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Environmental fate & pathways

Biodegradation in soil

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Description of key information

Aerobic Biodegradation in soil (OECD Guideline 307; USEPA Pesticide Assessment Guidelines. Subdivision N § 162 -1 Aerobic Soil Metabolism; PMRA DACO Number 8.2.3.4.2):

DT50 soil: 11.0 days at 25.1 °C (M-078563-01-2)

DT50 soil: 60.0 - 72.0 days at 25.1 °C (M-078931-01-2)

DT50 soil: 30.0 - 34.0 days at 19.8 °C (M-261658-01-2)

Anaerobic Biodegradation in soil (OECD 307; USEPA Pesticide Assessment Guidelines. Subdivision N§162 -2 Anaerobic Soil Metabolism; PMRA DACO Number 8.2.3.4.4):

DT50 soil: > 120 days at 20 °C (M-254009-02-1)

Key value for chemical safety assessment

Half-life in soil:
72 d
at the temperature of:
25.1 °C

Additional information

Aerobic Biodegradation in Soil

M-078563-01-2

The biotransformation of radiolabeled test substance [(5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl)methanone] was studied in a loamy sand soil (pH 5.6 [CaCl2], organic carbon content 1.2%) from Pikeville, North Carolina, USA for 358 days under aerobic conditions in the dark, at 25°C and 75% of 1/3 bar moisture (M-0778563-01-2). [phenyl-U-14C]-test substance and [pyrazole-3-14C]-test substance were applied at the rate of 0.14 mg a.i./kg soil. The experiment was conducted in accordance with the US EPA (Subdivision N, Section 162-1) and Canadian PMRA (Section C.1) guidelines, and in compliance with the OECD-GLP standards. The test systems consisted of Erlenmeyer flasks fitted with traps for the collection of CO2 and volatile organic compounds. Samples were analyzed at 0, 2, 4, 7, 10, 14, 21, 30, 39, 50, 65, 80, 100, 120, 168, 259, and 358 days of incubation. The soil samples were exhaustively extracted with acetonitrile/water (2/1, v/v) using 'mild' (40 °C / 100 bar) and 'aggravated' (100 °C / 100 bar) multicycle Accelerated Solvent Extraction (ASE) technique, and the test substance residues were analyzed by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Identification of transformation products was performed by co-chromatography with authentic reference material, and by coupled liquid chromatography – mass spectrometry (HPLC/MS/MS). Material balance was 101.3 ± 1.1% (range = 99.2 - 103.3%) of the applied radioactivity for the phenyl labeled test samples, and 100.9 ± 1.3% (range = 97.5 - 102.9%) for the pyrazole radiolabel series.
The parent compound decreased from 96.9% of the applied radioactivity at day 0 to 21.5% of the applied at the end of the study (mean of both label series). The DT50 of test substance in aerobic soil was 5 days, as determined from a degradation curve showing clear signs of bi-phasic decline kinetics. Simple first order modelling, therefore, returned a calculated half-life of 11 days, at limited quality of model fit (R² = 0.777). The major transformation product detected was the benzoic acid derivative test substance (2-methylsulfonyl-4-trifluoromethylbenzoic acid), reaching a maximum level of 12.2% of the applied amount, observed on the 7th day of incubation. The metabolite proved to be transient in nature, and was subject to rapid decline to 3.3% of applied at day 30. Unidentified minor transformation products were detected at maximum individual species abundances of ≤ 3.1% of applied in solvent extracts obtained under 'mild', or ≤ 0.9 % of applied in solvent extracts obtained under 'aggravated' conditions. The total unidentified radioactivity ranged from 0.6% to 14.1% of the applied amount (similar for both label positions). Extractable 14C-residues decreased from 99.3% (phenyl label) or 98.1% (pyrazole label) of the applied amount at day 0 to 38.5% or 36.2% of the applied, respectively, at study termination. Non-extractable 14C-residues increased from 1.7% (phenyl radiolabel) or 1.9% (pyrazole label) of the applied amount at day 0 to 43.2% or 44.8% of the applied, respectively, at the end of the study. At study termination, evolved 14CO2 accounted for 17.3% of the applied phenyl radiolabel, or 18.6% of the applied pyrazole radiolabel. No significant amounts of organic volatiles were detected for either label position (≤ 0.4% of applied for all samples). Sterilized soil control test systems showed absence of significant degradation of the test item. Within the observation period of 120 days, virtually all of the applied 14C-test substance could be recovered unchanged, with consequently marginal formation of non-extractable residues. With respect to extractability, however, a distinct gradual increase in soil adsorption upon aging was observed for the residues, as reflected in a steady decrease in radioactivity released from soil under 'mild' extraction conditions, and a subsequent corresponding increase in radioactivity found in the in the 'aggravated' condition extracts. The metabolic pathway of soil breakdown of test substance involves cleavage in between the phenyl and the pyrazole part of the molecule. The test substance benzoic acid metabolite was identified as single major metabolite derived from the phenyl moiety, being transient in nature and rapidly mineralised to 14CO2 or integrated into the soil matrix as non-extractable residues. For the pyrazole heterocycle moiety, no extractable metabolites of significant abundance could be observed, only formation of 14CO2 and non-extractable residues. As indicated by the behavior in sterile soil, breakdown of test substance is a microbially mediated process. Supplementary experiments were performed to investigate the observed bi-phasic decline kinetics of test substance in the test soil. By means of a modified extraction procedure including a 24 hours partitioning step in aqueous 0.01 M CaCl2 solution, a pronounced increase in soil adsorption of test substance was demonstrated upon aging. Mean partitioning coefficients Kd at day 50 and day 358 were 3.8 and 25.6 mL/g, corresponding to KOC values of about 320 and 2100 mL/g, respectively. In-line with these results, water-extractability of the remaining parent from soil was found significantly reduced after aging. A further experiment investigated the degradation when a second dose of test substance was applied to test systems that had been incubated for 80 days, already. Sampling and comparative analysis at day 100 (i.e. 20 days after second dosing) showed degradation of the additional dose to be similar to the degradation of the initial dose observed in the regular test systems at day 21. From both supportive experiments it was concluded that the biphasic degradation observed may most likely be attributed to a decrease in microbial bioavailability of the aged test substance residues, due to increase in their physical soil adsorption.

