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Environmental fate & pathways

Biodegradation in soil

Currently viewing:

Administrative data

Endpoint:
biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2001 to 20 April 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed to recognised testing standards in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: SETAC Document "Procedures for Assessing the Environmental Fate and Ecotoxicology of Pesticides (March 1995)".
Deviations:
no
GLP compliance:
yes
Test type:
laboratory

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
425-220-8
EC Name:
-
Cas Number:
5945-33-5
Molecular formula:
C39H34O8P2
IUPAC Name:
4-(2-{4-[(diphenoxyphosphoryl)oxy]phenyl}propan-2-yl)phenyl diphenyl phosphate; 4-{2-[4-({[4-(2-{4-[(diphenoxyphosphoryl)oxy]phenyl}propan-2-yl)phenoxy](phenoxy)phosphoryl}oxy)phenyl]propan-2-yl}phenyl diphenyl phosphate
Radiolabelling:
yes

Study design

Oxygen conditions:
aerobic
Soil classification:
other: Soil Survey and Land Research Centre (Cranfield University, Silsoe, Bedfordshire, UK)
Year:
2002
Soil propertiesopen allclose all
Soil no.:
#1
Soil type:
sandy loam
% Org. C:
1.2
pH:
4.3 - 5.8
CEC:
10.4 meq/100 g soil d.w.
Soil no.:
#2
Soil type:
clay loam
% Org. C:
1.5
pH:
7.5 - 7.8
CEC:
25.8 meq/100 g soil d.w.
Details on soil characteristics:
Two freshly collected soils were received from Landlook (Midlands, UK).

Soil 1: Sandy Loam (Landlook Soil Number 244; lnveresk Soil Code S380; route soil); received from Landlook on 05 November 2001.
Soil 2: Clay Loam (Landlook Soil Number 115; lnveresk Soil Code S382; rate soil); received from Landlook on 05 November 2001.

Soils were stored under aerobic conditions at ca +4°C upon receipt.

The moisture content and maximum water holding capacity (MWHC) were determined at lnveresk Research and the moisture content adjusted to 40% MWHC prior to use on the study.

Samples of each soil were characterised by Soil Survey and Land Research Centre (Cranfield University, Silsoe, Bedfordshire, UK) with respect to particle size and texture (BS 3882 and USDA), pH (water and 1M potassium chloride), cation exchange capacity, organic carbon and water holding capacity at applied pressure (0.33 bar).

Soil Characterisation Data is attached below under "attached background material".
Duration of test (contact time)open allclose all
Soil No.:
#1
Duration:
120 d
Soil No.:
#2
Duration:
120 d
Initial test substance concentrationopen allclose all
Soil No.:
#1
Initial conc.:
16.9 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#2
Initial conc.:
16.8 mg/kg soil d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Experimental conditionsopen allclose all
Soil No.:
#1
Temp.:
20±2°C
Microbial biomass:
ca 42.0 mg C.100 g-1 soil (oven dry equivalent)
Soil No.:
#2
Temp.:
20±2°C
Microbial biomass:
ca 67.6 mg C.100 g-1 soil (oven dry equivalent)
Details on experimental conditions:
Determination of Microbial Biomass
The microbial biomass of each soil was determined prior to the application of [14C]-DVP 506 and after the 120 day sampling interval in control soil samples, according to the method of Anderson and Domsch (Soil Biol. Biochem, 10, 215-221, 1978). Seven incubation units were set up with ca. 50g soil (oven dry equivalent) for each analysis occasion. Samples of each soil type were fortified with up to 500 mg of D-glucose (mixed with talcum powder). The fortified samples were transferred to incubation units where a stream of moist, carbon dioxide (CO2) free air was passed through each sample for up to 4 days. The levels of evolved CO2 were measured semi-continuously using an infrared gas analyser (Model; 225 Mark 3 Gas Analyser, Analytical Development Company), connected to a gas handling unit (Analytical Development Company).
The microbial biomass was then extrapolated from the experimentally determined respiration values, using the following equation (Anderson and Domsch):
Y = 40.04 X + 0.37

Where
X = respiration rate (ml CO2 100 g-1 oven dry equiv.h-1)
Y = mg microbial carbon. 100 g-1 oven dry equiv.

Microbial biomass values prior to initiation for sandy loam and clay loam test systems were ca 42.0 and 67.6 mg C.100 g-1 soil (oven dry equivalent) respectively. The corresponding termination values were ca 24.0 and 49.1 mg C.100 g-1 soil (oven dry equivalent), respectively.

