Chlorpyrifos

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4200 (Anaerobic Soil Metabolism)

Deviations:

no

GLP compliance:

yes

Test type:

laboratory

Specific details on test material used for the study:

Substance ID: INV304794
Lot Number: YH2-132673-071-te
Radiochemical Purity: 99.3%
Specific Activity: 30.3 mCi/mmol, 191,865 dpm/μg

Radiolabelling:

yes

Oxygen conditions:

anaerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2013

Soil no.:

#1

Soil type:

sandy loam

% Clay:

8

% Silt:

15

% Sand:

77

% Org. C:

2.8

pH:

7.7

CEC:

15.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.24

Soil no.:

#2

Soil type:

loam

% Clay:

27

% Silt:

30

% Sand:

43

% Org. C:

4.9

pH:

7.4

CEC:

27.1 meq/100 g soil d.w.

Bulk density (g/cm³):

1.09

Soil no.:

#3

Soil type:

clay

% Clay:

45

% Silt:

28

% Sand:

27

% Org. C:

2.2

pH:

7.8

CEC:

12.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.05

Soil no.:

#4

Soil type:

sandy loam

% Clay:

19

% Silt:

15

% Sand:

66

% Org. C:

2

pH:

6.8

CEC:

15.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.03

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
Longwoods (soil #1)
- Geographic location: Longwoods Quarry/Lincolnshire, UK
- Pesticide use history at the collection site: 16 years since arable use, No sprays or fertilizer since then
- Collection procedures: Sampling carried out according to ISO 10381-6
- Sampling depth (cm): 5-20 cm
- Storage conditions: 5ºC
- Storage length: 49 days
- Soil preparation: <2 mm sieved
- Field Moisture:
Capacity at 1/3 bar [pF 2.5] (%): 13.8
Capacity at 1/10 bar [pF 2.0] (%): 16.5
Capacity at 15 bar (%): 10.1

South Witham (soil #2)
- Geographic location: South Witham Quarry/Lincolnshire, UK
- Pesticide use history at the collection site: No pesticide used after 1995
- Collection procedures: Sampling carried out according to ISO 10381-6
- Sampling depth (cm): 5-15 cm
- Storage conditions: 5ºC
- Storage length: 48 days
- Soil preparation: <2 mm sieved
- Field Moisture:
Capacity at 1/3 bar [pF 2.5] (%): 27.7
Capacity at 1/10 bar [pF 2.0] (%): 33.7
Capacity at 15 bar (%): 20.8

Hareby (soil #3)
- Geographic location: Hareby/Lincolnshire, UK
- Pesticide use history at the collection site: No pesticide or fertilizer for some years
- Collection procedures: Sampling carried out according to ISO 10381-6
- Sampling depth (cm): 5-20 cm
- Storage conditions: 5ºC
- Storage length: 19 days
- Soil preparation: <2 mm sieved
- Field Moisture:
Capacity at 1/3 bar [pF 2.5] (%): 26.3
Capacity at 1/10 bar [pF 2.0] (%): 34.7
Capacity at 15 bar (%): 13.1

MSL (soil #4)
- Geographic location: Grand Forks, North Dakoda
- Pesticide use history at the collection site: No pesticide or fertilizer for the past 30 years
- Collection procedures: Shovel
- Sampling depth (cm): 0-6"
- Storage conditions: 5ºC
- Storage length: 24 days
- Soil preparation: <2 mm sieved
- Field Moisture:
Capacity at 1/3 bar [pF 2.5] (%): 21.0
Capacity at 1/10 bar [pF 2.0] (%): 28.7
Capacity at 15 bar (%): 13.7

Soil No.:

#1

Duration:

120 d

Soil No.:

#2

Duration:

120 d

Soil No.:

#3

Duration:

120 d

Soil No.:

#4

Duration:

120 d

Soil No.:

#1

Initial conc.:

1.5 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 1000 g a.i./ha or 0.9 lb a.i./acre

Soil No.:

#2

Initial conc.:

1.5 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 1000 g a.i./ha or 0.9 lb a.i./acre

Soil No.:

#3

Initial conc.:

1.5 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 1000 g a.i./ha or 0.9 lb a.i./acre

Soil No.:

#4

Initial conc.:

1.5 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 1000 g a.i./ha or 0.9 lb a.i./acre

Parameter followed for biodegradation estimation:

radiochem. meas.

Temp.:

20°C

Microbial biomass:

2.51 for Longwood soil, 2.71 for South Witham soil, 4.09 for Hareby soil, and 2.43 for MSL soil (for control)

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil condition: Fresh
- Soil (g/replicate): 50 g dry weight
- Control conditions: Delivery solvent treated for biomass and water quality analysis
- No. of replication controls: 2
- No. of replication treatments: 2
- Test apparatus (Type/material/volume): 250-mL glass screw-cap vessels with Teflon®-lined silicone septa, and contained approximately 50.0-g dry weight of soil.
- Details of traps for CO2 and organic volatile, if any: Ethylene glycol, KOH, and foam plug
- Identity and concentration of co-solvent: Acetonitrile, 4.8mL/kg soil

Test material application
- Volume of test solution used/treatment: 0.24 mL/treatment
- Application method: Applied on surface and homogeneous mixing
- Is the co-solvent evaporated: Yes

Experimental conditions
- Moisture maintenance method: Aerating moisturized air for 2 or 3 days
- Continuous darkness: Yes

Other details, if any: Flooding time: 3DAT for Longwood, South Witham, and Hareby soil; 4DAT for MSL soil

OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the aerobic conditions: The test systems were aerated continuously with hydrated laboratory air
- Methods used to create the anaerobic conditions: Following the aerobic incubation period, the test vessels were removed from the aerobic trapping system and flooded with 100 mL of nitrogen-purged purified reagent water. Test vessels were continuously blanketed with hydrated nitrogen gas.

SAMPLING DETAILS
- Sampling intervals: 0, 2, 3, 4, 5, 7, 14, 28, 45, 80 and 120 DAT (duplicate samples); 0 and 2 DAT – Aerobic test conditions; 3DAT – Flooding for only Longwoods, Haerby and South Witham soil samples; 4DAT – Flooding for only MSL soil sample
- Sampling method for soil samples: Aerobic soil extracted three times with acidified (0.1% HCl) 90:10 acetone:purified reagent water (v:v). For anaerobic soil system, water phase removed from the soil by pipette and transferred into a graduated cylinder, taking care not to disturb the soil and then adding acetone. Soil layer extracted three times with acidified (0.1% HCl) 90:10 acetone:purified reagent water (v:v).
- Method of collection of CO2 and volatile organic compounds: Aliquots (ethylene glycol and KOH) taken for radioassay by LSC at 2, 3, 4, 5, 7, 14, 28, 45, 80 and 120 DAT. Radiolabeled volatile organic carbon trapped in foam plug analyzed by LSC at 2, 3, 4, 5, 7, 14, 28, 45, 80 and 120 DAT.

