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Environmental fate & pathways

Biodegradation in soil

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Reference
Endpoint:
biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 June 1999 - 30 June 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
other: OECD II A 7.1.1
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Guidelines 161-2 and 163-1
Deviations:
no
GLP compliance:
yes
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
not specified
Soil no.:
#1
Soil type:
other: Silt loam, Commerce, Washington MS (M554)
% Clay:
13.6
% Silt:
56
% Sand:
30.4
% Org. C:
0.63
pH:
6.5
CEC:
9.74
Soil no.:
#2
Soil type:
other: Loam, Montmoreci, Benton IN (M555)
% Clay:
21.6
% Silt:
50
% Sand:
28.4
% Org. C:
1.1
pH:
5.2
CEC:
10.4
Soil no.:
#3
Soil type:
other: Silty clay loam, Yolo, Yolo CA (M556)
Details on soil characteristics:
SOIL COLLECTION AND STORAGE
- Storage conditions: Soils were stored at approximately 4 °C in a polyethylene bag stored within a fibre pack.
- Soil preparation (e.g., 2 mm sieved; air dried etc.): The soils were screened through a 2 mm sieve

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm (%): Silt loam: 18.55; Loam: 23.03; and Silty clay loam: 27.76

Approximately 50 g (oven dry weight equivalent) moist soil was weighed into the wide-mouth portion of biometer flasks. Enough distilled, deionised water was added to the soil to bring the moisture content to 75 % of 1/3 bar (field capacity) moisture. With the exception of the Day 0 samples, NaOH trapping solution (0.2 N, 100 mL) was added to the side-arms of the biometer flasks to trap CO₂. Soil flasks were sealed with greased stoppers, and side-arm flasks were connected to oxygen manifolds in a darkened incubator (25 °C) via expansion bulbs. Connection to the O₂ manifold with slight positive pressure provided a supply of oxygen to the samples throughout the study, ensuring continued aerobicity. The flasks were incubated in the dark at 25 °C for approximately one week before application, and then until the sampling date.
Soil No.:
#1
Duration:
365 d
Soil No.:
#2
Duration:
365 d
Soil No.:
#3
Duration:
365 d
Soil No.:
#1
Initial conc.:
0.25 ppm
Based on:
test mat.
Soil No.:
#2
Initial conc.:
0.25 ppm
Based on:
test mat.
Soil No.:
#3
Initial conc.:
0.25 ppm
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on experimental conditions:
PREPARATION AND APPLICATION OF THE TEST MATERIAL
- Preparation
Approximately one week prior to application, each radiolabelled test material (41 µCi each) was received in a separate volumetric flask (5.0 mL) and diluted to volume with acetone. Radioactive dichlorobenzoic acid (DCBA) was used to test the microbial viability of the soil at various time-points throughout the incubation period of the soils used for this study by monitoring the mineralisation of ¹⁴C-DCBA to ¹⁴C O₂.The ¹⁴C-DCBA (19 µCi) was also diluted with acetone in a volumetric flask (2.0 mL). Three aliquots (0.01 mL) were removed from each test material for LSC analysis to determine concentration.

- Application
The biometer soil samples were removed from the incubator and dosed with either the ¹⁴C-aniline (100 µL) or ¹⁴C-benzoyl (100 µL). Two direct spikes (100 µL) were taken during treatment of each soil type, for a total of six per radiolabel, to assess application rate. The target application rate was 0.25 ppm (µg/g). The dosed samples were returned to the pre-incubation conditions. Due to the inability of the liquid scintillation counter to accurately measure the amount of radioactivity in the direct spikes, the applied radioactivity (AR) was determined by summing the amount of radioactivity recovered in the samples analysed on the day of application.

At five time-points throughout the study, one biometer flask containing each soil type was treated with ¹⁴C-DCBA (50 or 80 µL). Three direct spikes (50 or 80 µL) were taken to determine application rate for each soil treatment. The target application rate was 0.25 ppm. The soil samples were weighed at the same time as those spiked with radiolabelled test material and incubated with the treated samples prior to dosing with ¹⁴C-DCBA.

