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Environmental fate & pathways

Biodegradation in soil

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Reference
Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to a well-documented method, not in compliance with GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
other: Environmental Assessment Technical Assistance Document 3.12, Aerobic biodegradation in soil. US Food and Drug Administration, Washington DC, PB87-175345, 1987.
Principles of method if other than guideline:
Degradability of radiolabeled test substance was followed in laboratory microcosms simulating sludge-amended soil, agricultural soil and river sediment.
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
not specified
Soil no.:
#1
Soil type:
other: Sludge amended soil
pH:
7
Soil no.:
#2
Soil type:
other: Agricultural soil
pH:
6.6
Details on soil characteristics:
SOIL COLLECTION AND STORAGE
- Geographic location:
#1. Sludge amended snely oil obtained from a farm in southern New Jersey, USA that routinely applies sludge from a local domestic wastewater treatment lant to its farmland
#2. Agricultural soil (not sludge amended) obtained from a farm in central New Jersey, USA.

Initial Characterisation soil #1 - Sludge amended soil:
PO4 as P : 3.3 mg/kg
NH3 as N: 26 mg/kg
Water holding capacity: 25.7%
% solids: 79.6%
pH initial: 7.0

Initial Characterisation soil #2 - Agricultural soil:
PO4 as P : <2.0 mg/kg
NH3 as N: 23 mg/kg
Water holding capacity: 29 %
% solids: 82.7%
pH initial: 6.6
Soil No.:
#1
Duration:
12 wk
Soil No.:
#2
Duration:
12 wk
Soil No.:
#1
Initial conc.:
10 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#1
Initial conc.:
0.2 other: µCi per gram soil dw
Based on:
other: radioacitivity
Soil No.:
#2
Initial conc.:
10 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#2
Initial conc.:
0.2 other: µCi per gram soil dw
Based on:
other: radioacitivity
Parameter followed for biodegradation estimation:
radiochem. meas.
test mat. analysis
Soil No.:
#1
Temp.:
22 -23 °C
Humidity:
75% of water holding capacity
Soil No.:
#2
Temp.:
22 -23 °C
Humidity:
75% of water holding capacity
Details on experimental conditions:
The fate of 14C-OTNE in soil was studied in microcosms according to protocols described in documents from the U.S. Food and Drug Administration of 1987. Sealed flasks with the two different soils were spiked with 10 µg test substance/g sediment dw (10 mg/kg) and incubated at laboratory ambient temperature (22-23 ºC) for 12 weeks. Periodically the headspace was flushed for oxygen replenishment and the effluent gas was drawn through a train of scintillation fluids to capture volatile organics and CO2 to be determined by liquid scintilation counting. Periodically also flasks were sacrificed and exhaustively extracted with hexane/acetone. An aliquot of the solvent fraction was used for thin layer chromatography for analysis of the test substance and metabolites including CO2.

TEST SYSTEM AND TEST CONDITIONS
- Test system: 34 air-tight serum vials (160 ml) with 20 g soil and 3 screw top Erlenmeyer flasks (250 ml) with 50 g of soil (dry weight).
- Composition of medium: soil and distilled water, to 75% water holding capacity
- Additional substrate: no additional nutrients were supplied
- Test temperature: 22-23ºC
- pH: 6.7 (initial)
- pH adjusted: no
- Aeration of dilution water: headspace air refreshment during sampling times
- Continuous darkness

- Details of trap for CO2 and volatile organics if used:
universal scintillation fluid (Liquiscint) to trap volatile organic metabolites;
phenethylamine-containing scintillation fluid (14C-Oxosol) for CO2.
Measurements by Pharmacia LKB 1209 Rackbeta liquid scintillation counter.

SAMPLING
- Sampling frequency: day 0, week 1, 2, 3, 4, 6, 8, 12.
- Sampling method: low vacuum on system with air-tight serum vials to sequential traps with specific scintillation fluid for organic metabolites and CO2.

