Pyrasulfotole

Biodegradation in soil

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Apr 2004 - 20 Jan 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Subdivision N, Section 162-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Canada PMRA DACO No. 8.2.3.4.4

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic/anaerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2005

Soil no.:

#1

Soil type:

loamy sand

% Clay:

7

% Silt:

14

% Sand:

79

% Org. C:

1.9

pH:

5.4

CEC:

6.2 meq/100 g soil d.w.

Bulk density (g/cm³):

1.12

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Pikeville, NC, USA
- Pesticide use history at the collection site: No pesticide used for greater than 5 years
- Collection procedures: Sample taken with a spade
- Sampling depth: 0-8 inches
- Storage conditions: Refrigerated
- Storage length: 25 days prior to use
- Soil preparation: 2 mm sieved; moisture determination

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm: 9.8%
- Bulk density: 1.12 g/cm³

Soil No.:

#1

Duration:

120 d

Soil No.:

#1

Initial conc.:

0.07 mg/kg soil d.w.

Based on:

act. ingr.

Soil No.:

#1

Initial conc.:

0.34 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

Metabolite Identification

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

20 ± 1 °C

Humidity:

15.7% soil moisture (31.3% water holding capacity)

Microbial biomass:

Test start: 1.20 x 10E+08 cells/g soil; middle of the study: 7.8 x 10E+07cells/mL water and 3.9 x 10E+08 cells/g soil; end of the study 6.2 x 10E+07 cells/mL water and 3.2 x 10E+08 cells/g soil.

Details on experimental conditions:

1. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): Refrigerated (mean <5 °C), typically 14 days,maximum 126 days
- Soil condition: Fresh
- Soil (g/replicate): 50 g dwt/replicate
- No. of replication controls: Duplicate
- No. of replication treatments: Duplicate
- Test apparatus (Type/material/volume): 250-mL Pyrex Erlenmeyer flask with side-arm and double-valve sealable top (anaerobic)
- Details of traps for CO2 and organic volatile, if any: Aerobic-- Soda lime and glass wool (biometer flask)
Anaerobic-- Flow through volatile traps: 2 M KOH, 2 M KOH, Ethylene Glycol, 1 M H2SO4
- Identity and concentration of co-solvent:
Acetonitrile: Milli-Q water (Phenyl; 2.2:97.8)
Acetonitrile: Milli-Q water (Pyrazole; 7.8:92.2)
Test material application
- Volume of test solution used/treatment:
[Phenyl-UL-14C]:
For the kinetic application solution, an aliquot (65 μL) of [phenyl-UL-14C]test substance (28.6 μCi/μMol; 1549 mg/mL, 122.3 μCi/mL) was diluted to 3.02 mL with water. Five 5-μL aliquots were radioassayed to determine the concentration. The chemical purity of the treatment application solution was determined by HPLC and identification confirmed by mass spectrometry. The concentration of the application solution was found to be 34.0 μg/mL. For the MID application solution, an aliquot (90 μL) of [phenyl-UL-14C]test substance was diluted to 840 μL with water. Five 5-μL aliquots were radioassayed to determine the concentration. The concentration of the application solution was found to be 154.2 μg/mL. For the [Phenyl-UL-14C] application 99 µL of the dosing solution were applied. For the MID treatment 109 µL were applied.
[Pyrazole-3-14C]:
For the kinetic application solution, an aliquot (235 μL) of [pyrazole-3-14C]test substance (55.3 μCi/μMol; 426 mg/mL, 65 μCi/mL) was diluted to 3.00 mL with water. Five 5-μL aliquots were radioassayed to determine the concentration. The chemical purity of the treatment application solution was determined by HPLC and identification confirmed by mass spectrometry. The concentration of the application solution was found to be 35.5 μg/mL. For the MID application solution, an aliquot (315 μL) of [pyrazole-3-14C]test substance was diluted to 800 μL with water. Five 5-μL aliquots were radioassayed to determine the concentration. The concentration of the application solution was found to be 180.3 μg/mL. For the [Pyrazole-3-14C] application 94 µL of the dosing solution were applied. For the MID treatment 93 µL were applied.
- Application method:
The application solution was applied in drops evenly across the surface of the soil using a 100-μL Hamilton Gastight® syringe. Soda lime traps were placed on each flask after applying grease on the ground glass joints of the traps. The flasks were labeled, wrapped in aluminum foil, weighed and stored in the environmental chamber.
- Is the co-solvent evaporated: No
Any indication of the test material adsorbing to the walls of the test apparatus: None
Experimental conditions (in addition to defined fields)
- Moisture maintenance method: 1 mL of milli-Q water was added to each test system 17 days after treatment
- Continuous darkness: Yes

2. OXYGEN CONDITIONS
- Methods used to create the an/aerobic conditions:
Aerobic Phase: Test systems were capped with soda-lime traps. The volatile traps contained a layer of glass wool followed by 10 g of soda-lime and topped with another layer of glass wool. The test systems were purged with nitrogen 30 days after treatment to force any possible CO2 into the soda-lime. Anaerobic Phase: The volatile trapping system contained six bubblers consisting of a blank, 30 mL of 2 M potassium hydroxide (KOH), 30 mL of 2 M KOH, 30 mL of ethylene glycol, 30 mL of 1 M sulfuric acid, and a blank. The volatiles were flushed at the time of sampling for approximately 10-15 minutes with nitrogen.
- Evidence that an/aerobic conditions were maintained during the experiment (e.g. redox potential): During the anaerobic phase of the study, the average redox potential (Eh) decreased from 441.5 mV at day 0 to 155.6 mV at day 33 and remained relatively constant through the end of the study. The average dissolved oxygen concentrations ranged from 3.5 mg/L at day 0 to 0.3 mg/L at day 33, and remained constant through the end of the study. Both parameters showed the systems reached anaerobic conditions by day 33. The average pH of the anaerobic test systems remained relatively stable between 5.3 and 6.3.