SAMPLE COLLECTION AND ANALYSIS
- Sample Collection
Samples were taken at 0 days after treatment (DAT), 14 DAT, and 1, 2, 4, 6, 9, and 12 months after treatment. At least one biometer flask for each soil type and each label was analysed at each sampling date. The initial analysis, through the acidic organic extraction, was typically completed on the date of sampling.

- Caustic Trap
After removing the samples from the incubator, an aliquot of the caustic trapping solution (approximately 20 mL) was removed by aspiration for analysis. Two aliquots (1 mL) of the trapping solution were analysed by LSC to determine the extent of mineralisation to ¹⁴CO₂. When greater than 10 % of the applied radioactivity was detected in the caustic trap, the presence of ¹⁴CO₂ was confirmed on an aliquot of the trapping solution by precipitation with BaCl₂ and Na₂CO₃. This procedure was conducted by mixing caustic sample (4 mL) with saturated BaCl₂ (4 mL) and adding saturated Na₂CO₃ (2 mL). After centrifugation, duplicate aliquots (1 mL) of the supernatant were analysed by LSC. In caustic, the ¹⁴CO₂ is converted to ¹⁴CO₃^-². The BaCl₂ precipitates the ¹⁴CO₃^-² from solution as BaCO₃ and the Na₂CO₃ increases precipitation due to its common ion effect.

- Calcium Chloride Extraction
The soil in the biometer flask was transferred to a labelled, tared glass jar. Calcium chloride (0.01 M, approximately 100 mL) was added to each soil sample, usually using the solution to rinse the biometer flask. After quickly breaking up the soil pellet by hand, the samples were shaken on a reciprocal shaker (approximately 180 excursions/minute) for five minutes. The samples were centrifuged and decanted into tared containers. The weight of the calcium chloride extract and post-extracted pellet was recorded. Aliquots were analysed by LSC and HPLC.

- Acidic Organic Extraction
Acidic acetonitrile (acetonitrile/0.1 N HCl, 80:20 v/v, approximately 150 mL) was added to the post-extracted soil. After breaking up the soil pellet by hand, the samples were shaken on low on a reciprocal shaker for 45 minutes, centrifuged, and the extract decanted into separate graduated cylinders. The extraction procedure was repeated two more times using approximately 100 mL extraction solvent. The extracts were pooled and the final volume recorded. Aliquots of the extract were analysed by LSC.
An aliquot of the acidic organic extract (20 - 50 mL) was concentrated to dryness on a rotary evaporator (50 °C water bath). The samples were reconstituted in acetonitrile (2.0 mL) with sonication, and diluted with water (3.0 mL), so the final volume was 5.0 mL. Aliquots (0.1 mL) were analysed by LSC to determine concentration recovery. An additional aliquot was analysed by HPLC.
The nine month samples were further extracted with acetonitrile/1.0 N HCl (80:20 v/v, 50 - 100 mL). The same extraction procedure outlined above was followed. An aliquot of the extract (100 mL) was concentrated on the rotary evaporator to approximately 20 mL, neutralised with NaOH (2N, approximately 8 mL), then concentrated to dryness. The sample was reconstituted in acetonitrile (4.0 mL) and diluted with water (6.0 mL) with sonication. Aliquots of the concentrated extract were analysed by LSC and HPLC.

