4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

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:

07 Jun 2016 to 06 Jul 2018

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

Deviations:

yes

Remarks:

The incubation temperature rose above 22 °C for approx. 4 days (max. 25.1 °C for a very short period). The deviation had no impact on the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4100 (Aerobic Soil Metabolism)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2016

Soil no.:

#1

Soil type:

silt loam

% Clay:

10

% Silt:

52

% Sand:

38

% Org. C:

1.8

pH:

7.2

CEC:

8.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#2

Soil type:

silt loam

% Clay:

6

% Silt:

54

% Sand:

40

% Org. C:

2

pH:

7

CEC:

9.1 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#3

Soil type:

sandy clay loam

% Clay:

24

% Silt:

22

% Sand:

54

% Org. C:

1.8

pH:

6.2

CEC:

16.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#4

Soil type:

sandy loam

% Clay:

6

% Silt:

24

% Sand:

70

% Org. C:

1.8

pH:

7

CEC:

12.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#5

Soil type:

loam

% Clay:

24

% Silt:

42

% Sand:

34

% Org. C:

2.2

pH:

6.5

CEC:

17.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#6

Soil type:

clay loam

% Clay:

36

% Silt:

30

% Sand:

34

% Org. C:

0.96

pH:

6.7

CEC:

20 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Details on soil characteristics:

Detailed Information of solil collection, accilimation and physio-chemical properties is provided in Table 1 in 'Any other information on materials and methods incl. tables'

SOIL STORAGE AND ACCLIMATION
- Storage: Upon arrival, the soils were kept moist and stored in the dark at approx. 4 °C
- Soil preparation: The soils were sieved using a 2 mm mesh sieve prior to analyses. The soils were acclimatised under aerobic conditions at 20 + 2 °C and at approximately the same moisture content used throughout the incubation period under darkness for 1 - 2 weeks. Following determination of the soil moisture content, aliquots of 100 g dry soil equivalent were dispensed into metabolism flasks. During incubation, moisture levels were observed at approximately fortnightly intervals, and water was added accordingly when necessary. Additional flasks containing treated soil were prepared to be used for the radio-validation and were incubated under the same conditions. In addition to the flasks containing treated soil, control soil samples were incubated under the same conditions as the treated samples. These served to determine the microbial biomass at the end of the incubation period. The microbial biomass was determined using the respiration method.

Duration:

120 d

Based on:

other: See Table 2 in 'Any other information on materials and methods incl. tables'

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

test mat. analysis

Soil No.:

#1

Temp.:

20 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 44.5

Microbial biomass:

35.79 mg C/100g soil at start and 31.33 mg C/100 soil at end of study

Soil No.:

#2

Temp.:

20 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 43.4

Microbial biomass:

38.11 mg C/100g soil at start and 29.68 mg C/100 soil at end of study

Soil No.:

#3

Temp.:

20 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 42.2

Microbial biomass:

54.59 mg C/100g soil at start and 39.13 mg C/100 soil at end of study

Soil No.:

#4

Temp.:

20 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 33.2

Microbial biomass:

38.20 mg C/100g soil at start and 24.19 mg C/100 soil at end of study

Soil No.:

#5

Temp.:

20 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 34.5

Microbial biomass:

46.35 mg C/100g soil at start and 29.52 mg C/100 soil at end of study

Soil No.:

#6

Temp.:

20 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 18.4

Microbial biomass:

20.52 mg C/100g soil at start and 21.64 mg C/100 soil at end of study

Details on experimental conditions:

- An overview of experimental design and sampling details are provided in Table 2 - Table 4 in 'Any other information on materials and methods incl. tables'

PREPARATION OF STOCK AND APPLICATION SOLUTIONS
The aim was to apply the radiolabelled test items at a target concentration of 20 µg test item per system, corresponding to 150 g test item/ha, assuming an incorporation depth of 5 cm and a bulk density of 1.5 g/cm3.
- Stock solution of [methylphenyl-U-14C]-labelled test substance: The entire radioactive test item (batch no. RDR-XXV- 30) was dissolved in 10 mL of acetonitrile. In order to determine the radioactive concentration of the stock solution, an aliquot of the stock solution was diluted (5 µL up to 10 mL with acetonitrile). Three 300 µL aliquots of the diluted solution were then radioassayed by LSC. The total radioactivity in the stock solution was determined to be 5’494’522’222 dpm, corresponding to 91.58 MBq, in 10 mL.
- Application solution of [methylphenyl-U-14C]-labelled test substance for Gartenacker, 18 Acres and East Anglia soils: An aliquot of the stock solution (1166 µL) was evaporated to dryness under a stream of nitrogen and dissolved in methanol/water 1/1 v/v to a volume of 100 mL. The radioactivity content of the application solution was determined by measuring triplicate 10 µL aliquots by LSC. The amount of radioactivity in the application solution was determined to be 613’440’000 dpm/100 mL. Hence, the concentration of the application solution was 0.0215 mg/mL. The exact amount of test substance applied to each vessel was determined from homogeneity checks taken before, during and after application.
- Application solution of [methylphenyl-U-14C]-labelled test substance for Sarpy and Capay soils: An aliquot of the stock solution (approximately 1 mL) was evaporated to dryness under a stream of nitrogen and dissolved in methanol/water 1/1 v/v to a volume of 100 mL. The radioactivity content of the application solution was determined by measuring triplicate 10 µL aliquots by LSC. The amount of radioactivity in the application solution was determined to be 532’710’000 dpm/100 mL. Hence, the concentration of the application solution was 0.0187 mg/mL. The exact amount of test substance applied to each vessel was determined from homogeneity checks taken before, during and after application.
- Stock solution of [halophenyl-U-14C]-labelled test substance for Gartenacker soil: The entire radioactive test item (batch no. RDR-XXVII-55) was dissolved in 2 mL of acetonitrile. In order to determine the radioactive concentration of the stock solution, an aliquot of the stock solution was diluted in triplicate (10 µL up to 10 mL with acetonitrile). Three 100 µL aliquots of the diluted solution were then radioassayed by LSC. The total radioactivity in the stock solution was determined to be 413’360’000 dpm, corresponding to 6.89 MBq, in 2 mL.
- Application solution of [halophenyl-U-14C]-labelled test substance for Gartenacker soil: An aliquot of the stock solution (1.69 mL) was evaporated to dryness under a stream of nitrogen and dissolved in methanol/water 7/5 v/v to a volume of 60 mL. The radioactivity content of the application solution was determined by measuring triplicate 10 µL aliquots by LSC. The amount of radioactivity in the application solution was determined to be 333’390’000 dpm/60 mL, corresponding to 0.978 mg/60 mL. Hence, the concentration of the application solution was 0.0160 mg/mL. The exact amount of test substance applied to each vessel was determined from homogeneity checks taken before, during and after application.
- Stock solution of [halophenyl-U-14C]-labelled test substance for East Anglia soil: The entire radioactive test item (batch no. BPM-LI-47-1) was dissolved in 10 mL of acetonitrile. In order to determine the radioactive concentration of the stock solution, an aliquot of the stock solution was diluted (5 µL up to 10 mL with acetonitrile). Three 500 µL aliquots of the diluted solution were then radioassayed by the test substance. The total radioactivity in the stock solution was determined to be 3’451’046’667 dpm, corresponding to 57.52 MBq, in 10 mL.
- Application solution of [halophenyl-U-14C]-labelled test substance for East Anglia soil: An aliquot of the stock solution (1 mL) was evaporated to dryness under a stream of nitrogen and dissolved in methanol/water 1/1 v/v to a volume of 50 mL. The radioactivity content of the application solution was determined by measuring triplicate 10 µL aliquots by the test substance. The amount of radioactivity in the application solution was determined to be 324’655’000 dpm/50 mL, corresponding to 0.978 mg/50 mL. Hence, the concentration of the application solution was 0.0196 mg/mL. The exact amount of test substance applied to each vessel was determined from homogeneity checks taken before, during and after application.
- Stock solution of [oxoisoxazolidinyl-4,5-14C]-labelled test substance: The entire radioactive test item(batch no. MGGIV-88-1) was dissolved in 20 mL of acetonitrile. In order to determine the radioactive concentration of the stock solution, an aliquot of the stock solution was diluted (5 µL up to 10 mL with acetonitrile). Three 1000 µL aliquots of the diluted solution were then radioassayed by LSC. The total radioactivity in the stock solution was determined to be 3’482’220’000 dpm, corresponding to 58.04 MBq, in 10 mL.
- Application solution of [oxoisoxazolidinyl-4,5-14C]-labelled test substance: An aliquot of the stock solution (2 mL) was evaporated to dryness under a stream of nitrogen and dissolved in methanol/water 1/1 v/v to a volume of 50 mL. The radioactivity content of the application solution was determined by measuring triplicate 10 µL aliquots by the test substance. The amount of radioactivity in the application solution was determined to be 333’455’000 dpm/50 mL, corresponding to 1.061 mg/50 mL. Hence, the concentration of the application solution was 0.0212 mg/mL. The exact amount of test substance applied to each vessel was determined from homogeneity checks taken before, during and after application.

APPLICATION OF RADIOLABELLED TEST ITEMS
An aliquot of 900 µL (for Gartenacker, 18 Acres and East Anglia soils treated with the methylphenyl-labelled test item) or 1000 µL (for Sarpy and Capay soils treated with the methylphenyl-labelled test item; and East Anglia soil treated with the halophenyl- and oxoisoxazolidinyl-radiolabelled test item) or 1250 µL (for Gartenacker treated with the halophenyl-radiolabelled test item) of the application solution of was applied to the surface of each soil sample using a syringe. The treated soil flasks were mixed thoroughly allowing the organic solvent to evaporate and to ensure uniform distribution of the test item applied. The amount of the organic solvent was ≤ 0.7 % v/w of the dry weight of soil. Prior to, during and after the application procedure, an aliquot of the application solution equal to the aliquot applied to the soil (i.e. 900 or 1000 µL or 1250 µL, respectively) was separately taken into a volumetric flask and made up to 20 mL with acetonitrile/water, 1/1 v/v and three 100 µL aliquots measured by LSC (homogeneity check). Untreated soil samples for the determination of the microbial biomass were treated in the same way using the same volume of solvent used in the treated samples but without test item.

INCUBATION CONDITION
For sampling on day 0, no volatile traps were set up, as the samples were worked-up immediately after application. All other flasks were connected to the gas-flow system and incubated in an incubation chamber. The mean temperature was 20.7 ± 0.5 °C between 14 June 2016 and 04 November 2016 (incubation of all soils and labels with the exception of Gartenacker soil treated with halophenyl labelled test item) and 20.3 ± 0.7 °C between 31 May 2017 and 28 September 2017 (incubation of Gartenacker soil treated with halophenyl labelled test item).
- Ventilation: During incubation, the system was continuously ventilated with moistened air to maintain aerobic conditions while minimizing water loss. Any volatile radioactivity was continuously flushed from the incubation vessels and collected in NaOH traps.

Remarks on result:

other: See Table 6 in 'Any other information on results incl. tables'

Key result

Soil No.:

#1

DT50:

56 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Remarks on result:

other: #1 and #2 soil are both Gartenacker (See Table 1 in 'Any other information on materials and methods incl. tables')

Key result

Soil No.:

#3

DT50:

125 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Key result

Soil No.:

#4

DT50:

80 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Key result

Soil No.:

#5

DT50:

112 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Key result

Soil No.:

#6

DT50:

293 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

No.:

#6

Details on transformation products:

RADIOACTIVE RESIDUES IN SOIL AQUEOUS ACETONITRILE EXTRACTS
- Methylphenyl label: Levels of parent compound in the treated soils decreased over the 120 day incubation period ranging from a mean level between 99.3 and 102.8% AR at time 0 and degrading to a mean level between 24.7 and 76.7% AR at the end of incubation (120 DAT). In four soils, up to two metabolites were present at levels of > 5% AR. In Capay soil, no metabolites accounted for mean levels > 5% AR. Two major metabolites were observed. Metabolite M2 was a major metabolite in four out of the five soils tested, first appearing after 14 days of incubation. In Gartenacker, 18 Acres, Sarpy and Capay soils, levels of M2 increased relatively slowly over time before reaching a plateau at approximately 63 DAT. Maximum mean levels in these soils were 13.2, 7.5, 8.4 and 3.2% AR, respectively. In East Anglia soil, M2 continued to increase steady throughout the entire incubation period, reaching a maximum mean level of 23.5% AR at 120 DAT.
A metabolite reached maximum levels towards the end of the incubation period (77 - 120 DAT) of 3.9, 6.5, 4.3, 10.8 and 1.3 % AR in Gartenacker, 18 Acres, East Anglia, Sarpy and Capay soils, respectively. This metabolite was shown to co-chromatograph with M8 by radio-HPLC. However, during the analysis of the 91 DAT methylphenyl labelled and 120 DAT halophenyl labelled Gartenacker soil extracts by LC-MS, another unidentified component was found to be present within the this metabolite radio-peak when analysed using HPLC. The 120 DAT halophenyl labelled Gartenacker soil extract was used to develop an additional HPLC method to separate M9 and the unidentified component to be able to accurately quantify them. Re-analysis of the 18 Acres and Sarpy soil extracts (where the metabolite was present at > 5% AR) confirmed that this metabolite comprised only of M9 in these soils. In the other three soils, this metabolite was found at levels < 5% AR, and therefore no further analysis was undertaken for these soils to determine whether this metabolite consisted solely of M9 or contained additional metabolite(s). No other metabolites accounted for mean levels > 5% AR including metabolites M49, M14, M5 and M6, which reached maximum levels of 4.7, 3.1, 3.0 and 4.8% across all soils, respectively.
- Halophenyl label: Levels of parent compound in the treated soils decreased over the 120 day incubation period from a mean level of 95.7% AR for Gartenacker soil and 97.4% AR for East Anglia soil at time 0 to a mean level of 30.4% AR in Gartenacker soil and 34.1% AR in East Anglia soil at the end of incubation (120 DAT). Only one metabolite was present at levels of > 5% AR. Metabolite M2, first appeared after 28 DAT in Gartenacker soil and after 14 DAT in East Anglia soil and reached maximum mean levels of 21.7 and 25.6% AR at 105 and 120 DAT in Gartenacker and East Anglia soils, respectively. No other metabolites accounted for mean levels > 5% AR, including M49, M14, M5, M6 and M9, which reached maximum levels of 4.4, 4.0, 3.8, 3.6 and 4.4% AR across both soils.
- Oxoisoxazolidinyl label: For East Anglia soil treated with the oxoisoxazolidinyl-labelled test item, levels of parent compound decreased over the 120 day incubation period from a mean level of 98.1% AR at time 0 to 42.0% AR at the end of incubation (120 DAT). No metabolites accounted for mean levels > 5% AR. Mean maximum levels for M14 and M5 were 2.8 and 2.4% AR, respectively.

PROPOSED METABOLIC PATHWAY
- In all soils, the test substance degraded primarily by opening of the oxoisoxazolidinyl ring to form either M14 or M5. Further hydrolysis of the amide in M5 could also result in further formation of the carboxylic acid M14. Elimination of the oxoisoxazolidinyl moiety yielded primary amide M49. Further amide hydrolysis yielded the carboxylic acid M2. Methylation of M2 yielded the methyl ester M9. Decarboxylation of the acid M2 yielded the phenol metabolite M6.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Remarks:

See Table 8 in 'Any other information on results incl. tables'

Residues:

yes

Remarks:

See 'Details on trasformation products'

Details on results:

An overview of the results is provided in Table 5 to Table 17 in 'Any other information on results incl. tables'.

MICROBIAL BIOMASS
The microbial biomass in all soils was ≥ 1.3% OC at the start and end of the study. Hence, the soils were deemed to be suitably microbially active for the purposes of the study.

RADIOCHEMICAL PURITY
The radiochemical purity of the application solutions determined by both HPLC and TLC was ≥ 98.9% before and after treatment, demonstrating the stability of the test item in the application solutions.

TREATMENT RATE
The application solutions remained homogeneous throughout the treatment processes (CV ≤1.2%). Based on the homogeneity radioassays (application controls), the actual amount of radioactivity and radiolabelled test items applied to the test soil samples is shown in Table 4 in 'Any other information on results incl. tables'.

MASS BALANCE
The mass balances (mean values of duplicate samples) in all soil samples ranged from 94.1 to 104.8% AR with an overall average per soil and label system ranging from 97.6% AR to 100.9% AR. The mass balance from all individual soil samples ranged from 89.7 to 105.7%.

VOLATILE DEGRADATION PRODUCTS
Formation of radioactive carbon dioxide reached mean levels between 0.2 and 6.7% AR at the end of the study for all soils applied with the test item labelled in methylphenyl position. In halophenyl labelled samples, levels of radioactivity present in the NaOH traps at the end of the study ranged from 1.1 - 2.3% AR. Formation of radioactive carbon dioxide was much higher for the oxoisoxazolidinyl label, reaching a mean of 19.9% AR at the end of the study. The identity of 14CO2 was confirmed by precipitation with barium hydroxide for Gartenacker soil (applied with the methylphenyl-labelled test item) and East Anglia soil (applied with the oxoisoxazolidinyl-labelled test item). Radioactivity trapped in selected foam bungs, that were included in the test soils treated with the halophenyl label, was < 0.1% AR. The foam bungs were confirmed to contain no significant radioactivity.

DISTRIBUTION OF RADIOACTIVE RESIDUES
The total amount of radioactivity extracted decreased slowly over time in all soils, ranging from 97.6 to 104.7% AR at 0 DAT and from 51.7 to 87.8% AR at 120 DAT. The majority of the radioactivity was recovered in the aqueous acetonitrile mixtures, which ranged from 95.7 - 102.8% AR for all soils at 0 DAT and from 50.3 to 85.4% AR at 120 DAT. The maximum amounts of radioactivity present in the THF and isohexane extracts at any time point were 4.1% and 0.6%, respectively. Unextracted residues (bound residues) increased slowly throughout the incubation, reaching maximum of 11.4 to 36.4% AR by the end of the incubation.

DT50 OF THE TEST SUBSTANCE IN SOIL
The rate of degradation in tested soils for the test substance was calculated using single first-order (SFO). The degradation half-life (DT50) of the test subtance was calculated to be 56, 125, 80, 112 and 293 days in Gartenacker, 18 Acres, East Anglia, Sarpy and Capay soil, respectively. The SFO kinetic model describes the degradation of the test substance with a χ2-square value ≤ 4.5, linear regression coefficient r2 ≥ 0.9175 and with an overall sufficient goodness of the fit as concluded from a visual assessment of the plots in all cases.


STEREOISOMERS OF THE TEST SUBSTANCE AND ITS MAJOR METABOLITES
The test substance is a chemical substance with two asymmetric carbon atoms, and therefore is comprised of four stereoisomers: “S, R”, “S, S”, “R, R” and “R, S”. According to the certificates of analysis of the three radiolabelled test items, the applied radiolabelled test substance consisted mainly of the “S, R” isomer, “Isomer A”, and accounting for 92.2 - 92.4% of the test substance. The “S, S” (“Isomer B”), “R, R” (“Isomer C”) and “R, S” (“Isomer D”) contributed 0.8 - 1.3%, 6.4 - 6.9% and < 0.1%, respectively. Application solutions for each radiolabelled test item, as well as soil extracts from 0 and 120 DAT for all soils and labels were analysed by chiral LC-MS to determine the isomer ratio of parent, two major metabolites, the acid M5 and the methyl ester M8. Additionally, soil extracts from selected intervals of 63, 105, 91, 63 and 63 DAT for Gartenacker, 18 Acres, East Anglia, Sarpy and Capay soils, respectively (all treated with the methylphenyl label) as well as the interval of 63 DAT for the two soils treated with the halophenyl label were analysed by chiral LC-MS to determine the isomer ratio of the amide M4. The ratio of Isomer A and other stereoisomers of the parent in the both the application solutions and 0 and 120 DAT soil extracts was confirmed to be consistent with that reported in the Certificates of Analysis for the test items. The isomer ratio for the three metabolites, M4, M5 and M8, were also found to be consistent with that predicted from the isomer ratio of the parent test items. Hence, it was confirmed that there was no change of stereochemistry during the incubation time of 120 days and hence, there was no evidence of either interconversion or stereoselective metabolism of either the test substance or its metabolites.

RADIOVALIDATION OF THE EXTRACTABLILITY OF THE RESIDUE ANALYTICAL METHODS
The data shows that the extractability of radioactive residues using the metabolism methodology and the residue analytical method were in good agreement, and that following chromatographic analysis of the extracted radioactivity, the levels of parent and its degradates detected were also in good agreement.

STORAGE STABILITY
In order to demonstrate the storage stability of the representative extracts during the interim period between initial and final analysis, chromatographic profiles obtained with HPLC initially, i.e. either 1 week or approx. 4 months after sampling were compared with profiles of the same extracts obtained approx. 5 or 9 - 12 months after sampling, respectively.
Comparison of initial and final radio-HPLC profiles showed that no significant changes in the profiles had occurred during the interim period of storage reflecting adequate stability in extracts stored during the study.

Table 5. Summary of Radiochemical Purity Determinations of Radiolabelled test substance

Test item radiolabel position	Soil	Radiochemical Purity (%)
		Pre-application	Post-application
Methylphenyl	Gartenacker	100.0	100.0
Methylphenyl	Gartenacker	99.5	98.9
Methylphenyl	18 Acres	100.0	100.0
Methylphenyl	18 Acres	99.5	98.9
Methylphenyl	East Anglia	100.0	100.0
Methylphenyl	East Anglia	99.5	98.9
Methylphenyl	Sarpy	99.7	99.7
Methylphenyl	Sarpy	100.0	100.0
Methylphenyl	Capay	99.7	99.7
Methylphenyl	Capay	100.0	100.0
Halophenyl	Gartenacker	99.8	99.5
Halophenyl	Gartenacker	99.0	99.3
Halophenyl	East Anglia	99.1	99.5
Halophenyl	East Anglia	99.4	99.4
Oxoisoxazolidinyl	East Anglia	100.0	100.0
Oxoisoxazolidinyl	East Anglia	100.0	100.0

Table 6. Mass balance

Total radioa ctivity	Sum of activity in soil extracts, residue on combustion and that trapped as14CO2in the 2M sodium hydroxide traps as well as that trapped in foam bungs, where applicable.
Mean Recovery at 0 DAT	Range 97.7 to 104.8% of applied dose
Overall recovery (all samples)	Range 89.7 to 105.7% of applied dose Gartenacker (methylphenyl label): Range = 97.0 - 105.7%, Mean = 100.7% Gartenacker (halophenyl label): Range = 95.8 - 99.8%, Mean = 97.6% 18 Acres (methylphenyl label): Range = 98.5 - 103.4%, Mean = 100.9% East Anglia (methylphenyl label): Range = 97.8 - 103.4%, Mean = 100.2% East Anglia (halophenyl label): Range = 95.3 - 102.2%, Mean = 98.7% East Anglia (oxoisoxazolidinyl label): Range = 96.1 - 102.0%, Mean = 99.6% Sarpy (methylphenyl label): Range = 89.7 - 102.6%, Mean = 98.4% Capay (methylphenyl label): Range = 96.6 - 103.8%, Mean = 99.8%

 

Table 7. Volatilisation

14CO2	Small to medium amounts of radioactivity were evolved as volatile products throughout the course of the study.
	14CO2 evolved at end of study	Gartenacker (methylphenyl label): 6.7% Gartenacker (halophenyl label): 2.3% 18 Acres (methylphenyl label): 1.3% East Anglia (methylphenyl label): 1.7% East Anglia (halophenyl label): 1.1% East Anglia (oxoisoxazolidinyl label): 19.9% Sarpy (methylphenyl label): 1.6% Capay (methylphenyl label): 0.2%
Other volatiles	No other volatiles were captured. Foam bungs were analysed for a single sample (Gartenacker, halophenyl label, 120 DAT; both replicates) and showed < 0.1% AR.

 

Table 8. Comparison of the Soil Metabolism (SM) and Residue (R) Extraction Methods (Radiovalidation of Residue Method): Distribution and Recovery of Radioactivity ([Methylphenyl- U-14C]-labelled test substance).

[Methylphenyl- U-14C]- labelled test substance (% AR)	Replicate	Present of applied radioactivity (%AR) by soil and Extraction method
		Gartenacker		18 Acres		East Anglia		Sarpy		Capay
		SM	R	SM	R	SM	R	SM	R	SM	R
MeCN:H2O extracts	A	55.6	54.8	78.6	80.4	78.3	73.7	75.0	78.4	86.8	84.4
	B	52.3	53.6	80.1	78.8	73.8	74.9	77.1	77.0	84.1	85.7
	Mean	54.0	54.2	79.3	79.6	76.1	74.3	76.1	77.7	85.4	85.1
THF extracts	A	1.7	n.p.	1.9	n.p.	1.6	n.p.	2.6	n.p.	2.2	n.p.
	B	2.0	n.p.	2.3	n.p.	1.6	n.p.	2.1	n.p.	2.1	n.p.
	Mean	1.8	n.p.	2.1	n.p.	1.6	n.p.	2.4	n.p.	2.1	n.p.
Isohexane extracts	A	0.3	n.p.	0.2	n.p.	0.1	n.p.	0.3	n.p.	0.2	n.p.
	B	0.3	n.p.	0.2	n.p.	0.1	n.p.	0.3	n.p.	0.3	n.p.
	Mean	0.3	n.p.	0.2	n.p.	0.1	n.p.	0.3	n.p.	0.2	n.p.
Total Extractables	A	57.6	54.8	80.6	80.4	80.0	73.7	77.9	78.4	89.2	84.4
	B	54.6	53.6	82.5	78.8	75.5	74.9	79.6	77.0	86.4	85.7
	Mean	56.1	54.2	81.6	79.6	77.7	74.3	78.7	77.7	87.8	85.1
Non- extractables	A	34.7	38.2	18.5	18.3	21.7	22.7	19.5	18.7	11.1	17.7
	B	38.2	37.1	17.2	20.2	21.7	20.6	18.5	25.4	11.7	17.9
	Mean	36.4	37.7	17.9	19.3	21.7	21.7	19.0	22.0	11.4	17.8
14CO2	A	6.7	7.2	1.4	1.0	1.6	1.7	1.7	1.3	0.2	0.2
	B	6.7	7.2	1.2	1.1	1.7	0.9	1.6	1.7	0.2	0.2
	Mean	6.7	7.2	1.3	1.1	1.7	1.3	1.6	1.5	0.2	0.2
Total % recovery	A	99.0	100.3	100.6	99.8	103.4	98.0	99.0	98.4	100.5	102.3
	B	99.5	98.0	100.9	100.2	98.9	96.4	99.7	104.1	98.3	103.8
	Mean	99.3	99.1	100.7	100.0	101.1	97.2	99.4	101.2	99.4	103.0

n.p. = not performed.