3. SAMPLING DETAILS
- Sampling intervals:
Aerobic phase: 0, 3, 9, and 30 days post application
Anaerobic phase: 0, 2, 5, 8, 15, 33, 61, 90, and 120 days postflooding
- Sampling method for soil samples: Entire sample filtered, extracted and air dried
- Method of collection of CO2 and volatile organic compounds: Aerobic: soda lime samples analyzed (for CO2) Anaerobic: Volatiles purged into series of traps (2 M KOH, 2 M KOH, Ethylene Glycol, 1 M H2SO4) with nitrogen gas at each sampling interval.
- Sampling intervals/times for:
> Moisture content: Approximately 1 mL of water was added after 17 days of aerobic incubation
> Redox potential/other: 0, 2, 5, 8, 15, 33, 61, 90, and 120 (days after flooding)
> Sample storage before analysis: Refrigerated (mean <5 °C), typically 14 days, maximum 126 days

Soil No.:

#1

Sampling day(s):

120 d

% Total extractable:

75.8

% Non extractable:

22.5

% CO2:

2.2

% Other volatiles:

0.1

Remarks on result:

other:

Remarks:

Mean material balance of radioactivity in the water/sediment system after 120 days. [14C]-U-phenyl. Total radioactivity recovered after 120 days: 100.5%

Soil No.:

#1

Sampling day(s):

120 d

% Total extractable:

62.2

% Non extractable:

24.9

% CO2:

6.6

% Other volatiles:

0.1

% Recovery:

93.8

Remarks on result:

other:

Remarks:

Mean material balance of radioactivity in the water/sediment system after 365 days. [14C]-3-pyrazole

Parent/product:

parent

Soil No.:

#1

% Degr.:

2.2

Parameter:

radiochem. meas.

Sampling time:

120 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [14C]-U-phenyl

Parent/product:

parent

Soil No.:

#1

% Degr.:

6.6

Parameter:

radiochem. meas.

Sampling time:

120 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [14C]-3-pyrazole

Soil No.:

#1

DT50:

> 120 d

Type:

other: non-linear first order

Temp.:

20 °C

Remarks on result:

other:

Remarks:

[phenyl-UL-14C]; k: 0.0009 d-1; r²: 0.57

Soil No.:

#1

DT50:

> 120 d

Type:

other: non-linear first order

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Calculated DT50 based on results at 20 °C.

Soil No.:

#1

DT50:

> 120 d

Type:

other: non-linear first order

Temp.:

20 °C

Remarks on result:

other:

Remarks:

[pyrazole-3-14C]; k: 0.0008 d-1; r²: 0.70

Transformation products:

yes

No.:

#1

Details on transformation products:

- Formation and decline of each transformation product during test: The only major transformation product formed in the aerobic phase was the benzoic acid of the test substance which formed 9.3% at day 30. No additional major transformation products were detected during the study. The residues of the metabolite 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 (B) consisted of less than 2.6 % of the applied amount throughout the study duration.
- Pathways for transformation: test substance to test substance benzoic acid

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity and anaerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
[phenyl-UL-14C]:
Water: 1.6 - 5.1% of AR on days 0 (of the anaerobic phase; day 30 of the aerobic phase) and day 15 (days after flooding), respectively.
Soil: 7.7 - 4.8 % of AR on days 0 (of the anaerobic phase; day 30 of the aerobic phase) and day 15 (days after flooding), respectively.
Total: 9.9 - 9.1% of AR on days 15 (days after flooding) and days 8 and 120, respectively.
[pyrazole-3-14C]
Water: No metabolite formed
Soil: No metabolite formed
Total: No metabolite formed

- Range of maximum concentrations in % of the applied amount at end of study period:
[phenyl-UL-14C]:
Water: 4.1 ± 0.7% of AR on day 120
Soil: 5.1 ± 0.5% of AR on day 120
Total: 9.1 ± 0.2% of AR on day 120
[pyrazole-3-14C]
Water: No metabolite formed
Soil: No metabolite formed
Total: No metabolite formed

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
[phenyl-UL-14C]:
Water: 0 - 1.3% of AR on days 5, 8 and 15 - day 120, respectively
Soil: 1.2% of AR on days 5, 8 and 15 - day 120, respectively
Total: 2.6% of AR on days 5, 8 and 15 - day 120, respectively
[pyrazole-3-14C]: Not applicable

EXTRACTABLE RESIDUES
- % of applied amount at day 0 (of the anaerobic phase; day 30 of the aerobic phase)
[phenyl-UL-14C]:
Water: 4.4 ± 0.8% of AR
Soil: 74.5 ± 0.8% of AR
Total: 78.8 ± 1.7% of AR
[pyrazole-3-14C]:
Water: 3.1 ± 0.0% of AR
Soil: 62.8 ± 0.8% of AR
Total: 65.9 ± 0.8% of AR
- % of applied amount at end of study period:
[phenyl-UL-14C]:
Water: 13.0 ± 2.1% of AR
Soil: 62.8 ± 2.3% of AR
Total: 75.8 ± 0.2% of AR
[pyrazole-3-14C]:
Water: 7.2 ± 0.2% of AR
Soil: 55.0 ± 0.1% of AR
Total: 62.2 ± 0.3% of AR

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0 (of the anaerobic phase; day 30 of the aerobic phase)
[phenyl-UL-14C]: 18.1 ± 0.5% of AR
[pyrazole-3-14C]: 23.8 ± 0.6% of AR
- % of applied amount at end of study period:
[phenyl-UL-14C]:22.5 ± 1.4% of AR
[pyrazole-3-14C]: 24.9 ± 1.5% of AR

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:
[phenyl-UL-14C]: 2.2 ± 0.3% of AR
[pyrazole-3-14C]: 6.6 ± 0.0% of AR

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:
[phenyl-UL-14C]: 0.1 ± 0.0% of AR
[pyrazole-3-14C]: 0.1 ± 0.0% of AR