- Post-extracted Soil Combustions
The post-extracted pellet was allowed to air-dry immediately following the final extraction. Portions of the air-dried pellet were combusted using a biological oxidiser. The results of the combustion analysis were used to determine material balance for each sample.
At two time points, one following the acetonitrile/0.1 N HCI extraction and another following the acetonitrile 1.0 N HCI extraction, the non-extractable residue was characterised further using an extraction that divided the bound material into fulvic, humic, and humin fractions. Fulvic acid is defined as the acid/base soluble portion, while humic acid is soluble in base but not in acid. The humin fraction is the material remaining associated with the soil after extraction with base.
Aliquots of the post-extracted soil (1 or 5 g) were extracted with sodium hydroxide (2 N, 5 or 25 mL), shaking on low for 15 - 20 minutes, centrifuging, and removing the extract. The procedure was repeated, pooling the extract. Aliquots of the extract were analysed by LSC to determine the combined amount of fulvic and humic acids. An aliquot of the sodium hydroxide extract was acidified to pH<2 (concentrated HCI) and allowed to react for approximately 15 minutes to precipitate the humic acid. After centrifugation, aliquots of the acidified extract were analysed by LSC to measure the amount of radioactivity associated with the fulvic acid, and by difference, determine the amount associated with the humic acid. Aliquots of the soil remaining after base extraction were combusted to determine the amount of radioactivity in the humin fraction.
Key result
Soil No.:
#1
DT50:
267 d
Type:
(pseudo-)first order (= half-life)
Temp.:
25 °C
Key result
Soil No.:
#2
DT50:
277 d
Type:
(pseudo-)first order (= half-life)
Temp.:
25 °C
Key result
Soil No.:
#3
DT50:
315 d
Type:
(pseudo-)first order (= half-life)
Temp.:
25 °C
Transformation products:
yes
Remarks:
Two metabolites were identified in the 14C-aniline-Iabelled samples: the urea and amine. Only 14CO2 was identified as a metabolite from the 14C-benzoyl-Iabelled samples.
No.:
#1
Details on results:
APPLICATION SUMMARY
Radiochemical purity of the test material prior to application was determined to be 99.4 and 98.6 % for the ¹⁴C-aniline and ¹⁴C-benzoyl test material, respectively, by HPLC. The specific activity was calculated to be 138 448 and 146 000 dpm/µg for the ¹⁴C-aniline and ¹⁴C-benzoyl test material, respectively. Radiochemical purity of the test materials post-application and through the extraction sequence was demonstrated by recovering an average of 98.0 and 98.9 % from the 0 DAT ¹⁴C-Aniline and ¹⁴C-Benzoyl samples, respectively.
The application rate is summarised in Table 1.

MICROBIAL VIABILITY TESTING OF SOIL
At five time points throughout the study, soil viability was determined by dosing each soil type with ¹⁴C-DCBA in acetone to give a soil concentration of 0.034 - 0.404 ppm. The application rate varied from the target rate of 0.25 ppm due to drying of the application solution and re-dissolving between applications. Caustic solution (100 mL of 0.2 N) was added to the side-arm of the biometer flask and these samples were incubated for 7 days at 25 °C. Evolved ¹⁴CO₂ was measured by counting triplicate 1.0 mL aliquots of the caustic solution. The average ability of the soil microbial population to mineralise 22.2, 24.4, and 8.8 % AR in the silt loam, loam and silty clay loam soils, respectively, indicates a viable microbial population for the duration of the study.

MATERIAL BALANCE AND DISTRIBUTION OF RADIOACTIVITY
- Material Balance
The material balance was based upon the 0 DAT samples rather than the direct spikes collected at application due to inability of the LSC to accurately measure the high amount of radioactivity in the direct spikes.
Instead, material balance was measured by the sum of radioactivity recovered in the caustic traps, in the extracts, and in the post-extracted soil pellet relative to the amount recovered in the samples for the same soil type and radiolabel at 0 DAT. Recoveries ranged from 88 to 106 % with an average recovery of 99.7 ± 3.7 % (Table 2). Two samples had recovery values below 94 %, both of which the containers broke during the organic extraction, lowering the amount of non-extractable residue.

- Radioactive Carbon Dioxide
The amount of radioactivity recovered as ¹⁴CO₂ was low (<5 % AR) for the ¹⁴C-aniline-labelled samples, while 38 - 46 % of the applied radioactivity was recovered in the caustic traps of the ¹⁴C-benzoyl-labelled samples at one year (Table 2). At 30 DAT, aliquots of the caustic traps from the ¹⁴C-benzoyl-labelled loam and silty clay loam soil samples were treated with barium chloride and sodium carbonate to precipitate CO₂. Background radioactive levels remained in solution after precipitation, indicating that all of the radioactivity was associated with ¹⁴CO₂. The procedure was repeated with the 60 DAT benzoyl silt loam soil samples and with the 9 month benzoyl samples from all soil types and >95 % of the radioactivity precipitated.