Table 9. Distribution and Recovery of Radioactivity: Gartenacker Soil (Halophenyl-U-14C]-labelled test substance)

[Halophenyl-U- 14C]-labelled test substance Gartenacker (% AR)	Replicate		Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	7	14	28	42	63	77	91	105	120
MeCN:H2O extracts	A	95.5	n.p.	n.p.	88.9	82.1	75.5	70.6	63.9	63.7	62.7
	B	95.9	n.p.	n.p.	87.8	84.2	76.7	73.5	62.1	64.5	61.6
	Mean	95.7	n.p.	n.p.	88.4	83.1	76.1	72.1	63.0	64.1	62.2
THF extracts	A	1.7	n.p.	n.p.	1.5	2.1	2.0	2.0	2.4	2.1	2.0
	B	1.7	n.p.	n.p.	1.7	2.1	2.0	1.8	2.4	2.1	2.1
	Mean	1.7	n.p.	n.p.	1.6	2.1	2.0	1.9	2.4	2.1	2.0
Isohexane extracts	A	0.3	n.p.	n.p.	0.3	0.5	0.4	0.4	0.5	0.5	0.4
	B	0.3	n.p.	n.p.	0.4	0.4	0.4	0.4	0.5	0.5	0.4
	Mean	0.3	n.p.	n.p.	0.4	0.4	0.4	0.4	0.5	0.5	0.4
Total Extractables	A	97.4	n.p.	n.p.	90.7	84.7	77.9	73.1	66.8	66.3	65.1
	B	97.8	n.p.	n.p.	89.9	86.6	79.0	75.7	65.0	67.1	64.1
	Mean	97.6	n.p.	n.p.	90.3	85.6	78.5	74.4	65.9	66.7	64.6
Non- extractables	A	<0.1	n.p.	n.p.	6.3	12.2	17.7	22.6	26.8	29.6	32.1
	B	<0.1	n.p.	n.p.	5.8	10.6	17.7	21.6	29.5	28.7	33.4
	Mean	<0.1	n.p.	n.p.	6.1	11.4	17.7	22.1	28.2	29.2	32.8
14CO2	A	n.p.	n.p.	n.p.	<0.1	0.2	0.9	1.3	2.2	2.7	2.6
	B	n.p.	n.p.	n.p.	0.1	0.2	0.9	1.5	2.3	2.6	2.0
	Mean	n.p.	n.p.	n.p.	<0.1	0.2	0.9	1.4	2.2	2.7	2.3
Foam bung trapped volatiles	A	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	<0.1
	B	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	<0.1
	Mean	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	n.p.	<0.1
Total % recovery	A	97.5	n.p.	n.p.	97.1	97.0	96.5	96.9	95.8	98.7	99.8
	B	97.9	n.p.	n.p.	95.9	97.4	97.6	98.8	96.8	98.5	99.5
	Mean	97.7	n.p.	n.p.	96.5	97.2	97.1	97.9	96.3	98.6	99.7
OVERALL MEAN ± SD		97.6 ± 1.2

n.p. = not performed.

Table 10. Distribution and Recovery of Radioactivity: East Anglia Soil ([Halophenyl-U-14C]-labelled test substance and [Oxoisoxazo- lidinyl-4,5-14C]- labelled test substance )

a)

[Halophenyl-U- 14C]-labelled test substance East Anglia (% AR)	Replicate		Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	7	14	28	42	63	77	91	105	120
MeCN:H2O extracts	A	97.8	100.2	97.4	88.5	87.0	85.0	81.6	78.4	74.6	73.3
	B	96.9	96.6	95.5	87.4	87.3	85.7	79.8	77.7	74.2	74.2
	Mean	97.4	98.4	96.5	87.9	87.2	85.3	80.7	78.0	74.4	73.7
THF extracts	A	1.4	0.8	0.8	0.9	0.9	1.5	1.5	1.7	1.6	2.1
	B	0.6	0.8	0.8	0.9	0.9	1.5	1.4	1.6	1.6	2.1
	Mean	1.0	0.8	0.8	0.9	0.9	1.5	1.5	1.6	1.6	2.1
Isohexane extracts	A	0.2	<0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
	B	<0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
	Mean	0.1	<0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Total Extractables	A	99.4	101.0	98.3	89.4	88.1	86.7	83.2	80.2	76.3	75.4
	B	97.6	97.5	96.3	88.3	88.3	87.3	81.4	79.4	75.9	76.5
	Mean	98.5	99.2	97.3	88.9	88.2	87.0	82.3	79.8	76.1	75.9
Non- extractables	A	<0.1	1.1	2.4	6.3	8.9	12.5	15.9	20.4	20.5	22.0
	B	<0.1	1.1	2.8	6.8	8.3	12.5	15.3	18.5	20.5	23.0
	Mean	<0.1	1.1	2.6	6.6	8.6	12.5	15.6	19.5	20.5	22.5
14CO2	A	n.p.	<0.1	<0.1	0.2	0.2	0.4	0.5	0.8	0.9	1.0
	B	n.p.	<0.1	<0.1	0.2	0.2	0.4	0.4	0.7	0.9	1.2
	Mean	n.p.	<0.1	<0.1	0.2	0.2	0.4	0.5	0.8	0.9	1.1
Total % recovery	A	99.5	102.2	100.9	95.9	97.1	99.5	99.5	101.4	97.7	98.4
	B	97.7	98.6	99.2	95.3	96.8	100.2	97.1	98.7	97.2	100.6
	Mean	98.6	100.4	100.0	95.6	97.0	99.9	98.3	100.1	97.5	99.5
OVERALL MEAN ± SD		98.7 ± 1.9

Note: Foam bang extraction/analysis was not performed.

b)

[Oxoisoxazo- lidinyl-4,5-14C]- labelled test substance East Anglia (% AR)	Replicate		Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	7	14	28	42	63	77	91	105	120
MeCN:H2O extracts	A	98.4	98.5	95.7	85.3	75.4	62.9	55.5	50.7	46.2	52.1
	B	97.7	97.7	95.0	81.7	79.1	65.9	57.9	50.5	55.9	48.5
	Mean	98.1	98.1	95.4	83.5	77.3	64.4	56.7	50.6	51.0	50.3
THF extracts	A	0.6	0.8	0.8	0.8	0.8	1.2	1.2	1.2	1.1	1.3
	B	0.6	0.9	0.8	0.8	0.8	1.2	1.2	1.2	1.1	1.3
	Mean	0.6	0.8	0.8	0.8	0.8	1.2	1.2	1.2	1.1	1.3
Isohexane extracts	A	<0.1	<0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
	B	0.1	<0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
	Mean	<0.1	<0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Total Extractables	A	99.1	99.4	96.6	86.2	76.4	64.2	56.8	52.0	47.3	53.6
	B	98.3	98.6	95.8	82.6	80.0	67.2	59.1	51.8	57.1	49.9
	Mean	98.7	99.0	96.2	84.4	78.2	65.7	58.0	51.9	52.2	51.7
Non- extractables	A	<0.1	1.7	3.5	8.0	14.2	22.6	24.1	28.1	30.3	27.8
	B	<0.1	1.9	4.3	9.3	12.0	20.5	27.3	27.7	26.4	29.5
	Mean	<0.1	1.8	3.9	8.6	13.1	21.6	25.7	27.9	28.4	28.7
14CO2	A	n.p.	0.8	1.5	3.6	6.9	14.1	16.8	18.4	22.1	19.0
	B	n.p.	0.7	1.6	4.3	5.7	12.0	15.7	18.4	17.9	20.7
	Mean	n.p.	0.8	1.5	3.9	6.3	13.0	16.2	18.4	20.0	19.9
Total % recovery	A	99.2	101.9	101.6	97.7	97.5	100.8	97.7	98.5	99.8	100.4
	B	98.4	101.2	101.7	96.1	97.7	99.7	102.0	98.0	101.4	100.0
	Mean	98.8	101.6	101.6	96.9	97.6	100.3	99.9	98.2	100.6	100.2
OVERALL MEAN ± SD		99.6 ± 1.8

n.p. = not performed.

Table 11.Comparison of the Soil Metabolism (SM) and Residue (R) Extraction Methods (Radiovalidation of Residue Method): Characterisation / Identification of Radioactive Residues in Soil Extracts ([MethylphenylU-14C]-labelled test substance).

[Methylphenyl- U-14C]-labelled test substance (% AR)	Replicate	Percent of Applied Radioactivity (% AR) by Soil and Extraction Method
		Gartenacker		18 Acres		East Anglia		Sarpy		Capay
		SM	R	SM	R	SM	R	SM	R	SM	R
Test substance	A	24.5	29.2	54.4	56.3	37.2	37.1	46.1	57.0	77.4	76.5
	B	24.9	27.8	57.0	54.1	36.3	40.4	51.7	45.7	76.0	75.0
	Mean	24.7	28.5	55.7	55.2	36.8	38.7	48.9	51.3	76.7	75.8
M4	A	3.1	3.9	3.8	2.3	4.3	3.4	3.9	2.7	2.7	2.6
	B	3.2	3.7	2.2	2.9	3.2	2.4	3.2	4.0	3.0	3.7
	Mean	3.1	3.8	3.0	2.6	3.7	2.9	3.5	3.4	2.9	3.1
M5	A	14.6	10.7	7.1	6.7	25.4	21.6	8.3	6.1	3.5	3.2
	B	11.6	13.4	7.8	6.0	21.6	21.7	6.2	8.6	2.8	3.6
	Mean	13.1	12.0	7.4	6.4	23.5	21.6	7.3	7.4	3.2	3.4
M6	A	2.1	3.3	2.1	1.7	2.1	1.4	1.8	1.5	3.2	2.1
	B	1.8	3.1	1.6	2.2	1.5	1.4	*	1.5	2.2	3.4
	Mean	2.0	3.2	1.9	2.0	1.8	1.4	0.9	1.5	2.7	2.8
M7	A	2.2	4.0	2.9	2.5	2.6	2.2	1.9	*	*	*
	B	3.2	2.8	2.2	2.9	2.0	1.6	3.1	2.4	*	*
	Mean	2.7	3.4	2.6	2.7	2.3	1.9	2.5	1.2	*	*
M9	A	3.9	3.8	2.2	1.5	1.3	2.8	2.1	*	*	*
	B	4.0	2.8	2.3	1.9	1.8	1.3	2.4	2.1	*	*
	Mean	4.0	3.3	2.2	1.7	1.6	2.1	2.2	1.0	*	*
Unidentified residue 1	A	*	*	*	2.3	*	*	*	1.3	*	*
	B	*	*	*	1.6	*	*	*	*	*	*
	Mean	*	*	*	1.9	*	*	*	0.7	*	*
Unidentified residue 2	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*

* = not detected.

