: 0.61; Soil II: 0.5
- Initial microbial biomass as % of total organic C: Soil I: 14.6 ; Soil II: 31.2
- Organic carbon content (% dry weight): Soil I: 0.588; Soil II: 1.2
- Nitrogen content (mg/100 g dry weight): Soil I: 0.9; Soil II: 0.8

DETAILS OF PREINCUBATION OF SOIL: The soils were stored for about 2 weeks under incubation conditions in closed plastic boxes. The soils were aerated at least every second day by thoroughly mixing until the determination of the microbial biomass which was prior to the start of the study

EFFECT PARAMETERS MEASURED:
In order to measure the short-term respiration of soil microbes, triplicate samples of 40-50 g of Soil I and 20 g of Soil II were taken from each treatment at 0-3 hours and 14 and 28 days after treatment. The samples were amended with a glucose-talc mixture, and the CO2 produced over a period of about 24 hours was measured.
Triplicate samples of 40g soil per treatment were taken from the lucerne-amended soil I at 0-3 hours and 14, 28, 56 and 91 days after treatment, and from soil II at 0-3 hours and 14 and 28 days after treatment. The samples were analysed for ammonia, nitrate and nitrite after extraction with 2N KCl solution.

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

Nominal: 0.20 and 2.0 mg/kg dry soil

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Remarks on result:

other: Highest concentration used

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other:

Remarks:

Highest concentration used

Details on results:

Test substance showed negligible effects in short-term respiration experiments up to a concentration in the soil corresponding to ten times the maximum recommended field rate, i.e. effects observed ranged from -2 to +8 % after 28 days of incubation. Furthermore, it was shown, that the ammonification in both soils was not affected by the test substance. Nitrication in the loamy soil was not affected. The slight stimulous effect still observed after 28 days on nitrification in the sandy loam soil at the highest treatment rate (N-min 21.3 % respect to control) was found to be transient, i.e. it disappeared after 91 days (N-min 1.5 % respect to control). It should be pointed out that the results from day 56 could not be used and hence this effect may have disappeared before than 91 days.

Reported statistics and error estimates:

The Dixon-test was used to eliminate outliers in the nitrification experiments. The test was performed with 3 values and a confidence interval of 10 %. The results observed in the respiration experiments after 28 days were statistically evaluated by the Dunnett-test (2n, 5%) to find significant variations.

Table 1. Effects of the test substance on glucose-induced short-term respiration

Treatment	0-3 hours		Day 14		Day 28
	Mean CO2(mg/hr/100gdwsoil)	Difference to control (%)	Mean CO2(mg/hr/100 gdw soil)	Difference to control (%)	Mean CO2(mg/hr/100 gdw soil)	Difference to control (%)
Soil I (Sandy loam)
Control	0.434	-	0.556	-	0.556	-
0.2 mg/kg	0.513	+ 18	0.641	+ 15	0.603	+ 8
2.0 mg/kg	0.474	+ 9	0.601	+ 8	0.585	+ 5
Soil II (Loam)
Control	1.159	-	1.006	-	0.966	-
0.2 mg/kg	1.090	- 6	1.104	+ 10	0.943	- 2
2.0 mg/kg	1.095	- 6	1.176	+ 17	0.984	+ 2

 

Table 2. Effects of the test substance on nitrification processes in sandy loam and loam soil

Treatment (mg/kg dry soil)	Day^a	Ammonium (NH4-N)		Nitrite (NO2-N)		Nitrate (NO3-N)		Total N
		mg/100 g dw soil	Differenceto control (%)	mg/100 g dw soil	Differenceto control (%)	mg/100 g dw soil	Differenceto control (%)	mg/100 g dw soil	Differenceto control (%)
Soil I (Sandy loam)
Control	0	0.28	-	<0.01	-	0.61	-	0.90	-
	14	0.05	-	0.03	-	0.19	-	0.26	-
	28	0.02	-	<0.01	-	0.83	-	0.86	-
	91	0.01	-	<0.01	-	4.18	-	4.21	-
0.2	0	0.22	-23.5	<0.01	0	0.58	-4.7	0.81	-10.5
	14	0.05	1.9	0.03	0.6	0.16	-14.0	0.24	-9.5
	28	0.02	8.6	<0.01	0	0.94	13.1	0.97	12.8
2.0	0	0.20	-29.0	<0.01	0	0.54	-11.9	0.75	-17.1
	14	0.04	-12.4	0.03	-0.6	0.14	-24.0	0.21	-19.2
	28	0.02	8.8	<0.01	0	1.01	21.9	1.04	21.3
	91	0.02	52.0	<0.01	0	4.24	1.4	4.27	1.5

 