- Calcium Chloride Extract
Throughout the 1-year sampling period, less than 8, 5, or 4 % AR was extracted with calcium chloride in the silt loam, loam and silty clay loam soils, respectively (Table 2). In the ¹⁴C-benzoyl-labelled samples, the test material was the only component detected. In the ¹⁴C-aniline-labelled samples, the amine degradate was detected in all soil types at up to 3.2 % AR at one year. The urea degradate was identified in only the ¹⁴C-aniline-labelled soil samples, at a maximum level of 0.61 % AR at 185 DAT in the silt loam soil.

- Organic Extracts
In general, a majority of the radioactive residue was extracted with acetonitrile/0.1 N HCl (80:20, v/v). A greater percentage of the total radioactivity was extracted from the aniline samples, generally 70 % at one year (Table 2). Less radioactivity, 30 - 40 %, was extractable from the ¹⁴C-benzoyl-labelled samples at one year. The difference between the two labels is primarily accounted for by the amount of radioactivity in the caustic traps, identified as ¹⁴CO₂.
As with the calcium chloride extracts, the ¹⁴C-benzoyl-labelled samples contained primarily the test material and no metabolites or solvent-front radioactivity exceeding 2.1 % AR. The ¹⁴C-aniline-labelled samples contained only the urea and amine metabolites. The urea metabolite was formed in approximately one month. In the silt loam soil, the urea degradate reached a maximum level of 6.1 % AR at 128 DAT, declined slightly, and accounted for 5.6 % AR at one year. In the loam soil, the urea metabolite accounted for 6.0 % AR at 30 DAT, began to decline, then was detected at 5.2 % AR at 274 DAT and 53 % AR at one year. The maximum amount of urea metabolite in the silty clay loam soil was 8.7 % AR at 30 DAT, then declined to 4.0 % AR by one year.
The amine metabolite was not detected in any of the soil types until the 58 DAT sample. The levels of the amine metabolite generally increased throughout the sampling period, reaching a maximum of 11 % AR at 274 DAT, 11 % AR at 365 DAT, and 13 % AR at 185 DAT, in the silt loam, loam and silty clay loam soils, respectively.
The nine-month samples were further extracted with acetonitrile/1.0 N HCl (80:20, v/v), which removed an additional 5 - 7 % AR from the ¹⁴C-aniline-labelled samples, and 1.5 - 2.5 % AR from the ¹⁴C-benzoyl-labelled samples (included in the values presented in Table 2). The aniline-labelled extracts were concentrated and analysed by HPLC. Although HPLC recoveries were low, the radioactivity eluted with the test material (2 % AR, loam soil) and the amine metabolite (5.2 % AR, silt loam soil). Therefore, extraction with acetonitrile/0.1 N HCl (80:20, v/v) was sufficient to remove the extractable test material and metabolites.

- Non-Extractable Residue
The non-extractable residues generally increased throughout the sampling period, to approximately 15 - 20 % in the ¹⁴C-benzoyl-labelled samples, and approximately 25 % in the ¹⁴C-aniline-labelled samples (Table 2). At 6 and 9 months, aliquots of the post-extracted soil were subjected to the bound residue determinations of humin, humic, and fulvic components. The results vary (Table 3), since the 9 month samples were extracted with acetonitrile/0.1 N HCl (80:20, v/v) then acetonitrile/1.0 N HCl (80:20, v/v) prior to bound residue analysis. Aside from one sample (9 month, silt loam Aniline), recoveries from these analyses were acceptable. The six-month samples showed that 40 - 83 % of the non-extractable residue was associated with humin. A lesser amount of radioactivity, 8 - 48 % was associated with fulvic acid, and less than 6 % was associated with humic acid. There were not significant differences between the two radiolabels. The nine-month sample results were similar, with 40 - 89 % of the radioactive residue generally associated with the humin fraction, 7 - 33 % associated with the fulvic acid, and 4 - 32 % associated with the humic acid. However, the stronger acidic extraction removed additional radioactivity, so that the proportions in each fraction varied from the six-month sample results.

