cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

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 1986 to 28 Oct 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: Guidelines of the Bureau of Plant Protection Products, Plant Disease Service, Wageningen, The Netherlands, Section H.4.1. and regarding the proposed modifications (Recommended Tests for Assessing the Side-Effects of Pesticides on the Soil Nicroflora)

Version / remarks:

May 1983

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

SOIL RESPIRATION TEST
- Treatment A: soil samples treated with deionised water to reach 35 % of the maximum water holding capacity and mixed with 0.5 % (w/w) lucerne meal.
- Treatment B: as A, additionally treated with 0.25 ppm test substance corresponding to a field dose of 0.12 kg/ ha (the concentrations were obtained when the highest recommended field dose (0.12 kg/ ha and ten times this amount) would be homogeneously distributed in the 5 cm top layer of the soil).
- Treatment C: as A, additionally treated with 2.5 ppm test substance corresponding to a field dose of 1.2 kg/ha.
- Application Solution of the test substance: Add 20.9 mg test substance in 500 mL deionised water.

- Application to soil:
- Treatment A: 800 g of air-dried soil were intensively stirred in the kitchen mixer with 104 mL deionised water and 4 g of lucerne meal (total duration about 30 minutes).
- Treatment B: 800 g of air-dried soil were intensively mixed with 50 mL deionised water, 4 g of lucerne meal, 5.0 mL of the application solution and finally 49 mL deionised water (total duration about 30 minutes).
- Treatment C: 800 g of air-dried soil were intensively mixed with 50 mL deionised water, 4 g of lucerne meal, 50 mL of the application solution and finally 4 mL deionised water (total duration about 30 minutes).

SOIL NITRIFICATION TEST
- Treatments A: Soil samples treated with deionised water to reach 35 % of the maximum water holding capacity.
- Treatment B: As Treatment A, amended with 38.8 mg ammonium sulphate/100 g dry soil (= 10.0 mg NH3)/100 g dry soil).
- Treatment C: As Treatment B, additionally treated with 0.25 ppm test substance corresponding to a field dose of 0.12 kg/ha (the concentrations were obtained when the highest recommended field dose (0.12 kg/ha and ten times this amount) would be homogeneously distributed in the 5 cm top layer of the soil).
- Treatment D: As Treatment B, additionally treated with 2.5 ppm test substance corresponding to a field dose of 1.2 kg/ha.
- Application Solutions: Add 20.9 mg test substance to 500 mL deionised water; 3880 mg ammonium sulphate in 100 mL deionised water(= 10.0 mg NH3/mL)

- Application to soil:
- Treatment A: 1400 g of air-dried soil were intensively stirred in the kitchen mixer with 181 mL deionised water (total duration about 30 min.).
- Treatment B: 1400 g of air-dried soil were intensively mixed with 167 mL deionised water and 14 mL ammonium sulphate solution (total duration about 30 minutes).
- Treatment C: 1400 g of air-dried soil were intensively mixed with 158 mL deionised water, 14 mL ammonium sulphate solution and 9 mL application solution of the test substance (total duration about 30 minutes).
- Treatment B: 1400 g of air-dried soil were intensively mixed with 79 mL deionised water, 14 mL ammonium sulphate solution and 88 mL application solution of test substance (total duration about 30 minutes).

Test organisms (inoculum):

soil

Total exposure duration:

35 d

Test temperature:

Soil resperation and nitrification: 22 ± 2 °C

Moisture:

Soil resperation and nitrification: 35 % of the maximum water holding capacity

Organic carbon content (% dry weight):

2.2

Nitrogen content (% dry weight):

13.1

Details on test conditions:

TEST SYSTEM (soil respiration)
- Testing facility: 500 mL Erlenmeyer flasks and connected tightly with the trapping apparatus
- Amount of soil: 227 g of soil (corresponding to 200 g air-dried soil)
- No. of replicates per concentration: 3
- No. of replicates per negative control: 1 (one Erlenmeyer flask containing no soil sample was connected with the system to be used as blank experiment)
- Others: The system was continuously ventilated with carbon dioxide free air at a rate of about 30 mL/ min. The outcoming air was passed through the collectors filled with 150 mL 0.2 N sodium hydroxide.