Reference 2

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 Jun 2002 - 22 Jun 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA: Pesticide Assessment Guidelines, Subdivision N - Chemistry: Environmental Fate. § 162-1 Aerobic Soil Metabolism Studies (12-Oct-1982)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: PMRA: Environmental Chemistry and Fate Guidelines for Registration of Pesticides in Canada. C.1 - Biotransformation, Soil (Laboratory) - Degradation Pathways and Persistence (15-Jul-1987) - [DACO number 8.2.3.4.2]

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2002

Soil no.:

#1

Soil type:

loamy sand

% Clay:

1

% Silt:

22

% Sand:

77

% Org. C:

1.2

pH:

6.2

CEC:

5.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.4

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Pikeville, North Carolina, USA
- Pesticide use history at the collection site: None > 10 years
- Collection procedures: Freshly sampled from field with shovel into bucket, immediate transport to sender's lab
- Sampling depth: 0 - 20.32 cm
- Storage conditions: Ventilated plastic bag, moist, at approx. 4 °C, in the dark
- Storage length: 34 days
- Soil preparation (e.g. air dried etc.): soil moisture partially reduced at ambient temperature, subsequently sieved to ≤ 2 mm.

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm: 9% disturbed
- Bulk density: 1.40 g/cm³

Soil No.:

#1

Duration:

358 d

Soil No.:

#1

Initial conc.:

0.137 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

[phenyl-U-14C]-test substance

Soil No.:

#1

Initial conc.:

0.143 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

[pyrazole-3-14C]-test substance

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

25.1 ± 0.1 °C

Humidity:

Soil moisture was maintained within the target range of 75 ± 10% of 1/3 bar water holding capacity via regular (approx. 2-4 week intervals) additions of autoclaved distilled water.

Microbial biomass:

Substrate Induced initial Respiratory response (SIR) indicated appropriate microbial soil activity at the start of the study and at day 129 after application. At day 365, however, a decrease in soil microbial activity was observed.

Details on experimental conditions:

1. EXPERIMENTAL DESIGN
- Soil preincubation conditions: The test systems were placed in a temperature controlled incubation chamber and subjected to pre-incubation for 10 days to allow for acclimatisation to the intended study incubation conditions (in the dark and at 25 °C, nominal).
- Soil condition: fresh soil
- Soil (g/replicate): 80 g dwt/replicate
- Control conditions: Sterile controls, biomass samples, microbial plate count samples.
- No. of replication controls, if used: two (one for each radiolabeleld position) and one biomass sample and one microbial plate count sample.
- No. of replication treatments: two replicates per treatment per sample day.
- Test apparatus: 300 mL Erlenmeyer glass flasks
- Details of traps for CO2 and organic volatile, if any: Each individual vessel was fitted with a separate trap attachment, in which sodalime and paraffin coated glass wool were used to trap carbon dioxide and organic volatile compounds, respectively.
- Identity and concentration of co-solvent: acetone/deionized autoclaved water (1/2 v/v)

Test material application
- Volume of test solution used/treatment: [14C]phenyl radiolabeled; 240 µL; [14C]pyrazole radiolabeled 270 µL
- Application method (e.g. applied on surface, homogeneous mixing etc.): 500 µL of the respective application solutions was made via small droplets applied directly to the soil via microliter pipette. Each flask was then gently shaken to incorporate the radiochemical.
- Is the co-solvent evaporated: No with the exception of passive evaporative loss following addition and mixing.

Any indication of the test material adsorbing to the walls of the test apparatus: No

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: addition of autoclaved deionized water
- Continuous darkness: Yes

Other details, if any:

2. OXYGEN CONDITIONS
- Methods used to create the an/aerobic conditions: passive exchange of air through the static volatile traps.

3. SUPPLEMENTARY EXPERIMENTS:

Additional experiments were performed on spare samples of viable soil, radiolabeled
test systems for supportive purposes:

Second Dosage Experiment
On day 80 after application, two spare test systems (no. 21 / 121 = one phenyl, one
pyrazole label) were treated with a second dose of radiolabeled test item each. The test systems were incubated a further 20
days, and subjected to standard processing and analysis on day 100. The results
obtained were compared with those of the regular test systems collected on day 80
and 100, to assess the degradation behaviour of the additional dose.

Soil Adsorption of Aged Test Substance Residues
Spare test systems (one per label position) were harvested on day 50 (no. 19 / 119)
and on day 358 (no. 22 / 122) after application, and subjected to an extended
aqueous phase partitioning / extraction procedures consisting of aqueous partitioning, aqueous extraction, 'mild' ASE solvent exraction and 'aggravated' ASE solvent extraction.

Moisture Increase Experiment
On day 57 after application, the soil moisture of two spare test systems (no. 20 / 120 = one phenyl, one pyrazole label) was increased to 45% MWHC by addition of distilled water. After continued incubation for a further 8 days, these samples were processed in parallel to the regular day 65 samples (no. 11 / 111), allowing for direct comparison of the analytical results.

4. SAMPLING DETAILS
- Sampling intervals: 0, 2, 4, 7, 10, 14, 21, 30, 39, 50, 65, 80, 100, 120, 168, 259, and 358 days post-application
- Sampling method for soil samples: Soil was extracted immediately after sampling of vessels. (except day 30 sample: stored frozen for one day)
- Method of collection of CO2 and volatile organic compounds: Solid trap containing sodalime for absorption of [14C]O2 and paraffin coated glass wool for volatile organic compounds.
- Sampling intervals/times for:
> Sterility check, if sterile controls are used: Sterilized soil: day 0, 21, and 65 for; microbial biomass day 1, 129, and 365, microbial plate count: 0, 121, and 358
> Moisture content: 2 - 4 week intervals
> Sample storage before analysis: Soil was extracted immediately after sampling of vessels (except day 30 sample: stored frozen for one day).