Soil No.:

#1

% Recovery:

98.5

Soil No.:

#2

% Recovery:

96.7

Soil No.:

#3

% Recovery:

97.6

Soil No.:

#4

% Recovery:

97.6

Key result

Soil No.:

#1

DT50:

3.6 d

Type:

other: DFOP

Temp.:

20 °C

Key result

Soil No.:

#2

DT50:

3.2 d

Type:

other: DFOP

Temp.:

20 °C

Key result

Soil No.:

#3

DT50:

4.9 d

Type:

other: DFOP

Temp.:

20 °C

Key result

Soil No.:

#4

DT50:

9.3 d

Type:

other: DFOP

Temp.:

20 °C

Transformation products:

yes

Remarks:

TCP (3,5,6-trichloro-2-pyridinol) and DCP (3,6-trichloro-2-pyridinol)

No.:

#1

No.:

#1

Volatile metabolites:

yes

Remarks:

CO2

Residues:

yes

Details on results:

MAJOR TRANSFORMATION PRODUCTS
- Water fraction:
Soil #1: The TCP degradate was first detected at an average of 2.00% AR at day 3, increased to 52.7% AR at day 45, and decreased to 12.5% AR at day 120. The DCP degradate was first detected at an average of 11.5% AR at day 28, increased to 50.3% AR at day 80, and decreased to 43.1% AR at day 120.
Soil #2: The TCP degradate was first detected at an average of 1.23% AR at day 3, increased to 29.9% AR at day 14, and decreased to 3.47 %AR at day 120. The DCP degradate was first detected at an average of 12.7% AR at day 28 and increased to 29.5% AR at day 120.
Soil #3: The TCP degradate was first detected at an average of 1.58% AR at day 3, increased to 48.1% AR at day 28, and decreased to 27.4 %AR at day 120. The DCP degradate was first detected at an average of 0.76% AR at day 28, increased to 17.3% AR at day 80, and decreased to 10.4% AR at day 120.
Soil #4: The TCP degradate was first detected at an average of 0.90% AR at day 4, increased to 31.6% AR at day 48 and decreased to 4.91 %AR at day 120. The DCP degradate was first detected at an average of 24.4% AR at day 80 and increased to 37.8% AR at day 120.
- Soil extractable fraction:
Soil #1: The TCP degradate was first detected at an average of 26.9% AR at day 2, increased to 39.8% AR at day 7, and decreased to 8.47% AR at day 120. The DCP degradate was first detected at an average of 11.9% AR at day 28, decreased to 2.28% AR at day 45, and increased to 16.1% AR at day 120.
Soil #2: The TCP degradate was first detected at an average of 30.0% AR at day 2, increased to 43.3% AR at day 14, and decreased to 5.19% AR at day 120. The DCP degradate was first detected at an average of 21.4% AR at day 28 and increased to 24.7% AR at day 120.
Soil #3: The TCP degradate was first detected at an average of 27.4% AR at day 2, increased to 37.7% AR at day 14, and decreased to 16.8% AR at day 120. The DCP degradate was first detected at an average of 0.76% AR at day 28, increased to 8.60% AR at day 80, and decreased to 5.63% AR at day 120.
Soil #4: Two major regions (≥ 5% AR) were detected in the MSL soil samples and were identified as TCP and DCP. The TCP degradate was first detected at an average of 15.1% AR at day 2, increased to 43.9% AR at day 28, and decreased to 4.73% AR at day 120. The DCP degradate was first detected at an average of 1.67% AR at day 48 and increased to 20.7% AR at day 120.
- Total soil test system (water and soil extractable fractions):
Soil #1: The TCP degradate was first detected at an average of 26.9% AR at day 2, increased to 80.2% AR at day 45, and decreased to 20.9% AR at day 120. The DCP degradate was first detected at an average of 23.4% AR at day 28 and increased to 59.2% AR at day 120.
Soil #2: The TCP degradate was first detected at an average of 30.0% AR at day 2, increased to 73.3% AR at day 14, and decreased to 8.66% AR at day 120. The DCP degradate was first detected at an average of 34.1% AR at day 28 and increased to 54.1% AR at day 120.
Soil #3: The TCP degradate was first detected at an average of 27.4% AR at day 2, increased to 82.1% AR at day 28, and decreased to 44.2% AR at day 120. The DCP degradate was first detected at an average of 1.52% AR at day 28, increased to 25.9% AR at day 80 and decreased to 16.0% AR at day 120.
Soil #4: The TCP degradate was first detected at an average of 15.1% AR at day 2, increased to 73.5% AR at day 28, and decreased to 9.64% AR at day 120. The DCP degradate was first detected at an average of 1.67% AR at day 48 and increased to 58.5% AR at day 120.

MINOR TRANSFORMATION PRODUCTS
- Several minor regions of radioactivity were observed in the chromatograms. Individual minor peaks did not exceed 5% of the applied radioactivity for the water fraction, soil extractable fraction or the total soil test system (water and soil extractable fractions). The minor transformation product was MTP (N-methyl-3,5,6-trichloro-2(1H)-pyridinone), formed at a maximum of 0.74% to 2.21% AR.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: 0.01% to 4.98% AR

EXTRACTABLE RESIDUES
Extractable 14C-residues decreased from 98.25% AR at Day 0 to 23.68% AR at the end of the study period.

NON-EXTRACTABLE RESIDUES
Radioactivity remaining in representative Day 120 non-extractable residue samples following the solvent extraction analyses were characterized by fractionation of the Longwoods, South Witham, Hareby and MSL soil humus into humin, humic acid and fulvic acid fractions. The radioactivity in the non-extractables was well distributed to the humin and fulvic acid. However, there is no radioactivity observed in humic acid fraction.
Soil #1: Day 120–430 % of applied radio activity: 7.77 (fulvic acid), 0.00 (humic acid), 9.29 (humin)
Soil #2: Day 120–472 % of applied radio activity: 10.75 (fulvic acid), 0.00 (humic acid), 15.33 (humin)
Soil #3: Day 120–517 % of applied radio activity: 10.52 (fulvic acid), 0.00 (humic acid), 11.16 (humin)
Soil #4: Day 120–539 % of applied radio activity: 6.49 (fulvic acid), 0.00 (humic acid), 7.34 (humin)
Non-extractable 14C residues increased from 0.32% AR at Day 0 to 26.08% AR at the end of the incubation period.

VOLATILIZATION
- The radioactivity in the 1 M KOH traps, corresponding to 14CO2, reached an accumulative average maximum of 2.06, 4.36, 5.42 and 5.52% at day 120 for the Longwoods soil, South Witham soil, Hareby soil and MSL soil, respectively. Negligible radioactivity was detected (≤0.02% AR) in the ethylene glycol volatile organic traps for all soils after 120 days of incubation.