KINETICS
The total levels of the test material and metabolites detected in each sample are summarised in Table 4. These levels represent the amount detected in both the calcium chloride and organic extracts. The urea metabolite never exceeded 9 % AR radioactivity in any soil, while the amine metabolite reached a maximum of 13.9 % AR in the silt loam soil. Degradation rates of metabolites were not able to be calculated since there was no consistent decline observed over the length of the study.
The degradation of the test material on each soil was evaluated assuming first-order kinetics. Half-life values ranged from 267 days on the Mississippi soil (silt loam) to 277 days on the Indiana soil (loam) and 315 days on the California soil (silty clay loam). Although there was variability in the data, for each soil type the data were scattered approximately equally above and below the trendline, indicating that a first-order model appropriately describes the degradation. Therefore, biphasic kinetic models were not evaluated.

AGED MOBILITY
The apparent (non-equilibrium) Kd for the test material parent was calculated to determine relative mobility information in-situ. Apparent Kd values were calculated assuming that the material in the calcium chloride extract had been present in the soil solution, whereas the material removed with the subsequent organic extraction represented the material that had been sorbed to the soil matrix. Only extractable material was used to determine apparent sorption coefficients; non-extractable material was assumed to be chemically distinct from the test material or any of the metabolites. Apparent Kd values therefore represent the actual partitioning of a chemical in soil at a given time. Apparent (non-equilibrium) Kd values differ from equilibrium Kd values which describe the partitioning of a chemical in soil after equilibrium has been achieved.

The observed apparent Kd values ranged from 25.9 to 265.5 mL/g, and averaged 96.3 mL/g for all three soil types (Table 5). There was not a correlation between apparent Kd and incubation time (r²(linear) = 034, 0.03, 0.03, for the silt loam, loam and silty clay loam soils, respectively). These Kd values indicate a low potential for leaching. Koc values ranged from 2357 to 21 940 mL/g, and averaged 10 199 mL/g for all three soil types (Table 5).
Mobility potentials for metabolites were calculated only for samples where it was detected in both phases. The apparent Kd values for the metabolites were comparable to slightly lower than for the test material, suggesting that the metabolites are immobile. The average apparent Kd values for urea and amine metabolites were 19.5 and 39.0 mL/g, respectively, with corresponding Koc values of 2478 and 3 983 mL/g. Therefore, the sorption coefficients for the test material and the urea and amine metabolites indicate that they will not be mobile in soil and are unlikely to pose a risk to groundwater.

METABOLITE CONFIRMATION
The presence of parent test material and the urea and amine metabolites were confirmed in the six-month aniline samples using normal-phase TLC.

DEGRADATION PATHWAY
The proposed metabolic pathway proceeds through the previously identified degradates. There is clear indication of bridge cleavage during aerobic soil metabolism. While the urea and amine were identified, the benzoyl portion of the molecule was not detected, and was presumed to be rapidly degraded to ¹⁴CO₂.

Table 1: Application Rate

Soil Type

Radiolabel

Total radioactivity Recovered* (dpm)

Application rate** (ppm)

Silt loam

Aniline

1 665 405.77

0.228

Benzoyl

1 686 088.27

0.243

Loam

Aniline

1 702 796.85

0.233

Benzoyl

1 686 403.85

0.243

Silty clay loam

Aniline

1 729 977.42

0.237

Benzoyl

1 723 625.44

0.249

* Total radioactivity recovered in calcium chloride extract, organic extract and post-extracted pellets of 0 DAT samples

** Application rate determined by subdividing direct spike samples into 4 subsamples, and summing radioactivity in each subsample were 0.215 and 0.223 ppm for the ¹⁴C-aniline and ¹⁴C-benzoyl, respectively.

 

Table 2: Radioactivity Distribution

Label

Day

% AR Values

Silt Loam

Loam

Silty Clay Loam

CO₂ Trap

CaCl₂ Ext.

Organic Ext.

Non-ext.

Total

CO₂ Trap

CaCl₂ Ext.

Organic Ext.

Non-ext.

Total

CO₂ Trap

CaCl₂ Ext.

Organic Ext.

Non-ext.