Adsorption of [14C]-DVP 506 to Test Apparatus
A stock solution of [14C]-DVP 506 in methanol was prepared. Aliquots of this solution were dispensed into amberlite glass jars, liquid scintillation vials and glass incubation units. An aliquot (10 ml) from the stock solution was filtered through a 0.2 μm filter and duplicate aliquots taken for liquid scintillation counting (LSC). The remaining filtrate was filtered again through a clean filter and duplicate aliquots taken for LSC. Duplicate aliquots (1 ml) were sampled from the stock solution for comparison of glass and Gilson Microman pipettes. The liquid scintillation vials and incubation units were left overnight at ambient temperature. The amber jars were left on an end-over-end shaker overnight.
Quantitative recoveries of radioactivity were obtained from the vessels sampled immediately and those left overnight. The filtered aliquots displayed no loss with each filtration indicating the test item did not adsorb to filters. This indicated that DVP 506 did not adsorb to the test apparatus. There was full recovery of the test item when using both types of pipette.

Test System
Test System Preparation
Samples of each soil type (50 g oven dry equivalent) were weighed into 250 ml Erlenmyer flasks. Ten incubates were formed for each soil type, including two incubates for contingency sampling if required. A further two incubates of sandy loam soil were prepared to investigate the evolution of
14CO2. Seven incubates of each soil type were prepared for initiation biomass measurements and seven incubates of each soil type for termination biomass measurements. The samples were adjusted to ca 40% maximum water olding capacity (MWHC).
A stream of moist CO2-free air, at a flow rate of ca 10-15 ml.min-1, was passed over the surface of each sample of soil. The gas mixture leaving each unit was passed through a series of 3 traps for the sandy loam soil. For the clay loam soil, the gas mixture leaving all units was combined and passed through a series of 3 bulk traps. For both soil types the first trap was a safety trap to prevent back flow, the second contained ethanediol to trap non-specific [14C]-organic volatiles and the third trap contained ethanolamine to trap liberated 14CO2. For the two incubates of sandy loam soil prepared to investigate the evolution of 14CO2, the traps consisted of a safety trap and 2 traps containing 0.1 M sodium hydroxide. Connections between traps and incubation units were made using a combination of glass connectors and PVC tubing.

Pre-incubation
The test samples were pre-incubated for 7 days under aerobic conditions in the dark at 20 ± 2°C.

Preparation of [14C]-DVP 506 Test Solution
500 μl of [14C]-DVP 506 stock solution in ethanol was transferred to a 5 ml volumetric flask containing 37 mg of non-labelled DVP 506. The ethanol was evaporated to dryness and the flask made up to volume with methanol. Aliquots (3 x 20 μl) were transferred to 10 ml volumetric flasks, which were made up to volume with methanol and aliquots (3 x 200 μl) taken for homogeneity checks by LSC.
The radiochemical purity of [14C]-DVP 506 in the test solution was confirmed by HPLC, prior to test item application.

Application of Test Solution
Dose solution (125 μl for both soil types) was applied drop wise onto the surface of the soil. To accurately quantify the amount of test item applied to each sample, the same volume of dose solution was dispensed into volumetric flasks (25 ml capacity) at regular intervals throughout dosing. These were then made to volume with methanol. Aliquots (2 x 200 μI) from each volumetric flask were submitted for liquid scintillation counting.
Following test item application, the samples were re-connected to the continuous gas flow system. The samples were then incubated in the dark at a nominal temperature of 20 ± 2°C for up to 120 days.
Following application, the soil moisture content was adjusted to ca 45% MWHC, and was maintained at 45c5% MWHC for the duration of the study.

Sampling
At each sampling interval (0, 2, 4, 8, 16, 32, 64, and 120 days after application), a single incubation flask was removed for analysis.
Traps were sampled and replenished at weekly intervals for the first four weeks and every four weeks thereafter. Traps were also sampled at the time of termination of the incubation units. Sodium hydroxide traps were removed at Day 64 and Day 120.