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: six serum vials were autoclaved for 1 hour and hen amended with mercuric chloride (500 mg/kg final concentration)
Soil No.:
#1
% Recovery:
85
Remarks on result:
other: Recovery week 3 - 6 - 8 - 12 was 85 - 85 - 83 - and 78%. For week 12 the extraction of the base fraction was 2 hours instead of overnight, likely showng an underestimate. Recovery in killed control was 108%.
Soil No.:
#2
% Recovery:
89
Remarks on result:
other: Recovery week 3 - 6 - 12 was 95 - 87 - 89 %. Recovery in killed control was 96%.
Soil No.:
#1
% Degr.:
77
Parameter:
test mat. analysis
Sampling time:
3 wk
Soil No.:
#1
% Degr.:
97.9
Parameter:
test mat. analysis
Sampling time:
6 wk
Soil No.:
#1
% Degr.:
99.7
Parameter:
test mat. analysis
Sampling time:
12 wk
Soil No.:
#1
% Degr.:
20.6
Parameter:
CO2 evolution
Sampling time:
3 wk
Soil No.:
#1
% Degr.:
48.4
Parameter:
CO2 evolution
Sampling time:
6 wk
Soil No.:
#1
% Degr.:
55.2
Parameter:
CO2 evolution
Sampling time:
8 wk
Soil No.:
#1
% Degr.:
61.7
Parameter:
CO2 evolution
Sampling time:
12 wk
Soil No.:
#2
% Degr.:
72
Parameter:
test mat. analysis
Sampling time:
3 wk
Soil No.:
#2
% Degr.:
98.9
Parameter:
test mat. analysis
Sampling time:
6 wk
Soil No.:
#2
% Degr.:
99.8
Parameter:
test mat. analysis
Sampling time:
12 wk
Soil No.:
#2
% Degr.:
30.7
Parameter:
CO2 evolution
Sampling time:
3 wk
Soil No.:
#2
% Degr.:
56.9
Parameter:
CO2 evolution
Sampling time:
6 wk
Soil No.:
#2
% Degr.:
67.4
Parameter:
CO2 evolution
Sampling time:
12 wk
Soil No.:
#1
DT50:
4.2 d
Type:
other: non-linear regression analysis
Remarks on result:
other: using the amount of parent substance remaining at each time point
Key result
Soil No.:
#2
DT50:
6 d
Type:
other: non-linear regression analysis
Remarks on result:
other: using the amount of parent substance remaining at each time point
Transformation products:
yes
Details on transformation products:
Results from TLC analyses on the soxhlet-extractable metabolites show that the parent migrated to 'region 5' whereas more polar metabolites were distributed in regions 1 to 4. During the first three weeks a significant portion of the total radioactivity was associated with these more polar metabolites. The majority migrated with the regions 3 and 4 (Rf = 0.38 - 0.56 and Rf 0.56 - 0.75, respectively) which is significantly separated from that of the parent in region 5 (Rf = 0.75 - 1.0).
In week 4 the percent total radioactivity associated with the polar metabolites began to decrease, indicating that the metabolites represent transient intermediates that can be further degraded into CO2, water soluble products and humic-bound products. The total percent of radioactivity recovered from the samples by soxhlet extraction in week 4, 8 and 12 decreased to 33, 5.3 and 5.3% in soil #1 and to 27, 3.6 and 3.8 % in soil #2, respectively. After week 12 only 3.0 % (#1) and 2.2% (#2) was recovered as the parent.
For the killed controls, the majority of the radioactivity migrated with the parent ("region 5"). The week 4 Killed control showed 94% (soil #1 and 104% (soil #2) of total radioactivity associated withthe parent whereas this was 79 % (#1) and 78% (#32) in week 12. This shows that there was very little abiotic loss of the parent substance over the 12-week time course.
Details on results:
Voltile organic compounds were trapped in specific scintillation fluids. However, the results do not suggest that they were detected as significant levels.
The total 14C-mass balance for the system was circa 83 - 89% for the two soils. The production of CO2 show that 62 % (soil #1) and 67 % (soil #2) of the total 14C-labeled parent substance was converted to CO2 after 12 weeks. After a lag period of approx. 7 days, the initial CO2 production rate was 1.4 % CO2 /day for the sludge amended soil (#1) and 1.8 % CO2 / day for the agricultural soil (#2). The rate of CO2 production began to decrease by day 42 for soil #1 and by day 35 for soil #2. The rapid CO2 evolution showed that microorganisms capable of mineralising the test substance are indigenous to the soil matrices, indicating that common soil micro-organisms are capable of mineralising OTNE. No nutrients were added, no pH correction was made and incubation was under aerobic conditions without any mixing.
Results with reference substance:
not included

14C-Mass balance analysis of test substance incubated in sludge amended agricultural soil (#1)

Measurement

Time of analysis

 

 

Killed – week (8 & 12)

Week 3

Week 6

Week 8

Week 12

Starting Parent substance added (µg)

200

200

200

200

200

Final Parent substance measured in microcosm (µg)

165

45

4.2

NA

0.55

%Parent substance remaining

83%

23%

2.1%

NA

0.28%

 

 

 

 

 

 

14-C recovered as CO2

0%

20.6%

48.4%

55.2%

61.7%

14-C recovered in aqueous fraction

5.1%

14.0%

8.2%

7.9%

4.4%

14-C recovered in methanol extraction

44.8%

17.2%

6.1%

4.1%

2.2%

14C-recovered in 2ndorganic extraction (hexane/acetone)

