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Environmental fate & pathways

Biodegradation in soil

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Reference
Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-07-25 - 1997-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: no guideline available
Principles of method if other than guideline:
CO2 Evolution when applied to soil
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
not specified
Soil classification:
not specified
Year:
1997
Soil no.:
#1
Soil type:
other: Gley humic, high aluminum, low clay activity, texture fine clayey (Cumilic Humaquept)
% Clay:
55
% Org. C:
4.9
pH:
3.7
CEC:
20.3 other: cmol/kg
Soil no.:
#2
Soil type:
other: Dusky red latosol distrofic, chric epipedon, texture very fine clayey (Rhodic Hapludox)
% Clay:
20
% Org. C:
1.2
pH:
4.6
CEC:
8.8 other: cmol/kg
Soil no.:
#3
Soil type:
other: Dark red latosol, high aluminum ,choric epipedon, texture fine loamy (Typic Hapludox)
% Clay:
70
% Org. C:
1.7
pH:
4.1
CEC:
12.1 other: cmol/kg
Details on soil characteristics:
see 'Any other information on materials and methods incl. tables'
Soil No.:
#1
Duration:
28 d
Soil No.:
#2
Duration:
28 d
Soil No.:
#3
Duration:
28 d
Soil No.:
#1
Initial conc.:
0.001 other: mg/g
Based on:
act. ingr.
Soil No.:
#2
Initial conc.:
0.001 other: mg/g
Based on:
act. ingr.
Soil No.:
#3
Initial conc.:
0.001 other: mg/g
Based on:
act. ingr.
Soil No.:
#1
Initial conc.:
0.01 other: mg/g
Based on:
act. ingr.
Soil No.:
#2
Initial conc.:
0.01 other: mg/g
Based on:
act. ingr.
Soil No.:
#3
Initial conc.:
0.01 other: mg/g
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on experimental conditions:
see 'Any other information on materials and methods incl. tables'
Key result
Soil No.:
#1
DT50:
< 30 d
Temp.:
25 °C
Key result
Soil No.:
#2
DT50:
< 30 d
Temp.:
25 °C
Key result
Soil No.:
#3
DT50:
< 30 d
Temp.:
25 °C
Transformation products:
no
Details on results:
Tables 2, 3 and 4 show the measurement of radioactivity evolved as 14CO2 from LE, LR and GH soils when two concentrations of active ingredient of Ferbam were added, at each sampling data.
The percentage of 14CO2 evolved from the test substance added to i the soil is shown on Table 5. It was calculated based on the radioactivity of the solution added to the soils. The cumulative percentage during 4 weeks incubation period is shown in Table 6.

Table 2: Measurement of radioactivity in desintegration per minute (dpm) on 14C-CO2 evolved from two concentrations of test substance applied on GH soil during 28 days (data from 4 replications)

Concentration
(ug/g)
Incubation time (days)
7 14 21 28
dpm
1.0 82830 12800 10805 4855
82945 11565 11095 4780
82510 10865 10405 7250
86290 11910 11440 7570
10.0 7338 14100 18875 6780
68660 14245 7210 6845
77665 15285 16265 8930
69115 15095 11930 9090

Standard 1.0 ug/g - 215260
Standard 10.0 ug/g - 278303

Table 3: Measurement of radioactivity in desintegration per minute (dpm) on 14C-CO2 evolved from two concentrations of test substance applied on LR soil during 28 days (data from 4 replications)

Concentration
(ug/g)
Incubation time (days)
7 14 21 28
dpm
1.0 81790 25605 18960 17920
81170 28740 22200 17820
83440 29025 21420 17560
85435 28885 22215 17915
10.0 58265 30740 29380 17515
58675 30480 27715 17885
53440 30870 29185 24080
53655 29705 28815 24810
Standard 1.0 ug/g - 215260
Standard 10.0 ug/g - 278303

Table 4: Measurement of radioactivity in desintegration per minute (dpm) on 14C-CO2 evolved from two concentrations of test substance applied on LE soil during 28 days (data from 4 replications)