Table 12.Summary of Characterisation / Identification of Radioactive Residues in Aqueous Acetonitrile Soil Extracts: Gartenacker Soil (Halophenyl-U-14C]-labelled test substance)

[Halophenyl-U-14C]-labelled test substance Gartenacker (% AR)	Replicate	Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	7	14	28	42	63	77	91	105	120
Test substance	A	95.5	n.p.	n.p.	72.8	56.6	47.8	40.8	34.1	24.7	30.9
	B	95.9	n.p.	n.p.	73.1	61.1	49.9	44.2	26.9	22.5	29.8
	Mean	95.7	n.p.	n.p.	73.0	58.8	48.9	42.5	30.5	23.6	30.4
M4	A	*	n.p.	n.p.	2.8	3.9	4.3	4.0	2.4	4.1	2.8
	B	*	n.p.	n.p.	2.6	4.3	4.5	3.9	3.4	3.1	3.3
	Mean	*	n.p.	n.p.	2.7	4.1	4.4	4.0	2.9	3.6	3.1
M5	A	*	n.p.	n.p.	4.2	10.2	11.6	12.1	15.5	20.6	15.4
	B	*	n.p.	n.p.	3.2	8.5	9.9	11.1	17.4	22.9	15.2
	Mean	*	n.p.	n.p.	3.7	9.4	10.8	11.6	16.5	21.7	15.3
M6	A	*	n.p.	n.p.	1.8	2.3	2.3	1.5	1.8	1.9	1.8
	B	*	n.p.	n.p.	1.5	2.2	1.9	2.0	1.5	2.5	2.1
	Mean	*	n.p.	n.p.	1.6	2.2	2.1	1.8	1.7	2.2	1.9
M7	A	*	n.p.	n.p.	1.5	2.0	2.0	2.3	2.4	3.0	2.8
	B	*	n.p.	n.p.	1.4	2.0	2.3	2.1	2.2	4.5	2.3
	Mean	*	n.p.	n.p.	1.5	2.0	2.2	2.2	2.3	3.8	2.5
M9	A	*	n.p.	n.p.	1.5	2.6	2.6	3.8	2.7	2.6	3.3
	B	*	n.p.	n.p.	1.5	1.6	2.7	3.5	4.0	3.1	3.5
	Mean	*	n.p.	n.p.	1.5	2.1	2.6	3.6	3.3	2.9	3.4
Unidentified residue 1	A	*	n.p.	n.p.	*	*	*	*	*	*	*
	B	*	n.p.	n.p.	*	*	*	*	*	*	*
	Mean	*	n.p.	n.p.	*	*	*	*	*	*	*
Unidentified residue 2	A	*	n.p.	n.p.	*	*	*	*	*	*	*
	B	*	n.p.	n.p.	*	*	*	*	*	*	*
	Mean	*	n.p.	n.p.	*	*	*	*	*	*	*
Unidentified residue 3	A	*	n.p.	n.p.	*	*	0.9	3.1	1.4	2.3	3.7
	B	*	n.p.	n.p.	*	*	1.6	2.8	2.7	1.7	3.0
	Mean	*	n.p.	n.p.	*	*	1.2	2.9	2.1	2.0	3.3
Unidentified residue 4	A	*	n.p.	n.p.	4.4	4.6	4.0	2.9	3.6	4.4	2.0
	B	*	n.p.	n.p.	4.5	4.5	3.8	3.9	4.1	4.2	2.4
	Mean	*	n.p.	n.p.	4.5	4.5	3.9	3.4	3.8	4.3	2.2
Unidentified residue 5	A	*	n.p.	n.p.	*	*	*	*	*	*	*
	B	*	n.p.	n.p.	*	*	*	*	*	*	*
	Mean	*	n.p.	n.p.	*	*	*	*	*	*	*
Total	A	95.5	n.p.	n.p.	88.9	82.1	75.5	70.6	63.9	63.7	62.7
	B	95.9	n.p.	n.p.	87.8	84.2	76.7	73.5	62.1	64.5	61.6

* = not detected.

 

Table 13.Summary of Characterisation / Identification of Radioactive Residues in Aqueous Acetonitrile Soil Extracts: East Anglia Soil (Halophenyl-U-14C]-labelled test substance).

[Halophenyl-U- 14C]-labelled test substance East Anglia (% AR)	Replicate	Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	7	14	28	42	63	77	91	105	120
Substance	A	97.8	100.2	86.0	75.4	69.4	58.1	52.2	37.9	37.6	35.8
	B	96.9	89.4	82.0	69.6	70.5	61.2	49.9	41.0	33.1	32.4
	Mean	97.4	94.8	84.0	72.5	70.0	59.7	51.0	39.5	35.3	34.1
M4	A	*	*	3.5	2.4	3.6	5.3	4.3	4.2	3.6	3.0
	B	*	*	3.0	5.3	3.6	3.0	3.4	3.5	4.0	3.8
	Mean	*	*	3.3	3.8	3.6	4.2	3.9	3.9	3.8	3.4
M5	A	*	*	2.6	4.1	7.9	12.1	16.4	20.7	21.2	23.7
	B	*	*	2.4	6.2	6.3	11.4	16.3	17.9	21.8	27.4
	Mean	*	*	2.5	5.1	7.1	11.7	16.4	19.3	21.5	25.6
M6	A	*	*	2.7	3.2	1.7	2.5	2.2	2.9	2.4	2.1
	B	*	7.2	5.3	2.9	1.9	2.7	2.0	2.2	2.4	1.6
	Mean	*	3.6	4.0	3.0	1.8	2.6	2.1	2.6	2.4	1.8
M7	A	*	*	*	*	1.4	1.5	2.0	2.5	2.7	2.8
	B	*	*	*	*	1.5	2.7	2.3	3.0	3.1	2.4
	Mean	*	*	*	*	1.5	2.1	2.2	2.7	2.9	2.6
M9	A	*	*	*	*	*	*	*	1.4	*	*
	B	*	*	*	*	*	*	*	1.8	2.4	1.7
	Mean	*	*	*	*	*	*	*	1.6	1.2	0.8
Unidentified residue 1	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unidentified residue 2	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unidentified residue 3	A	*	*	*	*	*	1.7	1.9	3.6	4.3	3.3
	B	*	*	*	*	*	1.5	1.1	3.3	4.5	3.2
	Mean	*	*	*	*	*	1.6	1.5	3.5	4.4	3.3
Unidentified residue 4	A	*	*	2.6	*	3.0	3.7	2.6	3.2	2.9	2.5
	B	*	*	2.8	*	3.5	3.2	3.2	3.0	2.9	1.8
	Mean	*	*	2.7	*	3.2	3.4	2.9	3.1	2.9	2.2
Unidentified residue 5	A	*	*	*	3.4	*	*	*	1.9	*	*
	B	*	*	*	3.4	*	*	1.5	2.1	*	*
	Mean	*	*	*	3.4	*	*	0.7	2.0	*	*
Total	A	97.8	100.2	97.4	88.5	87.1	85.0	81.6	78.4	74.6	73.3
	B	96.9	96.6	95.5	87.4	87.3	85.7	79.8	77.7	74.1	74.2

* = not detected.

Table 14. Summary of Characterisation / Identification of Radioactive Residues in Aqueous Acetonitrile Soil Extracts: East Anglia Soil (Oxoisoxazolidinyl-4,5-14C-labelled test substance).

[Oxoisoxazo- lidinyl-4,5-14C]-labelled test substance East Anglia (% AR)	Replicate	Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	7	14	28	42	63	77	91	105	120
Substance	A	98.4	98.5	91.2	85.3	69.6	55.4	45.7	42.2	37.9	44.7
	B	97.7	97.7	95.0	78.5	75.0	57.4	50.8	41.9	46.5	39.3
	Mean	98.1	98.1	93.1	81.9	72.3	56.4	48.3	42.1	42.2	42.0
M4	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
M5	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
M6	A	*	*	2.1	*	2.4	2.4	3.0	1.7	2.7	1.5
	B	*	*	*	*	1.6	3.1	2.2	2.5	2.0	2.1
	Mean	*	*	1.0	*	2.0	2.8	2.6	2.1	2.4	1.8
M7	A	*	*	*	*	1.7	2.0	2.6	2.2	1.6	2.0
	B	*	*	*	3.2	*	2.5	2.1	2.2	2.4	2.5
	Mean	*	*	*	1.6	0.8	2.2	2.4	2.2	2.0	2.2
Unidentified residue 6	A	*	*	*	*	*	*	*	*	1.3	1.5
	B	*	*	*	*	*	*	*	*	1.1	1.4
	Mean	*	*	*	*	*	*	*	*	1.2	1.5
Unidentified residue 1	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unidentified residue 2	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unidentified residue 3	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unidentified residue 4	A	*	*	2.4	*	1.8	3.0	2.6	3.1	*	2.4
	B	*	*	*	*	2.5	3.0	2.7	2.3	2.6	3.2
	Mean	*	*	1.2	*	2.1	3.0	2.7	2.7	1.3	2.8
Unidentified residue 5	A	*	*	*	*	*	*	1.5	1.5	2.7	*
	B	*	*	*	*	*	*	*	1.7	1.3	*
	Mean	*	*	*	*	*	*	0.7	1.6	2.0	*
Total	A	98.4	98.5	95.7	85.3	75.4	62.9	55.5	50.7	46.2	52.1
	B	97.7	97.7	95.0	81.7	79.1	65.9	57.9	50.5	55.9	48.5

* = not detected.

Table 15. Summary of DT50 and DT90 Values

	SFO model
Soil	DT50 [days]	DT90 [days]	K	χ2 error %	r2	Prob > t
Gartenacker	56	187	0.0123	4.5	0.9709	5.33E-026
18 Acres	125	416	0.005539	2.42	0.9737	1.36E-015
East Anglia	80	265	0.00868	2.37	0.9654	1.10E-041
Sarpy	112	373	0.006175	4.36	0.9386	2.95E-012
Capay	293	973	0.002367	1.78	0.9175	1.95E-011

Conclusions:

The key findings after application of the 14C-labelled test substance at three distinctive positions at an application rate of 0.2 mg/kg dry soil equivalent (approximately 150 g ai/ha) to five various soils and subsequent aerobic incubation (20 °C , dark and pF approx. 2) for 120 days are summarized below:
- The mass balance from all individual soil samples ranged from 89.7 to 105.7%.
- The test substance degraded steady over the course of the experiment. DT50 values were determined to range from 56 - 293 days.
- The carboxylic acid metabolite, M5, reached > 10% AR in both Gartenacker and East Anglia soils, with a maximum level of 25.6% AR after 120 days of incubation in East Anglia soil treated with halophenyl ring labelled test item. In 18 Acres, Sarpy and Capay soils, maximum levels of this metabolite ranged from 3.2 - 8.4% AR.

Executive summary:

The rate and route of degradation of the test substance, 14C-labelled in three distinctive positions, was investigated in five different soils: Gartenacker (silt loam), 18 Acres (sandy clay loam), East Anglia (sandy loam), Sarpy (loam) and Capay (clay loam). The degradation of 14C-methylphenyl ring labelled test substance was investigated in all five soils tested, whereas the degradation of 14C-halophenyl ring labelled test substance was investigated in Gartenacker and East Anglia soils and the degradation of 14C-oxoisoxazolidinyl ring labelled test substance was investigated in the East Anglia soil only. The study was conducted according to guidelines: OECD TG 307 and EPA 835.4100 and it was in compliance with GLP criteria. 14C-labelled test substance was applied at a target concentration of 20 µg of the test item per 100 g dry soil equivalent, corresponding to a single field application rate of 150 g test item/ha (assuming an incorporation depth of 5 cm and a bulk density of 1.5 g/cm3). The actual application rate achieved was 19.2 - 21.3 µg of the test item per system, corresponding to a single field application rate of 144 - 160 g test item/ha (95.8 - 106.7% of target). The soil was incubated in the dark under aerobic conditions in the laboratory at 20 °C for up to 120 days. Duplicate samples were taken for analysis at 0, 7, 14, 28, 42, 63, 77, 91, 105 and 120 days after treatment (DAT) with the exception of the Gartenacker soil treated with the 14C-halophenyl ring labelled test item, for which the samples were taken at 0, 28, 42, 63, 77, 91, 105 and 120 DAT.The soil samples were extracted eight times at room temperature using solvent of varying polarity: once with acetonitrile/water (50:50; v/v), twice acetonitrile/water (80:20; v/v), once with acidified acetonitrile/water (80:20; v/v; pH 3), twice with THF, and twice with isohexane. Aqueous acetonitrile extracts were pooled and analysed by RP-HPLC for parent compound and degradation products. Selected pooled aqueous acetonitrile extracts were analysed by 2D-NP-TLC and LC-MS/MS to confirm the identity of radioactive components. THF and isohexane extracts were not analysed further due to the low levels of radioactivity present. Application solutions for each radiolabelled test item, as well as soil aqueous acetonitrile extracts from 0 and 120 DAT for all soils and labels were analysed by chiral LC-MS to determine the isomer ratio of parent and the two major metabolites, M5 and M8. Additionally, soil extracts from selected intervals of 63, 105, 91, 63 and 63 DAT for Gartenacker, 18 Acres, East Anglia, Sarpy and Capay soils, respectively (all treated with the methylphenyl label) as well as the interval of 63 DAT for the two soils treated with the halophenyl label were analysed by chiral LC-MS to determine the isomer ratio of the amide M4. Any volatile radioactivity was continuously flushed from the vessels and collected in traps. Remaining unextracted residues were analysed by combustion and a mass balance determined for each sample.Radiovalidation of a separate residue method extraction procedure was performed within the study. At the selected sampling interval of 120 DAT, two replicates were extracted at room temperature following the residue method (two extractions with aqueous acetonitrile mixtures each for about 1 h at 250 rpm, followed by centrifugation, phase separation and LSC measurement). Further analysis of the samples was performed by HPLC.

