spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

Biodegradation in soil

Link to relevant study record(s)

Reference

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

Justification for type of information:

This study is classified as acceptable and satisfies the guideline requirements for both an aerobic biotransformation study in soil and a soil kinetic study.

Qualifier:

according to guideline

Guideline:

other: Official Journal of the European Communities No L 172, 95/36/EC Placing of the Plant Protection Products on the Market,

Version / remarks:

July 14,1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Pesticide Assessment Guideline, Subdivision N, §162-1

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: BBA Guidelines for Official Testing of Plant Protectants, Part IV, 4-1

Version / remarks:

1986

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Society of Environmental Toxicology and Chemistry (SETACEurope), Procedures for Assessing the Environmental Fate and Ecotoxicology of Pesticides

Version / remarks:

March 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

not specified

Year:

1999

Soil no.:

#1

Soil type:

sandy loam

% Clay:

8.8

% Silt:

28

% Sand:

63.2

% Org. C:

0.53

pH:

6.6

CEC:

5.18 meq/100 g soil d.w.

Bulk density (g/cm³):

1.63

Soil no.:

#2

Soil type:

sand

% Clay:

1

% Silt:

0.3

% Sand:

98.7

% Org. C:

0.22

pH:

6.4

CEC:

3.9 meq/100 g soil d.w.

Bulk density (g/cm³):

1.41

Soil no.:

#3

Soil type:

loamy sand

% Clay:

7.2

% Silt:

12.3

% Sand:

80.5

% Org. C:

2.48

pH:

6

CEC:

10 meq/100 g soil d.w.

Bulk density (g/cm³):

2.45

Soil no.:

#4

Soil type:

Silt

% Clay:

10.2

% Silt:

81.3

% Sand:

8.5

% Org. C:

2.62

pH:

7.8

CEC:

15 meq/100 g soil d.w.

Bulk density (g/cm³):

2.09

Details on soil characteristics:

#1 (Fresno)
- Geographic location: Bayer Research Farm, Fresno, CA (USA)
- Pesticide use history at the collection site: possibly Roundup in 1993
- Collection procedures: Soil was freshly collected for the test on 1/15/1997.
- Sampling depth (cm): top 6 inches
- Storage length: <1 month
- Soil preparation (e.g., 2 mm sieved; air dried etc.): sieved <2 mm, air dried
- Moisture at 1/3 atm (%): 8.1

#2 Vero beach
- Geographic location: Vero Beach, FL (USA)
- Pesticide use history at the collection site: no pesticides for last 5 years
- Collection procedures: Soil was freshly collected for the test on 1/27/1997.
- Sampling depth (cm): top 6 inches
- Storage length: <1 month
- Soil preparation (e.g., 2 mm sieved; air dried etc.): sieved <2 mm, air dried
- Moisture at 1/3 atm (%): 2.1

#3 BBA 2.2
- Geographic location: Hanhofen, Vorderpflaz Germany
- Pesticide use history at the collection site: Unknown prior to collection, but has been in the institute under natural vegetation for >5 years.
- Collection procedures: Soil BBA 2.2 was collected on originally on 9/29/1994 and stored under
natural vegetation in the Institute for Metabolism Research and Residue Analysis in order to preserve the soil microorganisms.
- Sampling depth (cm): top 30 cm (~12 inches)
- Storage length: <1 month
- Soil preparation (e.g., 2 mm sieved; air dried etc.): sieved <2 mm, air dried
- Moisture at 1/3 atm (%): 12

#4 Hoefchen
- Geographic location: Hohenseh 4A, Burscheid, Germany
- Pesticide use history at the collection site: No pesticides for > 5years
- Collection procedures: Soil was freshly collected for the test on 2/4/1997.
- Sampling depth (cm): top 20 cm (~8 inches)
- Storage length: <1 month
- Soil preparation (e.g., 2 mm sieved; air dried etc.): sieved <2 mm, air dried
- Moisture at 1/3 atm (%): 26.7

Soil No.:

#1

Duration:

360 d

Soil No.:

#2

Duration:

120 d

Soil No.:

#3

Duration:

120 d

Soil No.:

#4

Duration:

120 d

Soil No.:

#4

Initial conc.:

0.111 g/ha d.w.

Based on:

act. ingr.

Soil No.:

#1

Initial conc.:

0.112 g/ha d.w.

Based on:

act. ingr.

Soil No.:

#1

Initial conc.:

0.534 g/ha d.w.

Based on:

act. ingr.

Soil No.:

#2

Initial conc.:

0.111 g/ha d.w.

Based on:

act. ingr.

Soil No.:

#3

Initial conc.:

0.111 g/ha d.w.

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

Soil No.:

#4

Temp.:

Minimum: 19.90ºC
Average: 20.29ºC
Maximum: 20.90ºC

Humidity:

40% of max. water holding
capacity
Weights of the flasks were checked
regularly, and water was added as
needed to maintain soil moisture

Microbial biomass:

Microbial biomass/microbial
population of control soil: Initial: 927 Final: 506 Microbial biomass/microbial
population of treated soil, if
provided: Initial: - Final: 475

Soil No.:

#3

Temp.:

Minimum: 19.90ºC
Average: 20.29ºC
Maximum: 20.90ºC

Humidity:

40% of max. water holding
capacity
Weights of the flasks were checked
regularly, and water was added as
needed to maintain soil moisture

Microbial biomass:

Microbial biomass/microbial population of control soil: Initial: 253 Final: 183
Microbial biomass/microbial population of treated soil, if provided: Initial: - Final:174

Soil No.:

#1

Temp.:

Minimum: 19.90ºC
Average: 20.29ºC
Maximum: 20.90ºC

Humidity:

75% of 1/3 bar: Weights of the flasks were checked regularly, and water was added as needed to maintain soil moisture.

Microbial biomass:

Microbial biomass/microbial population of control soil: Initial: 216 (0 day) 107 (120 day)
83 (181 day) Final: 41.
Microbial biomass/microbial population of treated soil, if provided: Initial: 102 (120 day) 88 (181 day) Final: 38.