 

M-078931-01-2

The biotransformation of radiolabeled test substance [(5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl)methanone] was studied in a silt loam soil (pH 7.0 [CaCl2], organic carbon content 4.7%) from Grand Forks, North Dakota, USA for 358 days under aerobic conditions in the dark, at 25°C and 75% of 1/3 bar moisture (M-078931-01-2). [phenyl-U-14C]-test substance and [pyrazole-3-14C]-test substance were applied at the rate of 0.13 and 0.14 mg a.i./kg soil. The experiment was conducted in accordance with the US EPA (Subdivision N, Section 162-1) and Canadian PMRA (Section C.1) guidelines, and in compliance with the OECD-GLP standards. The test systems consisted of Erlenmeyer flasks fitted with traps for the collection of CO2 and volatile organic compounds. Samples were analyzed at 0, 3, 7, 14, 21, 30, 41, 50/51, 65, 80, 100, 120, 155, 190, 251, and 358 days of incubation. The soil samples were exhaustively extracted with acetonitrile/water (2/1, v/v) using 'mild' (40 °C / 100 bar) and 'aggravated' (100 °C / 100 bar) multicycle Accelerated Solvent Extraction (ASE) technique, and the test substance residues were analyzed by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Identification of transformation products was performed by co-chromatography with authentic reference material, and by coupled liquid chromatography – mass spectrometry (HPLC/MS/MS). Material balance was 98.8 ± 1.7% (range = 93.3 – 100.5%) of the applied radioactivity for the phenyl labeled test samples, or 99.4 ± 2.5% (range = 90.8 – 102.8%) for the pyrazole radiolabel series. The parent compound decreased from 95.7% of the applied radioactivity at day 0 to 23.3% of the applied at the end of the study (mean of both label series). The DT50 of test substance in aerobic soil was 60 days, as determined from a degradation curve showing slight deviation from exponential decline kinetics at advanced incubation time. Simple first order modelling, therefore, returned a calculated half-life of 72 days (R² = 0.976). No major transformation products were detected at ≥ 10% of AR level throughout study runtime. A minor metabolite was found, reaching a maximum abundance of 3.8% of the applied amount at day 30 after application, and identified as the benzoic acid derivative test substance (2-methylsulfonyl-4-trifluoromethylbenzoic acid). The concentration at study termination was 1.0% of applied. Other unidentified minor transformation products were detected at maximum individual species levels of 2.7% of applied in solvent extracts obtained under 'mild', or 1.2% of applied in solvent extracts obtained under 'aggravated' conditions. The total unidentified radioactivity ranged from 1.0% to 6.5% of the applied amount (similar for both label positions). Extractable 14C-residues decreased from 99.7% (phenyl label) or 99.0% (pyrazole label) of the applied amount at day 0 to 29.3% or 26.6% of the applied, respectively, at study termination. Non-extractable 14C-residues increased from 0.4% (phenyl radiolabel) or 1.1% (pyrazole label) of the applied amount at day 0 to 30.1% or 30.7% of the applied, respectively, at the end of the study. At study termination, evolved 14CO2 accounted for 40.5% of the applied phenyl radiolabel, or 33.5% of the applied pyrazole radiolabel. No significant amounts of organic volatiles were detected for either label position (< 0.1% of applied for all samples). Sterilized soil control test systems showed absence of