Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

07 Jun 2006 to 13 Jul 2006

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:

January 2000

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Version / remarks:

January 2000

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Application of Test Item: A stock solution was prepared by dissolving 9.1 mg of the test item in 3000 μL acetone. For the high dose an 1660-μL-aliquot and for the low dose an 500-μL-aliquot of the stock solution were added uniformly to 30 g of quartz sand (particle size about 0.1-0.3 mm). The solvent was completely evaporated to dryness in a stream of nitrogen. Thereafter, the sand mixture was homogenously mixed using a Vortex mixer. Before application the mixture was stored at about 4 °C for two days.
Each soil sample (150 g dry weight) received 1.5 g of the corresponding prepared quartz sand.
Intended amount [mg/kg dry soil] : 0.5 (low dose); 1.67 (high dose)
Amount applied [mg/kg dry soil]: 0.51 (low dose); 1.68 (high dose)
During application, the soil was thoroughly mixed by carefully swivelling the flasks. For the nitrification experiments, the soil samples were amended with 0.8 g lucerne meal (nitrogen content approximately 3%) after application. Finally, the soil moisture content of all samples was adjusted to 45% of the MWC by adding purified water.
Lucerne-grass meal
- Carbon: 40.6%
- Nitrogen: 2.5%
- C:N: 16.2:1

- Application of reference substances:
Dinoseb Acetate: Fortified sand was prepared at an amount corresponding to 3.78 mg dinoseb acetate per 1.5 g sand. Solutions of the reference item were prepared in acetone (39.35 mg dinoseb acetate in 1000 μL). An 960-μL-aliquot of the acetone solution containing the reference item was added uniformly to 15.0 g of quartz sand (particle size about 0.1- 0.3 mm). The solvent was completely evaporated to dryness in a stream of nitrogen. Thereafter the sand mixture was homogeneously mixed using a Vortex mixer. Portions of 1.5 g treated sand were filled into plastic vials and stored in a refrigerator (about 4°C) until application for two days. Each soil sample (150 g dry weight) received 1.5 g of the prepared quartz sand. The amount of sand represented 10 g per kg soil. The soil samples were mixed thoroughly after application by carefully swiveling the flasks. Following application, the soil samples were adjusted to 45% MWC and mixed thoroughly to achieve a uniform distribution of the test material applied.
Nitrapyrin: Fortified sand was prepared at an amount corresponding to 0.76 mg nitrapyrin per 1.5 g sand. Solutions of the reference item were prepared in acetone (8.48 mg nitrapyrin in 1000 μL). An 890-μL-aliquot of the acetone solution containing the reference item was added uniformly to 15.0 g of quartz sand (particle size about 0.1-0.3 mm). The sand mixture was homogeneously mixed using a Vortex mixer. Thereafter the solvent was completely evaporated to dryness in a stream of nitrogen. Portions of 1.5 g treated sand were filled into plastic vials and stored in a refrigerator (about 4°C) until application for two days. Each soil sample (150 g dry weight) received 1.5 g of the prepared quartz sand. The amount of sand represented 5.0 mg nitrapyrin per kg soil. The soil samples were mixed thoroughly after application by carefully swiveling the flasks. Following application and addition of lucerne meal for the nitrification samples, the soil samples were adjusted to 45% MWC.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2°C

Moisture:

45% of the soil MWC (i.e. 15.8 g water per 100 g dry soil).

Organic carbon content (% dry weight):

1.02

Nitrogen content (% dry weight):

0.06

Details on test conditions:

TEST SYSTEM
- Test container: 1-litre incubation flasks (stoppered with cotton wool plugs)
- Amount of soil: 150 g dry weight
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: bulk / series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site (latitude, longitude): Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), Speyer/Germany (latitude 49°11' N, longitude 8°11' E)
- Soil sampleing time: April 2006
- History of site: The soil has not been subjected to any pesticide or organic fertilizer treatment in the sampling year and four former years. In 2001 cucumber and in 2003 to 2005 pumpkins were planted. In 2002 it was fallow soil.
- % sand: 62.5
- % silt: 28.8
- % clay: 8.8
- Soil taxonomic classification: Sandy loam
- Soil classification system: USDA
- pH (in water): 7.4
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 37.3
- Maximum water holding capacity (in %): 35.0
- Cation exchange capacity (mmol/kg): 9
- Pretreatment of soil: The soil was sieved through a 2 mm sieve by the supplier. At the test facility, the soil was acclimatised at about 20°C for about one week.
- Initial microbial biomass as % of total organic C: 1.1