Soil No.:

#1

Temp.:

Minimum: 19.90ºC
Average: 20.29ºC
Maximum: 20.90ºC

Humidity:

75% of 1/3 bar. Weights of the flasks were checked regularly, and water was added as needed to
maintain soil moisture.

Microbial biomass:

HIGH DOSE
Microbial biomass/microbial population of control soil Initial: 216 (0 day) Final: -
Microbial biomass/microbial population of treated soil if provided: Initial: - Final: 93

Soil No.:

#2

Temp.:

Minimum: 19.90ºC
Average: 20.29ºC
Maximum: 20.90ºC

Humidity:

75% of 1/3 bar
Weights of the flasks were checked regularly, and water was added as needed to maintain soil
moisture.

Microbial biomass:

Microbial biomass/microbial population of control soil: Initial: 27 Final:12
Microbial biomass/microbial population of treated soil, if provided Initial: - Final: 93

Details on experimental conditions:

Soil #1 Fresno
Duration of test: 360 days
Soil condition: air dried, sieved =<2mm
Soil (g/replicate): 100
Test concentrations (mg a.i./kg soil) and equivalent g a.i./ha: 0.112 (250 g a.i./ha)
Control conditions, if used: Controls were only used for biomass determinations
Treatments: 32
Test apparatus (Type/material/volume): 300-mL Erlenmyer flask
Details of traps for CO2 and organic volatile, if any: Traps consisted of the following: polyurethane
plug, quartz wool (0.3 g) soda lime (10 g), quartz wool (0.2 g), soda lime (4 g) and quartz wool (0.2 g). See Appendix 6.
Identity and concentration of co-solvent: Acetonitrile

Test material application:
volume of test solution used / treatment: 460 µL per 3.7 kg of moist soil (~13.5 µL per 100 g dry soil)
Application method (eg: applied on surface, homogeneous mixing etc.): The application solution
was dispensed onto 20 or 50-g portions of dry soil in evaporating dishes. Treated subsamples of soil were mixed until the solvent had completely evaporated and then added to the total mass of
the corresponding soil. Subsequently, the gross mass of each soil was mixed on a tumbling mixer for 1 hour. Soil portions corresponding to 100 g dry material were then transferred to Erlenmyer flasks for incubation.
Is the cosolvent evaporated: Yes
Continuous darkness: Yes

Soil #1 Fresno HIGH DOSE:
Duration of test: 120 days
Soil condition: air dried, sieved =<2mm
Soil (g/replicate): 100
Test concentrations (mg a.i./kg soil) and equivalent g a.i./ha: 0.534 (1200 g a.i./ha; rate used in Japan only)
Control conditions, if used: Controls were only used for biomass determinations
Treatments: 15
Test apparatus (Type/material/volume): 300-mL Erlenmyer flask
Details of traps for CO2 and organic volatile, if any: Traps consisted of the following: polyurethane
plug, quartz wool (0.3 g) soda lime (10 g), quartz wool (0.2 g), soda lime (4 g) and quartz wool (0.2 g). See Appendix 6.
Identity and concentration of co-solvent: Acetonitrile

Test material application:
volume of test solution used / treatment: 1010 µL per 1.7 kg of moist soil (~64 µL per 100 g dry soil)
Application method (eg: applied on surface, homogeneous mixing etc.): The application solution
was dispensed onto 20 or 50-g portions of dry soil in evaporating dishes. Treated subsamples of soil were mixed until the solvent had completely evaporated and then added to the total mass of
the corresponding soil. Subsequently, the gross mass of each soil was mixed on a tumbling mixer for 1 hour. Soil portions corresponding to 100 g dry material were then transferred to Erlenmyer flasks for incubation.
Is the cosolvent evaporated: Yes
Continuous darkness: Yes

Soil #2 Vero Beach:
Duration of test: 120 days
Soil condition: air dried, sieved =<2mm
Soil (g/replicate): 100
Test concentrations (mg a.i./kg soil) and equivalent g a.i./ha: 0.111 (250 g a.i./ha)
Control conditions, if used: Controls were only used for biomass determinations
Treatments: 15
Test apparatus (Type/material/volume): 300-mL Erlenmyer flask
Details of traps for CO2 and organic volatile, if any: Traps consisted of the following: polyurethane
plug, quartz wool (0.3 g) soda lime (10 g), quartz wool (0.2 g), soda lime (4 g) and quartz wool (0.2 g). See Appendix 6.
Identity and concentration of co-solvent: Acetonitrile

Test material application:
volume of test solution used / treatment: 210 µL per 1.6 kg of moist soil (~13.4 µL per 100 g dry soil)
Application method (eg: applied on surface, homogeneous mixing etc.): The application solution
was dispensed onto 20 or 50-g portions of dry soil in evaporating dishes. Treated subsamples of soil were mixed until the solvent had completely evaporated and then added to the total mass of
the corresponding soil. Subsequently, the gross mass of each soil was mixed on a tumbling mixer for 1 hour. Soil portions corresponding to 100 g dry material were then transferred to Erlenmyer flasks for incubation.
Is the cosolvent evaporated: Yes
Continuous darkness: Yes

Soil #3 BBA 2.2
Duration of test: 120 days
Soil condition: air dried, sieved =<2mm
Soil (g/replicate): 100
Test concentrations (mg a.i./kg soil) and equivalent g a.i./ha: 0.111 (250 g a.i./ha)
Control conditions, if used: Controls were only used for biomass determinations
Treatments: 15
Test apparatus (Type/material/volume): 300-mL Erlenmyer flask
Details of traps for CO2 and organic volatile, if any: Traps consisted of the following: polyurethane
plug, quartz wool (0.3 g) soda lime (10 g), quartz wool (0.2 g), soda lime (4 g) and quartz wool (0.2 g). See Appendix 6.
Identity and concentration of co-solvent: Acetonitrile