The mass balances (mean values of duplicate samples) in all soil samples ranged from 94.1 to 104.8% AR with an overall average per soil and label system ranging from 97.6% AR to 100.9% AR. The mass balance from all individual soil samples ranged from 89.7 to 105.7%. The total amount of radioactivity extracted decreased slowly over time in all soils, ranging from 97.6 to 104.7% AR at 0 DAT and from 51.7 to 87.7% AR at 120 DAT. The majority of the radioactivity was recovered in the aqueous acetonitrile mixtures, which ranged from 95.7 to 102.8% AR for all soils at 0 DAT and from 50.3 to 85.4% AR at 120 DAT. The maximum amounts of radioactivity present in the THF and isohexane extracts at any time point were 4.1% and 0.6%, respectively.Unextracted residues (bound residues) increased slowly throughout the incubation, reaching maximum of 11.4 to 36.4% AR by the end of the incubation. Mineralisation to 14CO2 varied depending on the position of the radiolabel. In soils treated with methylphenyl and halophenyl labelled test item, 14CO2 reached a maximum of 6.7% and 2.3% AR, respectively, by the end of the incubation period. Formation of radiolabelled carbon dioxide was much higher for the oxoisoxazolidinyl label with a mean maximum of 19.9% AR at the end of the study.

For the Methylphenyl label, levels of parent compound in the total system decreased over the 120 day incubation period ranging from a mean level between 99.3 and 102.8% AR at time 0 and degrading to mean levels of 24.7 to 76.7% AR by the end of incubation (120 DAT).Two major metabolites were observed. For the Halophenyl label, levels of parent compound in the total system decreased over the 120 day incubation period from a mean level of 95.7% AR for Gartenacker soil and 97.4% AR for East Anglia soil at time 0 to a mean level of 30.4% AR in Gartenacker soil and 34.1% AR in East Anglia soil at the end of incubation (120 DAT). One metabolite was present at levels of >5% AR.

For Oxoisoxazolidinyl label, for East Anglia soil levels of parent compound in the total system decreased over the 120 day incubation period from a mean level of 98.1% AR at time 0 to 42.0% AR at the end of incubation (120 DAT). No metabolites accounted for mean levels of >5% AR. It was confirmed that there was no change of stereochemistry during the incubation time of 120 days and hence, there was no evidence of either interconversion or stereo-selective metabolism of either the test substance or its metabolites. Radiovalidation of a separate residue method extraction procedure was performed within the study. The data showed that the extractability of radioactive residues using the metabolism methodology and the residue analytical method were in good agreement, and that following chromatographic analysis of the extracted radioactivity, the levels of parent and its degradates detected were also in good agreement.

The key findings after application of [14C]-labelled test substance at three distinctive positions at an application rate of 0.2 mg/kg dry soil equivalent (approximately 150 g ai/ha) to five various soils and subsequent aerobic incubation (20 °C, dark and pF approx. 2) for 120 days are the following six points: 1)The mass balance from all individual soil samples ranged from 89.7 to 105.7%; 2) the test substance degraded steadily over the course of the experiment. DT50 values were determined to range from 56 - 293 days; 3) The carboxylic acid metabolite reached >10% AR in both Gartenacker and East Anglia soils, with a maximum residue level of 25.6% AR after 120 days of incubation in East Anglia soil treated with halophenyl ring labelled test item. In 18 Acres, Sarpy and Capay soils, maximum levels of this metabolite ranged from 3.2 - 8.4% AR. Methylation of the acid yielded another major metabolite, reaching a maximum of 10.8% and 6.5% AR at 120 DAT in Sarpy and 18 Acres soils, respectively. This metabolite was also present in Gartenacker, East Anglia and Capay soils, where levels were < 5% AR; 4) No other metabolites exceeded 5% AR. A number of other metabolites were identified which reached maximum levels of 4.7, 4.0, 3.8 and 4.8% AR, respectively; 5) Mineralisation to 14CO2 varied depending on the position of the radiolabel. In soils treated with methylphenyl and halophenyl test item, 14CO2 reached a maximum of 6.7% and 2.3% AR, respectively, by the end of the incubation period. Formation of radioactive carbon dioxide was much higher for the oxoisoxazolidinyl label with a mean maximum of 19.9% AR at the end of the study; 6) Unextracted residues (bound residues) increased slowly throughout the study, ranging from 11.4 to 36.4% AR by the end of the incubation.

Reference 2

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jul 2016 to 21 Jul 2018

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4100 (Aerobic Soil Metabolism)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2016

Soil no.:

#1

Soil type:

sandy clay

% Clay:

37

% Silt:

2

% Sand:

61

% Org. C:

2.7

pH:

3.9

CEC:

9.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#2

Soil type:

sandy clay loam

% Clay:

29

% Silt:

22

% Sand:

49

% Org. C:

2.4

pH:

5.4

CEC:

26.5 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#3

Soil type:

loamy sand

% Clay:

11

% Silt:

2

% Sand:

87

% Org. C:

10.2

pH:

4.5

CEC:

16 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Soil no.:

#4

Soil type:

sand

% Clay:

0

% Silt:

0

% Sand:

100

% Org. C:

0.78

pH:

4.2

CEC:

3.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.5

Details on soil characteristics:

Details of the soils, including pesticide treatment history, along with key physico-chemical properties of the soils are provided in Table 1 in 'Any other information on materials and methods incl. tabels'
- Soil preparation: The soils were sieved using a 2 mm mesh sieve prior to analyses and acclimation. Following determination of the soil moisture content, aliquots of 100 g dry soil equivalent were dispensed into metabolism flasks. The soils were acclimated under aerobic conditions at 20.9 ± 0.3 °C under darkness for about one week. Moisture levels were observed at approximately fortnightly intervals, and water was added accordingly when necessary. Additional flasks containing treated soil were prepared to be used for the radiovalidation and were incubated under the same conditions. In addition to the flasks containing treated soil, control soil samples were incubated under the same conditions as the treated samples. These served to determine the microbial biomass at the end of the incubation period. The microbial biomass was determined according to the fumigation extraction method.

Duration:

120 d

Based on:

other:

Remarks:

See Table 2 in 'Any other information on materials and methods incl. tables'

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

test mat. analysis

Soil No.:

#1

Temp.:

20.8 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 33.2

Microbial biomass:

107.54 mg C/100 g soil at the start and 64.07 mg C/100 g soil at the end of the study

Soil No.:

#2

Temp.:

20.8 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 32.8

Microbial biomass:

80.48 mg C/100 g soil at the start and 65.76 mg C/100 g soil at the end of the study

Soil No.:

#3

Temp.:

20.8 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 85.5

Microbial biomass:

198.06 mg C/100 g soil at the start and 126.57 mg C/100 g soil at the end of the study

Soil No.:

#4

Temp.:

20.8 °C

Humidity:

Moisture at pF2.0 (0.1bar, w/w %): 4.9

Microbial biomass:

22.98mg C/100 g soil at the start and 12.98 mg C/100 g soil at the end of the study

Details on experimental conditions:

An overview of the study design and sampling is provided in Table 2 and Table 3 in 'Any other information on materials and methods incl. tables'.

PREPARATION OF STOCK AND APPLICATION SOLUTIONS
The aim was to apply the radiolabelled test items at a target concentration of 20 µg test item per system, corresponding to 150 g test item/ha, assuming an incorporation depth of 5 cm and a bulk density of 1.5 g/cm3.
- Stock solution of [methylphenyl-U-14C]-labelled test substance: A stock solution was prepared in a separate study by adding the test item (batch no. RDRXXV-30; delivered in a glass ampule as a solution in acetonitrile) to a 10 mL volumetric flask. The flask was then filled to the mark by repeatedly rinsing the glass ampule with small volumes of acetonitrile. The stock solution was then transferred to a 10 mL glass vial and sealed with a septum cap. The radioactivity in the stock solution was determined by dilution of 5 µL of the stock solution to 10 mL with acetonitrile (in duplicate), followed by triplicate 300 µL LSC measurements of each replicate. Based on the measured mean amount of radioactivity, the stock solution contained 5’494’522’222 dpm (91.58 MBq). This stock solution was portioned for several studies and one of these aliquots (1650 µL) was used for the current study.

- Application solution of [methylphenyl-U-14C]-labelled test substance: An application solution was prepared by transferring the aliquot of 1650 µL of the stock solution to a 100 mL volumetric flask, rinsing the stock solution aliquot flask with methanol (1 mL), adding the rinse to the application solution flask and filling to the mark with methanol/water (1/1; v/v). To determine the radioactivity content in the application solution, a 700 µL aliquot of the application solution was diluted to 100 mL with acetonitrile/water (1/1; v/v) and the radioactivity quantified by quintuplicate LSC measurements of 1 mL aliquots. Based on the measured mean amount of radioactivity, the concentration of the application solution was 6’406’900 dpm/700 µL and accounted for a radioactivity content of 915’271’429 dpm (15.25 MBq), corresponding to 3.20 mg of the test substance in 100 mL of the application solution.

- Stock solution of [halophenyl-U-14C]-labelled test substance: A stock solution was prepared in a separate study by adding the test item (batch no. BPMLI-47-1; delivered in a glass ampule as a solution in acetonitrile) to a 10 mL volumetric flask. The flask was then filled to the mark by repeatedly rinsing the glass ampule with small volumes of acetonitrile. The stock solution was then transferred to a 10 mL glass vial and sealed with a septum cap. The radioactivity in the stock solution was determined by dilution of 5 µL of the stock solution to 10 mL with acetonitrile (in duplicate), followed by triplicate 500 µL LSC measurements of each replicate. Based on the measured mean amount of radioactivity, the stock solution contained 3’451’046’667 dpm (57.52 MBq). This stock solution was portioned for several studies and one of these aliquots (1000 µL) was used for the current study.

- Application solution of [halophenyl-U-14C]-labelled test substance: An application solution was prepared by transferring the aliquot of 1000 µL of the stock solution to a 25 mL volumetric flask, rinsing the stock solution aliquot flask with methanol (1 mL), adding the rinse to the application solution flask and filling to the mark with methanol/water (1/1; v/v). To determine the radioactivity content in the application solution, a 500 µL aliquot of the application solution was diluted to 100 mL with acetonitrile/water (1/1; v/v) and the radioactivity quantified by quintuplicate LSC measurements of 1 mL aliquots. Based on the measured mean amount of radioactivity, the concentration of the application solution was 6’782’660 dpm/500 µL and accounted for a radioactivity content of 339’133’000 dpm (5.65 MBq), corresponding to 1.02 mg of SYN547407 in the 25 mL application solution.

- Stock solution of [oxoisoxazolidinyl-4,5-14C]-labelled test substance: A stock solution was prepared in a separate study by adding the test item (batch no.MGG-IV-88-1; delivered in a glass ampule as a solution in acetonitrile) to a 20 mL volumetric flask. Similarly to the previous two stock solutions, the flask was then filled to the mark by repeatedly rinsing the glass ampule with small volumes of acetonitrile. The stock solution was then transferred to a 20 mL glass vial and sealed with a septum cap. The radioactivity in the stock solution was determined by dilution of 5 µL of the stock solution to 10 mL with acetonitrile (in duplicate), followed by triplicate 1000 µL LSC measurements of each replicate. Based on the measured mean amount of radioactivity, the stock solution contained 3’482’220’000 dpm (58.04 MBq). This stock solution was portioned for several studies and one of these aliquots (1500 µL) was used for the current study.

- Application solution of [oxoisoxazolidinyl-4,5-14C]-labelled test substance: An application solution was prepared by transferring the aliquot of 1500 µL of the stock solution to a 25 mL volumetric flask, rinsing the stock solution aliquot flask with methanol (1 mL), adding the rinse to the application solution flask and filling to the mark with methanol/water (1/1; v/v). To determine the radioactivity content in the application solution, a 500 µL aliquot of the application solution was diluted to 100 mL with acetonitrile/water (1/1; v/v) and the radioactivity quantified by quintuplicate LSC measurements of 1 mL aliquots. Based on the measured mean amount of radioactivity, the concentration of the application solution was 4’970’340 dpm/500 µL and accounted for a radioactivity content of 248’517’000 dpm (4.14 MBq), corresponding to 0.79 mg of SYN547407 in the 25 mL application solution.

APPLICATION OF RADIOLABELLED TEST IETAMS
An aliquot of 640 µL of the [methylphenyl-U-14C]-labelled test substance application solution was applied to the surface of each soil sample (all four soils, including radiovalidation samples). An aliquot of 500 µL of the [halophenyl-U-14C]-labelled test substance application solution was applied to the surface of each soil sample (Argissolo soil only). An aliquot of 640 µL of the [oxoisoxazolidinyl-4,5-14C]-labelled test substance application solution was applied to the surface of each soil sample (Argissolo soil only). Applications were performed using a Hamilton syringe by dosing the application solutions drop-wise to the surface of the soil samples. The treated soil flasks were mixed thoroughly, allowing the organic solvent to evaporate and to ensure uniform distribution of the test item applied. The amount of the organic solvent applied was ≤0.25% v/w of the dry weight of soil.