SOURCE AND PROPERTIES OF SUBSTRATE (soil respiration)
- Geographical reference of sampling site: Field soil from Flaach (canton of Zurich, Switzerland) was freshly collected by the test facility, Field Trial Department and immediately transported to the testing facility.
- Organic matter content: 3.8%
- Organic carbon content: 2.2%
- pH: 7.6
- % Lime: 7.8%
- % sand: 68.5%
- % silt: 19.4%
- % clay (< 20 µm): 12.1%
- Soil taxonomic classification: Sandy loam soil of low organic matter content
- Ammonium: < 0.2 mg/100 g soil
- Maximum water holding capacity: 37.1 ± 0.5%
- Nitrite: < 0.01 mg/100g soil
- Nitrate: 13.1 mg/100 g soil

SOIL INCUBATION (soil respiration)
At room temperature in dark

DETAILS OF PREINCUBATION OF SOIL (soil respiration)
Immediately after receipt, about 5 kg of the soil were air-dried and sifted by passing it through a 36 mesh/cm2 sieve. The soil was then used for the respiration study without further pretreatment. An additional amount of about 15 kg of soil was stored in a refrigerator at about 5˚C and open to air, until it was used for the investigation of soil nitrification.

EFFECT PARAMETERS MEASURED (soil respiration)
The amount of CO2 liberated by the soils was determined after 2, 4, 7, 10, 14, 21, 28 and 35 days.

TEST SYSTEM (soil nitrification)
- Testing facility: 300 mL Erlenmeyer flasks and closed with a plug of cotton.
- Amount of soil: 113 g of soil (corresponding to 100 g air-dried soil)
- No. of replicates per concentration: 12

SOIL INCUBATION (soil nitrification)
The Erlenmeyer flasks were stored at 22 ± 2 ˚C in the dark. Twice a week, the soil samples were brought to the original moisture content by adding deionised water.

EFFECT PARAMETERS MEASURED (soil nitrification)
- Sampling: Two soil samples from each treatment a - d were analysed immediately after the preparation, as well as after 3 and 7 days
- Extraction of Soil: The soil samples were mixed with 100 mL 2 N potassium chloride solution, shaken for 15 minutes on the reciprocal shaking machine and then centrifuged at 3000 rpm for 5 minutes. The clear supernatant solution was filtered through a fluted filter paper into a 200 mL volumetric flask. The procedure was repeated with another 100 mL of 2 N potassium chloride solution and filtered into the same flask. The combined aqueous extracts were made up to the mark with 2 N potassium chloride solution.

Determination of Ammonia
An aliquot of 2.0 mL of the soil extract was diluted to 50 mL with 2 N potassium chloride solution and the amount of ammonia was determined using the Spectroquant method No. 14752.
5.0 mL of the diluted extract solution were mixed with 0.65 mL of the reagent NH4 -1B and one spoonful of the reagent NH4 -2B and shaken vigorously. After 5 minutes, four drops of the reagent NH4-3B were added and, after shaking, allowed to react for 5 minutes. Within one hour, the yellow or green solution was transferred into the cell and assayed in the spectrophotometer between 450 nm and 700 nm against an extract from the treatment a which was reacted in the same way.
The maximum absorption at about 690 nm, corrected for the baseline in the absorption minimum between 470 and 530 nm, was read and the concentration of mg NH3/100 g dry soil, was calculated from the calibration curve.