Conclusions:

The major transformation products detected were TCP (3,5,6-trichloro-2-pyridinol) and DCP (3,6-trichloro-2-pyridinol), with maximum concentrations of 82.70% and 70.98% AR, observed on day 45 and day 120 of incubation, respectively. The corresponding concentrations at the end of the study were 28.17% and 70.98% AR, respectively. The test substance was first metabolized by hydrolysis to produce TCP. TCP was further degraded to DCP by dechlorination.

Executive summary:

The biotransformation of radiolabeled test substance was studied in Longwoods (sandy loam soil from UK, pH 7.7, organic carbon 2.8%), South Witham (loam soil from UK, pH 7.4, organic carbon 4.9%), Hareby (clay soil from UK, pH 7.8, organic carbon 2.2%) and MSL (sandy loam soil from USA, pH 6.8, organic carbon 2.0%) soils for 120 days under anaerobic conditions in the dark at 20°C, and soil moisture was adjusted to a pF soil moisture value of 2.0 (16.5% for Longwoods soil, 33.7% for South Witham soil, 34.7% for Hareby soil and 28.7% for MSL soil). The test substance was applied at the rate of 1.5 mg a.i./kg soil, equivalent to 1000 g a.i./ha or 0.9 lb a.i./acre. The experiment was conducted in accordance with the US EPA guideline OPPTS 835.4200 and the OECD guideline 307.

Samples were initially incubated for 2 to 3 days under aerobic conditions in the dark at 20°C then samples were flooded with water. After flooding, samples were maintained in the dark in an incubator set at 20°C with nitrogen flowing across the samples to encourage anaerobic conditions. The bottles containing the treated, flooded soils were connected to a bottle of 1 N potassium hydroxide (KOH) for the collection of CO2.

Samples were analyzed at 0 and at 2 or 3 days (0 and 2 days for Longwood soil only) after application (DAT) then at 3 or 4, 10 or 11, 24 or 25, 41 or 42, 76 or 77, and 116 or 117 days after flooding (DAF). The anaerobic samples were centrifuged to separate the aqueous layer which was analyzed by liquid scintillation counting (LSC) and high performance liquid chromatography with radiochemical detection (HPLC/RAM). The soil samples were extracted on a horizontal shaker at low speed with acidified (0.1% hydrochloric acid, HCl) 90:10 acetone:purified reagent water (PRW) (v:v), and the radioactive residues were analyzed by LSC and HPLC/RAM. Identification of the transformation products was performed by co-chromatography with authentic standards and liquid chromatography with mass spectrometry (LC/MS).

Average material balance values for the four tested soils were 97.59% of applied radioactivity (AR). The concentration of the parent compound decreased from 97.11% AR at day 0 to 3.50% AR at the end of the study period. The DT50 of the test substance in anaerobic soil was 5.3 days.

The major transformation products detected were TCP (3,5,6-trichloro-2-pyridinol) and DCP (3,6-trichloro-2-pyridinol), with maximum concentrations of 82.70% and 70.98% AR, observed on day 45 and day 120 of incubation, respectively. The corresponding concentrations at the end of the study were 28.17% and 70.98% AR, respectively. The minor transformation product was MTP (N-methyl-3,5,6-trichloro-2(1H)-pyridinone), formed at a maximum of 0.74% to 2.21% AR. The total unidentified radioactivity ranged from 0.01% to 4.98% AR. Extractable 14C-residues decreased from 98.25% AR at Day 0 to 23.68% AR at the end of the study period. Non-extractable 14C residues increased from 0.32% AR at Day 0 to 26.08% AR at the end of the incubation period. At study termination, evolved 14CO2 and volatile organics were 5.52% and 0.02% AR, respectively.

The radioactivity in the non-extractables was well distributed to the humin (15.33% AR) and fulvic acid (10.75% AR) at the end of the incubation period. However, there is no radioactivity observed in the humic acid fraction.

The test substance was first metabolized by hydrolysis to produce TCP. TCP was further degraded to DCP by dechlorination.

Reference 2

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4100 (Aerobic Soil Metabolism)

Deviations:

no

GLP compliance:

yes

Test type:

laboratory

Specific details on test material used for the study:

Substance ID: [14C]-chlorpyrifos
Lot Number: YE1-132849-021
Radiochemical Purity: 98.6%
Specific Activity: 29.7 mCi/mmol

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Soil no.:

#1

Soil type:

silt loam

% Clay:

17

% Silt:

63

% Sand:

20

% Org. C:

1.6

pH:

5.2

CEC:

9.7 meq/100 g soil d.w.

Bulk density (g/cm³):

0.91

Soil no.:

#2

Soil type:

sandy clay loam

% Clay:

23

% Silt:

18

% Sand:

59

% Org. C:

0.65

pH:

8

CEC:

17 meq/100 g soil d.w.

Bulk density (g/cm³):

1.12

Soil no.:

#3

Soil type:

sandy loam

% Clay:

20

% Silt:

19

% Sand:

61

% Org. C:

1.7

pH:

6.4

CEC:

16.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1.08

Soil no.:

#4

Soil type:

clay loam

% Clay:

29

% Silt:

40

% Sand:

31

% Org. C:

1.3

pH:

6.7

CEC:

19.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1.17

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Four soils were collected from differing areas of the U.S.A. The soil samples were obtained from Boone County, Missouri (silt loam), Willacy County, Texas (sandy clay loam), Grand Forks County, North Dakota (sandy loam), and Tehama County, California (clay loam).
- Storage conditions: Approximately 4°C in the dark in closed bags
- Storage length: 16 to 68 days prior to dosing
- Soil preparation: Prior to use, the test soils were sieved through an approximately 2-mm mesh sieve.

Soil No.:

#1

Duration:

120 d

Soil No.:

#2

Duration:

120 d

Soil No.:

#3

Duration:

120 d

Soil No.:

#4

Duration:

120 d

Soil No.:

#1

Initial conc.:

1.55 other: μg/g dry soil

Based on:

act. ingr.

Soil No.:

#2

Initial conc.:

1.55 other: μg/g dry soil

Based on:

act. ingr.

Soil No.:

#3

Initial conc.:

1.55 other: μg/g dry soil

Based on:

act. ingr.

Soil No.:

#4

Initial conc.:

1.55 other: μg/g dry soil

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

20°C

Humidity:

75.7% Moisture at 0 bar

Microbial biomass:

Initial: 651.4 μg/g; Final: 657.6 μg/g

Soil No.:

#2

Temp.:

20°C

Humidity:

67.3% Moisture at 0 bar

Microbial biomass:

Initial: 473.4 μg/g; Final: 509.0 μg/g

Soil No.:

#3

Temp.:

20°C

Humidity:

68.0% Moisture at 0 bar

Microbial biomass:

Initial: 614.9 μg/g; Final: 602.9 μg/g

Soil No.:

#4

Temp.:

20°C

Humidity:

59.0% Moisture at 0 bar

Microbial biomass:

Initial: 482.2 μg/g; Final: 546.0 μg/g

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS: A short 5-day preliminary test was performed exactly as described for the definitive test and showed acceptable mass accountability and chromatography, which indicated that the test system design and methods for extraction and analysis were acceptable for use in the definitive test.