Test material application:
volume of test solution used / treatment: 210 µL per 1.8 kg of moist soil (~13.4 µL per 100 g dry soil)
Application method (eg: applied on surface, homogeneous mixing etc.): The application solution
was dispensed onto 20 or 50-g portions of dry soil in evaporating dishes. Treated subsamples of soil were mixed until the solvent had completely evaporated and then added to the total mass of
the corresponding soil. Subsequently, the gross mass of each soil was mixed on a tumbling mixer for 1 hour. Soil portions corresponding to 100 g dry material were then transferred to Erlenmyer flasks for incubation.
Is the cosolvent evaporated: Yes
Continuous darkness: Yes

Soil #4 Hoefchen
Duration of test: 120 days
Soil condition: air dried, sieved =<2mm
Soil (g/replicate): 100
Test concentrations (mg a.i./kg soil) and equivalent g a.i./ha: 0.111 (250 g a.i./ha)
Control conditions, if used: Controls were only used for biomass determinations
Treatments: 15
Test apparatus (Type/material/volume): 300-mL Erlenmyer flask
Details of traps for CO2 and organic volatile, if any: Traps consisted of the following: polyurethane
plug, quartz wool (0.3 g) soda lime (10 g), quartz wool (0.2 g), soda lime (4 g) and quartz wool (0.2 g). See Appendix 6.
Identity and concentration of co-solvent: Acetonitrile

Test material application:
volume of test solution used / treatment: 210 µL per 1.9 kg of moist soil (~13.4 µL per 100 g dry soil)
Application method (eg: applied on surface, homogeneous mixing etc.): The application solution
was dispensed onto 20 or 50-g portions of dry soil in evaporating dishes. Treated subsamples of soil were mixed until the solvent had completely evaporated and then added to the total mass of
the corresponding soil. Subsequently, the gross mass of each soil was mixed on a tumbling mixer for 1 hour. Soil portions corresponding to 100 g dry material were then transferred to Erlenmyer flasks for incubation.
Is the cosolvent evaporated: Yes
Continuous darkness: Yes

Aerobic conditions: The samples contained traps that were open to the atmosphere.
Supplementary experiments: In order to produce sufficient amounts of the metabolites for
structure elucidation, the sandy loam soil Fresno (1 kg dry soil) and sand soil Vero Beach (200 g
dry soil) were treated with an exaggerated rate of a.i. (ca. 20 x the proposed application rate, see
Table 4). Mixtures of labeled and non-labeled test substances were used to treat both soils.
Sampling details in Table 5 below.

Soil No.:

#1

% Total extractable:

>= 96.9 - <= 102.7

% Non extractable:

>= 1.9 - <= 8.5

% CO2:

76

% Other volatiles:

< 0.1

% Recovery:

100.1

Remarks on result:

other: CO2 after 360 days incubation

Soil No.:

#1

% Total extractable:

>= 98.1 - <= 103.2

% Non extractable:

>= 1.6 - <= 9.2

% CO2:

55.3

% Other volatiles:

< 0.1

% Recovery:

100

Remarks on result:

other: High Dose, CO2 after 120 days incubation

Soil No.:

#2

% Total extractable:

>= 98 - <= 105.6

% Non extractable:

>= 1.9 - <= 14.4

% CO2:

22.5

% Other volatiles:

< 0.1

% Recovery:

102.8

Remarks on result:

other: CO2 after 120 days incubation

Soil No.:

#3

% Total extractable:

>= 97.1 - <= 103.2

% Non extractable:

>= 2 - <= 8

% CO2:

86.3

% Other volatiles:

< 0.1

% Recovery:

100.4

Remarks on result:

other:

Soil No.:

#4

% Total extractable:

>= 94.1 - <= 101.8

% Non extractable:

>= 2.1 - <= 6.8

% CO2:

93.1

% Other volatiles:

< 0.1

% Recovery:

99.2

Remarks on result:

other: CO2 after 120 days incubation

Parent/product:

parent

Key result

Soil No.:

#4

% Degr.:

> 50

Parameter:

CO2 evolution

Sampling time:

1 d

Parent/product:

parent

Key result

Soil No.:

#3

% Degr.:

> 50

Parameter:

CO2 evolution

Sampling time:

6 d

Parent/product:

parent

Key result

Soil No.:

#2

% Degr.:

> 50

Parameter:

CO2 evolution

Sampling time:

9 d

Parent/product:

parent

Key result

Soil No.:

#1

% Degr.:

> 50

Parameter:

CO2 evolution

Sampling time:

13 d

Remarks on result:

other: HIGH DOSE

Parent/product:

parent

Key result

Soil No.:

#1

% Degr.:

> 50

Parameter:

CO2 evolution

Sampling time:

9 d

Key result

Soil No.:

#4

DT50:

0.5 d

Type:

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

Temp.:

20 °C

Key result

Soil No.:

#3

DT50:

3.9 d

Type:

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

Temp.:

20 °C

Key result

Soil No.:

#2

DT50:

5.5 d

Type:

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

Temp.:

20 °C

Key result

Soil No.:

#1

DT50:

6 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: HIGH DOSE

Key result

Soil No.:

#1

DT50:

5.3 d

Type:

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

Temp.:

20 °C

Transformation products:

yes

No.:

#4

No.:

#3

No.:

#2

No.:

#1

Details on transformation products:

Fresno soil: The major transformation products detected were Spirodiclofen-Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en-2-one], Spirodiclofen -Ketohydroxy [3-(2,4-dichlorophenyl)-3-hydroxy-1-oxaspiro[4.5]decane-2,4- dione], and Spirodiclofen -Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[4.5]decan-2- one] with maximum concentrations of 17.3, 27.9, and 16.4% of the applied amount, observed on the 6th, 20th, and 120th day of incubation, respectively. At the end of the study period, the corresponding concentrations were 0, 1.5, and 11.1% of the applied amount, respectively. The minor transformation product, 2,4-dichlorobenzoic acid (FHW0107B), was formed at a maximum concentration of 2% of the applied amount. The total unidentified radioactivity was 0.8 to 7.1% of the applied amount.