Results and discussion

Material (mass) balanceopen allclose all
Soil No.:
#1
% Recovery:
99
St. dev.:
4
Soil No.:
#2
% Recovery:
94
St. dev.:
4
% Degradationopen allclose all
Key result
Soil No.:
#1
% Degr.:
0
Parameter:
radiochem. meas.
Sampling time:
120 d
Key result
Soil No.:
#2
% Degr.:
0
Parameter:
radiochem. meas.
Sampling time:
120 d
Transformation products:
no
Details on transformation products:
None specified
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
Details on results:
Recovery and Distribution of Applied Radioactivity
Following administration of [14C]-DVP 506 to sandy loam soil, quantitative recoveries of radioactivity (ca 95-103%) were obtained at all sampling time points. Greater than 90% of the applied radioactivity was recovered in methanol extracts of soil (ca 90.5-97.7%) with non-solvent extractable residues accounting for between ca 4-6% of the applied radioactivity. No significant production of 14CO2 or 14C-organic volatiles was observed.
A similar pattern of recovery of total radioactivity was observed following administration of [14C]-DVP 506 to clay loam soil. Although no soil residues were analysed and no mass balance calculated, recovery of radioactivity in methanolic extract of soil varied from ca 90-98%. A very minor amount of the applied radioactivity (0.83%) was measured as 14CO2 in the bulk amine traps over the 120 day incubation period. However it is not considered that this constitutes a significant mineralisation of [14C]-DVP 506.

Chromatographic Analysis
Analysis of soil extracts by HPLC and TLC indicated one major component in each sample. [14C]-DVP 506 accounted for almost 100% of the radioactivity in each extract (equivalent to ca 89.4-97.7% applied radioactivity in sandy loam soil and ca 88.5-98.0% applied radioactivity in clay loam soil).
Following analysis by radio-HPLC, very minor quantities of uncharacterised components were detected in occasional samples. These components accounted for ca 0.7-1.7% of applied radioactivity for Unknown A (retention time ca 7 min) and 0.5% applied radioactivity for Unknown B (retention time ca 16 min). Unknown B was only observed in sandy loam soil. The pattern of appearance of these very minor components did not indicate any trend in the degradation of the test item. Similarly, analysis by radio-TLC also indicated occasional very minor components. These were not considered significant.

Calculation of DT50 and DT90 Values
No pattern of decrease in the amount of [14C]-DVP 506 present in soil samples could be observed throughout the period of the experiment. As no degradation has occurred, no attempt has been made to calculate the DT50 and DT90 values.
Results with reference substance:
Reference substance not utilised in this study.

Any other information on results incl. tables

Recovery of Total Radioactivity Following Administration of [14C]-DVP 506 to Sandy Loam Soil.

Results expressed at % of total radioactivity applied

Sampling Interval

Percentage of Applied Radioactivity as:

Soil Extracts in Methanol

Soil

Non-extractable Residue

[14C]-Organic Volatiles

14CO2

Apparatus Wash

Total

1

2

Total

Zero time

77.77

19.91

97.69

5.14

NS

NS

0.01*

102.84

Day 2

72.80

19.14

91.94

6.20

0.00*

0.02

0.00*

98.17

Day 4

71.88

18.66

90.54

4.20

0.00*

0.03

0.00*

94.77

Day 8

72.43

18.78

91.21

4.56

0.00*

0.03*

0.00*

95.80

Day 16

73.60

18.71

92.31

4.81

0.00

0.05*

0.00*

97.16

Day 32

74.97

19.33

94.30

5.23

0.00*

0.07*

0.00*

99.60

Day 64

74.53

21.17

95.70

5.54

0.00*

0.20

0.05

101.49

Day 120

73.79

20.27

94.06

6.00

0.00*

0.30

0.01*

100.38

 (*) = Results calculated from data less than 30 d.p.m. above background

NS = No sample

 

Recovery of Total Radioactivity Following Administration of [14C]-DVP 506 to Clay Loam Soil.

Results expressed at % of total radioactivity applied

Sampling Interval

Percentage of Applied Radioactivity as:

Soil Extracts in Methanol

[14c]-Organic Volatiles

14CO2

Apparatus Wash

1

2

Total

Zero time

75.54

22.48

98.02

NS

NS

0.01*

Day 2

71.44

21.56

93.00

0.00*

0.00*

0.00*

Day 4

70.48

19.59

90.07

0.00

0.01

0.00*

Day 8

69.43

20.20

89.63

0.00

0.02

0.00*

Day 16

69.51

20.01

89.52

0.00

0.03

0.11

Day 32

70.61

20.36

90.97

0.00

0.24

0.01*

Day 64

72.60

21.28

93.88

0.00

0.41

0.00*

Day 120

73.88

19.65

93.53

0.00

0.83

0.00*

(*) = Results calculated from data less than 30 d.p.m. above background

NS = No sample collected.