DETAILS OF PREINCUBATION OF SOIL
Before use the soil moisture content was determined. Thereafter, the soil was filled into incubation flasks and equilibrated at 20 ± 2°C in the dark for six days. The soil moisture content was adjusted to just below 45% of the maximum water holding capacity (MWC) prior to application. Furthermore, all samples used for the determination of the nitrification process were amended with lucerne meal as a source of organic nitrogen. The lucerne meal contained approximately 3% nitrogen. The pH of the soil at start and at end of incubation in water was 7.38 and 7.43, respectively.

EFFECT PARAMETERS MEASURED
- Sampling Interval: Respiration and nitrification were determined for all treatments at the intervals of 0 - 3 hours, 6 days (for the nitrification), 7 days (for the respiration), 14 and 28 days after treatment.
- Microbial Biomass: Bulk samples of untreated soil were prepared for microbial biomass analysis by sieving and adjusting moisture content to 45% of its MWC. Seven subsamples of 40 g wet soil each (corresponding to 34.6 g dry soil) were mixed with different concentrations of glucose and talc. The talc served as an inert ingredient to improve the homogeneous distribution of the glucose in the soil. For the biomass determination, only those glucose concentrations providing the three highest initial and constant CO2 production rates (substrate saturation conditions) were considered (mixtures 4-6). The CO2 production rates were calculated as an average value (Vco2) from the rate determinations during the constant initial respiration period (lag phase). The microbial biomass was then calculated according to Anderson & Domsch.
- Glucose Induced Short-Term Respiration (Carbon Mineralisation Test): For the short-term respiration experiment the glucose concentration, which exerted a maximum respiration response (mixture 5) was added to the soil samples after sampling. Triplicate aliquots of 40 g wet soil (corresponding to 34.56 g dry soil) were amended with the glucose-talc mixtures, namely 1.16 g glucose/kg dry soil. After amending the soil samples with glucose, the short-term respiration rates were measured by analysing evolved CO2 for about 19 to 23 hours. In order to evaluate the influence of the test item on carbon mineralisation in soil, the respiration rates of treated and control soil during the first 12 consecutive hours were compared. The dinoseb acetate treated samples were compared to the control samples in order to show the sensitivity of the test system and method.
- Nitrification of Lucerne Meal - Determination of Nitrite and Nitrate: After adjustment to the target moisture, triplicate aliquots of about 47 g wet soil (corresponding to 40 g dry soil) of the sample were taken at each sampling day. These aliquots were extracted for 15 minutes with 2 M KCI (29 mL) by shaking them at room temperature on a horizontal shaker. After centrifugation at about 2000 x gmax for 10 minutes, the supernatant liquid was decanted through fluted filter paper. The extraction process was repeated using 29 mL 2 M KCI. The combined aqueous extracts were adjusted to a final volume of 50 mL with 2M KCI. The concentrations of nitrite- and nitrate- nitrogen were then determined in triplicate using a Flow Injection Analyser. The system was calibrated using standard solutions. The initial nitrogen content (nitrate and nitrite) of the soil was measured in unamended and untreated soil samples at Day 0.
- Nitrite (NO2-) Analysis: The determination of nitrite is based on a colorimetric method. In this method, the nitrite ion reacts with sulfanilamides under acidic conqitions to yield a diazo compound which is coupled with N-1-naphthylethylene-diamine dihydrochloride to form a soluble dye. The concentration is measured colorimetrically at a wavelength of 550 nm. The limit of quantitation is 0.02 mg/L extract, corresponding to 0.08 mg/kg dry soil.
- Nitrate (NO3-) Analysis: Nitrate is reduced to nitrite in a cadmium reductor. The nitrite is then determined as described above. The method determines the sum of free nitrite and nitrate simultaneously [NO2-) + (NO3-)]. Hence, the NO3- concentrations are calculated by subtracting NO2- as measured above from the [(NO2-) + (NO3-)] concentration. The limit of quantitation is 0.1 mg/L extract, corresponding to 0.55 mg/kg dry soil.