significant degradation of the test item. Within the observation period of 120 days, virtually all of the applied 14C-test substance could be recovered unchanged, with consequently marginal formation of non-extractable residues. However, with respect to extractability a distinct gradual increase in soil adsorption upon aging was observed for the residues, as seen by a steady decrease in extractable radioactivity in the 'mild' extraction, and a subsequent increase in extractable radioactivity in the 'aggravated' extraction. The metabolic pathway of soil breakdown of test substance involves cleavage in between the phenyl and the pyrazole part of the molecule. The benzoic acid derivative of the test substance was identified as single extractable metabolite derived from the phenyl moiety, being transient in nature and rapidly mineralised to 14CO2 or integrated into the soil matrix as non-extractable residues. For the pyrazole heterocycle moiety, no extractable metabolites of significant abundance could be observed, only formation of 14CO2 and non-extractable residues. As indicated by the behavior in sterile soil, breakdown of test substance is a microbially mediated process. A supplementary experiment was performed to investigate the observed increase in soil adsorption of test substance with incubation time in the test soil. By means of a modified extraction procedure including a 24 hours partitioning step in aqueous 0.01 M CaCl2 solution, a pronounced increase in soil adsorption of test substance was demonstrated upon aging. The mean partitioning coefficient Kd at day 363 was 4.1 mL/g, corresponding to a KOC of about 88 mL/g. In-line with these results, water extractability of the remaining parent from soil was found reduced after aging.

 

M-261658-01-2

The biotransformation of radiolabeled test substance [(5-hydroxy-1,3-dimethylpyrazol- 4-yl)(2-mesyl-4-trifluoromethylphenyl)methanone] was studied in a sandy loam soil (pH 6.1 in CaCl2, 1.4% organic carbon) from Monheim, Germany for 120 d under aerobic conditions in the dark, at 20 °C and 50% of maximum water holding capacity. [Phenyl-UL-14C]test substance and [pyrazole-3-14C]test substance were applied at the target rate of 0.13 mg a.i./kg soil assuming homogeneous distribution in 2.5 cm topsoil layer. The experiment was conducted in accordance with the OECD 307, and in compliance with GLP. The test systems consisted of Erlenmeyer flasks, each containing 80 g of soil dry matter equivalents, attached with solid phase traps for the collection of 14CO2 and volatile organics (static test system design). Samples were analyzed at 0, 1, 3, 7, 14, 21, 29, 41, 62, 86, and 120 days of incubation. Two replicate test systems per radiolabel were harvested at each sampling interval. The entire soil per flask was extracted with acetonitrile / water (4/1, v/v) by six cycles of Accelerated Solvent Extraction (ASE) conducted at 40 °C and 103 bar pressure (‘mild’ conditions), followed by two additional cycles conducted at 100 °C and 103 bar pressure (‘aggressive’ conditions). Extracts were analyzed by reversed phase HPLC with flow-through radioactivity detection, and for exemplary result verification also by normal phase TLC with digital radioimaging. Identification of the test item residues and transformation products was achieved by check of co-elution with certified unlabeled reference items in the two contrasting chromatography systems, and confirmed by HPLC-MS/MS mass spectrometry.