In order to confirm the concentrations and homogeneity of the application solutions during application procedure, the volume of application solution applied per soil flask was given into acetonitrile/water (1/1, v/v) and the obtained dilutions were analysed by LSC. For samples treated with [methylphenyl-U-14C]-labelled test substance(all four soils), an aliquot of the application solution equal to the aliquot applied to the soil (i.e. 640 µL) was separately applied to 100 mL of acetonitrile/water (1/1, v/v) before, during and after the application procedure. For samples treated with [halophenyl-U-14C]-labelled test substance (Argissolo soil only), an aliquot of the application solution equal to the aliquot applied to the soil (i.e. 500 µL) was separately applied to 100 mL of acetonitrile/water (1/1, v/v) before and after the application procedure. For samples treated with [oxoisoxazolidinyl-4,5-14C]-labelled test substance (Argissolo only), an aliquot of the application solution equal to the aliquot applied to the soil (i.e. 640 µL) was separately applied to 100 mL of acetonitrile/water (1/1, v/v) before and after the application procedure. In each case, five 1 mL aliquots of the diluted solutions were measured by LSC (homogeneity check).
Untreated soil samples for the determination of the microbial biomass were treated in the same way using an equivalent volume of solvent corresponding to the highest volume applied to samples but without test item (640 µL of methanol/water (1/1, v/v)). Each flask was uniquely identified to ensure unmistakable identification. For sampling on day 0, no volatile traps were set up, as the samples were worked-up immediately after application. All other flasks were connected to the gas-flow system and incubated in an incubation chamber. The mean temperature during the incubation period was 20.8 ± 0.3 °C.

During incubation, the system was continuously ventilated with moistened air to maintain aerobic conditions while minimizing water loss. Any volatile radioactivity was continuously flushed from the incubation vessels and collected in NaOH traps. For flasks applied with [halophenyl-U-14C]-labelled test substance, polyurethane foam “traps” were additionally placed inside of each test flask outlet arm to ensure all possible volatiles were captured.

Remarks on result:

other: See Table 4 in 'Any other information on results incl. tables'

Key result

Soil No.:

#1

DT50:

257 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20.8 °C

Key result

Soil No.:

#2

DT50:

300 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20.8 °C

Key result

Soil No.:

#3

DT50:

244 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20.8 °C

Key result

Soil No.:

#4

DT50:

723 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20.8 °C

Transformation products:

yes

No.:

#1

No.:

#2

Details on transformation products:

RADIOACTIVE RESIDUES IN SOIL EXTRACTS
- Methylphenyl label: In all soils treated with [methylphenyl-U-14C]-labelled test substance, the test substance degraded slowly. Immediately after application (0 DAT), the total amount of the test substance present in the acetonitrile/water and THF (where applicable) extractions represented 98.0%, 99.3%, 97.9% and 99.6% AR in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively. By the end of the study (120 DAT), the test substance represented 74.9%, 76.4%, 70.4% and 87.7% AR in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively. The majority of the parent extracted was recovered in the acetonitrile/water extracts, with only a relatively small proportion (up to 8.4%) recovered in the THF extracts. In the isohexane extracts of soils treated with [methylphenyl-U-14C]-labelled test substance (Gleissolo soil only), the test substance represented only a very minor fraction of 1.5% AR at 120 DAT, the only interval analysed by HPLC. Only a single metabolite was present at levels of > 5%. The single major metabolite observed, Metabolite M2, was a major metabolite in Latossolo and Gleissolo soils only, first appearing after 47 days of incubation. In these two soils, levels of M2 increased relatively slowly over time reaching maximum mean levels of 7.7 and 6.0% AR at 120 and 89 DAT in Latossolo and Gleissolo soils, respectively, as recovered from acetonitrile/water and THF extracts together. In Argissolo and Neossolo soils, mean levels of M2 were ≤ 3% AR throughout the incubation period. No other metabolites accounted for mean levels of > 5% AR, including metabolite M49 which reached maximum levels of 3.3, 2.1, 0.4 and 3.1% AR in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively, as recovered from acetonitrile/water and THF extracts together.
- Halophenyl label: In Argissolo soil treated with [halophenyl-U-14C]-labelled test substance, the total amount of the test substance recovered in both the acetonitrile/water and THF extracts declined from 98.1% at 0 DAT to 76.8% at 120 DAT. The majority of the test substance was recovered in the acetonitrile/water extracts with only ≤ 6.2% (mean) AR recovered in THF extracts. No single metabolite was present at levels of > 5%, including metabolite M49 and metabolite M2, which reached maximum levels of 3.0 and 3.6% AR, respectively, as recovered from acetonitrile/water and THF extracts together.
- Oxoisoxazolidinyl label: In Argissolo soil treated with [oxoisoxazolidinyl-4,5-14C]-labelled test substance, the total amount of the test substance recovered in both the acetonitrile/water and THF extracts declined from 98.9% AR at 0 DAT to 74.1% AR at 120 DAT. The majority of the test substance was recovered in the acetonitrile/water extracts with only ≤ 7.3% (mean) AR recovered in THF extracts. No metabolites were detected in either acetonitrile/water or THF extracts when analyzed by HPLC and TLC.

PROPOSED METABOLIC PATHWAT
The degradation profiles indicated that the test substance degraded primarily by elimination of the oxoisoxazolidinyl moiety yielding either the amide metabolite M49 or the acid metabolite M5. Further amide hydrolysis also yielded the carboxylic acid M2.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Residues:

yes

Details on results:

An overview of the results is provided in Table 5 - Table 13 in 'Any other information on results incl. tables'.

MICROBIAL BIOMASS
The microbial biomass was ≥ 1.2% OC at the start and end of the study for all the soils. Hence, the soils were deemed to be suitably microbially active for the purposes of the study.

TREATMENT RATE
See Table 4 in 'Any other information on materials and methods incl. tables'

MASS BALANCE
For soil samples treated with [methylphenyl-U-14C]-labelled test substance, the overall mass balance was determined to be 100.1 ± 2.1%, 99.4 ± 1.6%, 100.5 ± 2.0% and 99.5 ± 2.0% of the applied radioactivity (AR) for Latossolo, Argissolo, Gleissolo and Neossolo, respectively.
Individual values ranged from 94.5 to 104.3% AR. For soil samples treated with [halophenyl-U-14C]-labelled test substance (Argissolo soil only), the overall mass balance was determined to be 100.8 ± 1.8% AR. Individual values ranged from 97.2 to 103.9% AR. For soil samples treated with [oxoisoxazolidinyl-4,5-14C]-SYN547407 (Argissolo soil only), the overall mass balance was determined to be 100.3 ± 2.2% AR. Individual values ranged from 94.6 to 102.7% AR.

VOLATILE DEGRADATION PRODUCTS
Formation of radioactive carbon dioxide in soils treated with the test item labelled in either the methylphenyl position (all soils) or halophenyl position (Argissolo soil only) reached means of 0.2 to 3.1% AR by the end of the study. For Argissolo soil treated with test item labelled in the oxoisoxazolidinyl position, 14CO2 reached a mean of 5.9% AR by the end of the study. The identity of 14CO2 was confirmed by precipitation with barium hydroxide for Argissolo soil labelled in the oxoisoxazolidinyl position. The polyurethane foam traps which were incorporated in the test apparatus of samples treated with [halophenyl-U-14C]-labelled test substance in order to capture possible volatiles, were not further analysed since the complete mass balance was obtained from all other fractions quantified.

DISTRIBUTION OF RADIOACTIBE RESIDUES
At time 0, for soil samples treated with [methylphenyl-U-14C]-labelled test substance, the mean extractable radioactivity in acetonitrile/water extracts was 96.8%, 96.2%, 91.6% and 99.6% AR in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively. A general decrease in radioactivity found in the acetonitrile/water extracts was observed during the study. At the end of the 120-day incubation period, the corresponding values were 84.7%, 76.8%, 65.7% and 90.2% AR in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively. Additional room temperature extractions with tetrahydrofuran (THF) yielded only a small additional percentage of applied radioactivity. At time 0, for soil samples treated with [methylphenyl-U-14C]-labelled test substance, the mean extractable radioactivity found in THF extracts was 1.2%, 3.2%, 6.2% and 0.2% AR in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively. The levels of THF-extractable radioactivity remained stable during the incubation period and at the end of the 120-day period the corresponding values were 2.6%, 3.6%, 8.7% and 0.9% AR in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively. Further room temperature extractions with isohexane yielded an even smaller percentage of radioactivity. At time 0, for soil samples treated with [methylphenyl-U-14C]-labelled test substance, the mean extractable radioactivity found in isohexane extracts was below the limit of quantification (LOQ; 0.176% AR) in Latossolo and Neossolo soils, and 0.3% and 1.1% AR in Argissolo and Gleissolo soils, respectively. At the end of the 120-day incubation period, the corresponding values were 0.3%, 0.5% and 2.1% AR in Latossolo, Argissolo and Gleissolo soils, respectively, while remaining below the LOQ for Neossolo soil.
At time 0, for soil samples treated with [halophenyl-U-14C]-labelled test substance (Argissolo soil only), the mean extractable radioactivity in acetonitrile/water extracts was 96.0% AR. A general decrease in radioactivity found in the acetonitrile/water extracts was observed for this radiolabelled test substance, similar to [methylphenyl-U-14C]-labelled test substance. At the end of the 120-day incubation period the corresponding value was 78.5% AR. Additional room temperature extractions with THF yielded only a small additional percentage of applied radioactivity, ranging from 2.0 to 6.6% AR. Likewise, further room temperature extractions with isohexane yielded an even smaller percentage of radioactivity, ranging from 0.3 to 1.1% AR.
At time 0, for soil samples treated with [oxoisoxazolidinyl-4,5-14C]-the test substance (Argissolo soil only), the mean extractable radioactivity in acetonitrile/water extracts was 97.2% AR. A general decrease in radioactivity found in the acetonitrile/water extracts was observed for this radiolabelled the test substance similar to the other two radiolabeled test items used. At the end of the 120-day incubation period the corresponding value was 70.7% AR. Additional room temperature extractions with THF yielded only a small additional percentage of applied radioactivity, ranging from 1.8 to 7.3% AR. Likewise, further room temperature extractions with isohexane yielded an even smaller percentage of radioactivity, ranging from 0.4 to 1.3% AR. Unextracted residues (bound residues) increased slowly throughout the incubation, reaching maximum of 11.0 to 18.5% AR for Latossolo, Argissolo and Gleissolo soils (treated with all radiolabelled test items) and 3.2% AR for Neossolo soil by the end of the incubation.

RATE OF DEGRADATION ON THE TEST SUBSTANCE IN SOIL
The rate of degradation of the test substance was calculated using single first-order (SFO) kinetics and expressed for the parent found in acetonitrile/water and THF extracts. Degradation kinetics for each of the three soils (Latossolo, Gleissolo and Neossolo) treated with only [methylphenyl-U-14C]-labelled test substance were calculated individually. Degradation kinetics for Argissolo soil, which was treated separately with each of the three radiolabelled test items, were calculated after first combining results for the parent test item for each of the three labels. The SFO kinetic model describes the degradation of the test substance with a χ2-square value ≤ 1.95, linear regression coefficient r2 ≥ 0.8384 and with an overall sufficient goodness of the fit as concluded from a visual assessment of the plots in all cases. The degradation half-life (DT50) of the test substance was calculated to be 257, 300, 244 and 723 days in Latossolo, Argissolo, Gleissolo and Neossolo soils, respectively.

ANYALYSIS OF STEREOISOMERS OF THE TEST SUBSTANCE AND ITS MAJOR METABOLITE M5
The test substance is a chemical substance with two asymmetric carbon atoms, and therefore is comprised of four stereoisomers. According to the certificates of analysis of the three radiolabelled test items, the applied radiolabelled the test substance consisted mainly of the (S, R isomer) “Isomer A”, and accounting for 92.2 - 92.4% of the test substance. The (S, S isomer) “Isomer B”, (R, R isomer) “Isomer C” and (R, S isomer) “Isomer D”, contributed 0.9 - 1.3%, 6.4 - 6.9% and < 0.1%, respectively.
Application solutions for each radiolabelled test item, as well as soil acetonitrile/water and THF extracts from 0 and 120 DAT for all soils and labels were analysed by chiral LC-MS to determine the isomer ratio of parent and the major (> 5%) metabolite, M5. Additionally, soil extracts from a selected interval of 89 DAT for Gleissolo (treated with the methylphenyl label and containing the highest amounts of M5 at any time point in this soil) were analysed by chiral LC-MS to determine the isomer ratios.
The ratio of Isomer A and other stereoisomers of the test substance in the both the application solutions and 0, 89 and 120 DAT soil extracts (both solvent systems) was confirmed to be consistent with that reported in the Certificates of Analysis for the test items. The isomer ratio for M5 was also found to be consistent with that predicted from the isomer ratio of the parent test items. Hence, it was confirmed that there was no change of stereochemistry during the incubation time of 120 days and hence, there was no evidence of either interconversion or stereo-selective metabolism of either the test substance or its metabolite, M5.