VEHICLE CONTROL PERFORMED: No

Nominal and measured concentrations:

- Low dose: 0.5 mg/kg soil dw
- High dose: 1.67 mg/kg soil dw

Reference substance (positive control):

yes

Remarks:

Dinoseb acetate (respiration); Nitrapyrin (nitrification)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1.67 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

respiration rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1.67 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

nitrate formation rate

Details on results:

An overview of the results is provided in Table 1 - Table 3 in 'Any other information on results incl. tables'
MICROBIAL BIOMASS AND OPTIMUM GLUCOSE AMENDMENT
The maximum rate of initial CO2 evolution from untreated soil was 0.262 mL per hour and 100 g dry soil. By applying the formula of Anderson and Domsch (1978), the microbial biomass, expressed as microbial carbon per kg of dry weight soil (microbial C/kg dry weight soil) was calculated to be 108.6 mg microbial carbon (C). Amendments with different amounts of glucose/kg dry soil resulted in a maximum and constant respiration response. The lowest amount of glucose resulting in a maximum CO2 production, i.e. 1.16 g/kg dry soil was used for the short-term respiration experiment.

GLUCOSE INDUCED SHORT-TERM RESPIRATION
Respiration can be regarded as a measure of the general turn-over of organic matter in soil. Respiration levels were determined by monitoring glucose-induced evolution of CO2 resulting from microbial activity during short-term experiments (for about 19 to 23 hours each). The respiration rates during 12 consecutive hours, for treated and control soil, were compared in order to determine possible side effects of the test item on microorganisms. In the present study, the influence of the test item on glucose-induced soil respiration was monitored in short-term experiments over an incubation period of 28 days. The rate of respiration at test initiation was 8.54 mg CO2/h per kg dry soil for the control, 8.44 mg CO2/h for the low dose and 8.31 mg CO2/h for the high dose treated samples. In comparison to the control, decreases in respiration of 1.2% and 2.7% were observed for the low and high dose treated samples, respectively. On Day 7 mean decreases of 1.4% and 3. 7% were observed for the two dose rates in comparison to the control. By Day 14 mean respiration was between 1.7% and 3.0% below the mean control respiration. After 28 days of incubation, the rate of respiration was 10.56 mg CO2/h for the control, 10.54 mg and 10.37 mg CO2/h for the low and high dose treated soil samples, respectively. This resulted in a deviation of -0.2% and -1.8% from the control for the low and high dose treated samples, respectively. Thus, no influence of the test substance on microbial respiration in soil Speyer 2.3 was observed for the two treatments.

NITRIFICATION OF LUCERNE MEAL
The microbial conversion of organic nitrogen to nitrate is a multi-step process. In a primary reaction, soil organic matter is mineralised to ammonia (ammonification, mineralisation). A wide range of possible sinks of ammonia are reported, e.g. immobilisation by soil microorganisms by incorporation and formation of amino acids, and ion exchange or adsorption with soil constituents. Furthermore, ammonia is converted via nitrite to nitrate, a process which is designated as nitrification. This step represents the second step in organic matter conversion. The nitrification processes are considered to be important for soil fertility. Chemicals may affect the various micro-organism species/populations, which carry out the conversion of organic matter to ammonium, nitrite and nitrate. Potential effects of chemicals on nitrification may be assessed by comparing the concentrations of nitrite and nitrate formation in control and treated soils.
- Nitrification: Initially, no nitrite (NO2-) and 37.31 mg nitrate (NO3-) per kg dry soil were detected in untreated and unamended soil, respectively. On Day 0, the N02- concentration increased to 2.1 % for the low and decreased to 0.5% for the high dose treated samples, respectively. Thereafter, the N02- concentration was below the limit of determination (< 0.08 mg/kg dry soil) in all samples. On Day 6, the N02- concentration was in single samples just above the limit of determination. Therefore, calculated deviations in percent are not significant. Thus, the treatment with the test item had no influence on the nitrite formation and transformation. In the nitrapyrin treated samples the amount of No2- per kg dry soil was 0.21 mg NO2-/kg dry soil and was thereafter below the limit of determination. The mean initial concentration of nitrate was 41.7 mg for the control, 41.3 mg and 41.8 mg for the low and high dose treated samples, respectively (Table 4 and Figure 7). Mean nitrate levels decreased until Day 6 and increased thereafter in all sample types, reaching 72.4, 70.4 and 83.1 mg, respectively, on Day 28. The calculated deviation to control was -2.7% and 14.8%, respectively. Thus, after 28 days no influence of the test item on nitrate formation was observed up to 10 times the maximum expected concentration in the field.
The values obtained for the low dose on Days 6 to 28 and for the high dose on Days 14 and 28, were significantly different from the control (Dunnett-test, two sided, a = 0.05). However, the deviation from control was far below the trigger value of 25% given by the test guideline.