Fresno soil (high dose): The major transformation products detected were Spirodiclofen -Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en-2-one], Spirodiclofen - Ketohydroxy [3-(2,4-dichlorophenyl)-3-hydroxy-1-oxaspiro[4.5]decane-2,4- dione], and Spirodiclofen Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro [4.5] decan-2-one] with maximum concentrations of 14.5, 29.1, and 16.2% of the applied amount, observed on the 6th, 20th, and 120th day of incubation, respectively. At the end of the study period, the corresponding concentrations were 0.6, 8.1, and 16.2% of the applied amount, respectively. The minor transformation product, 2,4-dichlorobenzoic acid (FHW0107B), was formed at a maximum concentration of 4.3% of the applied amount. The total unidentified radioactivity was 1.4 to 6.4% of the applied amount.

Vero Beach soil: The major transformation products detected were Spirodiclofen -Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en-2-one], Spirodiclofen -Ketohydroxy [3-(2,4-dichlorophenyl)-3-hydroxy-1-oxaspiro[4.5]decane-2,4- dione], and 2,4 dichlorobenzoic acid (FHW0107B) with maximum concentrations of 38.4, 44.4, and 39.6% of the applied amount, observed on the 9th, 30th, and 120th day of incubation, respectively. At the end of the study period, the corresponding concentrations were 0.8, 10.9, and 39.6% of the applied amount, respectively. The minor transformation product, Spirodiclofen -Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[ 4.5] decan-2-one], was formed at a maximum of 4.8% of the applied amount. The total unidentified radioactivity was 0.6 to 9.5% of the applied amount.

BBA 2.2 soil: The major transformation products detected were Spirodiclofen -Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en-2-one] and Spirodiclofen-Ketohydroxy [3-(2,4-dichlorophenyl)-3-hydroxy-1-oxaspiro[4.5]decane-2,4- dione] with maximum concentrations of 19.1 and 20.7% of the applied amount, observed on the 2nd and 9th day of incubation, respectively. At the end of the study period, the corresponding concentrations were 0.4 and 1.2% of the applied amount, respectively. The minor transformation products were Spirodiclofen-Dihydroxy [3-(2,4-dichlorophenyl)-3,4- dihydroxy-1-oxaspiro[4.5]decan-2-one] and 2,4-dichlorobenzoic acid (FHW0107B) formed at a maximum of 2.5 and 3.0% of the applied amount, respectively. The total unidentified radioactivity was 1.2 to 6.6% of the applied amount.

Hoefchen soil: The major transformation products detected were Spirodiclofen-Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en-2-one] and Spirodiclofen -Ketohydroxy [3-(2,4-dichlorophenyl)-3-hydroxy-1-oxaspiro[4.5]decane-2,4- dione] with maximum concentrations of 51.9 and 10.6% of the applied amount, observed on the 2nd and 3rd day of incubation, respectively. At the end of the study period, the corresponding concentrations were both 0% of the applied amount. The minor transformation products were Spirodiclofen -Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[ 4.5] decan- 2-one] and 2,4 dichlorobenzoic acid (FHW0107B) formed at a maximum of 1.0 and 5.3% of the applied amount, respectively. The total unidentified radioactivity was 0.5 to 6.9% of the applied amount.

Transformation pathway:
The initial biotransformation of Spirodiclofen involves hydrolysis of the ester side-chain to produce Spirodiclofen-Enol. The Enol is further metabolized to Spirodiclofen- Ketohydroxy by oxidation. Subsequently, the Ketohydroxy is degraded to 2,4-dichlorobenzoic acid. The Ketohydroxy is also reduced to Spirodiclofen-Dihydroxy. Eventually, all of the degradates are mineralized to carbon dioxide. Spirodiclofen Enol and Spirodiclofen-Ketohydroxy were detected in all soils in concentrations >10% of the applied radioactivity. Spirodiclofen-Dihydroxy and 2,4-dichlorobenzoic acid were >10% of the applied radioactivity in only the Fresno and Vero Beach soils, respectively. See figure attached to end of this DER.

Table 5: Biotransformation of [dihydrofuranon-3-14C]Spirodiclofen, expressed as percentage of applied radioactivity, in Fresno soil (sandy loam) under aerobic conditions Table 1/2

	Sampling times (days)
	0	1	2	3	6	9	13
Spirodiclofen	91.9	82.9	75.4	71.4	57.4	47.4	36.0
Spirodiclofen- Enol	1.3	10.0	15.7	17.1	17.3	15.0	13.5
Spirodiclofen- Keto hydroxy	0.4	1.7	5.4	8.8	16.6	23.3	27.9
Spirodiclofen- Dihydroxy	0.0	0.0	0.0	0.5	1.8	3.3	5.1
2,4-Di chlorobenzoic acid	0.0	0.0	0.0	0.0	0.0	0.6	1.5
2.0Unidentified radioactivity	1.4	0.8	1.6	2.2	3.1	4.8	4.1
Total ext. residues	95.0	95.4	98.1	100.0	96.2	94.4	87.3
CO2	0.0	0.2	0.3	0.5	1.6	3.8	7.2
Total volatile organics	0.0	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Non-ext. residues	1.9	2.3	2.3	2.3	3.4	4.0	4.6
Total % recovery	96.9	97.9	100.8	102.7	101.2	102.2	99.1

Table 2/2 

	Sampling times (days)
	20	30	59	120	181	272	360
Spirodiclofen	25.2	20.1	8.4	4.7	2.9	2.5	2.3
Spirodiclofen- Enol	7.8	4.3	2.6	0.5	0.3	0.1	0.0
Spirodiclofen- Keto hydroxy	27.9	27.0	15.8	7.2	3.7	2.1	1.5
Spirodiclofen- Dihydroxy	8.5	11.2	15.7	16.4	14.4	11.4	11.1
2,4-Di chlorobenzoic acid	1.5	2.0	0.0	0.0	0.0	0.0	0.0
Unidentified radioactivity	7.1	3.3	5.9	3.3	2.9	3.0	2.2
Total ext. residues	78.0	67.9	48.4	32.1	24.2	19.1	17.1
CO2	14.2	23.1	42.3	60.1	65.6	71.7	76.0
Total volatile organics	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Non-ext. residues	6.4	7.8	9.7	10.1	9.7	9.	8.5
Total % recovery	98.6	98.8	100.4	102.2	99.5	100.1	101.6