Soil No.:

#1

Sampling day(s):

358 d

% Total extractable:

38.5

% Non extractable:

43.2

% CO2:

17.3

% Other volatiles:

0.2

% Recovery:

99.2

Remarks on result:

other:

Remarks:

Material balance of radioactivity in the water/sediment system after 365 days. [14C]-U-phenyl

Soil No.:

#1

Sampling day(s):

358 d

% Total extractable:

36.2

% Non extractable:

44.8

% CO2:

18.6

% Other volatiles:

0.3

% Recovery:

99.9

Remarks on result:

other:

Remarks:

Material balance of radioactivity in the water/sediment system after 365 days. [14C]-3-pyrazole

Parent/product:

parent

Soil No.:

#1

% Degr.:

17.3

Parameter:

radiochem. meas.

Sampling time:

358 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [14C]-U-phenyl

Parent/product:

parent

Soil No.:

#1

% Degr.:

18.6

Parameter:

radiochem. meas.

Sampling time:

358 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [14C]-3-pyrazole

Soil No.:

#1

DT50:

11 d

Type:

other: Single compartment first-order

Temp.:

25.1 °C

Remarks on result:

other: r²: 0.777

Soil No.:

#1

DT50:

36.95 d

Type:

other: Single compartment first-order

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Calculated DT50 based on results at 25.1 °C.

Transformation products:

yes

No.:

#1

Details on transformation products:

- Pathways for transformation: test substance to 2-methylsulfonyl-4-trifluoromethylbenzoic acid or CO2 + NER
- Formation and decline of each transformation product during test: The major transformation product was the benzoic acid derivative if the test item, reaching a maximum of 12.2% of the applied amount on the 7th day of incubation. The metabolite was transient in nature and rapidly declined to 3.3% of applied by day 30. The amount of individual, unidentified minor transformation products was <= 3.1% of applied in solvent extracts obtained under 'mild' and <= 0.9% of applied radioactivity under 'aggravated' conditions.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Remarks:

For the [14C]-U-phenyl test substance NER equalled 43.2% AR at Day 358. For [14C] -3-pyrazole test substance NER equalled 44.8% AR at day 358.

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: [14C]-3-phenyl-benzoic acid derivative metabolite 12.2 - 1.9% on Days 7 and 50, respectively.
- Range of maximum concentrations in % of the applied amount at end of study period: 4.2% at Day 358

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
For [14C]-U-phenyl 14.1% at Day 358; For [14C]-3-pyrazole 13.3% at Day 358

EXTRACTABLE RESIDUES
- % of applied amount at day 0: For [14C]-U-phenyl 99.3%; For [14C]-3-pyrazole 98.1%
- % of applied amount at end of study period: For [14C]-U-phenyl 38.5%; For [14C]-3-pyrazole 36.2%

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: For [14C]-U-phenyl 1.7%; For [14C]-3-pyrazole 1.9%
- % of applied amount at end of study period: For [14C]-U-phenyl 43.2%; For [14C]-3-pyrazole 44.8%

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: For [14C]-U-phenyl 17.3%; For [14C]-3-pyrazole 18.6%

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: For [14C]-U-phenyl 0.2%; For [14C]-3-pyrazole 0.3%

STERILE TREATMENTS (if used)
- Transformation of the parent compound: For [14C]-U-phenyl 95.4% of AR remained as parent on Day 120; For [14C]-3-pyrazole 94.8% of AR remained as parent on Day 120
- Formation of transformation products: For [14C]-3-phenyl benzoic acid derivative 3.2% of AR on Day 120
- Formation of extractable and non-extractable residues: For [14C]-U-phenyl 99.3% of AR as extractable, 2.7% AR as non-extractable on Day 120; For [14C]-3-pyrazole 95.6% of AR as extractable, 3.5% AR as non-extractable on Day 120
- Volatilization: For [14C]-U-phenyl <0.1% of AR on Day 120; For [14C]-3-pyrazole <0.1% of AR on Day 120

RESULTS OF SUPPLEMENTARY EXPERIMENT (if any):
Whereas only insignificant further degradation of the residual test substance was observed inbetween day 80 to 100 for the regular samples (approx. -1% AR), a distinct breakdown of about 55-60% of the test substance applied as a second dose occurred in the repeated dose test systems. The degree of decline, as well as the metabolic profile, were found comparable to the fate of the initial dose within the first 21 days of the study. Soil adsorption of the test substance residue was found to be subject to tremendous increase with aging time. At the end of the study, a mean Kd of 25.6 mL/g - corresponding to a KOC value of about 2100 mL/g - indicated a significant reduction in mobility of parent soil residues. A major portion of the residual parent did not participate in aqueous partitioning, but was only released upon multistep ASE solvent extraction. The above results suggest that the biphasic decline behavior of the test substance observed in this soil might be attributed to a gradual decrease in bioavailability of the compound to the soil microflora, caused by increase in soil adsorption. No significant influence of the higher soil moisture level on degradation of the residual the test substance could be found. Such result are in agreement with the hypothesis assuming a hindered desorption of parent from soil into water once aged.

Reference 3

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 Jun 2002 - 22 Jun 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4100 (Aerobic Soil Metabolism)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USEPA Pesticide Assessment Guidelines, Subdivision N - Chemistry: Environmental Fate. § 162-1 Aerobic Soil Metabolism Studies (12-Oct-1982)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: PMRA Environmntal Chemistry and Fate Guidelines for Registration of Pesticides in Canada. C1 - Biotransformation, Soil (Laboratory) - Degradation Pathways and Persistence (15-Jul-1987) - [DACO number 8.2.3.4.2]

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden, Germany

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2004

Soil no.:

#1

Soil type:

silt loam

% Clay:

17

% Silt:

54

% Sand:

29

% Org. C:

4.7

pH:

7.3

CEC:

26 meq/100 g soil d.w.