Results with reference substance (positive control):

An overview of the results is provided in Table 1 - Table 3 in 'Any other information on results incl. tables'
The dinoseb acetate treated samples showed an increasing inhibiting effect when compared to the non-treated samples. The deviation from the control was -45.4% on Day 0, -30.8% (Day 7), -49.8% (Day 14) and -57.6% (Day 28). Thus, the reference item dinoseb acetate showed a clear inhibitory effect.
The nitrate concentration on Day 28 for the nitrapyrin treated samples was 12.6 mg NO3-/kg dry soil. This resulted in a deviation of -82.6% to the control. Thus, the reference item nitrapyrin showed a strong inhibiting effect.

Reported statistics and error estimates:

In the respiration and nitrification experiments, the mean of individual values at the end of their respective incubation period were statistically evaluated on a 5%-significance level (a= 0.05, two-sided) by the Dunnett-test (Days 0 to 28) to find significant differences between control and treated samples (Dunnett, 1955 and 1964; Sachs, 1999).

Table 1. Influence of the test item and dinoseb acetate on glucose-induced short-term respiration of Soil Speyer 2.3.

	Incubation time [days]	Respiration rates
		[mg CO2/h per kg dry soil]
		Replicates					%SD	% Deviation from control
		a	b	C	Mean	[SD]
Control	0	7.99	8.88	8.75	8.54	0.48	5.6	-
	7	8.66	9.12	9.10	8.96	0.26	2.9	-
	14	8.22	8.84	8.69	8.58	0.33	3.8	-
	28	10.18	10.75	10.73	10.56	0.32	3.1	-
Low dose	0	8.54	8.48	8.30	8.44	0.13	1.5	-1.2
	7	9.00	8.73	8.77	8.83	0.15	1.7	-1.4
	14	8.60	8.40	8.30	8.44	0.15	1.8	-1.7
	28	10.68	10.34	10.59	10.54	0.18	1.7	-0.2
High dose	0	8.27	8.36	8.31	8.31	0.05	0.5	-2.7
	7	8.65	8.66	8.59	8.63	0.04	0.5	-3.7
	14	8.28	8.36	8.34	8.33	0.04	0.5	-3.0
	28	10.48	10.34	10.28	10.37	0.10	1.0	-1.8
Dinoseb acetate	0	4.98	4.48	4.53	4.66*	0.27	5.9	-45.4
	7	6.36	5.99	6.27	6.21*	0.20	3.1	-30.8
	14	4.58	4.26	4.10	4.31*	0.25	5.7	-49.8
	28	4.50	4.43	4.51	4.48*	0.04	1.0	-57.6

SD: Standard deviation

* Value is significantly different from the control (Dunnett-test, two-sided, a = 0.05)

Table 2. Influence of the test item and nitrapyrin on nitrite formation after amendment of lucerne meal. 

	Incubation time (days)	Nitrite [mg NO2- / kg dry soil]
		Replicates						% Deviation from control
		a	b	C	Mean	SD	%SD
Control	0	0.19	0.19	0.20	0.19	0.005	2.5	- - - -
	6	0.09	<0.08	0.09	<0.08	0.050	86.6
	14	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.
	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.
Low dose	0	0.19	0.20	0.20	0.20	0.004	2.1	2.1
	6	0.08	0.09	0.09	0.09	0.004	4.8	48.3
	14	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
High dose	0	0.19	0.19	0.19	0.19	0.002	1.2	-0.5
	6	0.09	0.08	<0.08	<0.08	0.050	86.9	<0.1
	14	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
Nitrapyrin	0	0.21	0.20	0.21	0.21*	0.006	3.0	7.3
	6	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	-100.0
	14	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.
	28	<0.08	<0.08	<0.08	<0.08	<0.001	n.a.	n.a.