2. EXPERIMENTAL DESIGN
- Soil preincubation conditions: seven days prior to definitive testing dosing initiation at 20°C
- Soil (g/replicate): 50 g (dry weight) moist soil for test samples; 250 g (dry weight) for biomass samples
- Controls: Since radiolabeled test substance was used, there was no need to use control substance.
- No. of replication treatments: 22 samples per soil type, enough for duplicate samples at 9 time points plus two contingency sets
- Test apparatus: Air was pulled through a reagent water pre-trap humidifier and a blank trap, then into the series of test vessels. Air exiting the series of test vessels was then passed through a blank trap for overflow, and a single trap to collect volatiles
- Details of traps for CO2 and organic volatile: One 2 N KOH trap (180 mL)
- Identity of solvent: Acetonitrile

Test material application
- Volume of test solution used/treatment: 67 μL
- Application method: Pipette
- Is the co-solvent evaporated: NA, minimal amount

Any indication of the test material adsorbing to the walls of the test apparatus: None (verified by mass balance results)

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: Soil sample weights were adjusted to 50% of moisture-holding capacity at 0 bar with reagent water. During the study, representative test vessels were weighed at each sampling interval. Any weight loss relative to Day 0 was attributed to soil moisture loss, and the appropriate amount of reagent water (usually < 1.0 g) was added to any samples that showed moisture loss to bring the moisture content to approximately 50% of 0 bar moisture.
- Continuous darkness: Yes (except during general use of the chamber).

3. OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: Aerobic conditions were maintained in all samples by drawing humidified air through the series containing the soil samples.

4. SAMPLING DETAILS
- Sampling intervals: Days 0, 3, 7, 10, 14, 30, 59, 90, and 120 days
- Method of collection of CO2 and volatile organic compounds: The volatile traps designated for 14CO2 for each sample train were collected at the same time intervals (excluding Day 0)
- Sample storage before analysis: Samples were extracted on the sampling day. Extracts were stored frozen (approximately -20°C) and initially analyzed by LSC the day of sampling.

Soil No.:

#1

% Recovery:

96.6

St. dev.:

3.9

Remarks on result:

other: silt loam

Soil No.:

#2

% Recovery:

97.5

St. dev.:

2.5

Remarks on result:

other: sandy clay loam

Soil No.:

#3

% Recovery:

97.4

St. dev.:

2.9

Remarks on result:

other: sandy loam

Soil No.:

#4

% Recovery:

96.4

St. dev.:

3.4

Remarks on result:

other: clay loam

Key result

Soil No.:

#1

DT50:

22 d

Type:

other: DFOP

Temp.:

20 °C

Remarks on result:

other: silt loam

Key result

Soil No.:

#2

DT50:

6 d

Type:

other: SFO

Temp.:

20 °C

Remarks on result:

other: sandy clay loam

Key result

Soil No.:

#3

DT50:

10 d

Type:

other: DFOP

Temp.:

20 °C

Remarks on result:

other: sandy loam

Key result

Soil No.:

#4

DT50:

37 d

Type:

other: DFOP

Temp.:

20 °C

Remarks on result:

other: clay loam

Transformation products:

yes

Remarks:

TCP (3,5,6-trichloro-2-pyridinol)

No.:

#1

Details on transformation products:

The results obtained in this study indicate that the test substance will degrade in a microbially viable soil to TCP and other known minor metabolites. TCP, in turn is finally degraded to CO2, as well as to un-extractable residues.

Volatile metabolites:

yes

Remarks:

CO2

Residues:

yes

Details on results:

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

MAJOR TRANSFORMATION PRODUCTS
- Refer table 1 under 'Any other information on results incl. tables'

MINOR TRANSFORMATION PRODUCTS
- Refer table 1 under 'Any other information on results incl. tables'

EXTRACTABLE and NON-EXTRACTABLE RESIDUES
- Boone Soil (silt loam): Mean extractability values decreased from 99.7% AR at Day 0 to 22.0% AR at Day 120. The mean level of non-extractable residue increased to 14.4% AR at Day 120, and the amount of 14CO2 captured in the KOH trap reached a maximum of 51.7% AR at Day 120.
- Raymondville Soil (sandy clay loam): Mean extractability values decreased from 100.3% AR at Day 0 to 14.4% AR at Day 120. The mean level of non-extractable residue increased to 26.9% AR at Day 120, and the amount of 14CO2 captured in the KOH trap reached a maximum of 54.2% AR at Day 120.
- MSL-PF Soil (sandy loam): Mean extractability values decreased from 99.0% AR at Day 0 to 24.7% AR at Day 120. The mean level of non-extractable residue increased to 16.1% AR at Day 120, and the amount of 14CO2 captured in the KOH trap reached a maximum of 52.2% AR at Day 120.
- Tehama Soil (clay loam): Mean extractability values decreased from 98.5% AR at Day 0 to 27.1% AR at Day 120. The mean level of non-extractable residue increased to 22.5% AR at Day 120, and the amount of 14CO2 captured in the KOH trap reached a maximum of 43.3% AR at Day 120.

VOLATILIZATION
- CO2 volatiles after 120 days ranged from 43.3 to 54.2% AR

Table 1:  Mean Maximum %Applied Radioactivity of Transformation products

Soil	Mean Maximum %Applied Radioactivity
	TCP	TMP	MTCP
Boone (silt loam)	16.6 (Day 30)	1.1 (Day 59)	3.2 (Day 120)
Raymondville (sandy clay loam)	59.7 (Day 14)	1.5 (Day 14)	1.0 (Day 30)
MSL-PF (sandy loam)	43.3 (Day 14)	2.9 (Day 59)	3.9 (Day 90)
Tehama (clay loam)	14.8 (Day 10)	2.1 (Day 59)	1.4 (Day 59)

 

Conclusions:

At 20°C, the DT50 values for [14C]-test substance were 22, 6, 10, and 37 days for the Boone, Raymondville, MSL-PF, and Tehama soils, respectively.

Executive summary:

The biodegradation of the test substance in aerobic soil was investigated in four aerobic soils. Samples were incubated for up to 120 days under aerobic conditions in the dark at 20°C ± 2°C. [14C]-test substance was applied at the rate of 1.5 μg a.i./g. The experiment was conducted in accordance with OECD 307 and US EPA OPPTS 835.4100.

[14C]- test substance was applied to a silt loam from Boone County, Missouri, USA; a sandy clay loam from Willacy County, Texas, USA; a sandy loam from Grand Forks County, North Dakota, USA; and a clay loam from Tehama County, California, USA at a dose rate of 1.5 μg a.i./g (equivalent to 1 kg/ha), and the soil samples were incubated at 20 ± 2°C in the dark. Humidified air was drawn through the test systems and then through traps designed to retain radiolabeled volatile components to demonstrate a radiochemical balance.