Bulk density (g/cm³):

0.86

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Grand Forks, North Dakota, USA
- Pesticide use history at the collection site: No pesticide treatment (ever)
- Collection procedures: Freshly sampled from field with shovel into bucket, immediate transport to sender's lab
- Sampling depth: 0 - 15.24 cm
- Storage conditions: in ventilated plastic bag, moist, at approx. 4 °C, in the dark
- Storage length: 70 days
- Soil preparation: removal of plant material and stones, partial reduction of soil moisture at ambient temperature, sieving to ≤ 2mm

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm: 45.5%
- Bulk density: 0.86 g/cm3

Soil No.:

#1

Duration:

358 d

Soil No.:

#1

Initial conc.:

0.129 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

[phenyl-U-14C]-test substance

Soil No.:

#2

Initial conc.:

0.136 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

[pyrazole-3-14C]-test substance

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

25.1 °C

Humidity:

45.5%

Microbial biomass:

day 1: 113.6 ± 5.8 mg/100 g soil (dry weight) day 121: 123.6 ± 3.6 mg/100 g soil (dry weight) day 357: 72.9 ± 10.4 mg/100 g soil (dry weight)

Details on experimental conditions:

1. EXPERIMENTAL DESIGN
- Soil preincubation conditions: 70 days; in ventilated plastic bag, moist, at approx. 4°C, in the dark
- Soil condition: fresh
- Soil: 50 g/replicate
- No. of replication controls, if used: two sterile soil controls; one biomass and one microbial plate count
- No. of replication treatments: two (one per radiolabel treatment)
- Test apparatus: 300 mL Erlenmeyer glass flask
- Details of traps for CO2 and organic volatile, if any: Sodalime and glass wool saturated with paraffin oil, open static system
- Identity and concentration of co-solvent: acetone and deionized, autoclaved water (1/1, v/v)

Test material application
- Volume of test solution used/treatment: 500 µL/test system
- Application method (e.g. applied on surface, homogeneous mixing etc.): Small droplets applied directly to soil using a microliter pipette. Each flask was then gently shaken to incorporate the radiochemical and to allow for solvent evaporation.
- Is the co-solvent evaporated: None with the exception of passive evaporative loss during incorporation/shaking

Any indication of the test material adsorbing to the walls of the test apparatus: No

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: Evaporation losses were compensated for by addition of autoclaved distilled water on gravimetric basis (26 times).
- Continuous darkness: Yes

2. OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: passive diffusion through open CO2 traps

3. SUPPLEMENTARY EXPERIMENTS:
Soil adsorption of the test substance residues was found subject to increase with aging time, as concluded from a gradual change in extraction behavior noticed for the sterile soil control test systems. At the end of the study a mean Kd value of 4.1 mL/g - equivalent to a KOC of 88 mL/g - indicated significant reduction in mobility of the aged residues of the test substance in soil.

4. SAMPLING DETAILS
- Sampling intervals: Duplicate samples, consisting of one phenyl label test system and one pyrazole label test system, were collected at 16 intervals throughout the incubation period. Sampling timepoints were day 0, 3, 7, 14, 21, 30, 41, 50/51, 65, 80, 100, 120, 155, 190, 251, and 358 after application.
- Sampling method for soil samples: Soil was extracted immediately after sampling of vessels.
- Method of collection of CO2 and volatile organic compounds: Solid trap containing sodalime for absorption of [14C]O2 and paraffin coated glass wool for volatile organic compounds
- Sampling intervals/times for:
> Sterility check, if sterile controls are used: 7, 21, 41, 80, and 120 days postapplication
> Moisture content: At regular intervals (about 2-4 weeks) during incubation period the test system moisture was checked gravimetrically. Small amounts of autoclaved distilled water were added for 26 times during study runtime, to keep soil moisture at 75±10% of 1/3 bar water holding capacity level.
> Sample storage before analysis: Soil of radiolabeled test systems was extracted on the day of sampling or within a few days from preparation. When not in use, extracts were stored in a freezer. Adequate stability of stored extracts until their final use (e.g. in
confirmational experiments) was demonstrated by re-analysis of representative samples after storage intervals of up to approx. eleven months.
- Other observations, if any:
Microbial biomass determination: 1, 121, and 357 days after application
Microbial plate count analysis: 0, 120, and 358 days after application
Sterility checks: colony forming units plate count analysis of sterilized soil control test systems at days 0, 21, 80

Soil No.:

#1

Sampling day(s):

358 d

% Total extractable:

29.3

% Non extractable:

30.1

% CO2:

40.5

% Other volatiles:

< 0.1

% Recovery:

99.9

Remarks on result:

other:

Remarks:

Mean material balance of radioactivity in the water/sediment system after 358 days. [phenyl-U-14C]-test substance

Soil No.:

#1

Sampling day(s):

358 d

% Total extractable:

26.6

% Non extractable:

30.7

% CO2:

33.5

% Other volatiles:

< 0.1

% Recovery:

90.8

Remarks on result:

other:

Remarks:

Mean material balance of radioactivity in the water/sediment system after 358 days. [pyrazole-3-14C]-test substance

Parent/product:

parent

Soil No.:

#1

% Degr.:

40.5

Parameter:

radiochem. meas.

Sampling time:

358 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [phenyl-U-14C]-test substance

Parent/product:

parent

Soil No.:

#1

% Degr.:

33.5

Parameter:

radiochem. meas.

Sampling time:

358 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [pyrazole-3-14C]-test substance

Soil No.:

#1

DT50:

72 d

Type:

other: Simple first order modeling calculated half-life

Temp.:

25.1 °C

Soil No.:

#1

DT50:

241.9 d

Type:

other: Simple first order modeling calculated half-life

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Calculated DT50 based on results at 25.1 °C. [phenyl-U-14C]-test substance

Soil No.:

#1

DT50:

60 d

Type:

other: determined from a degradation curve

Temp.:

25.1 °C

Soil No.:

#1

DT50:

201.5 d

Type:

other: Simple first order modeling calculated half-life

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Calculated DT50 based on results at 25.1 °C. [pyrazole-3-14C]-test substance

Transformation products:

no

Remarks:

No major transformation products were detected.