SD:Standard deviation

n.a.: not applicable

* Value is significantly different from the control (Dunnett-test, two-sided, a = 0.05)

Table 3. Influence of the test item and nitrapyrin on nitrate formation after amendment of lucerne meal.

	Incubation time [days]	Nitrate [mg NO3-/ kg dry soil]
		Replicates						% Deviation from control
		a	b	C	Mean	SD	%SD
	0	42.5	41.2	41.3	41.7	0.8	1.8	- - - -
Control	6	10.4	10.4	10.3	10.4	0.1	0.6
	14	32.0	32.1	32.1	32.1	0.1	0.3
	28	73.1	72.4	71.6	72.4	0.7	1.0
	0	40.3	41.5	42.1	41.3	0.9	2.3	-0.8
Low dose	6	11.4	11.2	10.9	11.2*	0.2	2.0	7.8
	14	31.0	31.2	31.1	31.1*	0.1	0.3	-3.1
	28	69.8	70.9	70.5	70.4*	0.6	0.8	-2.7
	0	42.1	41.2	42.0	41.8	0.5	1.2	0.3
High dose	6	10.2	10.2	9.7	10.0	0.3	2.7	-3.2
	14	36.1	35.3	36.1	35.8*	0.5	1.4	11.7
	28	83.2	83.3	82.7	83.1*	0.3	0.4	14.8
	0	40.3	40.2	41.5	40.7	0.7	1.8	-2.4
Nitrapyrin	6	3.5	3.6	3.6	3.6*	<0.1	0.9	-65.6
	14	4.8	4.8	4.7	4.8*	0.1	1.1	-85.1
	28	12.9	12.6	12.4	12.6*	0.3	2.0	-82.6

SD: Standard deviation

Value is significantly different from the control (Dunnett-test, two-sided, a = 0.05)

Validity criteria fulfilled:

yes

Remarks:

variation between replicate control samples < 15% and variation between control and treated soil samples < 25%

Conclusions:

In a toxicity test to soil microorganisms, performed in accordance with OECD TG 216 and 217, the test substance caused no effects on soil respiration (measured as CO2 production) and on nitrogen transformation (measured as NO2-N and NO3-N formation) by the end of the 28-day incubation period at the highest treatment tested (1.67 mg/kg dry soil).

Executive summary:

The influence of the test substance on soil microorganisms was determined by measuring the soil respiration (CO2 production) and nitrification processes (NO2-N and NO3-N formation) according to the OECD TG 216 and 217. The study was conducted in compliance with GLP criteria. The test substance was added to quartz sand as a solution in acetone. After evaporation of the acetone the treated quartz sand was mixed into batches of a sandy loam soil (pH (water) 7.4, 1.02% OC, microbial biomass 108.6 mg C/kg (i.e. 1.1% of the total soil organic carbon)). The treatment rates were 0.5 and 1.67 mg/kg soil d.w. Control soil was treated in the same way, but without the addition of test substance. Positive controls, Dinoseb acetate (25 mg/kg soil d.w.) and Nitrapyrin (5 mg/kg soil d.w.) were included for respiration and nitrification tests, respectively. Aliquots (150 g d.w.) were dispensed into 1 L incubation flasks closed with cotton wool plugs (3 replicate per treatment/control), which were incubated at 20 ± 2°C. Soil moisture was maintained at 45% soil MWHC. For nitrification, soil was amended with lucerne meal (0.5%). Respiration and nitrification were determined for all treatments at the intervals of 0-3 hours, 6 days (for the nitrification), 7 days (for the respiration), 14 and 28 days after treatment.