- Calibration Curve: Aliquots of 2.0 mL of the soil extract from the treatment a were pipetted into 50 ml volumetric flasks. Each 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 ml of an ammonium sulphate solution containing 3.88 mg in 100 ml 2 N potassium chloride solution (by dilution of 1 mL of the application solution to 1000 mL with deionised water) were added and made up to volume with 2 N potassium chloride solution.
Aliquots of 5.0 mL of these solutions which contained between 0.1 and 20 µg NH (corresponding to 0.1 and 20 mg/100 g dry soil) were reacted using the Spectroquant method and measured as described above. The so obtained absorption values were plotted against the calculated NH3 concentrations in µg / 5 ml solution or in mg/100 g dry soil.
- Limit of determination: 0.1 mg NH3/100 g dry soil
- Range of linearity: 0.1 - 20 mg NH3/100 g dry soil
- Precision: ± 5%
DETERMINATION OF NITRITE
An aliquot of 5.0 mL of the soil extract was mixed with a spoonful of the Spectroquant nitrite reagent, shaken vigorously and allowed to react for 10 minutes. The violet solution was transferred into the cell and assayed in the spectrophotometer between 410 and 700 nm against an extract from the treatment a which was treated in the same way.
The maximum absorption between 520 and 550 nm was read and corrected for the baseline (obtained by drawing the absorption minimum line between 410 and 700 nm). The concentration of the nitrite as mg NO2/100 g dry soil, was calculated from the calibration curve.
- Calibration Curve: The following standard sodium nitrite solutions were prepared:
- Standard solution 1: 75 mg sodium nitrite in 1000 mL deionised water= 50 µg NO2-/ml
- Standard solution 2: 10 mL of solution 1 in 100 mL deionised water = 5 µg NO2-/mL
- Standard solution 3: 10 mL of solution 2 in 100 mL deionised water = 0.5 µg NO2/Ml
Aliquots of 1.0 mL of the standard solution 1, respectively 4.0, 2.0 and 1.0 mL of the standard solution 2, as well as 4.0 and 2.0 mL of the standard solution 3 were pipetted into 20 mL volumetric flasks. Aliquots of 16 mL of the soil extract from treatment a were added and the flasks were made up to volume with deionised water.
Aliquots of 5.0 ml of these solutions which contained 12.5, 5, 2.5, 1.25, 0.5 or 0.25 µg NO2-/5 mL (corresponding 0.5 to 0.01 mg NO2-/100 g dry soil) were reacted with the Spectroquant reagent and measured as described above. The so obtained absorption values were plotted against the calculated NO2- concentrations in µg/5 mL solution or in mg/100 g dry soil
- Limit of determination: 0.01 mg NO2-/100 g dry soil
- Range of linearity: 0.01 - 0.5 mg NO2-/100 g dry soil
- Precision: ± 5%

DETERMINATION OF NITRATE
An aliquot of 2.0 mL of the soil extract was diluted to 50 mL with 2 N potassium chloride solution. 5.0 mL of this solution were pipetted into a reduction column which has been filled with amalgamated cadmium powder to a height of about 15 cm, and allowed to react for 10 minutes. The nitrite so obtained was washed through the column with five lots of about 10 mL deionised water and collected in a 50 mL volumetric flask. The flask was made up to volume with deionised water. An aliquot of 5.0 mL of this solution was mixed with the Spectroquant reagent and treated in the same way as described for the determination of the nitrite content. The blank solution was prepared from 5.0 mL of 2 N potassium chloride solution which has been taken through the procedure.
The calculation was done by the following equation: NO3(mg/100 g dry soil) = S x D x E
S = mg NO2-/ 100 g soil read from the nitrite calibration curve
D = dilution factor, in this case D = 250
E = calculation factor for NO3-, E = 1.348.
- Limit of determination: 2.5 mg NO3-/100 g dry soil
- Range of linearity: 2.5 - 12.5 mg NO3-/100 g dry soil
- Precision: ±10%

Nominal and measured concentrations:

- Nominal concentrations (respiration and nitrification): 0 (control), 0.25 mg/kg dry soil, 2.5 mg/kg dry soil

Reference substance (positive control):

no

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Respiration and nitrification

Details on results:

An overview of the results is provided in Table 1 – Table 4 in ‘Any other information on results incl. tables’

SOIL RESPIRATION
Without any test substance treatment (samples A), the total quantities of CO2 evolved within 5 weeks were between 470 mg and 965 mg per 200 g air-dried soil. The average of the three replicates was 744 mg with a standard deviation of 251 mg. This figure represents the value of the control experiment and it is therefore taken as 100% (relative standard deviation of 34%). After application of 0.25 ppm test substance (treatment B) to the soil the CO2 formation resulted in cumulative amounts of 301 mg to 1039 mg. The average amount was calculated to be 790 mg or 106% relative to the control experiment (standard deviation of 423 mg, respectively relative standard deviation of 57%). The total quantities of CO2 yielded from the soil fortified with 2.5 ppm (treatment C) were in the range between 658 mg and 1044 mg, the average being 911 ± 219 mg(= 122 ± 29 % relative to the control experiment).
These figures let us conclude that the average quantities of carbon dioxide formed in the test substance treated soil samples do not significantly differ from those produced in the untreated ones. Regarding the single values from the corresponding three replicates, a relative large error can be seen. However, since the "outliers" exist in all three treatments, we conclude that these deviations are caused by the experimental conditions and are not a consequence of the test substance treatment.