Results for Radiolabel #A - [phenyl-UL-14C] test substance:

Material balance was 101.4 ± 2.3 % (mean ± SD, range = 96.9 - 104.3 %) of the applied radioactivity. The parent compound decreased from 100.0 % of the applied amount at day 0 to 18.9 % of the applied at the end of the incubation period. The simple first order half-life of [phenyl-UL-14C]test substance in the test soil was 34 days (R² = 0.96). No major transformation products (> 10 % of applied) were observed. A minor product was identified as test substance-benzoic acid (2-methylsulfonyl-4-trifluoromethylbenzoic acid), with maximum concentration of 8.9 % of the applied radioactivity observed at the 14th day of incubation. The corresponding concentration at the end of the study period was 2.3 % of the applied. The total unidentified radioactivity ranged from <LOD to 2.1 % of the applied amount, and consisted of multiple (up to 3) minor peaks corresponding to typically ≤ 1.2% of the applied each. Extractable [14C] residues decreased from 101.3% of the applied amount at day 0 to 23.3 % of the applied at the end of study period. Non-extractable [14C] residues increased from 2.0 % of applied at day 0 to a maximum of 60.1 % at the end of the study. Exemplary humic substance fractionation indicated these non-extractable residues to be of heterogeneous nature, associated with humic and fulvic acids, as well as with humin. At study termination, evolved 14CO2 accounted for 16.3 % of the applied radioactivity. No significant amounts of volatile organics were detected at any sampling timepoint (≤ 0.1 % of applied).

Results for Radiolabel #B - [pyrazole-3-14C] test substance:

Material balance was 99.6 ± 2.9% (mean ± SD, range = 93.9 - 104.9%) of the applied radioactivity. The parent compound decreased from 98.4 % of the applied amount at day 0 to 17.3% of the applied at the end of the incubation period. The simple first order half-life of [pyrazole-3-14C]test substance in the test soil was 30 days (R² = 0.95). No transformation products of significance were observed. The total unidentified radioactivity ranged from 0.2% to 2.6% of the applied amount, and consisted of multiple (up to 5) minor peaks corresponding to typically < 1% of the applied each. Extractable [14C] residues decreased from 98.6% of the applied amount at day 0 to 19.0% of the applied at the end of study period. Non-extractable [14C] residues increased from 2.7% of applied at day 0 to a maximum of 62.1% at the end of the study. Exemplary humic substance fractionation indicated these non-extractable residues to be of heterogeneous nature, associated with humic and fulvic acids, as well as with humin. At study termination, evolved 14CO2 accounted for 18.0% of the applied radioactivity. No significant amounts of volatile organics were detected at any sampling timepoint (≤ 0.1% of applied). Concluded from the metabolic profiles observed for the two radiolabels in the test, breakdown pathways of test substance in aerobic soil involve cleavage between the phenyl and the pyrazole moiety of the molecule. Test substance-benzoic acid, the single metabolite of significance derived from the phenyl moiety, was transient and mineralised to 14CO2 or substantially integrated into the soil matrix as non-extractable residues. For the pyrazole heterocycle moiety, no significant abundance of any extractable degradates could be observed, only formation of 14CO2 and non-extractable residues. The initial attack to the parent molecule therefore seems to represent the rate determining step in the metabolic breakdown of test substance, so that downstream intermediates cannot accumulate to levels of concern. The mean first order half-life of test substance in the test soil, averaged for both radiolabels, was 32 days (R² = 0.95). Based on these study results, neither the parent active ingredient test substance, nor any of its transformation products will persist in a viable soil environment.

Anaerobic Biodegradation in Soil

The aerobic/anaerobic biotransformation of radiolabeled test substance, (5-hydroxy-1,3-dimethyl-1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethylphenyl) methanone, was studied in a loamy sand (pH 5.4 (water) 4.8 (CaCl2), organic carbon 1.9%) from Pikeville, NC, USA (M-254009-02-1). During the first phase of the study, the soil was maintained under aerobic conditions for 30 days in the dark at 20 ± 1 °C and 15.7% soil moisture (31.3% water holding capacity). Following the aerobic phase, the samples were flooded with water (water/soil ratio 1:1 w/w) and maintained in the dark under anaerobic conditions for 120 days at 20 ± 1 °C. test substance was applied at the rate of 0.07μg a.i./g, equivalent to 50 g a.i./ha (soil depth of 5 cm). The experiment was conducted in accordance with OECD 307 and in compliance with the US EPA Subdivision N, Section 162-2 and GLP standards (40 CFR part 160). The test system consisted of forty-eight (twenty-four for each radiolabel) 250 mL Erlenmeyer flasks attached to traps for the collection of CO2 and volatile organic compounds during the aerobic phase. Samples were analyzed at 0, 3, 9 and 30 days of aerobic incubation, and at 0, 2, 5, 8, 15, 33, 61, 90 and 120 days of incubation following flooding of the samples (anaerobic). The water samples were not extracted with solvent, and the soil samples were extracted with an accelerated solvent extractor (ASE) using acetonitrile/water (65:35), three times at ambient (40 °C) temperature and two times at 100 °C. test substance residues were analyzed by HPLC using a flow-through 14C detector. Identification of the transformation product was accomplished by co-chromatography and mass spectral (LC-MS) analysis.