Table 6: Biotransformation of [dihydrofuranon-3-14C]Spirodiclofen, expressed as percentage of applied radioactivity, in Fresno soil (sandy loam) under aerobic conditions (high dose experiment). (1/2)

	Sampling times (days)
	0	1	2	3	6	9	13
Spirodiclofen	94.3	87.0	77.1	71.9	63.7	52.0	44.5
Spirodiclofen- Enol	1.3	7.9	11.9	13.6	14.5	13.1	10.5
Spirodiclofen- Keto hydroxy	0.2	1.3	4.7	7.5	15.5	21.3	26.4
Spirodiclofen- Dihydroxy	0.0	0.0	0.0	0.5	1.4	2.6	4.3
2,4-Di chlorobenzoic acid	0.0	0.0	0.0	0.0	0.4	0.8	1.9
Unidentified radioactivity	1.4	1.5	2.3	2.4	3.4	3.4	4.7
Total ext. residues	97.1	97.7	96.0	95.9	98.9	93.2	92.3
CO2	0.0	0.2	0.3	0.4	1.3	2.6	4.9
Total volatile organics	0.0	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Non-ext. residues	1.6	2.3	1.9	2.2	3.0	3.6	4.5
Total % recovery	98.7	100.1	98.1	98.6	103.2	99.4	101.7

2/2

	Sampling times (days)
	13	20	30	59	120
Spirodiclofen	44.5	31.8	24.4	11.2	5.3
Spirodiclofen- Enol	10.5	7.5	4.5	2.5	0.6
Spirodiclofen- Keto hydroxy	26.4	29.1	27.6	18.4	8.1
Spirodiclofen- Dihydroxy	4.3	7.5	9.7	15.2	16.2
2,4-Di chlorobenzoic acid	1.9	2.9	4.3	0.4	0.0
Unidentified radioactivity	4.7	6.4	4.5	5.6	4.7
Total ext. residues	92.3	85.2	75.0	53.3	34.9
CO2	4.9	9.9	17.7	38.5	55.3
Total volatile organics	<0.1	<0.1	<0.1	<0.1	<0.1
Non-ext. residues	4.5	5.4	6.8	9.1	9.2
Total % recovery	101.7	100.6	99.5	101.0	99.4

Table 7: Biotransformation of [dihydrofuranon-3-14C]Spirodiclofen, expressed as percentage of applied radioactivity, in Vero Beach soil (sand) under aerobic conditions.(1/2)

	Sampling times (days)
	0	1	2	3	6	9	13
Spirodiclofen	96.0	88.6	74.9	68.4	53.8	38.3	28.4
Spirodiclofen- Enol	1.9	10.8	20.0	27.0	36.0	38.4	37.8
Spirodiclofen- Keto hydroxy	0.2	0.7	3.5	4.4	10.3	17.1	25.5
Spirodiclofen- Dihydroxy	0.0	0.0	0.0	0.0	0.1	0.2	0.3
2,4-Di chlorobenzoic acid	0.0	0.0	0.0	0.0	0.0	0.8	1.9
Unidentified radioactivity	1.4	0.9	1.6	0.6	1.8	3.4	4.0
Total ext. residues	99.1	101.0	100.0	100.4	102.0	98.2	97.9
CO2	0.0	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Total volatile organics	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Non-ext. residues	1.9	2.6	2.5	2.9	3.3	4.4	5.1
Total % recovery	101.0	103.6	102.5	103.3	105.4	102.7	103.3

2/2

	Sampling times (days)
	20	30	59	120
Spirodiclofen	14.6	7.8	1.9	1.0
Spirodiclofen- Enol	35.9	20.3	5.5	0.8
Spirodiclofen- Keto hydroxy	32.9	44.4	31.7	10.9
Spirodiclofen- Dihydroxy	1.3	2.5	4.6	4.8
2,4-Di chlorobenzoic acid	4.2	11.1	33.0	39.6
Unidentified radioactivity	7.2	7.3	9.5	4.0
Total ext. residues	96.1	93.4	86.2	61.1
CO2	0.5	1.7	6.8	22.5
Total volatile organics	<0.1	<0.1	<0.1	<0.1
Non-ext. residues	6.2	7.9	12.6	14.4
Total % recovery	102.7	102.9	105.6	98.0

 

Table 8: Biotransformation of [dihydrofuranon-3-14C]Spirodiclofen, expressed as percentage of applied radioactivity, in BBA 2.2 soil (loamy sand) under aerobic conditions(1/2)

	Sampling times (days)
	0	1	2	3	6	9	13
Spirodiclofen	92.5	80.4	66.1	60.1	46.9	34.5	26.3
Spirodiclofen- Enol	2.2	12.4	19.1	17.3	16.0	13.7	8.5
Spirodiclofen- Keto hydroxy	0.5	3.1	9.0	12.4	19.0	20.7	19.6
Spirodiclofen- Dihydroxy	0.0	0.0	0.0	0.0	1.2	1.7	2.2
2,4-Di chlorobenzoic acid	0.0	0.0	0.8	1.7	2.2	2.7	3.0
Unidentified radioactivity	1.4	1.2	1.6	4.8	5.6	4.3	6.4
Total ext. residues	96.9	97.1	96.6	96.3	90.9	77.6	66.0
CO2	0.0	0.2	0.8	1.6	8.1	17.1	30.0
Total volatile organics	0.0	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Non-ext. residues	2.0	3.3	2.7	3.0	4.2	4.9	5.9
Total % recovery	98.9	100.6	100.2	100.9	103.2	99.6	101.9

 

2/2

	Sampling times (days)
	20	30	59	120
Spirodiclofen	15.0	10.7	4.2	3.4
Spirodiclofen- Enol	5.1	2.2	1.3	0.4
Spirodiclofen- Keto hydroxy	13.1	7.6	2.8	1.2
Spirodiclofen- Dihydroxy	2.5	1.6	1.0	0.0
2,4-Di chlorobenzoic acid	2.2	0.9	0.3	2.0
Unidentified radioactivity	6.6	4.6	1.9	0.2
Total ext. residues	44.5	27.6	11.5	7.2
CO2
Total volatile organics	49.6	64.9	78.3	86.3
Non-ext. residues	6.9	7.3	7.3	8.0
Total % recovery	101.1	99.7	97.1	101.5

 

Table 10.Best fit DT50 and DT90 values for the degradation of Spirodiclofen in soils using the method of Timme et al. (1986).