Details on transformation products:

- Formation and decline of each transformation product during test: No major metabolite was formed (< 10%).
[phenyl-U-14C]-test substance
benzoic acid metabolite
Range 1.0 - 3.8% of test substance AR on days 120 and 30, respectively
- Pathways for transformation: Test substance to benzoic acid metabolite and/or CO2 + NER

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Remarks:

30.1 and 30.7% of AR at day 358, [14C-U-Phenyl] and [14C-3-pyrazole], labeled, respectively

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: [phenyl-U-14C] test material benzoic acid metabolite: 1.0 and 3.8% on days 120 and 30, respectively. [pyrazole-3-14C] test material benzoic acid metabolite: Not detected.
- Range of maximum concentrations in % of the applied amount at end of study period: [phenyl-U-14C] test material benzoic acid metabolite: 1.0% on day 358. [pyrazole-3-14C] test material benzoic acid metabolite: Not detected.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
[phenyl-U-14C] test material: maximum 6.5% at day 155 that subsequently declined to 3.5% on day 358
[pyrazole-3-14C] test material: maximum 6.1% at day 155 that subsequently declined to 4.6% on day 358

EXTRACTABLE RESIDUES
- % of applied amount at day 0: [phenyl-U-14C] test material: 99.7%; [pyrazole-3-14C] test material: 99.0%
- % of applied amount at end of study period: [phenyl-U-14C] test material: 29.3%; [pyrazole-3-14C] test material: 26.2%

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: [phenyl-U-14C] test material: 0.4%; [pyrazole-3-14C] test material: 1.1%
- % of applied amount at end of study period: [phenyl-U-14C] test material: 30.1%; [pyrazole-3-14C] test material: 30.7%

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: [phenyl-U-14C] test material: 40.5%; [pyrazole-3-14C] test material: 33.5%

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: [phenyl-U-14C] test material: <0.1%; [pyrazole-3-14C] test material: <0.1%

STERILE TREATMENTS (if used)
- Transformation of the parent compound: At all sampling timepoints, extracts of the sterilized soil test systems were found to contain unchanged residual parent, as almost exclusive radiolabeled constituent (≥93.6% AR). A small amount (2.6 – 3.1% AR at days 0 – 41, rising to 3.7% AR at day 120) of the benzoic acid metabolite present throughout the phenyl radiolabel series was attributed to a slight impurity by this compound in the application solution, or to the activity of re-populating soil microbes.

RESULTS OF SUPPLEMENTARY EXPERIMENT:
Soil adsorption of the test material residues was found subject to increase with aging time, as concluded from a gradual change in extraction behavior noticed for the sterile soil control test systems. At the end of the study a mean Kd value of 4.1 mL/g - equivalent to a KOC of 88 mL/g - indicated significant reduction in mobility of the aged residues of the test material in soil.

Reference 4

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 Apr - 17 Nov 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2005

Soil no.:

#1

Soil type:

sandy loam

% Clay:

11

% Silt:

20

% Sand:

69

% Org. C:

1.4

pH:

6.5

CEC:

9.1 meq/100 g soil d.w.

Bulk density (g/cm³):

1.26

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Monheim/Northrine-Westfalia, Germany
- Pesticide use history at the collection site: 1999-2004: no pesticides used; 2005: ‘Roundup’, 5.0 L/ha (= glyphosate)
- Collection procedures: Five subsamples taken from field with spade and combined into one plastic bag (total amount ca. 30 kg).
- Sampling depth: 0 - 20 cm
- Storage conditions: Direct soil preparation, thereafter short-term storage at approx. +4 °C in aerated plastic bag until use for test system preparation.
- Storage length: 2005-04-14 to 2005-04-26 (12 days) from field sampling until test item application, thereof 8 days refrigeration storage.
- Soil preparation: Plant material and stones were removed; soil was passed through a 2 mm sieve.

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm: 10.4%
- Bulk density: 1.26 g/cm3

Soil No.:

#1

Duration:

120 d

Soil No.:

#1

Initial conc.:

0.133 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

[14C-UL-phenyl]

Soil No.:

#1

Initial conc.:

0.122 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

[14C-3-pyrazole]

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

20 °C

Humidity:

approx. 50% of maximum water holding capacity

Microbial biomass:

367 mg Cbiomass/kg (test start); 213 mg Cbiomass/kg (test end)

Details on experimental conditions:

1. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): Short-term storage, 12 days at approx. +4 °C in aerated plastic bag until use for test system preparation.
- Soil condition: Fresh
- Soil (g/replicate): 80 g/replicate
- Control conditions: sames as test with native soil, and soil treated with application solvent (without test item)
- No. of replication controls: Single flask at day 0, duplicate flasks (native soil/solvent control) at day 120.
- No. of replication treatments: Duplicate [phenyl-UL-14C]-test substance; duplicate [pyrazole-3-14C]-test substance
- Test apparatus: 300 mL glass Erlenmeyer flask
- Details of traps for CO2 and organic volatile: Static volatiles trap attachment for CO2 (soda lime) and organic volatiles (polyurethane foam), permeable for atmospheric oxygen.
- If no traps were used, is the system closed/open: system is open due to passive diffusion of the O2 containing atmosphere through the CO2 traps
- Identity and concentration of co-solvent: deionized water / methanol (85/15, v/v)

Test material application
- Volume of test solution used/treatment: 1000 μL per flask
- Application method: Dropwise application to the soil surface using a micropipette, further distribution in the soil by gentle shaking.
- Is the co-solvent evaporated: passive evaporation assumed to have occurred between application and soil mixing

Any indication of the test material adsorbing to the walls of the test apparatus: No

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: Maintained constant weight of test systems by periodically adding deionized water.
- Continuous darkness: Yes

2. OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: Passive diffusion of atmosphere containing O2 through the open CO2 traps

3. SAMPLING DETAILS
- Sampling intervals: Sampling timepoints were days 0, 1, 3, 7, 14, 21, 29, 41, 62, 86, and 120 after application.
- Sampling method for soil samples: At each sampling interval, duplicate test systems per radiolabel were removed from the incubation chamber, and processed.
- Method of collection of CO2 and volatile organic compounds: Acidic liberation of [14C]O2 from sodalime of trap system, solvent extraction of organic volatiles from polyurethane foam.
- Sampling intervals/times for:
> Moisture content: Day -4, 0, 1,3, 7, 14, 21, 29, 41, 49, 62, 86 and 120
> Sample storage before analysis: Soil was immediately extracted. No storage stability experiments were therefore conducted for storage of non-extracted soil.