At zero time, 3, 7, 10, 14, 30, 59, 90, and 120 days after application, duplicate soil samples of each soil type were extracted with 90:10 acetonitrile:0.1 N HCl (v/v). The extracts were concentrated and analyzed by HPLC-UV. Non-extractable (bound) residues were determined by combustion analysis.

There were a couple instances towards the end of the study (after the DT75 of the compound) that were outside of the desired mass balance range of 90 and 110%. The mass balance for all the replicates included in degradation modeling was maintained between 88 and 102% AR. Over the 120-day incubation period, test substance degraded into TCP (≤59.7% AR) and various other metabolites, none of which individually accounted for more than 5% AR. CO2 accounted for 43.3 to 54.2% AR at 120 days. No volatile metabolites, other than CO2, were observed.

At 20°C, the DT50 values for [14C]- test substance were 22, 6, 10, and 37 days for the Boone, Raymondville, MSL-PF, and Tehama soils, respectively. The SFO kinetic fit of the data provided the best fit for the Raymondville soil system whereas the DFOP kinetic fit of the data provided the best fit for all other soil systems. At 20°C, the DT50 values from SFO kinetic fit for [14C]-TCP from [14C]-test substance were 10, 24, 21, and 10 days for the Boone, Raymondville, MSL-PF, and Tehama soils, respectively.

Reference 3

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

other: EU Commission Directive 95/36 EC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC-Europe Procedures for assessing the environmental fate and ecotoxicity of pesticides

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: German BBA Guideline section IV, 4-1

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Draft Document entitled 'Aerobic and anaerobic transformation in soil'

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Dutch (CTB) Guideline G.1.1

Deviations:

no

GLP compliance:

yes

Test type:

laboratory

Specific details on test material used for the study:

Substance ID: INV1533
Lot Number: F0571-39a
Radiochemical Purity: >99%
Specific Activity: 25.2 mCi/mmol

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Soil no.:

#1

Soil type:

sandy clay loam

% Org. C:

1.7

pH:

8.3

CEC:

21.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1.2

Soil no.:

#2

Soil type:

silty clay loam

% Org. C:

1

pH:

8

CEC:

16.2 meq/100 g soil d.w.

Bulk density (g/cm³):

1.3

Soil no.:

#3

Soil type:

sand

% Org. C:

1.2

pH:

6.8

CEC:

6.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.4

Soil no.:

#4

Soil type:

loam

% Org. C:

0.8

pH:

8.2

CEC:

10.3 meq/100 g soil d.w.

Bulk density (g/cm³):

1.2

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
Marcham (soil #1)
- Geographic location: Marcham, UK
- Dry matter % (m/m): 98.2
- Water content % (m/m): 1.8
- 63 µm-2 mm (%): 61.50
- 20-63 µm (%): 6.79
- 2-20 µm (%): 10.28
- <2 µm (%): 21.43
- 53 µm-2mm (%): 61.54
- 20-53 µm (%): 7.12
- 2-20 µm: 10.04
- <2 µm (%): 21.29
- Organic matter (%): 2.4
- WHCmax % (m/m): 45.6
- WHC0.33 bar % (m/m): 20.8
- WHC15 bar % (m/m): 10.9

Charentilly (soil #2)
- Geographic location: Charentilly, France
- Dry matter % (m/m): 98.7
- Water content % (m/m): 1.3
- 63 µm-2 mm (%): 11.10
- 20-63 µm (%): 32.40
- 2-20 µm (%): 28.87
- <2 µm (%): 27.62
- 53 µm-2mm (%): 13.24
- 20-53 µm (%): 32.32
- 2-20 µm: 28.12
- <2 µm (%): 26.32
- Organic matter (%): 1.7
- WHCmax % (m/m): 44.3
- WHC0.33 bar % (m/m): 24.4
- WHC15 bar % (m/m): 10.0

Cuckney (soil #3)
- Geographic location: Cuckney, UK
- Dry matter % (m/m): 99.2
- Water content % (m/m): 0.8
- 63 µm-2 mm (%): 88.00
- 20-63 µm (%): 2.66
- 2-20 µm (%): 3.48
- <2 µm (%): 5.86
- 53 µm-2mm (%): 88.79
- 20-53 µm (%): 2.32
- 2-20 µm: 3.32
- <2 µm (%): 5.56
- Organic matter (%): 1.5
- WHCmax % (m/m): 34.9
- WHC0.33 bar % (m/m): 8.1
- WHC15 bar % (m/m): 5.5

Thessaloniki (soil #4)
- Geographic location: Thessaloniki, Greece
- Dry matter % (m/m): 98.7
- Water content % (m/m): 1.4
- 63 µm-2 mm (%): 38.98
- 20-63 µm (%): 26.00
- 2-20 µm (%): 21.97
- <2 µm (%): 13.06
- 53 µm-2mm (%): 42.46
- 20-53 µm (%): 24.42
- 2-20 µm: 20.71
- <2 µm (%): 12.41
- Organic matter (%): 1.3
- WHCmax % (m/m): 43.4
- WHC0.33 bar % (m/m): 19.4
- WHC15 bar % (m/m): 6.7

SOIL PRE-INCUBATION: The soils were partially air-dried until friable and sieved (2 mm) and the appropriate amount of each soil was pre-incubated at ca. 20°C (and 10°C) for at about 4 weeks at a moisture content of 40% (and 10%) of the maximum water holding capacity (MWHC) before use.

Soil No.:

#1

Duration:

120 d

Soil No.:

#2

Duration:

120 d

Soil No.:

#3

Duration:

120 d

Soil No.:

#4

Duration:

120 d

Soil No.:

#1

Initial conc.:

1.28 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 960 g a.s./ha

Soil No.:

#2

Initial conc.:

1.28 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 960 g a.s./ha

Soil No.:

#3

Initial conc.:

1.28 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 960 g a.s./ha

Soil No.:

#4

Initial conc.:

1.28 mg/kg soil d.w.

Based on:

act. ingr.

Remarks:

equivalent to 960 g a.s./ha

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

20°C

Humidity:

40%

Microbial biomass:

Initial: 298 mg/kg; Final: 183 mg/kg

Soil No.:

#1

Temp.:

10°C

Humidity:

40%

Microbial biomass:

Initial: 288 mg/kg; Final: 216 mg/kg

Soil No.:

#1

Temp.:

20°C

Humidity:

10%

Microbial biomass:

Initial: 185 mg/kg; Final: 54 mg/kg

Soil No.:

#1

Temp.:

20°C

Humidity:

40%

Microbial biomass:

Initial: 0 mg/kg; Final: 0 mg/kg

Soil No.:

#2

Temp.:

20°C

Humidity:

40%

Microbial biomass:

Initial: 57 mg/kg; Final: 56 mg/kg

Soil No.:

#3

Temp.:

20°C

Humidity:

40%

Microbial biomass:

Initial: 78 mg/kg; Final: 45 mg/kg

Soil No.:

#4

Temp.:

20°C

Humidity:

40%

Microbial biomass:

Initial: 44 mg/kg; Final: 29 mg/kg

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS
- First test: The extraction and analysis procedure were based on methods supplied by the sponsor, and were developed and validated in two preliminary tests. For this purpose two of the soils were used, i.e. the sandy clay loam soil (representing a higher pH value) and the sand soil (representing a lower pH value; the test substance hydrolyzes more rapidly at higher pH values).
Four portions of 50 g (dry weight equivalent) of each soil at 40% MWHC were placed into test flasks and 0.430 ml of the dosing solution added. Two of the flasks with each soil were immediately extracted according to the following method: The soils were shaken with 100 ml of acetone/0.1 M HCl (9:1, v/v) for 20 minutes on a mechanical shaker. After centrifugation for 10 min. (ca. 2000 rpm), the supernatant was removed and the remaining soil extracted with further portions of the extraction solvent until <3% of the applied radioactivity was found in the extracts. The volume of each extract was measured, and the radioactivity content of duplicate aliquots (1 ml) determined by LSC, using Ultima-Gold XR (5 ml) as scintillation fluid. The non-extractable soil residues were determined.
The soil extracts were combined and acetone was evaporated with the aid of a rotary evaporator at ca. 40°C. After evaporation, the remaining aqueous phase was acidified with 20 µL 18% of HCl and shaken with 100 ml of dichloromethane. After separation of the two layers, the organic layer was removed and the aqueous phase was extracted again with 50 ml of dichloromethane. The dichloromethane extracts were pooled and dichloromethane was removed with the aid of a rotary evaporator at ca. 40°C. The remaining solvent was evaporated to almost dryness by a gentle stream of nitrogen using a heating block at ca. 40°C. The resulting concentrate was diluted to ca. 6 ml with acetonitrile for HPLC analysis. The bound residues were determined in order to establish the mass balance.
- Second test: For the second preliminary test, two of the soils i.e. the sandy clay loam soil and the sand soil were used. Two portions of 50 g of each soil at 40% MWHC were placed in test flasks and the appropriate concentration of the radiolabeled test substance was added.
The duplicate flasks of each soil were immediately extracted. Since the recovery after the concentration procedure was less than 90%, the method was somewhat changed. The extracts were combined and the acetone was evaporated using a rotary evaporator at a temperature of ca. 40°C. The aqueous phase left after evaporation was acidified with 200 µL of 18% HCI and shaken with 100 ml of dichloromethane. After separation of the two layers, the organic layer was removed and I ml of ethanediol was added to serve as a 'keeper' for organic volatiles. The aqueous phase was extracted again with 50 ml of dichloromethane and the two dichloromethane extracts were pooled. The amount of radioactivity was determined by weighing 1 ml of the dichloromethane extract in a scintillation vial and followed by addition of I ml Ultima gold XR and LSC counting. The solvent was evaporated using a rotation evaporator till ca. 5 ml of the extract was left. No HPLC analysis were performed for the second preliminary test.

2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): 20°C (and 10°C) for at about 4 weeks
- Soil condition: Partially air-dried until friable
- Soil (g/replicate): 50
- No. of replication treatments: 10 flasks for each measurement date and one reseve flask
- Test apparatus (Type/material/volume): Conical flasks fitted with a soda lime column for trapping evolved CO2
- Identity and concentration of co-solvent: Ethanol/toluene

Test material application
- Volume of test solution used/treatment: 0.430 mL
- Application method: The test substance was added to the soil samples in each flask.
- Is the co-solvent evaporated: Yes

Experimental conditions
- Moisture maintenance method: Maintained during the experiment by addition (if required) of deionized water at regular (2 week) intervals
- Continuous darkness: Yes

3. SAMPLING DETAILS
- Sampling intervals: One test flask each from the sandy clay loam and the loam soils was taken for analysis after 0, 1, 2, 3, 6, 9, 16 (14 days for sandy clay loam at 10°C), 23, 42, 63 and 84 days after treatment. One sample of the silty clay loam and the sand soil was taken for analysis after 0, 1,3, 7, 14, 21, 42, 63, 84 and 120 days. The sampling intervals were based upon the results of the preliminary tests. The sterilized sandy clay loam soil was analyzed after 0, 21, 152 and 161 days.
- Sampling method for soil samples: The soil samples were transferred to plastic bottles, and the test flasks rinsed with 100 ml of acetone/0.1 M HCl (9:1 v/v). The combined soil and acetone/0.1 M HCl solution was shaken for 20 min. on a mechanical shaker. After shaking, the plastic bottles were centrifuged for 10 min. (ca. 2000 rpm). The supernatant layer was then decanted. The extraction of the soil samples with 100 ml of acetone/0.1 M HCl (9:1 v/v) was repeated (4 - 5 times) until the radioactivity in the extract was less than 3% of the applied radioactivity. The extracts were pooled, the total volume was measured and the amount of radioactivity was determined by adding 1 ml in duplicate to scintillation vials containing 10 ml of Ultima Gold XR. The extracts were stored frozen until they were concentrated to appropriate volumes by means of a rotary evaporator.
- Method of collection of CO2 and volatile organic compounds: Before opening the flasks by removing the soda lime traps, moist air was blown through to force volatile compounds present in the head space into the trap. The paraffin oil-covered quartz wool was then removed and placed in a scintillation vial together with scintillation liquid (Ultima Gold). The amount of trapped carbon dioxide evolved from the soil was determined by dissolving the lower soda lime layer in 18% hydrochloric acid solution in a closed system. The 14CO2 released by the acid was carried by a flow of nitrogen gas through wash bottles containing a solution of Carbo sorb (CO2-adsorbent) and Permafluor E+ (scintillation fluid) (4:5 v/v). The radioactivity collected in the wash bottle solutions was analysed by scintillation counting.

Soil No.:

#1

% Recovery:

99.6

St. dev.:

2.2

Remarks on result:

other: Sandy clay loam soil (40% MWHC and 20°C)

Soil No.:

#1

% Recovery:

100

St. dev.:

3.4

Remarks on result:

other: Sandy clay loam soil (40% MWHC and 10°C)

Soil No.:

#1

% Recovery:

100

St. dev.:

2.6

Remarks on result:

other: Sandy clay loam soil (10% MWHC and 20°C)

Soil No.:

#1

% Recovery:

98.7

St. dev.:

1.8

Remarks on result:

other: Sterile sandy clay loam soil (40% MWHC and 20°C)

Soil No.:

#2

% Recovery:

97.2

St. dev.:

5.7

Remarks on result:

other: Silty clay loam soil (40% MWHC and 20°C)

Soil No.:

#3

% Recovery:

96.6

St. dev.:

4.6

Remarks on result:

other: Sand soil (40% MWHC and 20°C)

Soil No.:

#4

% Recovery:

100

St. dev.:

1.7

Remarks on result:

other: Loam soil (40% MWHC and 20°C)

Key result

Soil No.:

#1

DT50:

43 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 40% MWHC

Key result

Soil No.:

#1

DT50:

80 d

Type:

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

Temp.:

10 °C

Remarks on result:

other: 40% MWHC

Key result

Soil No.:

#1

DT50:

126 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 10% MWHC

Key result

Soil No.:

#1

DT50:

21 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 40% MWHC (sterile)

Key result

Soil No.:

#2

DT50:

95 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 40% MWHC

Key result

Soil No.:

#3

DT50:

111 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 40% MWHC

Key result

Soil No.:

#4

DT50:

46 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 40% MWHC

Transformation products:

yes

Remarks:

TCP (3,5,6-trichloro-2-pyridinol) and TMP (3,5,6-trichloro-2-methoxypyridine)

No.:

#1

Details on transformation products:

HPLC analysis of the extractable radioactivity showed that the test substance was converted mainly to TCP (more than 10% of the applied radioactivity, maximum amount observed was ca. 40% after 84 days of incubation), which is in agreement with literature values. Subsequently, TCP was degraded to TMP and an unknown metabolite, but the quantity of both metabolites formed was less than 10% of the applied radioactivity, and so these can be considered minor. The amount of non-extractable radioactivity varied between approximately 3% and 25% of applied at the end of the test.

Volatile metabolites:

yes

Remarks:

CO2

Residues:

yes

Details on results:

MAJOR AND MINOR TRANSFORMATION PRODUCTS
- Sandy clay loam soil (40% MWHC and 20°C): Concentrations of the test substance were 95.4% of the applied radioactivity at day-0 which declined to 34.3% of the applied radioactivity by day-84. The primary metabolite, TCP, was not detected at day-0 and increased to a maximum of 39.1% of the applied radioactivity by day-23. Two other metabolites i.e. TMP and an unknown metabolite were observed. TMP was only observed from day-42 to day-84 and reached 7.5% of the applied radioactivity. The unknown metabolite was seen more routinely, but reached only 3.2% of the applied radioactivity at day-63. Both of these can be considered minor.
- Sandy clay loam soil (40% MWHC and 10°C): Concentrations of the test substance were 103.1% of the applied radioactivity at day-0 which declined to 54.8% of the applied radioactivity by day-84. The primary metabolite, TCP, was not detected at day-0 and increased to a maximum of 40.1% of the applied radioactivity by day-84. Two minor metabolites i.e. TMP and an unknown metabolite were also observed. TMP was only seen on day-42 and day-84 and reached 2.4% of the applied radioactivity. The unknown metabolite was seen from day-42 to day-84, but reached only 1.7% of the applied radioactivity at day-84.
- Sandy clay loam soil (40% MWHC and 10°C): Concentrations of the test substance were 105.2% of the applied radioactivity at day-0 which declined to 64.8% of the applied radioactivity by day-84. The primary metabolite, TCP, was not detected at day-0 and increased to a maximum of 29.6% of the applied radioactivity by day-84. No other metabolites were found.
- Sterile sandy clay loam soil (40% MWHC and 20°C): Concentrations of the test substance were 95.6% of the applied radioactivity at day-0 which declined to 22.0% of the applied radioactivity by day-161. The primary metabolite, TCP, was not detected at day-0 and increased to a maximum of 71.8% of the applied radioactivity by day-161. No other metabolites were found.
- Silty clay loam soil (40% MWHC and 20°C): Concentrations of the test substance were 106.2% of the applied radioactivity at day-0 and declined to 44.1% of the applied radioactivity by day-120. The primary metabolite, TCP, was not detected at day-0 and increased to a maximum of 5.5% of the applied radioactivity by day-42. Two minor metabolites i.e. TMP and an unknown metabolite were also observed. TMP was only observed from day-21 to day-120 and reached 6.4% of the applied radioactivity. The unknown metabolite was only detectable on two time points and reached only 1.4% of the applied radioactivity at day-42.
- Sand soil (40% MWHC and 20°C): Concentrations of the test substance were 107.0% of the applied radioactivity at day-0 and declined to 45.6% ofthe applied radioactivity by day-120. The primary metabolite, TCP, was not detected at day-0 and increased to a maximum of 6.5% of the applied radioactivity by day-21. Two minor metabolites i.e. TMP and an unknown metabolite were also observed. TMP was only observed on day-42 and reached only 0.9% of the applied radioactivity. The unknown metabolite was detectable on two time points and reached only 1.2% of the applied radioactivity at day-42.
- Loam soil (40% MWHC and 20°C): Concentrations of the test substance were 102.5% of the applied radioactivity at day-0 and declined to 37.0% of the applied radioactivity by day-120. The primary metabolite, TCP, was not detected at day-0 and increased to a maximum of 41.1% of the applied radioactivity by day-63. Two minor metabolites i.e. TMP and an unknown metabolite were also observed. TMP was observed from day-42 to day 84 and reached 7.4% of the applied radioactivity. The unknown metabolite was also detectable from day-42 to day 84, but reached only 1.5% of the applied radioactivity at day-42.


EXTRACTABLE RESIDUES
- Sandy clay loam soil (40% MWHC and 20°C): The extractable radioactivity gradually declined throughout the study and reached a level of 81.1% of the applied radioactivity by day-84
- Sandy clay loam soil (40% MWHC and 10°C): The extractable radioactivity varied between 90.3% (day-63) and 107.5% (day-2) of the applied radioactivity
- Sandy clay loam soil (40% MWHC and 10°C): The extractable radioactivity varied between 105.2% (day-0) and 94.5% (day-84) of the applied radioactivity
- Sterile sandy clay loam soil (40% MWHC and 20°C): The extractable radioactivity varied between 95.6% (day-0) and 91.3% (day-21) of the applied radioactivity
- Silty clay loam soil (40% MWHC and 20°C): The extractable radioactivity gradually declined throughout the study and reached a level of 53.0% of the applied radioactivity by day-120
- Sand soil (40% MWHC and 20°C): The extractable radioactivity gradually declined throughout the study and reached a level of 49.6% of the applied radioactivity by day-120
- Loam soil (40% MWHC and 20°C): The extractable radioactivity gradually declined throughout the study and reached a level of 79.4% of the applied radioactivity by day-120

NON-EXTRACTABLE RESIDUES
- Sandy clay loam soil (40% MWHC and 20°C): Non-extractable residues increased to a level of 12.0% of the applied radioactivity by day-63
- Sandy clay loam soil (40% MWHC and 10°C): Non-extractable residues increased to a level of 7.2% of the applied radioactivity by day-63
- Sandy clay loam soil (40% MWHC and 10°C): Non-extractable residues increased to a level of 7.0% of the applied radioactivity by day-42
- Sterile sandy clay loam soil (40% MWHC and 20°C): Non-extractable residues increased to a level of 7.6% of the applied radioactivity by day-21
- Silty clay loam soil (40% MWHC and 20°C): Non-extractable residues increased to a level of 11.0% of the applied radioactivity by day-120
- Sand soil (40% MWHC and 20°C): Non-extractable residues increased to a level of 25.0% of the applied radioactivity by day-120
- Loam soil (40% MWHC and 20°C): Non-extractable residues increased to a level of 9.9% of the applied radioactivity by day-63