Soils	DT50 (days)	DT90 (Days)	For the calculation of kinetic parameters refer to the following Appendix
Fresno	5.3	58.6	82
Fresno, high dose	6	66.4	83
Vero Beach	5.5	28.5	84
BBA 2.2	3.9	43.4	85
Hoefchen	0.5	13.6	86

HALF-LIFE using first-order kinetics: The mathematical assessment of the metabolism data was performed utilizing the ACSL Optimise Software package (MGA Software, 1996). All reaction steps were assumed to be of first order since this has become the EPA’s accepted method for determination of degradation kinetics. The evaluation of the results yielded the DT50 and DT90 values that are shown in Table 11 and 12 (below).

For the calculation of kinetic data of metabolites based on metabolism studies, a knowledge of the degradation pathway is necessary. A simple degradation scheme was established in the present study which was based on the proposed degradation pathway proposed by Oi & Bornatsch (current study).

Spirodiclofen –K1-> Spirodiclofen-Enol –K2-> Spirodiclofen –K3-> Bound Residues & CO2 (BRC)

Figure 1: Degradation pathway of Spirodiclofen applied to the calculations where k1 to k3 are the reaction rate constants.

Assuming first order kinetics for all reaction steps, the following rate equations can be deduced:

rl = kl * CSpirodiclofen(1)

r2 = k2 * CSpirodiclofen-Enol (2)

r3 = k3 * CSpirodiclofen-Ketohydroxy (3)

with r representing the reaction rate and C the concentration of the specified compound. This leads to mass balance equations for each compound. Since the reaction of one radiolabeled molecule produces only one radiolabeled molecule, no stoichiometric coefficients have to be taken into account for the mass balance equations (Equation 4 to 7). The concentrations can be expressed as a percent of applied radioactivity as follows:

dCSpirodiclofen / dt = - kl * CSpirodiclofen (4)

dCSpirodiclofen-Enol / dt = k1 * CSpirodiclofen - k2 * CSpirodiclofen-Enol (5)

dCSpirodiclofen-Ketohydroxy / dt = k2 * CSpirodiclofen-Enol - k3 * CSpirodiclofen-Ketohydroxy (6)

dCBRC / dt = k3 * CSpirodiclofen-Ketohydroxy (7)

Where t represents the time and dC/dt represents the rate of change in concentration differentiated by the time. The coupled differential equation system (Equation 4 to 7) was solved numerically. The calculated concentrations were fitted to experimental data by adjusting the parameters ki. The results of the current soil metabolism study was used to provide the experimental data. The fitting algorithm and the numerical solution of the differential equations were computed utilizing the ACSL Optimise program package (MGA Software, 1996). The definition of the differential equation systems within ACSL. Optimise is based on ACSL (Advanced Continuous Simulation Language). The results of the parameter fitting are summarized in an ACSL report, containing information on the parameter estimation, a comparison of observed and predicted data, a statistical summary, the correlation matrix, and the variance-covariance matrix (see Appendix 2 to 5 in Bayer Corporation report no. 110867). From the rate constants, the DT50 were calculated as:

DT50 Spirodiclofen = ln(2) / kl (8)

DT50 Spirodiclofen-Enol = ln(2) / k2 (9)

DT50 Spirodiclofen- Ketohydroxy = ln(2) / k3 (10)

The corresponding DT90 values were obtained by replacing ln(2) by ln(10) in equations 8 to 10.

Executive summary:

The biotransformation of [dihydrofuranon-3-14C]Spirodiclofen was studied in a sandy loam soil (Fresno, CA, USA; pH 6.6, organic carbon 0.53%) for 360 days under aerobic conditions in the dark at 20 + 1ºC. The biotransformation and degradation kinetics were further investigated in the sandy loam (Fresno), a sand (Vero Beach, FL, USA; pH 6.4, organic carbon 0.22%), a loamy sand (BBA 2.2; Hanhofen, Voderpflaz, Germany; pH 6.0, organic carbon 2.48%), and a silt soil (Hoefchen; Hohenseh 4A, Burscheid, Germany; pH 7.8, organic carbon 2.62%) for 120 days under aerobic conditions in the dark at 20 + 1ºC, and 75% of 1/3 bar moisture for the Fresno and Vero Beach soils and 40% of the maximum water holding capacity for the BBA 2.2 and Hoefchen soils. [Dihydrofuranon-3-14C]SPIRODICLOFEN was applied at the rate of 0.111 mg a.i./kg soil, equivalent to 250 g a.i./ha. A “high dose” study was conducted solely for Japanese registration at a rate of 0.533 mg a.i./kg soil which was equivalent to 1200 g a.i./ha. The study was conducted in accordance with EPA-Guidelines for aerobic soil metabolism studies [ref l in section V of the DER], the BBA Guidelines for Official Testing of Plant Protectants, Part IV [ref 2 in section V of DER], the EPA Addendum on data reporting for aerobic soil metabolism studies [ref 3 in section V of DER], the EPA "Standard Evaluation Procedure, Aerobic Soil Metabolism Studies" [ref 4 in section V], EPA’s Environmental Fate Rejection Rate Analysis [ref 5 in section V], and relevant EU Guidelines [ref 6 and 7 in section V], and in compliance with the OECD– GLP standards. The test system consisted of 300-mL Erlenmeyer flasks with attached traps for the collection of CO2 and volatile organics. For biotransformation, samples were analysed at 0, 1, 2, 3, 6, 9, 13, 20, 30, 59, 120, 181, 272, and 360 days of incubation. For the kinetic experiments, samples were analyzed at 0, 1, 2, 3, 6, 9, 13, 20, 30, 59, and 120 days of incubation. The soil samples were extracted with acetonitrile followed by acetonitrile/buffer mixtures, and the [dihydrofuranon-3-14C]SPIRODICLOFEN residues were analyzed by reverse phase TLC using autoradiography obtained with a bio-imaging analyzer (BAS 200, Fuji Co.) for radiodetection. A second TLC system was used for confirmation. Unknown degradates were identified by GC/MS, LC/MS, and/or 1H-NMR.