The respiration rate in the control was 8.54 mL CO2/hour/kg soil d.w. on day 0. This rate increased to 10.56 mL CO2/hour/kg soil d.w. on day 28. Respiration of treated soil showed negligible differences (≤ 3%) compared to the untreated soil at all sampling points in both low and high dosages. The positive control showed as low as 57.6 % inhibition on the respiration on day 28 compare to the control group. Initially, no nitrite (NO2-) and 37.31 mg nitrate (NO3-) per kg dry soil were detected in untreated and unamended soil, respectively. On Day 0, the NO2- concentration increased to 2.1 % for the low dose treatment, and decreased to 0.5% for the high dose treated samples. Thereafter, the NO2- concentration was below the limit of determination (< 0.08 mg/kg dry soil) in all samples. On Day 6, the NO2- concentration in two replicates was just above the limit of determination. Therefore, the calculated deviations in percent were not significant. Thus, the treatment with the test item had no influence on the nitrite formation and transformation. In the nitrapyrin treated samples the amount of nitrite was 0.21 mg/kg dry soil and was thereafter below the limit of determination. The nitrate formation level of the control replicates was around 42 mg/kg soil dw on day 0. This number increased to 72 mg/kd soil dw on day 28. Compare to the control, the test substance treated groups showed 2.7% inhibition and 14.8 stimulation on day 28 for the low dose and high does, respectively. The positive control showed 82.6% inhibition on nitrate formation on day 28 compare to the control. Based on the findings, it is concluded that the NOEC value for the respiration and nitrogen transformation is ≥ 1.67 mg/kg soil dw.

Description of key information

All available data was assessed. The study representing the lowest endpoint was included here and its effect value was used as the key value. Another study is included as supporting information.

28-d NOEC ≥ 1.67 mg/kg dry soil (sandy loam), respiration and nitrification, OECD TG 216 and 217, Völkel 2001

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1.67 mg/kg soil dw

Additional information

Two studies are available for this endpoint, both in accordance with standard guidelines and GLP compliant. In the first study, the influence of the test substance on soil microorganisms was determined by measuring the soil respiration (CO2 production) and nitrification processes (NO2-N and NO3-N formation) according to OECD TG 216 and 217. The test substance was added to quartz sand as a solution in acetone. After evaporation of the acetone, the treated quartz sand was mixed into batches of a sandy loam soil (1.02% OC, microbial biomass 108.6 mg C/kg). The treatment rates were 0.5 and 1.67 mg/kg soil dw. Control soil was treated in the same way, but without the addition of test substance. Positive controls, Dinoseb acetate (25 mg/kg soil dw) and Nitrapyrin (5 mg/kg soil dw) were included for respiration and nitrification tests, respectively. Aliquots (150 g soil dw) were dispensed into 1 L incubation flasks closed with cotton wool plugs, which were incubated at 20 ± 2°C. Soil moisture was maintained at 45% soil MWHC. For nitrification, soil was amended with lucerne meal (0.5%). After 28 days incubation, the test substance showed no significant effects on the respiration and nitrification processes driven by soil micro-organisms in the test soil at concentrations up to the highest treatment rate of 1.67 mg/kg dry soil. Thus, the NOEC for respiration and nitrification was determined to be ≥ 1.67 mg/kg dry soil (Völkel 2001). This result represents the worst-case (i.e. showed the lowest NOEC value). In addition, studying the adverse effects of a substance on the process of carbon and nitrogen transformation over 28 days is the preferred test for the soil microorganism toxicity endpoint according to Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7c: Endpoint specific guidance (version June 2017). Therefore, this study is selected to be the key study.

In the second study, the effects of the test substance on the growth of several fungi cultures in a laboratory test were assessed over a test period of maximum 4 days of exposure according to IOBC guideline (modified for soil fungi). The fungi (Mucor circinelloides and Phytophthora nicotianae) were exposed to 0.125, 0.25, 0.5, 1.0, 2.0 and 40.0 mg/kg soil dw. Two days before test start, the test item was mixed with sterilized soil (autoclaved) and subsequently incubated for 2 days at 20 - 25 °C in an incubator. At test start, the test soil was weighed into sterilized petri dishes (10 g soil dry weight/petri dish). Nutrient agar was placed on top of the soil, and then a piece of mycelium inoculum (5 mm diameter) was placed to the center of each plate. The inoculated petri dishes were then incubated at 20 - 25 °C in an incubator for 3 days (M. circinelloides) and 4 days (P. nicotianae). The growth of both fungal species was not significantly reduced in ≤ 2.0 mg/kg soil dw groups. Thus, the 28-d NOEC was determined to be 2.0 mg/kg soil dw for the growth of both fungi species (Schulz 2009).