Treatment (mg/kg dry soil)	Day^a	Ammonium (NH4-N)		Nitrite (NO2-N)		Nitrate (NO3-N)		Total
		mg/100 g dw soil	Differenceto control (%)	mg/100 g dw soil	Differenceto control (%)	mg/100 g dw soil	Differenceto control (%)	mg/100 g dw soil	Differenceto control (%)
Soil II (Loam)
Control	0	0.2	-	<0.01	-	0.5	-	0.8	-
	14	0.04	-	0.03	-	0.08	-	0.15	-
	28	0.0	-	<0.01	-	0.99	-	1.03	-
0.2	0	0.2	-32.9	<0.013	0	0.6	4.2	0.82	-7.9
	14	0.04	-0.4	0.030	0	0.06	-30.1	0.13	-16.8
	28	0.02	-12.7	<0.013	0	0.93	-6.4	0.96	-6.5
2.0	0	0.18	-38.1	<0.01	0	0.60	1.1	0.79	-11.6
	14	0.04	0.4	0.03	1.1	0.06	-32.8	0.13	-17.8
	28	0.0	-28.4	<0.01	0	1.05	6.1	1.08	5.1

a The samples taken on day 56 from soil I were analysed, but ambiguous results were obtained due to a technical defect of FIA- Analyser. Hence only the results on day 91 are reported.

Validity criteria fulfilled:

yes

Conclusions:

In a soil respiration and nitrification test, performed in accordance with the BBA Part VI 1-1guideline, the test substance had no adverse effects on the respiration and nitrification processes driven by soil micro-organisms in two agricultural soils at concentrations up to and including 2.0 mg/kg dry soil, i.e. the highest concentration tested.

Executive summary:

The influence of the test substance on soil microorganisms was determined by measuring the CO2 evolved during short-term respiration experiments after glucose amendments. The test was performed according to the BBA Part VI 1-1 guideline and in compliance with GLP. Furthermore, the amounts of NH4-N, NO2-N and NO3-N which were formed during the nitrification/mineralization of lucerne meal, were investigated. For this purpose, two agricultural soils, a sandy loam (Soil l) and a loam (Soil ll) were incubated with the test substance at 20 ± 2 °C for at least 28 days.

The test substance was applied as a solution prepared in acetone. A field rate of 150 g test substance/ha corresponding to 200 µg test substance per kg dry soil was applied. The respective concentration for the treatment corresponding to 10 times the field rate was 2000 µg/kg dry soil.

The test substance showed negligible effects in short-term respiration experiments up to a concentration in the soil corresponding to ten times the maximum recommended field rate, i.e. effects observed ranged from -2 to +8 % after 28 days of incubation.

Furthermore, it was shown, that the ammonification in both soils was not affected by the test substance. Nitrification in the loam was not affected. The slight stimulous effect still observed after 28 days on nitrification in the sandy loam soil at the highest treatment rate (N-min 21.3 % respect to control) was found to be transient, i.e. it disappeared after 91 days (N-min 1.5 % respect to control). It should be pointed out that the results from day 56 could not be used and hence this effect may have disappeared before than 91 days. Therefore, no negative effect of the test compound on organic matter turnover and hence on the soil fertility may be expected from the use of the test substance.

The test substance had no adverse effects on the respiration and nitrification processes driven by soil micro-organisms in two agricultural soils at concentrations up to and including 2.0 mg/kg dry soil, ie the highest concentration tested.

Description of key information

All available data was assessed. One study was available addressing the toxicity to soil microorganisms and its effects value was used as the key value:

Toxicity to soil microorganisms, NOEC 2.0 mg/kg dw soil, absence of effects, BBA VI, 1 -1, Wüthrich 1990

Additional information

One study is available for this endpoint, which was selected as key study. The influence of the test substance on soil microorganisms was determined by measuring the CO2 evolved during short-term respiration experiments after glucose amendments. The test was performed according to the BBA Part VI 1-1 guideline and in compliance with GLP. Furthermore, the amounts of NH4-N, NO2-N and NO3-N which were formed during the nitrification/mineralization of lucerne meal were investigated. For this purpose, two agricultural soils, a sandy loam (Soil l) and a loam (Soil ll) were incubated with the test substance at 20 ± 2 °C for at least 28 days. The test substance was applied as a solution prepared in acetone. A field rate of 150 g test substance/ha corresponding to 200 µg test substance per kg dry soil was applied. The respective concentration for the treatment corresponding to 10 times the field rate was 2000 µg/kg dry soil. The test substance had no adverse effects on the respiration and nitrification processes driven by soil micro-organisms in two agricultural soils at concentrations up to and including 2.0 mg/kg dry soil, ie the highest concentration tested.

Metabolites - additional information

In addition to the studies with the test substance, three toxicity studies on soil micro-organisms are available with the major degradation products (i.e. M4, M6 and M7), all performed in accordance with OECD TG 216 and OECD TG 217, and in compliance with GLP requirements. As the dossier has been prepared to address the test substance itself, these studies are not summarized as endpoint study records but are briefly discussed here. For metabolite M4 no significant effects on nitrogen and carbon transformation were observed at concentrations up to 0.155 mg/kg soil (Voelkel, 2004). For metabolite M6 no significant effects on nitrogen and carbon transformation were observed at concentrations up to 0.15 mg/kg soil (Van der Kolk, 2003). For metabolite M7 no significant effects on nitrogen and carbon transformation were observed at concentrations up to 0.123 mg/kg soil (Voelkel, 2001).