 

Distribution of Total Radioactivity Determined by Radio-HPLC Analysis of

Sandy Loam Soil Extracts Following Administration of [14C]-DVP 506

Sampling Interval

Flask Number

Sample Component as a Percentage of Applied Radioactivity

[14C]-DVP 506

Unknown A

(RT ca 7 min)

Unknown B

(RT ca 16 min)

Zero time

1

97.7

ND

ND

Day 2

2

91.9

ND

ND

Day 4

3

89.4

0.7

0.5

Day 8

4

91.2

ND

ND

Day 16

5

91.8

ND

0.5

Day 32

6

94.3

ND

ND

Day 64

7

95.7

ND

ND

Day 120

8

94.1

ND

ND

ND = Not detected

 

Distribution of Total Radioactivity Determined by Radio-HPLC Analysis of

Clay Loam Soil Extracts Following Administration of [14C]-DVP 506

Sampling Interval

Flask Number

Sample Component as a Percentage of Applied Radioactivity

[14C]-DVP 506

Unknown A

(RT ca 7 min)

Unknown B

(RT ca 16 min)

Zero time

20

98.0

ND

ND

Day 2

21

91.7

1.3

ND

Day 4

22

89.4

0.8

ND

Day 8

23

89.6

0.8

ND

Day 16

24

88.5

1.1

ND

Day 32

25

91.0

ND

ND

Day 64

26

93.9

ND

ND

Day 120

27

91.9

1.7

ND

ND = Not detected

 

TLC Characterisation of Radioactivity Extracted from Sandy Loam Soil

Following Administration of [14C]-DVP 506

Sampling Interval

Flask Number

Component as a Percentage of Applied Radioactivity

[14C]-DVP 506

Unknown 1

Origin Bound

Zero time

1

97.1

0.4

0.2

Day 2

2

92.0

ND

ND

Day 4

3

90.5

ND

ND

Day 8

4

91.2

ND

ND

Day 16

5

92.3

ND

ND

Day 32

6

94.3

ND

ND

Day 64

7

95.7

ND

ND

Day 120

8

94.1

ND

ND

ND = Not detected

 

TLC Characterisation of Radioactivity Extracted from Clay Loam Soil

Following Administration of [14C]-DVP 506

Sampling Interval

Flask Number

Component as a Percentage of Applied Radioactivity

[14C]-DVP 506

Unknown 1

Origin Bound

Zero time

20

97.4

0.6

ND

Day 2

21

93.0

ND

ND

Day 4

22

90.1

ND

ND

Day 8

23

89.6

ND

ND

Day 16

24

89.5

ND

ND

Day 32

25

91.0

ND

ND

Day 64

26

93.9

ND

ND

Day 120

27

93.4

ND

0.1

ND = Not detected

Applicant's summary and conclusion

Conclusions:
As there was no evidence of significant degradation of [14C]-DVP 506 in soil, it was therefore not possible to calculate a value for the DT50 or DT90 of [14C]-DVP 506.
Executive summary:

The objective of this study was to investigate the route and rate of degradation of [14C]-DVP 506, under aerobic conditions following application to 2 fresh field soils.

[14C]-DVP 506 was applied to samples (ca 50 g oven dry weight equivalent) of sandy loam and clay loam at a concentration of ca 16.8 mg.kg-1(of soil at 45% of maximum water holding capacity), equivalent to the predicted environmental concentration (PEC). Samples were incubated in the dark at a nominal temperature of 20±2°C for up to 120 days. Ethanediol and monoethanolamine traps were used to collect any evolved [14C]-volatile components including14CO2. At each sampling interval soils were extracted with methanol and the total radioactivity in each extract determined by liquid scintillation counting. The amount of non-solvent extractable radioactivity remaining in the sandy loam soil residue was also determined.

The total recoveries of applied radioactivity (mass balance) from sandy loam soil were quantitative (ca 95-103%) with greater than 90% of the applied radioactivity recovered from the methanol extracts (90.5-97.7%). The amount of radioactivity remaining in sandy loam soil residues accounted from between ca 4%-6% of the applied radioactivity. Mass balances were not performed on the clay loam soil, but methanol extracts recovered between ca 90-98% of the applied radioactivity, similar to the sandy loam soil. No significant amounts of14CO2or14C-organic volatiles were recovered from either soil type.

Chromatographic analysis of soil extracts revealed only one major component, [14C]-DVP 506 accounting for between ca 89-98% in both sandy loam and clay loam soils. In occasional extracts, very minor amounts (<2%) of 2 further unknown components were detected.

As there was no evidence of significant degradation of [14C]-DVP 506 in soil, it was therefore not possible to calculate a value for the DT50or DT90of [14C]-DVP 506.