Fresno soil:
The metabolism/biotransformation of SPIRODICLOFEN was investigated in Fresno soil for 360 days at a rate that corresponded to a field use rate of 250 g a.i./ha. The material balance was 100.1 +1.8% of the applied amount. The concentration of the parent compound decreased from 91.9% of the applied amount at day 0, to 2.3% of the applied at the end of the study period. The DT50 for SPIRODICLOFEN in Fresno aerobic soil was 5.3 days. SPIRODICLOFEN was rapidly degraded in Fresno soil by hydrolysis of the ester side-chain to initially produce SPIRODICLOFEN-Enol. The major transformation products were SPIRODICLOFEN-Enol [3-(2,4 dichlorophenyl)-4- hydroxy-1-oxaspiro[4.5]dec-3-en-2-one], SPIRODICLOFEN-Ketohydroxy [3-(2,4-dichloro-phenyl)-3-hydroxy-1-oxaspiro[4.5]decane-2,4-dione], and SPIRODICLOFEN-Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[4.5]decan-2-one] with maximum concentrations of 17.3, 27.9, and 16.4% of the applied amount, observed on the 6th, 20th, and 120th day of incubation, respectively. At study termination (360 days), the corresponding concentrations were 0, 1.5, and 11.1% of the applied amount, respectively. The minor transformation product, 2,4-dichlorobenzoic acid (FHW0107B), was formed at a maximum concentration of 2% of the applied amount. The total unidentified radioactivity ranged from 0.8 to 7.1% of the applied amount. Extractable [14C]residues decreased from 95.0% of the applied amount at day 0 to 17.1% of the applied amount at study termination (the decrease in extractable residues was due to the mineralization of the majority of the radioactivity). Non-extractable [14C]residues increased from 1.9% of the applied amount at day 0 to 8.5% of the applied at the end of incubation period. At study termination, 76.0 and <0.1% of the applied radioactivity was present as CO2 and volatile organic compounds, respectively. Fresno soil (high dose): The rate of degradation was studied in aerobic Fresno soil using an application rate that corresponded to a field rate of 1200 g a.i./ha which was a proposed rate for use in Japan. The material balance was 100.0 + 1.5% of the applied amount. The concentration of the parent compound decreased from 94.3% of the applied amount at day 0, to 5.3% of the applied at the end of the study period. The DT50 for SPIRODICLOFEN in aerobic Fresno soil (using a 4.8x rate) was 6.0 days. Even at the higher rate, SPIRODICLOFEN was rapidly degraded initially to the Enol in soil. The major transformation products were SPIRODICLOFEN-Enol [3-(2,4-dichlorophenyl)-4- hydroxy-1-oxaspiro[4.5]dec-3-en-2-one], SPIRODICLOFEN-Ketohydroxy [3-(2,4- dichlorophenyl)-3-hydroxy-1 oxaspiro[4.5]decane-2,4-dione], and SPIRODICLOFEN-Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[4.5]decan-2-one] with maximum
concentrations of 14.5, 29.1, and 16.2% of the applied amount, observed on the 6th, 20th, and 120th day of incubation, respectively. At study termination (120 days), the corresponding concentrations were 0.6, 8.1, and 16.2% of the applied amount, respectively. The minor transformation product, 2,4-dichlorobenzoic acid (FHW0107B), was formed at a maximum concentration of 4.3% of the applied amount. The total unidentified radioactivity ranged from 1.4 to 6.4% of the applied amount. Extractable [14C]residues decreased from 97.1% of the
applied amount at day 0 to 34.9% of the applied, at study termination (the decrease in extractable residues was due to the mineralization of the majority of the radioactivity). Nonextractable [14C]residues increased from 1.6% of the applied amount at day 0 to 9.2% of the applied at the end of incubation period. At study termination, 55.3 and <0.1% of the applied radioactivity was present as CO2 and volatile organic compounds, respectively.

Vero Beach soil:
The rate of degradation of SPIRODICLOFEN was investigated in Vero Beach soil for 120 days at a rate that corresponded to a field use rate of 250 g a.i./ha. The material balance was 102.8 + 2.1% of the applied amount. The concentration of the parent compound decreased from 96.0% of the applied amount at day 0, to 1.0% of the applied at the end of the study period. The DT50 for SPIRODICLOFEN in Vero Beach aerobic soil was 5.5 days. SPIRODICLOFEN was rapidly degraded initially to
the Enol in Vero Beach soil. The major transformation products were SPIRODICLOFEN-Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en-2-one], SPIRODICLOFEN-Ketohydroxy [3-(2,4- dichlorophenyl)-3-hydroxy-1-oxaspiro[4.5]decane-2,4-dione], and 2,4-dichlorobenzoic acid (FHW0107B) with maximum concentrations of 38.4, 44.4, and 39.6% of the applied amount,
observed on the 9th, 30th, and 120th day of incubation, respectively. At study termination (120 days), the corresponding concentrations were 0.8, 10.9, and 39.6% of the applied amount, respectively. The minor transformation product, SPIRODICLOFEN-Dihydroxy [3-(2,4- dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[4.5]decan-2-one], was formed at a maximum of 4.8% of the applied amount. The total unidentified radioactivity ranged from 0.6 to 9.5% of the
applied amount. Extractable [14C]residues decreased from 99.1% of the applied amount at day 0 to 61.1% of the applied, at study termination (the decrease in extractable residues was due to the mineralization of the majority of the radioactivity). Non-extractable [14C]residues increased from 1.9% of the applied amount at day 0 to 14.4% of the applied at the end of incubation period. At study termination, 22.5 and <0.1% of the applied radioactivity was present as CO2
and volatile organic compounds, respectively.