MINERALISATION
- Sandy clay loam soil (40% MWHC and 20°C): Captured radioactive CO2 reached a maximum of 8.0% of the applied radioactivity at day-84
- Sandy clay loam soil (40% MWHC and 10°C): Captured radioactive CO2 reached a maximum of 3.1 % of the applied radioactivity at day-84
- Sandy clay loam soil (40% MWHC and 10°C): Little or no 14CO2 was produced using these environmental conditions, since a maximum of only 0.1% of the applied radioactivity was found as CO2 at day-42.
- Sterile sandy clay loam soil (40% MWHC and 20°C): Captured radioactive CO2 reached a maximum of 2.7% of the applied radioactivity at day-161
- Silty clay loam soil (40% MWHC and 20°C): Captured radioactive CO2 reached a maximum of 23.6% of the applied radioactivity at day-120
- Sand soil (40% MWHC and 20°C): Captured radioactive CO2 reached a maximum of 26.9% of the applied radioactivity at day-120
- Loam soil (40% MWHC and 20°C): Captured radioactive CO2 reached a maximum of 13.6% of the applied radioactivity at day-120

VOLATILIZATION
- Sandy clay loam soil (40% MWHC and 20°C): The maximum radioactivity observed as volatile organics (oil covered glass wool) was 0.1% of the applied radioactivity and was observed at day-6
- Sandy clay loam soil (40% MWHC and 10°C): The maximum radioactivity observed as volatile organics (oil covered glass wool) was 0.9% of the applied radioactivity and was observed at day-3
- Sandy clay loam soil (40% MWHC and 10°C): The maximum radioactivity observed as volatile organics (oil covered glass wool) was 0.2% of the applied radioactivity and was observed at day-3
- Sterile sandy clay loam soil (40% MWHC and 20°C): The maximum radioactivity observed as volatile organics (oil covered glass wool) was 0.1% of the applied radioactivity and was observed at day-21
- Silty clay loam soil (40% MWHC and 20°C): The maximum radioactivity observed as volatile organics (oil covered glass wool) was 0.4% of the applied radioactivity and was observed at day-84
- Sand soil (40% MWHC and 20°C): The maximum radioactivity observed as volatile organics (oil covered glass wool) was 0.2% of the applied radioactivity and was observed at day-84
- Loam soil (40% MWHC and 20°C): The maximum radioactivity observed as volatile organics (oil covered glass wool) was 0.2% of the applied radioactivity and was observed on two time point

The results of the calculations with [14C]-test substance:

Soil type	Exposure Conditions	k (per day)	R2	T1/2 DT50 (days)	DT90 (Days)
Sandy clay loam	40% MWHC, 20°C 40% MWHC, 10°C 10% MWHC, 20°C 40% MWHC, 20°C, Sterile	0.0163 0.0087 0.0055 0.0333	0.837 0.828 0.810 0.834	43 80 126 21	141 264 419 69
Silty clay loam	40% MWHC, 20°C	0.0073	0.965	95	315
Sand	40% MWHC, 20°C	0.0064	0.953	111	369
Loam	40% MWHC, 20°C	0.015	0.954	46	153

Conclusions:

The test substance was degraded in soil under standard conditions with a DT50 of 43 to 126 days (mean 74 days), depending on soil type.

Executive summary:

The route and rate of soil degradation of [14C]-test substance was studied under aerobic conditions in the laboratory. The test substance was applied to four European soils (Marcham sandy clay loam (UK), Charentilly silty clay loam (France), Cuckney sand (UK) and Thessaloniki loam (Greece)) at a nominal rate of 1.28 mg/kg), equivalent to 960 g a.s/ha. The effects of moisture, temperature, and biological activity were also investigated by analyzing the route and rate of degradation of [14C]-test substance on sandy clay loam soil either at a lower temperature (10°C vs. 20°C), at lower humidity (10% MWHC vs. 40% MWHC), or in gamma-radiation sterilized soil. The soils were incubated in the dark under aerobic conditions for up to 120 days. At different time points, the following parameters were determined: CO2 evolution, extractable and non-extractable soil radioactivity, and distribution of the extractable radioactivity between parent compound and metabolite(s) by HPLC and confirmed by TLC.

The loss of [14C]-test substance (as % of applied radioactivity) in the various soils were fitted to a first order model to determine t1/2 (DT50) and DT90 values.

The degradation rates in sandy clay loam soil and the loam soil are similar (t1/2 43 and 46 days, respectively), which could be explained by the higher pH values ofthe soils (7.7 and 7.9, respectively). The results and the pH values of the other soils are also comparable. Therefore, the main initial degradation process in these soils can be described as chemical hydrolysis which is pH dependent. This was confirmed by the test with sterile sandy clay loam where the test substance degraded with a comparable DT50 to the non-sterile soil.

The result of the test substance degradation in the sandy clay loam soil at 10°C showed a slower degradation rate. The difference in the rate of degradation between 10°C and 20°C (when considered in terms of t1/2) was in a ratio expected from known temperature effects on chemical degradation. The results of the sandy clay loam soil with 10% of the maximum water holding capacity (MWHC) at 20°C demonstrated much slower degradation than under standard conditions (40% MWHC). This difference is likely due to the reduced amounts of water, which in turn reduces chemical hydrolysis. The degradation in the silty clay loam and sand soils is a combination of hydrolysis and biodegradation, since the amount of 14CO2 mineralized (ca. 23%) is more than in the first two soils (ca. 10%).

The recovery of radioactivity in all four soils under the different conditions varied between 87.7% and 108.8% of applied, but averaged 90-110% for each soil type under each exposure condition.

HPLC analysis of the extractable radioactivity showed that the test substance was converted mainly to TCP (more than 10% ofthe applied radioactivity, maximum amount observed was ca. 41% after 84 days of incubation), which is in agreement with literature values. Subsequently, TCP was degraded to TMP and an unknown metabolite, but the quantity of both metabolites formed was less than 10% of the applied radioactivity, and so these can be considered minor. The amount of non-extractable radioactivity varied between approximately 3% and 25% of applied at the end of the test.

Description of key information

Study Type	Study Details	Value	Guideline	Reliability
Anaerobic metabolism	Longwood, South Witham, Hareby, MSL soil for up to 120 days	DT50 = 3.6 days (Longwood soil) DT50 = 3.2 days (South Witham soil) DT50 = 4.9 days (Hareby soil) DT50 = 9.3 days (MSL soil)	OECD 307 OPPTS 835.4200	1
Aerobic metabolism	Sandy clay loam soil ; Silty clay loam soil; sand soil and Loam soil	DT50 values were <126, 315, 111 and 46 days	SETAC giudance	1
Aerobic metabolism	Boone, Raymondville, MSL-PF, and Tehama soils	DT50 values for [14C]-chlorpyrifos were 22, 6, 10, and 37 days	OECD 307	1