Table 5. Mass balance

Total radioactivity	Sum of radioactivity in soil extracts, residue on combustion and 14CO2 trapped in the 2N sodium hydroxide solutions
Mean Recovery at 0 DAT	Range 98.3% to 100.2% of applied dose
Overall recovery (all samples)	Range 94.5% to 104.3% of applied dose Latossolo (methylphenyl label): Mean 100.1 ± 2.1% Argissolo (methylphenyl label): Mean 99.4 ± 1.6% Gleissolo (methylphenyl label): Mean 100.5 ± 2.0% Neossolo (methylphenyl label): Mean 99.5 ± 2.0% Argissolo (halophenyl label): Mean 100.8 ± 1.8% Argissolo (oxoisoxazolidinyl label): Mean 100.3 ± 2.2%

Table 6. Volatile Degradation Products

14CO2	Small amounts of radioactivity were evolved as volatile products throughout the course of the study.
	14CO2 evolved at end of study	Latossolo (methylphenyl label): Mean 1.9% Argissolo (methylphenyl label): Mean 3.1% Gleissolo (methylphenyl label): Mean 1.1% Neossolo (methylphenyl label): Mean 0.2% Argissolo (halophenyl label): Mean 0.8% Argissolo (oxoisoxazolidinyl label): Mean 5.9%
Other volatiles	No other volatiles were captured.

Table 7. Comparison of the Soil Metabolism (SM) and Residue (R) Extraction Methods (Radiovalidation of Residue Method): Distribution and Recovery of Radioactivity (Soil metabolism samples were taken after 120 days of incubation; residue method samples were taken after 125 days of incubation).

[Methylphenyl- U-14C]-labelled test substance	Replicate	Soils
		Latossolo (soil 1)		Argissolo (soil 2)		Gleissolo (soil 3)		Neossolo (soil 4)
(% AR)		SM	R	SM	R	SM	R	SM	R
Acetonitrile/water extracts	A	83.5	78.2	75.3	65.0	65.1	65.4	90.2	89.1
	B	85.9	77.4	78.2	67.1	66.3	64.7	90.3	89.0
	Mean	84.7	77.8	76.8	66.1	65.7	65.0	90.2	89.0
THF extracts	A	2.8	n.p.	3.6	n.p.	8.5	n.p.	0.9	n.p.
	B	2.4	n.p.	3.5	n.p.	8.8	n.p.	0.8	n.p.
	Mean	2.6	n.p.	3.6	n.p.	8.7	n.p.	0.9	n.p.
Isohexane extracts	A	0.3	n.p.	0.5	n.p.	2.2	n.p.	<LOQ	n.p.
	B	0.3	n.p.	0.6	n.p.	1.9	n.p.	<LOQ	n.p.
	Mean	0.3	n.p.	0.5	n.p.	2.1	n.p.	<LOQ	n.p.
Total extractables	A	86.7	78.2	79.4	65.0	75.9	65.4	91.2	89.1
	B	88.6	77.4	82.3	67.1	77.0	64.7	91.2	89.0
	Mean	87.6	77.8	80.8	66.1	76.5	65.0	91.2	89.0
Non-extractables	A	11.2	14.2	18.2	24.4	18.9	27.1	3.2	3.9
	B	10.8	14.4	16.9	21.2	18.1	27.2	3.2	4.5
	Mean	11.0	14.3	17.6	22.8	18.5	27.1	3.2	4.2
14CO2	A	1.9	1.5	3.5	3.3	1.1	1.0	0.2	0.2
	B	1.8	1.5	2.6	4.0	1.1	0.8	0.2	0.2
	Mean	1.9	1.5	3.1	3.7	1.1	0.9	0.2	0.2
T O T A L	A	99.8	93.9	101.1	92.8	95.9	93.5	94.5	93.2
	B	101.2	93.3	101.9	92.4	96.2	92.6	94.6	93.6
	Mean	100.5	93.6	101.5	92.6	96.0	93.0	94.6	93.4

n.p. = not performed

Table 8. Distribution and Recovery of Radioactivity (% AR): Argissolo Soil Treated with [Halophenyl-U-14C]-labelled test substance

[Halophenyl-U-14C]-labelled test substance Soil 2 (Argissolo)	Replicate	Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	8	14	28	47	62	75	89	105	120
Acetonitrile/water extracts	A	95.9	94.8	93.5	91.4	88.3	87.3	83.5	84.6	82.1	79.1
	B	96.2	91.9	94.7	87.1	84.5	86.7	88.2	85.1	82.4	78.0
	Mean	96.0	93.4	94.1	89.3	86.4	87.0	85.8	84.9	82.2	78.5
THF extracts	A	2.1	2.4	2.9	4.4	6.2	5.2	6.1	4.0	3.0	4.5
	B	2.0	2.6	3.0	6.3	7.0	3.7	3.1	3.3	3.1	4.2
	Mean	2.0	2.5	3.0	5.4	6.6	4.4	4.6	3.6	3.0	4.3
Isohexane extracts	A	0.4	0.5	0.4	0.6	0.9	0.8	1.2	0.7	0.7	0.6
	B	0.2	0.5	0.4	0.8	1.3	0.5	0.4	0.7	0.9	0.8
	Mean	0.3	0.5	0.4	0.7	1.1	0.7	0.8	0.7	0.8	0.7
Total extractables	A	98.4	97.7	96.8	96.4	95.4	93.3	90.7	89.2	85.7	84.1
	B	98.5	95.0	98.1	94.2	92.9	90.9	91.6	89.2	86.4	83.0
	Mean	98.4	96.4	97.4	95.3	94.1	92.1	91.2	89.2	86.0	83.5
Non-extractables	A	0.7	3.0	3.7	5.5	8.3	10.2	10.9	11.8	14.0	12.5
	B	0.6	3.1	3.8	6.9	8.5	9.7	9.9	11.9	13.4	13.4
	Mean	0.6	3.1	3.8	6.2	8.4	9.9	10.4	11.8	13.7	13.0
14CO2	A	n.p.	<0.1	0.1	0.2	0.3	0.4	0.4	0.6	0.8	0.9
	B	n.p.	<0.1	0.1	0.2	0.3	0.4	0.4	0.6	0.7	0.8
	Mean	n.p.	<0.1	0.1	0.2	0.3	0.4	0.4	0.6	0.8	0.8
T O T A L	A	99.0	100.8	100.7	102.1	103.9	103.9	102.0	101.7	100.6	97.5
	B	99.0	98.2	102.0	101.2	101.6	101.0	101.8	101.7	100.5	97.2
	Mean	99.0	99.5	101.3	101.7	102.8	102.4	101.9	101.7	100.5	97.3
MEAN ± SD		100.8 ± 1.8

 n.p. = not performed

Table 9. Distribution and Recovery of Radioactivity (% AR): Argissolo Soil Treated with [Oxoisoxazolidinyl-4,5-14C]-labelled test substance

[Oxoisoxazolidinyl -4,5-14C]- labelled test substance Soil 2 (Argissolo)	Replicate	Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	8	14	28	47	62	75	89	105	120
Acetonitrile/water extracts	A	96.0	93.2	91.1	86.0	75.8	82.7	80.9	77.2	76.7	70.9
	B	98.3	92.1	89.0	88.3	81.3	83.0	79.9	78.5	74.8	70.5
	Mean	97.2	92.6	90.1	87.1	78.5	82.9	80.4	77.8	75.8	70.7
THF extracts	A	1.7	3.3	2.8	5.3	9.7	3.3	3.0	3.5	2.6	3.4
	B	1.8	2.8	3.6	4.0	5.0	2.8	2.6	3.0	2.6	3.4
	Mean	1.8	3.1	3.2	4.7	7.3	3.1	2.8	3.2	2.6	3.4
Isohexane extracts	A	0.3	0.7	0.4	1.0	1.6	0.6	0.7	0.6	0.5	0.5
	B	0.4	0.6	0.5	0.8	1.0	0.6	0.5	0.6	0.3	0.5
	Mean	0.4	0.6	0.5	0.9	1.3	0.6	0.6	0.6	0.4	0.5
Total extractables	A	98.1	97.2	94.4	92.3	87.0	86.7	84.6	81.3	79.8	74.8
	B	100.5	95.5	93.2	93.2	87.3	86.4	83.0	82.0	77.7	74.4
	Mean	99.3	96.3	93.8	92.7	87.1	86.5	83.8	81.7	78.8	74.6
Non-extractables	A	<LOQ	3.9	4.8	7.5	10.4	10.2	12.1	12.8	14.0	14.4
	B	<LOQ	3.9	5.2	6.9	9.5	11.1	12.3	13.0	13.7	14.7
	Mean	<LOQ	3.9	5.0	7.2	9.9	10.6	12.2	12.9	13.9	14.5
14CO2	A	n.p.	0.9	1.7	2.7	2.8	4.6	5.2	6.4	6.9	6.2
	B	n.p.	1.0	1.7	2.7	3.8	5.3	5.4	6.6	6.8	5.6
	Mean	n.p.	1.0	1.7	2.7	3.3	4.9	5.3	6.5	6.8	5.9
T O T A L	A	98.1	102.0	100.9	102.4	100.2	101.4	102.0	100.5	100.7	95.4
	B	100.5	100.4	100.1	102.7	100.6	102.7	100.7	101.6	98.2	94.6
	Mean	99.3	101.2	100.5	102.6	100.4	102.1	101.3	101.0	99.5	95.0
MEAN ± SD		100.3 ± 2.2

 n.p. = not performed

Table 10 Comparison of the Soil Metabolism (SM) and Residue (R) Extraction Methods (Radiovalidation of Residue Method): Characterisation / Identification of Radioactive Residues in Soil Extracts (Soil metabolism samples were taken after 120 days of incubation; residue method samples were taken after 125 days of incubation)

[Methylphenyl-U- 14C]-labelled test substance	Replicate	Soils
		Latossolo (soil 1)		Argissolo (soil 2)		Gleissolo (soil 3)		Neossolo (soil 4)
(% AR)		SM	R	SM	R	SM	R	SM	R
The test substance	A	75.9	68.0	74.3	65.0	69.9	59.1	87.2	78.7
	B	73.9	67.3	78.5	67.1	70.9	59.5	88.2	79.3
	Mean	74.9	67.7	76.4	66.1	70.4	59.3	87.7	79.0
M4	A	2.5	*	*	*	0.4	*	3.0	6.5
	B	3.9	*	*	*	0.3	*	2.0	4.2
	Mean	3.2	*	*	*	0.4	*	2.5	5.3
M5	A	8.0	10.2	0.2	*	5.1	6.3	*	3.9
	B	7.4	10.1	0.3	*	5.4	5.2	*	5.4
	Mean	7.7	10.2	0.3	*	5.2	5.7	*	4.7
Unidentified residue 1	A	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*
Unidentified residue 2	A	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*
Unidentified residue 3	A	*	*	4.3	*	*	*	*	*
	B	3.2	*	2.9	*	*	*	*	*
	Mean	1.6	*	3.6	*	*	*	*	*
Unidentified residue 4	A	*	*	*	*	0.4	*	*	*
	B	*	*	*	*	0.5	*	*	*
	Mean	*	*	*	*	0.5	*	*	*

* Not detected

Table 11.Summary of Characterisation / Identification of Radioactive Residues in Argissolo Soil Treated with [Halophenyl-U-14C]-labelled the test substance (Total Extractables) (Values represent the sum of residues found in acetonitrile/water, THF, and isohexane extracts where applicable)  

[Halophenyl-U-14C]- labelled test substance Soil 2 (Argissolo) Total extracts	Replicate	Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	8	14	28	47	62	75	89	105	120
The test substance	A	97.9	97.2	92.9	88.2	89.3	85.4	80.5	81.0	77.9	78.2
	B	98.2	91.2	93.2	87.9	87.1	85.7	86.6	80.1	79.9	75.4
	Mean	98.1	94.2	93.1	88.0	88.2	85.6	83.5	80.5	78.9	76.8
M4	A	*	*	1.8	2.5	1.7	4.4	2.6	*	*	0.2
	B	*	3.3	2.1	1.6	2.1	1.6	*	1.7	*	0.2
	Mean	*	1.6	1.9	2.0	1.9	3.0	1.3	0.8	*	0.2
M5	A	*	*	1.8	3.1	1.9	0.3	3.6	3.3	3.4	3.0
	B	*	*	2.4	1.8	2.3	3.1	2.8	3.9	2.5	3.8
	Mean	*	*	2.1	2.5	2.1	1.7	3.2	3.6	3.0	3.4
Unidentified residue 1	A	*	*	*	*	*	*	*	2.2	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	1.1	*	*
Unidentified residue 2	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unidentified residue 3	A	*	*	*	2.1	1.6	*	2.9	2.1	3.7	2.1
	B	*	*	*	2.0	*	*	1.9	2.9	3.2	2.8
	Mean	*	*	*	2.1	0.8	*	2.4	2.5	3.5	2.4
Unidentified residue 4	A	*	*	*	*	*	2.5	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	1.2	*	*	*	*