SOIL NITRIFICATION
All soil samples, the untreated as well the treated ones, contained an average quantity between 9.3 and 9.9 mg NH3/100 g dry soil immediately after fortification. After 3 days, the level of ammonia in the untreated samples declined to 3.8 mg, that one in the treated samples to 5.8 - 6.1 mg. 4 days later, the concentrations of NH3 were below the limit of determination in all samples analysed (< 0.2 mg NH3/100 g soil). Nitrite was detected in measurable but very low quantities only at the beginning of the experiment and after 3 days. Then, it was rapidly oxidised to nitrate and thus was no longer present in all the soils sampled 7 days after fortification (< 0.01 mg/100 dry soil).

The formation of nitrate paralleled the degradation of ammonia under all conditions tested (the reported and tabulated figures for NO3- give the amounts exceeding the naturally present amounts in the soil (= 13.1 mg/100 g dry soil). 3 days after treatment, the concentration of nitrate was between 13.2 and 16.8 mg (36 % - 46 % relative to the amounts of NH3 applied). At the end of the experiment, the concentration of nitrate produced was in good accordance with that ones expected from the actual concentrations of ammonia added at the beginning of the study (88 - 95 %) (10 mg NH3 yield 36.5 mg NO3- at a complete conversion rate).
We conclude that the conversion of the incorporated ammonia is very rapid and being completed within less than 7 days under all conditions tested. It is not significantly affected in the soils fortified with 0.25 and 2.5 ppm test substance. Furthermore, the formation of nitrite which is an intermediate of the nitrification process is very low. This observation is quite normal, if the nitrification process is not inhibited. The subsequent oxidation process to nitrate, the end-product of the nitrogen cycle, is found to occur in almost quantitative yield.
In conclusion it can be pointed out that treatment of the soil with 0.25 and 2.5 ppm of the test substance shows the absence of any toxic and inhibiting factors towards nitrification of microorganisms.

Table 1. Soil Respiration of Lucerne Meal Amended Flaach Soil (Control, Without test substance Treatment); Values Given Represent mg CO2 evolved/ 200 g Dry Soil

Intervals(days)	mg carbon dioxide evolved/200 g dry soil
	sample A1	sample A2	sample A3	Average A
0 - 2	273	41	66	127
2 - 4	118	37	87	81
4 - 7	143	69	134	115
7 - 10	89	32	99	73
10 - 14	86	80	107	91
14 - 21	107	114	128	116
21 - 28	47	40	90	59
28 - 35	102	57	87	82
0 - 35	965	470	789	741 ± 251
% of average	130	63	106	100 ± 34

 

Table 2. Soil Respiration of Lucerne Meal Amended Flaach Soil (Soil treated with 0.25 mg a.i./kg dry soil test substance); Values Given Represent mg CO2 evolved/ 200 g Dry Soil

Intervals(days)	mg carbon dioxide evolved/200 g dry soil
	sample A1	sample A2	sample A3	Average A
0 - 2	260	44	273	192
2 - 4	129	24	123	92
4 - 7	129	77	128	111
7 - 10	87	21	103	70
10 - 14	131	51	108	97
14 - 21	125	50	129	101
21 - 28	64	0	82	49
28 - 35	114	34	84	77
0 - 35	1039	301	1030	790 ± 423
% of control	140	41	139	107 ± 57

Table 3. Soil Respiration of Lucerne Meal Amended Flaach Soil (Soil treated with 2.5 mg a.i./kg dry soil test substance); Values Given Represent mg CO2 evolved/ 200 g Dry Soil

Intervals(days)	mg carbon dioxide evolved/200 g dry soil
	sample A1	sample A2	sample A3	Average A
0 - 2	253	89	239	194
2 - 4	132	80	133	115
4 - 7	140	130	133	134
7 - 10	103	78	105	95
10 - 14	105	97	110	104
14 - 21	139	84	128	117
21 - 28	80	25	92	66
28 - 35	92	75	92	86
0 - 35	1044	658	1032	911± 219
% of control	141	89	139	123±29

 

Table 4: Influence of the test substance on Nitrification of Ammonium Sulphate Amended Flaach Soil; Values Given Represent mg of Ammonia, Nitrite and Nitrate/100 g Dry Soil.

Treatment with a.i. of test substance	Untreated	0.25 mg/kg dry soil	2.5 mg/kg dry soil
Ammonia 0 days 3 days 7 days	9.3 3.8 < 0.2	9.9 5.8 < 0.2	9.5 6.1 < 0.2
Nitrite 0 days 3 days 7 days	0.06 0.08 < 0.01	0.06 0.10 < 0.01	0.02 0. 11 < 0.01
Nitrate * 0 days 3 days 7 days	0.7 16.8 32.2	0.1 15.6 34.5	< 0 .1 13.2 34.0

 *the reported and tabulated figures for NO3- give the amounts exceeding the naturally present amounts in the soil (= 13.1 mg/100 g dry soil)

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the NOEC was determined to be 2.5 mg a.i./kg soil dw.