Total

¹⁴C-Aniline

0

NA

3.5

96.0

0.4

100

NA

2.9

96.8

0.3

100

NA

1.1

98.4

0.6

100

14

0.1

7.0

93.9

1.1

102

0.1

2.9

99.3

1.3

104

0.1

2.3

96.9

1.4

101

30

0.2

7.6

96.1

2.1

106

0.3

2.6

97.0

4.9

105

0.2

1.7

96.9

4.2

103

58

0.4

5.8

92.7

4.6

103

0.7

4.7

83.4

12.5

101

0.5

3.8

94.3

7.3

106

128

1.3

2.2

83.1

15.0

102

-1.4

2.3

80.7

15.2

99.5

1.0

1.2

82.7

13.9

98.8

128†

1.4

2.2

83.7

12.1

99.4

-

-

-

-

-

1.4

1.7

75.4

19.0

97.6

185

1.9

3.6

84.6

13.8

104

2.7

2.0

71.4

22.9

99.0

1.2

2.1

81.8

14.6

99.7

275

3.1

2.2

85.4*

7.0

97.7

3.5

3.7

79.6*

13.9

101

2.1

3.0

78.4*

11.7

95.2

365

4.2

5.4

69.1

26.9

106

4.6

4.1

68.0

26.7

103

2.6

1.8

72.8

25.8

103

¹⁴C-Benzoyl

0

NA

3.3

96.4

0.4

100

NA

1.2

98.4

0.3

100

NA

1.1

98.5

0.5

100

14

1.0

7.7

89.1

1.5

99.3

3.1

2.5

90.2

4.0

99.8

2.3

3.3

89.5

5.1

100

30

3.4

4.3

92.0

3.8

103

8.1

2.7

86.5

7.9

105

12.2

1.9

72.7

13.8

101

58

8.9

6.6

80.1

6.1

102

15.6

5.0

66.5

11.6

98.7

19.2

2.8

58.8

17.6

98.4

128

20.4

1.3

63.7

11.4

96.8

24.5

0.9

57.9

14.1

97.4

26.1

1.2

47.8

19.6

94.7

128†

22.7

1.0

61.0

13.9

98.6

24.8

1.3

57.3

14.5

97.9

28.7

1.2

44.3

20.4

94.5

185

24.3

0.8

59.2

11.6

95.9

27.3

1.2

54.5

14.0

96.9

27.5

1.1

43.2

16.5

88.2

275

34.9

0.9

51.1*

8.3

95.2

35.1

1.9

50.9*

13.0

101

28.6

1.2

57.4*

14.1

101

365

45.7

1.7

33.4

16.7

97.4

38.2

1.7

38.6

17.7

96.2

39.5

0.7

32.9

15.5

88.5

Average

SD

 

 

 

 

100.4

3.2

 

 

 

 

100.3

2.7

 

 

 

 

98.3

4.6

Ext. - Extracted

† For the silty clay loam soil, this measurement was taken on day 185

* The 275 DAT samples were sequentially extracted with 80:20 acetonitrile/0.1 N HCl then 80:20 acetonitrile/1.0 N HCl and the sum is reported

 

Table 3: Bound Residue Analysis of Post-extracted Soil

Soil Type

Sample Identification

Fulvic (%)

Humic (%)

Humin (%)

Recovery (%)

6 Month Samples

Silt loam

Aniline 1

Aniline 2

22.8

19.7

2.3

2.6

82.6

59.3

108

82

Benzoyl 1

Benzoyl 2

31.1

35.7

5.4

3.0

52.2

66.7

89

105

Loam

Aniline 1

Aniline 2

28.0

26.6

0.2

2.5

68.2

61.6

96

91

Benzoyl 1

Benzoyl 2

45.7

48.3

2.2

5.6

47.2

39.8

95

94

Silty clay loam

Aniline 1

Aniline 2

11.1

8.4

0.9

4.8

82.7

80.2

95

93

Benzoyl 1

Benzoyl 2

21.2

22.9

4.3

0.8

77.7

63.7

103

87

9 Month Samples

Silt loam

Aniline

29.6

31.5

129

191

Benzoyl

32.5

7.6

53.8

94

Loam

Aniline

16.4

21.0

59.2

97

Benzoyl

32.8

12.4

42.5

88

Silty clay loam

Aniline

7.2

4.4

88.9

101

Benzoyl

17.1

6.1

76.8

100

 