* Not detected

Table 12. Summary of Characterisation / Identification of Radioactive Residues in Argissolo Soil Treated with [Oxoisoxazolidinyl-4,5-14C]-labelled test substance (Total Extractables). (Values represent the sum of residues found in acetonitrile/water, THF, and isohexane extracts where applicable)

[Oxoisoxazolidinyl-4,5- 14C]-labelled test substance Soil 2 (Argissolo) Total extracts	Replicate	Percent of Applied Radioactivity (% AR) by Incubation Time in Days after Treatment (DAT)
		0	8	14	28	47	62	75	89	105	120
The test substance	A	97.8	96.5	93.9	91.3	85.5	86.1	83.9	80.7	79.3	74.3
	B	100.1	94.9	92.7	92.4	86.2	85.8	82.5	81.5	77.4	73.9
	Mean	98.9	95.7	93.3	91.8	85.9	85.9	83.2	81.1	78.4	74.1
M4	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
M5	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unknown metabolite 1	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unknown metabolite 2	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unknown metabolite 3	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*
Unknown metabolite 4	A	*	*	*	*	*	*	*	*	*	*
	B	*	*	*	*	*	*	*	*	*	*
	Mean	*	*	*	*	*	*	*	*	*	*

* Not detected

Table 13. Summary of DT50 Values

Soil (Model)	Degradation Kinetics for Test Item
	DT50 [days]	Parameter	χ2 error %	r2	Prob > t
Latossolo (SFO)^a	257	k = 0.002698	1.95	0.9122	4.14E-011
Argissolo (SFO)^a	300	k = 0.002308	1.1	0.8447	5.26E-029
Gleissolo (SFO)^a	244	k = 0.002845	1.47	0.9724	1.32E-015
Neossolo (SFO)^b	723	k = 9.59E-004	1.17	0.8384	8.60E-009

SFO: single first-order kinetics; non-linear method

DT50: Calculated degradation half-life of parent.χ2: chi-square statistical value. r2: linear regression coefficient relating goodness of fit as value approaches unity.

a Calculations based on the sum of HPLC results for acetonitrile/H2O and THF extracts.

b Calculation based on HPLC results for acetonitrile/H2O extracts (THF extracts were not analysed by HPLC due to residue levels ≤ 0.9% AR).

Conclusions:

The key findings after application of [14C]-labelled test substance at three distinctive positions to four various Brazilian soils at an application rate of 0.2 mg/kg dry soil equivalent (approximately 150 g ai/ha) and subsequent aerobic incubation (20.8 ± 0.3 °C, dark and pF 2) for 120 days are summarized below:
- The mass balance from the soil samples ranged from 94.5% to 104.3% applied radioactivity (AR).
- The test substance degraded slowly, but consistently in all study soils. The DT50 values of the test substance were determined to range from 244 to 723 days.
- The carboxylic acid metabolite, M5, reached maximum levels of 7.7% AR (mean) throughout the incubation period.
- No other metabolites exceeded 5% AR.
- Mineralisation to 14CO2 was relatively low (≤ 6.8% AR) in all soils throughout the incubation period, irrespective of labelling.
- Unextracted residues (bound residues) increased slowly throughout the study, reaching maximum levels of 18.5% AR (mean) by the end of the incubation.

Executive summary:

The rate and route of degradation of the test substance, 14C-labelled in three distinctive positions, was investigated in four different Brazilian soils: Latossolo (sandy clay), Argissolo (sandy clay loam), Gleissolo (loamy sand) and Neossolo (sand).The study was conducted in according to guidelines: OECD TG 307 and EPA 835.4100 and in compliance with GLP criteria. The degradation of 14C- methylphenyl ring labelled test substance was investigated in all four soils tested, whereas the degradation of 14C-halophenyl ring and 14C-oxoisoxazolidinyl ring labelled the test substance was investigated in the Argissolo soil only. 14C-labelled test substance was applied at a target concentration of 20 µg of the test item per system, equivalent to a single field application rate of 150 g test item/ha (assuming an incorporation depth of 5 cm and a bulk density of 1.5 g/cm3). The actual application rate achieved was 20.6 µg, 20.4 µg and 20.6 µg of the test item per system treated with methylphenyl, halophenyl and oxoisoxazolidinyl radiolabelled test items, respectively, which is equivalent to a single field application rate of 154.5, 153.0 and 154.5 g test item/ha (102.8%, 101.8% and 103.1% of target) for samples treated with methylphenyl, halophenyl and oxoisoxazolidinyl radiolabelled test items, respectively. The soil samples were incubated in the dark in the laboratory at 20.8 ± 0.3 °C for 120 days. Duplicate samples were taken for analysis at 0, 8, 14, 28, 47, 62, 75, 89, 105 and 120 days after treatment (DAT).

The soil samples were extracted a total of eight times at room temperature using solvents of varying polarity: once with acetonitrile/water (50:50; v/v), twice with acetonitrile/water (80:20; v/v) and once with acidified (formic acid) acetonitrile/water (80:20; v/v; pH 3), twice with THF, and twice with isohexane. Aqueous acetonitrile extracts were pooled and analysed by RP-HPLC for parent compound and degradation products. Selected pooled aqueous acetonitrile extracts were analysed by 2D-TLC and LC-MS/MS to confirm the identity of radioactive components. THF and isohexane extracts were analysed in a similar fashion, where applicable. Application solutions for each radiolabelled test item, as well as selected (acetonitrile/water and THF) soil extracts from 0, 89 and 120 DAT for all soils and labels were analysed by chiral LC-MS to determine the isomer ratio of parent and the major metabolite, M5. Any volatile radioactivity was continuously flushed from the vessels and collected in traps. Remaining unextracted residues were analysed by combustion and a mass balance determined for each sample. Radiovalidation of a separate residue extraction method was performed within the study. At the selected sampling interval of 125 DAT, two replicates of each soil treated with the methylphenyl labelled test item were extracted at room temperature following the residue method (two extractions with aqueous acetonitrile mixtures each for about 1 h at 250 rpm, followed by centrifugation, phase separation and LSC measurement). Further analysis of the samples was performed by HPLC.

For soil samples treated with [methylphenyl-U-14C]-labelled test substance (all four soils), individual mass balance values ranged from 94.5% to 104.3% applied radioactivity (AR). For soil samples treated with [halophenyl-U-14C]-labelled test substance (Argissolo soil only), individual mass balances ranged from 97.2% to 103.9% AR. For soil samples treated with [oxoisoxazolidinyl-4,5-14C]-labelled test substance (Argissolo soil only), individual mass balance values ranged from 94.6% to 102.7% AR. The total amount of radioactivity extracted decreased slowly over time in all soils, ranging from 98.1 to 99.9% AR at 0 DAT and from 74.6 to 91.2% AR at 120 DAT. The majority of the radioactivity was recovered in the aqueous acetonitrile mixtures, which ranged from 91.6 to 99.6% AR for all soils at 0 DAT and from 65.7 to 90.2% AR at 120 DAT. The maximum amounts of radioactivity present in the THF and isohexane extracts at any time point were 8.7% and 2.1%, respectively. Following treatment of all four soils with [methylphenyl-U-14C]-labelled test substance, the total amount of parent present in the acetonitrile/water and THF extracts declined slowly over time from 97.9 – 99.6% AR at 0 DAT to 74.9, 76.4, 70.4 and 87.7% AR at 120 DAT in Latossolo, Argissolo, Gleissolo and Neosollo soils, respectively. The majority of the parent extracted was recovered in the acetonitrile/water extracts, with only a relatively small proportion (up to 8.4%) recovered in the THF extracts. In the isohexane extracts of soils treated with [methylphenyl-U-14C]-labelled test substance (Gleissolo soil only), the test substance represented only a very minor fraction of 1.5% AR at 120 DAT, the only interval analysed by HPLC.

Only one metabolite (M2) was present at levels of > 5%. In Latossolo and Gleissolo soils, levels of metabolite M2 increased relatively slowly over time reaching maximum mean levels of 7.7 and 6.0% AR at 120 and 89 DAT, respectively. In Argissolo and Neossolo soils, it remained at levels of < 3% AR throughout the incubation period. No other metabolites was present at levels of >5% AR, including metabolite M49, which reached maximum levels of 3.3, 2.1, 0.4 and 3.1% AR in Latossolo, Argissolo, Gleisollo and Neossolo soils, respectively. Following treatment of Argissolo soil with [halophenyl-U-14C]-labelled test substance, the total amount of parent present in the acetonitrile/water and THF extracts declined slowly over time from 98.1% at 0 DAT to 76.8% at 120 DAT. The majority of the test substance was recovered in the acetonitrile/water extracts with only ≤ 6.2% (mean) AR recovered in THF extracts. No single metabolite was present at levels of > 5%, including metabolite M4 and metabolite M2, which reached maximum levels of 3.0 and 3.6% AR, respectively, as recovered from acetonitrile/water and THF extracts together.

Following treatment of Argissolo soil with [oxoisoxazolidinyl-4,5-14C]-labelled test substance, the total amount of parent present in the acetonitrile/water and THF extracts declined slowly over time from 98.9% AR at 0 DAT to 74.1% AR at 120 DAT. The majority of the the test substance was recovered in the acetonitrile/water extracts with only ≤ 7.3% (mean) AR recovered in THF extracts. No metabolites were detected in either the acetonitrile/water or THF extracts. Unextracted residues (bound residues) increased slowly throughout the incubation, reaching maximums of 11.0 to 18.5% AR for Latossolo, Argissolo and Gleissolo soils and 3.2% AR for Neossolo soil by the end of the incubation. Formation of radioactive carbon dioxide in soils treated with the test item labelled in the methylphenyl position reached a mean maximum of 3.1% AR. For Argissolo soil treated in the halophenyl position, 14CO2 reached a mean maximum of 0.8% AR. For Argissolo soil treated in the oxoisoxazolidinyl position, 14CO2 reached a mean maximum of 6.8% AR. The rate of degradation in tested soils for the test substance was calculated using single first-order (SFO) kinetics and the amount of parent found in acetonitrile/water and THF extracts and under consideration of all three labelling positions (for Argissolo soil only). Unextracted residues (bound residues) increased slowly throughout the study, reaching maximum levels of 18.5% AR (mean) by the end of the incubation.

The stereoisomer ratio of the parent compound in application solutions, 0 and 120 DAT soil extracts (all soils) and in the 89 DAT soil extracts of Gleissolo soil was confirmed to be consistent with that reported in the test item Certificates of Analysis (CoAs). The isomer ratio of the metabolite M2 was also consistent with that predicted from the provided CoAs. Hence, it was confirmed that there was no change of stereochemistry during the incubation time of 120 days and hence, there was no evidence of either interconversion or stereo-selective metabolism of either the test substance or its metabolite, M5. Additional soil samples treated with [methylphenyl-U-14C]-labelled test substance (all soils) were extracted by following a residue analytical extraction method at 125 DAT (radiovalidation method). The data showed that the extractability of radioactive residues using the metabolism methodologies and the residue analytical method were in good agreement and that following analysis of the extracted radioactivity, the levels of parent and metabolites detected by HPLC were also in agreement.

Based on the findings, the degradation half-life (DT50) of the test substance in the tested soil Latossolo, Argissolo, Gleissolo and Neossolo is calculated to be 257 days, 300 days, 244 days and 723 day, respectively.

Description of key information

Geometric mean DT50 in soil = 184.68 d, 20 °C, OECD TG 307, Piskorski 2018; Blüthgen-Schiller 2018.

Key value for chemical safety assessment

Half-life in soil:

184.68 d

at the temperature of:

20 °C

Additional information

The biodegradation of the radiolabelled test substance was tested under GLP to OECD TG 307 in two reliable and valid soil simulation studies (Piskorski 2018, Blüthgen-Schiller 2018). Ten soils covering different soil types from Europe, USA and Brazil (including loams, silt loams, sandy loams, sandy clay loams, sandy clay and sand) were used in the tests, which also covered a wide range of organic carbon content (0.78 to 10.2%) and pH (4.2 to 7.4). The studies were conducted at a temperature of about 20 °C, with a soil moisture content ranging from 4.9 to 85.5% (the majority of soils were tested at ca. 30% moisture). The dissipation half-life in the soils ranged from 56 to 723 days, and the geometric mean of the dissipation half-lives was calculated to be 184.7 days (n = 10).