BBA 2.2 soil:
The rate of degradation of SPIRODICLOFEN was investigated in aerobic BBA 2.2 soil for 120 days at a rate that corresponded to a field use rate of 250 g a.i./ha. The material balance was 100.4 + 1.6% of the applied amount. The concentration of the parent compound decreased from 92.5% of the applied amount at day 0, to 3.4% of the applied at the end of the study period. The DT50 for SPIRODICLOFEN in aerobic BBA 2.2 soil was 3.9 days. SPIRODICLOFEN was rapidly degraded initially to
the Enol in BBA 2.2 soil.
The major transformation products were SPIRODICLOFEN-Enol [3-(2,4 dichlorophenyl)-4- hydroxy-1-oxaspiro[4.5]dec-3-en-2-one] and SPIRODICLOFEN-Ketohydroxy [3-(2,4- dichlorophenyl)-3-hydroxy-1 oxaspiro[4.5]decane-2,4-dione] with maximum concentrations of 19.1 and 20.7% of the applied amount, observed on the 2nd and 9th day of incubation,
respectively. At study termination (120 days), the corresponding concentrations were 0.4 and 1.2% of the applied amount, respectively. The minor transformation products were SPIRODICLOFEN- Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[4.5]decan-2-one] and 2,4-dichlorobenzoic acid (FHW0107B) formed at maximum concentrations of 2.5 and 3.0% of the applied amount, respectively. The total unidentified radioactivity ranged from 1.2 to 6.6% of the applied amount. Extractable [14C]residues decreased from 96.9% of the applied amount at day 0 to 7.2% of the applied at study termination (the decrease in extractable residues was due to the mineralization of the majority of the radioactivity). Non-extractable [14C]residues increased from 2.0% of the applied amount at day 0 to 8.0% of the applied at the end of incubation period. At study termination, 86.3 and <0.1% of the applied radioactivity was
present as CO2 and volatile organic compounds, respectively.

Hoefchen soil:
The rate of degradation of SPIRODICLOFEN was investigated in aerobic BBA 2.2 soil for 120 days at a rate that corresponded to a field use rate of 250 g a.i./ha. The material balance was 99.2 + 2.6% of the applied amount (two sampling intervals were excluded from this calculation due to errors in sampling ¹⁴CO₂). The concentration of the parent compound decreased from 86.3% of the applied amount at day 0 to 1.0% of the applied at the end of the study period. The DT50 for SPIRODICLOFEN in aerobic Hoefchen soil was 0.5 days. SPIRODICLOFEN was rapidly degraded initially to the Enol in Hoefchen soil.
The major transformation products were SPIRODICLOFEN-Enol [3-(2,4-dichlorophenyl)-4- hydroxy-1-oxaspiro[4.5]dec-3-en-2-one] and SPIRODICLOFEN-Ketohydroxy [3-(2,4- dichlorophenyl)-3-hydroxy-1 oxaspiro[4.5]decane-2,4-dione] with maximum concentrations of
51.9 and 10.6% of the applied amount, observed on the 2nd and 3rd day of incubation, respectively. At study termination (120 days), the corresponding concentrations were both 0% of the applied amount. The minor transformation products were SPIRODICLOFEN-Dihydroxy [3-(2,4-dichlorophenyl)-3,4-dihydroxy-1-oxaspiro[4.5]decan-2-one] and 2,4- dichlorobenzoic acid (FHW0107B) formed at maximum concentrations of 1.0 and 5.3% of the
applied amount, respectively. The total unidentified radioactivity ranged from 0.5 to 6.9% of the applied amount. Extractable [14C]residues decreased from 97.9% of the applied amount at day 0 to 1.5% of the applied, at study termination (the decrease in extractable residues was due to the mineralization of the majority of the radioactivity). Non-extractable [14C]residues increased from 2.1% of the applied amount at day 0 to 6.8% of the applied at the end of incubation period. At study termination, 93.1 and <0.1% of the applied radioactivity was
present as CO2 and volatile organic compounds, respectively.
No sterile treatments were used in the current study.

The initial biotransformation involves hydrolysis of the ester side-chain of SPIRODICLOFEN to produce SPIRODICLOFEN-Enol. The Enol is further metabolized to SPIRODICLOFEN-Ketohydroxy by oxidation.
Subsequently, the Ketohydroxy is degraded to 2,4-dichlorobenzoic acid. The Ketohydroxy is also reduced to SPIRODICLOFEN-Dihydroxy. Eventually, all of the degradates are mineralized to carbon dioxide. The SPIRODICLOFEN-Enol and SPIRODICLOFEN-Ketohydroxy were detected in all soils in concentrations >10% of the applied radioactivity. SPIRODICLOFEN-Dihydroxy and 2,4-dichlorobenzoic acid were >10% of the applied radioactivity in only the Fresno and Vero Beach soils, respectively.

 

Description of key information

Aerobic degradation and metabolism of dihydrofuranone-labelled spirodiclofen was investigated under controlled laboratory conditions in four soils (aerobic, dark, 20°C) for up to 360 days DT50 values of 0.5-5.5 days were calculated for the different soil types.
Metabolism and mineralisation was observed over 12 days (23-93%) and 360 days (76%).
The arithmetric mean of the different soilt types was calculated for the endpoint summary (excluding ther high dose).

The half-life in soil was converted from 3.8 d at 20°C to 8.07 at 12°C (using Arrhenius equation).

Key value for chemical safety assessment

Half-life in soil:

8.07 d

at the temperature of:

12 °C