Table 4: Total Test Material and Metabolite levels

Day

Silt Loam

Loam

Silty Clay Loam

¹⁴C-Benzoyl

¹⁴C-Aniline

Urea*

Amine*

¹⁴C-Benzoyl

¹⁴C-Aniline

Urea*

Amine*

¹⁴C-Benzoyl

¹⁴C-Aniline

Urea*

Amine*

0

99.6

99.6

ND

ND

94.9

97.6

ND

ND

99.5

99.4

ND

ND

14

96.7

99.8

ND

ND

94.3

96.2

4.0

ND

91.8

95.8

3.2

ND

30

96.2

99.6

0.4

0.5

90.9

91.9

6.0

ND

74.2

88.4

8.7

0.2

58

86.7

91.8

3.0

3.3

70.3

77.8

4.8

5.3

61.3

83.1

8.2

5.8

128

63.9

66.1

6.6

9.2

56.4

70.6

3.3

6.5

47.8

64.9

6.0

11.3

128†

59.6

70.3

4.5

7.5

55.7

-

-

-

44.8

55.2

6.2

13.8

185

58.8

72.0

3.4

8.0

53.2

58.5

3.6

8.5

43.5

64.7

4.7

13.7

275

46.3

60.0

3.0

11.6

48.4

36.2

5.2

6.9

53.2

59.1

3.4

10.5

365

30.3

48.3

5.6

13.9

36.7

48.8

5.8

11.0

31.8

54.4

4.5

10.7

† For the silty clay loam soil, this measurement was taken on day 185

* ¹⁴C-aniline labelled

 

Table 5: Apparent Kd and Koc Values for the Test Material

Time (DAT)

Radiolabel

Silt Loam

Loam

Silty Clay Loam

Kd (mL/g)

Koc (mL/g)

Kd (mL/g)

Koc (mL/g)

Kd (mL/g)

Koc (mL/g)

0

Aniline

58.3

9260

72.1

6558

202.9

16 771

Benzoyl

65.4

10 381

169.5

15 406

196.8

16 261

14

Aniline

27.8

4408

74.8

6803

97.5

8060

Benzoyl

25.3

4009

83.2

7565

83.9

6930

30

Aniline

29.6

4699

84.5

7681

133.1

10 988

Benzoyl

45.7

7258

75.1

6829

106.6

8814

58

Aniline

35.8

5684

40.2

3654

52.7

4352

Benzoyl

25.7

4073

29.0

2640

50.0

4130

128

Aniline

112.4

117.8

17 847

18 697

130.3

-

11 845

-

158.0

-

13 059

-

Benzoyl

104.6

129.8

16 596

20 603

133.6

93.7

12 148

8516

93.1

-

7698

-

185

Aniline

69.3

-

11 004

-

124.5

-

11 322

-

150.1

161.1

12 404

13 312

Benzoyl

156.9

-

24 899

-

139.8

-

12 706

-

83.8

112.7

6926

9315

1274

Aniline

97.9

15 536

25.9

2357

71.6

5919

Benzoyl

117.5

18 643

51.5

4686

92.4

7635

365

Aniline

95.5

15 152

44.8

4068

265.5

21 940

Benzoyl

84.9

13 478

99.0

9002

120.9

9992

Average

 

77.8

12 346

86.6

7870

124.0

10 251

Average for all 3 soils: Kd: 96.3 mL/g; Koc: 10 199 mL/g

Conclusions:
The half-life of the test material in soil under aerobic conditions ranged from 267 to 315 days.
Executive summary:

The degradation of the test material in soil was investigated in accordance with the standardised guidelines PA 161-2 and 163-1 and OECD II 7.1.1 under GLP conditions.