Executive summary:

The influence of the test substance on soil microorganisms was determined by measuring the soil respiration and nitrification processes according to the guidelines of the Bureau of Plant Protection Products, Plant Disease Service, Wageningen, The Netherlands, and the modifications proposed in the paper “Recommended Tests for Assessing the Side-Effects of Pesticides on the Soil Microflora, (1985)”. The study was conducted in compliance with GLP criteria. A freshly collected field soil (sandy loam, 2.2% OC; air-dried and sieved using DIN No. 6 (36 mesh/cm2)) was watered with deionized water to 35% MWHC and treated with 0.25 and 2.5 mg a.i./kg soil dry weight (corresponding to 0.12 kg/ha and ten times this amount) and incubated in the dark at 22 ± 2˚C during 35 and 7 days for respiration and nitrification, respectively. For respiration processes, the potential effects of the test substance on soil microorganisms were determined at 8 samplings by measuring the CO2 released after a lucerne amendment (0.5 %). For nitrification processes, the potential effects of the test substance on soil microorganisms were determined by measuring the presence of NH4-H, NO2-N and NO3-N formed during the mineralisation and nitrification of ammonium sulphate (38.8 mg/100 g dry soil).

For respiration, the results showed a mean deviation of total CO2 released of 5.8 and 18.3% compared to the control at study end (day 35) for the 0.25 and the 2.5 mg/kg soil treatment, respectively. The conversion of ammonia to nitrate was complete and very rapid (taking less than 7 days at all the tested concentrations). Results showed deviations of 7.0 and 5.6 % compared to the control in the 0.25 and 2.5 mg/kg soil treatment groups, respectively, at the end of 7 days incubation period. These deviations were below the 25 % effect trigger compared to the controls in both respiration and nitrification processes. Accordingly, no adverse effects of the test substance were observed on soil microbial activity. Based on the findings, the NOEC was determined to be 2.5 mg a.i./kg soil dw.

Description of key information

35-d NOEC = 2.5 mg a.i./kg dry soil (sandy loam), respiration and nitrification, Bureau of Plant Protection Products guideline from the Netherlands, Wisson 1987

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

2.5 mg/kg soil dw

Additional information

The influence of the test substance on soil microorganisms was determined by measuring the soil respiration and nitrification processes according to the guidelines of the Bureau of Plant Protection Products, Plant Disease Service, Wageningen, The Netherlands, and the modifications proposed in the paper “Recommended Tests for Assessing the Side-Effects of Pesticides on the Soil Microflora, (1985)”. The study was conducted in compliance with GLP criteria. A freshly collected field soil (sandy loam, 2.2% OC; air-dried and sieved using DIN No. 6 (36 mesh/cm2)) was watered with deionized water to 35% MWHC and treated with 0.25 and 2.5 mg a.i./kg soil dry weight (corresponding to 0.12 kg/ha and ten times this amount) and incubated in the dark at 22 ± 2˚C during 35 and 7 days for respiration and nitrification, respectively. For respiration processes, the potential effects of the test substance on soil microorganisms were determined at 8 samplings by measuring the CO2 released after a lucerne amendment (0.5 %). For nitrification processes, the potential effects of the test substance on soil microorganisms were determined by measuring the presence of NH4-H, NO2-N and NO3-N formed during the mineralisation and nitrification of ammonium sulphate (38.8 mg/100 g dry soil).

For respiration, the results showed a mean deviation of total CO2 released of 5.8 and 18.3% compared to the control at study end (day 35) for the 0.25 and the 2.5 mg/kg soil treatment, respectively. The conversion of ammonia to nitrate was complete and very rapid (taking less than 7 days at all the tested concentrations). Results showed deviations of 7.0 and 5.6 % compared to the control in the 0.25 and 2.5 mg/kg soil treatment groups, respectively, at the end of 7 days incubation period. These deviations were below the 25 % effect trigger compared to the controls in both respiration and nitrification processes. Accordingly, no adverse effects of the test substance were observed on soil microbial activity. Based on the findings, the NOEC was determined to be 2.5 mg a.i./kg soil dw.