Soil No.:

#1

Sampling day(s):

120 d

% Total extractable:

23.3

% Non extractable:

60.1

% CO2:

16.3

% Other volatiles:

< 0.1

% Recovery:

99.7

Remarks on result:

other:

Remarks:

Mean material balance of radioactivity in the water/sediment system after 358 days. [phenyl-UL-14C]-test substance

Soil No.:

#1

Sampling day(s):

120 d

% Total extractable:

19

% Non extractable:

62.1

% CO2:

18

% Other volatiles:

< 0.1

% Recovery:

99.1

Remarks on result:

other:

Remarks:

Mean material balance of radioactivity in the water/sediment system after 358 days. [pyrazole-3-14C]-test substance

Parent/product:

parent

Soil No.:

#1

% Degr.:

16.3

Parameter:

radiochem. meas.

Sampling time:

120 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [phenyl-UL-14C]-test substance

Parent/product:

parent

Soil No.:

#1

% Degr.:

18

Parameter:

radiochem. meas.

Sampling time:

120 d

Remarks on result:

other:

Remarks:

Mineralization to 14CO2 after 365 days. [pyrazole-3-14C]-test substance

Soil No.:

#1

DT50:

34 d

Type:

other: Single Compartment First Order

Temp.:

19.8 °C

Remarks on result:

other: [phenyl-UL-14C]

Soil No.:

#1

DT50:

70.9 d

Type:

other: Single Compartment First Order

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Calculated DT50 based on results at 25.1 °C. [phenyl-UL-14C]-test substance

Soil No.:

#1

DT50:

30 d

Type:

other: Single Compartment First Order

Temp.:

19.8 °C

Remarks on result:

other: [pyrazole-3-14C]

Soil No.:

#1

DT50:

62.5 d

Type:

other: Single Compartment First Order

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Calculated DT50 based on results at 25.1 °C. [pyrazole-3-14C]-test substance

Transformation products:

no

Remarks:

No major transformation products were detected.

Details on transformation products:

- Formation and decline of each transformation product during test: Identity of residual parent and its degradate test substance-benzoic acid in soil extracts was confirmed by demonsting co-elution with spikes of the corresponding authentic reference items, in two contrasting chromatography separation systems (reversed-phase HPLC, normal-phase TLC. For an exemplary sample ('mild extract' of day 14, [phenyl-UL-14C] test system), identity of residual parent and metabolite-benzoic acid was also confirmed by HPLC-MS/MS (spectrum of metabolite-benzoic acid compared with spectrum of the corresponding authentic reference metabolite-benzoic acid).
- Pathways for transformation: Concluded from the metabolic profiles observed for the two radiolabels in the test, [phenyl-UL-14C] and [pyrazole-3-14C] breakdown pathways of parent in aerobic soil involve cleavage between the phenyl and the pyrazole moiety of the molecule. Test substance-benzoic acid, the single degradate of significance derived from the phenyl moiety, was transient and mineralised to [14C]O2 or was 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 throughout the study, only formation of [14C]O2 and non-extractable residues. The initial attack to the parent molecule therefore seems to represent the rate determining step in the metabolic breakdown of the test substance in soil, which prevents any downstream intermediates from accumulation to levels of concern. Finally, both ring systems of the test substance are either mineralized, or included into the soil matrix as heterogenous non-extractable residues.

[phenyl-UL-14C]:
- No major transformation products (> 10 % of applied) were observed. A minor product was identified as [test substance]-benzoic 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
[pyrazole-3-14C]:
- 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.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: [phenyl-UL-14C]; test substance benzoic acid (2-methylsulfonyl-4-trifluoromethylbenzoic acid) 8.9 - 1.3% of AR on days 14 and 0, respectively [pyrazole-3-14C]: No extractable transformation products of significance were observed at any sampling interval. 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.
- Range of maximum concentrations in % of the applied amount at end of study period:
[phenyl-UL-14C]: test substance benzoic acid 2.3 ± 0.1% of AR on day 120
[pyrazole-3-14C]: No extractable transformation products of significance were observed.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
[phenyl-UL-14C]: Non-detectable - 2.1% (Sum of 2-3 minor compounds, each ≤ 1.2 % of applied) on days 0 and 120, respectively.
[pyrazole-3-14C]: 0.2 - 1.7% on days 0 and 120, respectively.

EXTRACTABLE RESIDUES
- % of applied amount at day 0:
[phenyl-UL-14C]: 101.3% AR
[pyrazole-3-14C]: 98.6% AR
- % of applied amount at end of study period:
[phenyl-UL-14C]: 23.3% AR
[pyrazole-3-14C]: 19.0% AR

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:
[phenyl-UL-14C]: 2.0% AR
[pyrazole-3-14C]: 2.7% AR
- % of applied amount at end of study period:
[phenyl-UL-14C]: 60.1% AR
[pyrazole-3-14C]: 62.1% AR

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:
[phenyl-UL-14C]: 16.3% AR
[pyrazole-3-14C]: 18.0% AR

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:
[phenyl-UL-14C]: < 0.1% AR
[pyrazole-3-14C]: < 0.1% AR

Reference 5

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Sep 2020 - 05 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Version / remarks:

2002

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Soil no.:

#1

Soil type:

silty clay loam

% Clay:

25.8

% Silt:

61.8

% Sand:

12.4

% Org. C:

2.2

pH:

6.5

CEC:

16 meq/100 g soil d.w.