Degradation of ¹⁴C-aniline and ¹⁴C-benzoyl labelled test material was studied on three U. S. soils: Commerce silt loam from Mississippi, Montmorenci silt loam from Indiana, and Yolo silty clay loam from California. The test material was applied to moist soil (50 g oven dry weight equivalent) at an approximate rate of 0.25 ppm. Samples were incubated in the dark at 25 °C and 75 % of 1/3 bar (field capacity) moisture. Samples were analysed at eight time points with incubation periods between 0 and 365 days.

First order half-life values ranged from 267 to 315 days. Two metabolites approached or exceeded 10 % of applied radioactivity, the urea, and the amine. Both metabolites were observed only from the degradation of ¹⁴C-aniline test material. Significant amounts of ¹⁴CO₂ were recovered from only the ¹⁴C-benzoyl test material treated soils, reaching 38 - 46 % of applied radioactivity at one year. Bound radioactive residues increased throughout the incubation period, reaching 15 - 27 % by one year, and were qualitatively determined to be associated with humin, humic acid and fulvic acid, and therefore did not contain additional amounts of parent test material or metabolites. Aged mobility data of the test material and its metabolites were also generated. Apparent (non-equilibrium) Kd values for the parent ranged from 25 to 266 mL/g and averaged 96 mL/g. Metabolite apparent Kd values were slightly lower than parent values. Observed partition coefficients for the test material and metabolites did not change over time.

In conclusion, the half-life of the test material in soil under aerobic conditions ranged from 267 to 315 days. Two metabolites were identified in the ¹⁴C-aniline-labelled samples: the urea and amine. Only ¹⁴CO₂ was identified as a metabolite from the ¹⁴C-benzoyl-labelled samples. Bound residues increased steadily and were qualitatively characterized as humin, humic acid, and fulvic acid, and therefore did not contain additional amounts of parent test material or metabolites. The test material is not considered to be at risk for leaching due to its high apparent Kd (96 mL/g) and low water solubility.

Description of key information

The half-life of the test material in soil under aerobic conditions ranged from 267 to 315 days.

Key value for chemical safety assessment

Half-life in soil:
315 d
at the temperature of:
25 °C

Additional information

The degradation of the test material in soil was investigated in accordance with the standardised guidelines PA 161-2 and 163-1 and OECD II 7.1.1 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Degradation of ¹⁴C-aniline and ¹⁴C-benzoyl labelled test material was studied on three U. S. soils: Commerce silt loam from Mississippi, Montmorenci silt loam from Indiana, and Yolo silty clay loam from California. The test material was applied to moist soil (50 g oven dry weight equivalent) at an approximate rate of 0.25 ppm. Samples were incubated in the dark at 25 °C and 75 % of 1/3 bar (field capacity) moisture. Samples were analysed at eight time points with incubation periods between 0 and 365 days.

First order half-life values ranged from 267 to 315 days. Two metabolites approached or exceeded 10 % of applied radioactivity, the urea, and the amine. Both metabolites were observed only from the degradation of ¹⁴C-aniline test material. Significant amounts of ¹⁴CO₂ were recovered from only the ¹⁴C-benzoyl test material treated soils, reaching 38 - 46 % of applied radioactivity at one year. Bound radioactive residues increased throughout the incubation period, reaching 15 - 27 % by one year, and were qualitatively determined to be associated with humin, humic acid and fulvic acid, and therefore did not contain additional amounts of parent test material or metabolites. Aged mobility data of the test material and its metabolites were also generated. Apparent (non-equilibrium) Kd values for the parent ranged from 25 to 266 mL/g and averaged 96 mL/g. Metabolite apparent Kd values were slightly lower than parent values. Observed partition coefficients for the test material and metabolites did not change over time.

In conclusion, the half-life of the test material in soil under aerobic conditions ranged from 267 to 315 days. Two metabolites were identified in the ¹⁴C-aniline-labelled samples: the urea and amine. Only ¹⁴CO₂ was identified as a metabolite from the ¹⁴C-benzoyl-labelled samples. Bound residues increased steadily and were qualitatively characterized as humin, humic acid, and fulvic acid, and therefore did not contain additional amounts of parent test material or metabolites. The test material is not considered to be at risk for leaching due to its high apparent Kd (96 mL/g) and low water solubility.