56.7%

26.9%

8.1%

2.6%

3.7%

14-c recovered in base extraction (humic fraction)

1.1%

6.3%

13.9%

13.2%

5.7%

TOTAL 14-C recovered

108%

85%

85%

83%

78%

 

 

 

 

 

 

14C-Mass balance analysis of test substance incubated in agricultural soil (#2)

Measurement

Time of analysis

 

Killed – week (8 & 12)

Week 3

Week 6

Week 12

Starting Parent substance added (µg)

200

200

200

200

Final Parent substance measured in microcosm (µg)

164

55

2.2

0.35

%Parent substance remaining

82%

28%

1.1%

0.28%

 

 

 

 

 

14-C recovered as CO2

0%

30.7%

56.9%

67.4%

14-C recovered in aqueous fraction

8.9%

15.8%

4.3%

1.8%

14-C recovered in methanol extraction

58.1%

22.2%

4.9%

2.4%

14C-recovered in 2ndorganic extraction (hexane/acetone)

25.4%

7.9%

2.3%

1.3%

14-c recovered in base extraction (humic fraction)

3.5%

18.1%

18.3%

16.0%

TOTAL 14-C recovered

96%

95%

87%

89%

Conclusions:
Radiolabeled OTNE in soil is almost completely degraded after 6 weeks. After 6 weeks the mineralisation (CO2 evolution) is circa 50%. After 1 to 2 weeks a range of more polar metabolites was found. After a lag time of approx. 7 days the initial rate of CO2 production was 1.4 - 1.8% /day in the sludge amended soil and in the agricultural soil, respectively. The half-life of the parent substance was estimated at 6.0 and 4.2 days in the sludge amended soil and in the agricultural soil, respectively.
Executive summary:

The fate of14C-OTNE in agricultural soils was studied in microcosms according to protocols described in documents from the U.S. Food and Drug Administration of 1987. Samples were taken from an agricultural soil and a sludge amended agricultural soil from farms in New Jersey, USA.

Sealed flasks with the soil, spiked with 10 µg test substance/g sediment dw (10 mg/kg) were incubated at laboratory ambient temperature for 12 weeks. Periodically the headspace was flushed for oxygen replenishment and the effluent gas was drawn through a train of scintillation fluids to capture volatile organics and CO2to be determined by liquid scintilation counting. Periodically also flasks were sacrificed and exhaustively extracted with hexane/acetone. An aliquot of the solvent fraction was used for thin layer chromatography for analysis of the test substance and metabolites. The total 14C-mass balance was established for the parent and metabolites based on extraction.

Radiolabeled OTNE in soil is almost completely degraded after 6 weeks. After 6 weeks the mineralisation (CO2 evolution) is circa 50%. After 1 to 2 weeks a range of more polar metabolites was found. After a lag time of approx. 7 days the initial rate of CO2 production was 1.4 - 1.8% /day in the sludge amended soil and in the agricultural soil, respectively. The half-life of the parent substance was estimated at 6.0 and 4.2 days in the sludge amended soil and in the agricultural soil, respectively.

Description of key information

The fate of 14C-OTNE in agricultural soils was studied in microcosms according to protocols described in documents from the U.S. Food and Drug Administration of 1987. Samples were taken from an agricultural soil and a sludge amended agricultural soil from farms in New Jersey, USA.
Sealed flasks with the soil, spiked with 10 µg test substance/g sediment dw (10 mg/kg) were incubated at laboratory ambient temperature for 12 weeks. Periodically the headspace was flushed for oxygen replenishment and the effluent gas was drawn through a train of scintillation fluids to capture volatile organics and CO2 to be determined by liquid scintillation counting. Periodically also flasks were sacrificed and exhaustively extracted with hexane/acetone. An aliquot of the solvent fraction was used for thin layer chromatography for analysis of the test substance and metabolites. The total 14C-mass balance was established for the parent and metabolites based on extraction.
Radiolabeled OTNE in soil is almost completely degraded after 6 weeks. After 6 weeks the mineralisation (CO2 evolution) is circa 50%. After 1 to 2 weeks a range of more polar metabolites was found. After a lag time of approx. 7 days the initial rate of CO2 production was 1.4 - 1.8% /day in the sludge amended soil and in the agricultural soil, respectively. The half-life of the parent substance was estimated at 6.0 and 4.2 days in the sludge amended soil and in the agricultural soil, respectively.

Key value for chemical safety assessment

Half-life in soil:
6 d
at the temperature of:
22 °C

Additional information