Phenyl label: The total material balance in the soil/water system was 98.8% ± 1.5% of the applied radioactivity. In the aerobic phase, extractable [14C] residues in soil decreased from 99.2% at day 0 to 74.5% by day 30. Non-extractable (bound) residues in soil increased from 0.8% at day 0 to 18.1% at day 30. At the end of the aerobic phase 1.3% and 0.0% of the applied radioactivity was present as CO2 and volatile organic compounds, respectively.

The concentration of test substance in the aerobic phase decreased from 97.3% at day 0 to 68.4% of the applied amount at day 30. The only major transformation product formed in the aerobic phase was test substance benzoic acid derivative which formed at 9.3% of the applied radioactivity at day 30. During the anaerobic phase, radioactivity in the water increased from 4.4% at day 0 to 17.3% at day 33, and then decreased to 13.0% of the applied radioactivity at the end of the study. Extractable [14C] residues in soil decreased from 74.5% at day 0 to 62.8% of the applied radioactivity at the end of the study. Non-extractable residues in soil increased from 18.1% at day 0 to 22.5% of the applied amount at day 120. No more than 1.4% of additional CO2 and volatile organic compounds were produced throughout the anaerobic phase of the study.

During the anaerobic phase, the concentration of test substance in water increased from 2.4% at day 0 to 11.7% at day 33 and then decreased to 7.6% of the applied amount at study termination. The concentration of test substance in the soil decreased from 66.0% at day 0 to 56.5% of the applied amount at study termination. No additional major transformation products were detected during the study. The test substance benzoic acid derivative residues remained relatively stable during the anaerobic phase, ranging from 9.1 to 9.9% of the applied radioactivity in the total system. The minor unidentified transformation product consisted of less than 2.6% of the applied amount throughout the study duration.

Pyrazole label: Total material balance in the soil/water system was 94.6% ± 2.4% of the applied radioactivity. During the aerobic phase, the extractable [14C] residues in soil decreased from 98.0% at day 0 to 62.8% by day 30. Non-extractable (bound) residues in soil increased from 2.0% at day 0 to 23.8% at day 30. At the end of the aerobic phase, 5.5% and 0.0% of the applied radioactivity was present as CO2 and volatile organic compounds, respectively. The concentration of test substance in the aerobic phase decreased from 93.5% at day 0 to 62.8% of the applied amount at day 30. There were no major transformation products formed.

During the anaerobic phase, radioactivity in the water increased from 3.1% at day 0 to 14.2% at day 33, and then decreased to 7.2% of the applied radioactivity at the end of the study. Extractable [14C] residues in soil decreased from 62.8% at day 0 to 55.0% of the applied radioactivity at the end of the study. Non-extractable residues in soil decreased from 23.8% at day 0 to 18.4% by Day 8 then increased to 24.9% of the applied amount at day 120. No more than 1.2% of additional CO2 and volatile organic compounds were produced throughout the anaerobic phase of the study. During the anaerobic phase, the concentration of test substance in water increased from 3.1% at day 0 to 14.2% at day 33 and then decreased to 7.2% of the applied amount at study termination. The concentration of test substance in the soil decreased from 62.8% at day 0 to 55.0% of the applied amount at study termination. No major transformation products were detected during the anaerobic phase of the study with the pyrazole radiolabel. The half-life of [phenyl-UL-14C]test substance in the anaerobic soil/water system using nonlinear first-order kinetics was determined to be >120 days (k = 0.0009 day-1; R²=0.57). The half-life of [pyrazole-3-14C]test substance in the anaerobic soil/water system using nonlinear first-order kinetics was determined to be >120 days (k = 0.0008 day-1; R²=0.70).