Bulk density (g/cm³):

0.96

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Sarpy County, Louisville, Nebraska
- Pesticide use history at the collection site: No pesticides since 2000
- Collection procedures: The soil was sampled freshly from the field.
- Sampling depth: 0-6 inches
- Storage conditions: Refrigerated after receiving (T< 4 °C)
- Storage length: 10 days under refrigeration at the receiving facility

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at pF 2.0 (%): 38.4
- Bulk density (g/cm3): 1.5

Soil No.:

#1

Duration:

119 d

Soil No.:

#1

Initial conc.:

0.237 mg/kg soil d.w.

Based on:

test mat.

Remarks:

equivalent to a field application rate of 100 g/ha

Soil No.:

#1

Initial conc.:

0.251 mg/kg soil d.w.

Based on:

test mat.

Remarks:

equivalent to a field application rate of 100 g/ha

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

20 ± 0.001 °C

Humidity:

55 ± 5% of MWHC

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS: No

2. EXPERIMENTAL DESIGN
- Soil preincubation conditions: The test systems were equilibrated at study conditions for 5 days prior to application of active material.
- Soil (g/replicate): 50 ± 2
- Control conditions: Test Systems for determination of soil microbial biomass was untreated soil without application solvent and treated soil with application solvent.
- No. of replication controls, if used: 6 for biomass determination (start, middle, and end of study)
- No. of replication treatments: 2
- Test apparatus: The test systems consisted of cylindrical bottles connected to a continuous supply of humidified air, regulated by peristaltic pump heads.
- Details of traps for CO2 and organic volatile, if any: Flow-through system with ethylene glycol, 2 M KOH and 1 M H2SO4
- Identity of co-solvent: Acetonitrile was evaporated under a stream of nitrogen and then the test material was resolved in MeOH:H2O (1:1).

Test material application
- Volume of test solution used/treatment: 180 µL
- Application method: Application solution was applied drop-wise onto the soil surface of the respective equilibrated test systems using a 250 μL Hamilton gas-tight syringe.
- Is the co-solvent evaporated: No

Experimental conditions
- Moisture maintenance method: Water loss from evaporation was determined by weighing all test systems for kinetic samples at study start, biweekly, and the respective test systems at each sampling interval. Moisture was adjusted by adding water to obtain initial test system weight as needed throughout the incubation period.
- Continuous darkness: Yes

3. OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the an/aerobic conditions: Aerobic (air flowing through trap attachments permeable to oxygen).

4. SUPPLEMENTARY EXPERIMENTS: No

5. SAMPLING DETAILS
- Sampling intervals: Replicate samples were processed and analyzed at 0, 2, 3, 4, 6, 14, 27, 56, 95 and, 119 days after treatment.
- Sampling method for soil samples: The entire soil content of each test system was transferred into a bottle using the extraction solvent (ACN/H2O 4:1).
- Method of collection of CO2 and volatile organic compounds: The volume of the ethylene glycol and 1 M H2SO4 traps were recorded. The two 2M KOH traps were combined, and volume was recorded. Three 0.5 mL aliquots of each were radioassayed by LSC to determine the total radioactivity trapped.
- Sampling intervals/times for:
> Moisture content: At study start, biweekly, and the respective test systems at each sampling interval.
- Sample storage before analysis: The soils were processed (extracted) immediately after sampling; first HPLC/radiodetection analysis of soil extracts were performed within 3 days for most samples. Storage stability data has been provided for the samples that were stored more than 3 days.

Soil No.:

#1

Sampling day(s):

119 d

% Total extractable:

7.5

% Non extractable:

69.9

% CO2:

22.6

% Other volatiles:

0

% Recovery:

100

Parent/product:

parent

Soil No.:

#1

% Degr.:

22.6

Parameter:

radiochem. meas.

Sampling time:

119 d

Remarks on result:

other:

Remarks:

Mineralization of parent substance to 14CO2

Soil No.:

#1

DT50:

3.06 d

Type:

other: First oder multi compartment (FOMC)

Temp.:

20 °C

Soil No.:

#1

DT50:

6.5 d

Type:

other: First oder multi compartment (FOMC)

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Calculated DT50, based on results at 20 °C.

Transformation products:

not specified

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Description of key information

DT50 of parent substance in soil: 6.5 days at 12 °C (aerobic, OECD 307, one soil, 22.6% mineralization after 119 days)

 DT50 of parent substance in soil: >120 days at 12 °C (anaerobic, OECD 307, one soil, 2.2% mineralization after 120 days)

Additional information

In the key study, the biodegradation of radiolabeled [phenyl-UL-14C]-(5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl)methanone was studied in a silty clay loam (pH 6.5, organic carbon content 2.2%) for 119 days at 20 °C under aerobic conditions (M-765810-01-1). The study was conducted according to OECD Guideline 307 (2002) and GLP. After an incubation period of 119 days 22.6% of the applied radioactivity were mineralized to 14CO2. The degradation half-life of the parent substance was calculated using a first order multi compartment model (FOMC) and accounted for 3.06 days (6.5 days, recalculated to 12 °C).

 

A second key study investigated the aerobic/anaerobic biotransformation of radiolabeled test substance, (5-hydroxy-1,3-dimethyl-1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethylphenyl) methanone 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. 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).

Phenyl label:. 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 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. No more than 1.4% of additional CO2 and volatile organic compounds were produced throughout the anaerobic phase of the study.

Pyrazole label: 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. There were no major transformation products formed.

No more than 1.2% of additional CO2 and volatile organic compounds were produced throughout the anaerobic phase of the study.. 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).

These results are supported by three more supporting studies.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-0778563-01-2The 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 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. 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

 

In a second supporting study, 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). 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 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). At study termination, evolved 14CO2 accounted for 40.5% of the applied phenyl radiolabel, or 33.5% of the applied pyrazole radiolabel.

 

In a third supporting study, 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. The experiment was conducted in accordance with the OECD 307, and in compliance with GLP.

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