Concentration
(ug/g)
Incubation time (days)
7 14 21 28
dpm
1.0 116730 11960 12475 24135
121620 10490 12150 24040
122765 10300 12290 11810
119680 10165 12230 11670
10.0 75385 14730 12515 22990
91415 15415 12305 22745
85325 15900 12625 22990
83935 16895 12410 22660
Standard 1.0 ug/g - 215260
Standard 10.0 ug/g - 278303

Table 5: Percentage of 14C-CO2 evolved from two concentrations of test substance applied on GH, LR and LE soils during 28 days of incubation (data from 4 replications)

Concentration
(ug/g)
days
7 14 21 28
GH soil
1.0 38,5 6,0 5,0 2,3
38,5 5,4 5,2 2,2
38,3 5,1 4,8 3,4
40,1 5,5 5,3 3,5
10.0 26,4 5,1 6,8 2,4
24,7 5,1 2,6 2,5
27,9 5,5 5,8 3,2
24,8 5,4 4,3 3,3
LR soil
1.0 38,0 11,9 8,8 8,3
37,7 13,4 10,3 8,3
38,8 13,5 10,0 8,2
39,7 13,4 10,3 8,3
10.0 20,9 11,1 10,6 6,3
21,1 11,0 10,0 6,4
19,2 11,1 10,5 8,7
19,3 10,7 10,4 8,9
LE soil
1.0 54,2 5,6 5,8 11,2
56,5 4,9 5,6 11,2
57,0 4,8 5,7 5,5
55,6 4,7 5,7 5,4
10.0 27,1 5,3 4,8 8,3
32,9 5,5 4,4 8,2
30,7 5,7 4,5 8,3
30,2 6,1 4,5 8,1

Table 6: Accumulative 14C-CO2 percentage evolved from two concentrations of test substance applied to GH, LR and LE soils during 28 days of incubation (data from 4 replications)

Concentration
(ug/g)
days
7 14 21 28
GH soil
1.0 38,5 44,4 49,4 51,7
38,5 43,9 49,1 51,3
38,3 43,4 48,2 51,6
40,1 45,6 50,9 54,5
10.0 26,4 31,4 38,2 40,7
24,7 29,8 32,4 34,8
27,9 33,4 39,2 42,5
24,8 30,3 36,6 37,8
LR soil
1.0 38,0 49,9 58,7 67,0
37,7 51,1 61,4 69,7
38,8 52,3 62,2 70,4
39,7 53,1 63,4 71,8
10.0 20,9 32,0 42,5 48,8
21,1 32,0 42,0 48,4
19,2 30,3 40,8 49,4
19,3 30,0 40,3 49,2
LE soil
1.0 54,2 59,8 65,6 76,8
56,5 61,4 67,0 78,2
57,0 61,8 67,5 73,0
55,6 60,3 66,0 71,4
10.0 27,1 32,4 36,9 45,1
32,9 38,4 42,8 51,0
30,7 36,4 40,9 49,2
30,2 36,2 40,7 48,8

Table 7: Glucose -14C consumed by soil microorganisms

Concentrations
(ug/g soil)
Glucose consumed
(mmol glucose / g soil* hour)
GH LR LE
Control# 11,3 4,6 10,0
11,3 4,5 10,7
1.0## 5,2 1,0 3,3
5,3 1,3 3,0
10.0## 9,0 1,5 3,1
6,9 1,2 3,2
# - Sample analyzed at the beginning of assy (zero day)
## - Sample analyzed at the end of the assay (28 days)

Table 8: Biodegradability classification of test substance according to MHM/SEMA, 1990

14C-CO2 Classification
0 - 1 % High persistent (half-life higher tahn 180 days)
1 - 10 % Medium persistent (half-life between 90 and 180 days)
10 - 25 % Reduced persistent (half-life between 30 and 90 days)
> 25 % no persistent (half-life lower than 30 days)
Conclusions:
According to the results, for tested concentrations 1.0 and 10.0ug/g, the percentage of CO2 evolved from GH soil were 52.3 and 38.9%, from LR were 69.7 and 49.0% and from LE soil were 74.9 and 48.5% respectively. Therefore the substance could be classified as not persistent to soil (half-life lower than 30 days)
The microbial activity of GH, LR and LE soils decreased with the time using 1.0 and 10.0 ug of test substance/g of soil.
Executive summary:

This study was conducted to determine the amount of Ferbam (ferbam) mineralized to CO2 when applied to LE (Typic Hapludox), LR (Rhodic Hapludox) and GH (Cumulic Humaquept) soils. This assay was carried out in Bartha biométrie flasks containing 50 g of soil. Two concentrations of the active ingredient were added in duplicate resulting in concentrations of 1.0 and 10.0 ug/g of soil. To trap the CO2 evolved, from the test substance, 10 mL of a 0.1 N KOH solution was added in the lateral compartment of the flask. Every week, the KOH solution was renewed. The sampled solutions were transferred into scintillation vials and radioanalysed weekly. The biométrie flasks were incubated at 25±2°C in dark room for 28 days.

According to the results, for tested concentrations 1.0 and 10.0 ug/g, the percentage of CO2 evolved from GH soil were 52.3 and 38.9 %, from LR were 69.7 and 49.0 % and from LE soil were 74.9 and 48.5 % respectively. Therefore the substance could be classified as not persistent to soil (half-life lower than 30 days)

The microbial activity of GH, LR and LE soils decreased with the time using 1.0 and 10.0 ug of test substance/g of soil.

Description of key information

This study was not conducted according to GLP and a guideline, but in accordance with generally accepted scientific standards and described in sufficient detail. According to the results, for tested concentrations 1.0 and 10.0 µg/g, the percentage of CO2 evolved from GH soil were 52.3 and 38.9%, from LR were 69.7 and 49.0% and from LE soil were 74.9 and 48.5% respectively. Therefore the substance could be classified as not persistent to soil (half-life lower than 30 days) .

The microbial activity of GH, LR and LE soils decreased with the time using 1.0 and 10.0 µg of test substance/g of soil.

Key value for chemical safety assessment

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

Additional information

Key: da Silva, 1997

This study was conducted to determine the amount of Ferbam (ferbam) mineralized to CO2 when applied to LE (Typic Hapludox), LR (Rhodic Hapludox) and GH (Cumulic Humaquept) soils. This assay was carried out in Bartha biométrie flasks containing 50 g of soil. Two concentrations of the active ingredient were added in duplicate resulting in concentrations of 1.0 and 10.0 µg/g of soil. To trap the CO2 evolved, from the test substance, 10 mL of a 0.1 N KOH solution was added in the lateral compartment of the flask. Every week, the KOH solution was renewed. The sampled solutions were transferred into scintillation vials and radioanalysed weekly. The biometrie flasks were incubated at 25±2°C in dark room for 28 days.

According to the results, for tested concentrations 1.0 and 10.0 µg/g, the percentage of CO2 evolved from GH soil were 52.3 and 38.9 %, from LR were 69.7 and 49.0 % and from LE soil were 74.9 and 48.5 % respectively. Therefore the substance could be classified as not persistent to soil (half-life lower than 30 days)

The microbial activity of GH, LR and LE soils decreased with the time using 1.0 and 10.0 ug of test substance/g of soil.

Supporting information: Nixon, 1997

This study was designed and conducted according to U.S. EPA Pesticide Assessment Guidelines, Subdivision N, Series 162-1 to evaluate the degradation rate of [14C]ferbam in sandy loam under aerobic metabolism conditions and to quantitate any degradation products formed.

Ferbam was unstable in both reverse- and normal-phase high performance liquid chromatography (HPLC) and in thin-layer chromatography (TLC) to such a large degree as to preclude these methods for determination of purity of the test substance. As a result of this instability, the purity of [I4C]ferbam was determined in a chloroform-d3 solution by proton nuclear magnetic resonance spectroscopy (NMR). Purity of [14C]ferbam in chloroform-d3 solution was likewise determined by NMR. Just prior to dosing, aliquots of [14C]ferbam and [13C]ferbam solutions were transferred to a foil-covered, silylated, glass 22-ml vial.

Aliquots of the final dose solution containing 90.69 µg (13.07 uCi) of [14C]ferbam and 134 µg of [13C]ferbam were applied to 20 g of sandy loam in incubation flasks. The final concentration of 11.2 ppm was based on a mean of radioassays of three diluted aliquots of the dose solution; one taken prior to dosing, one taken midway through dosing and one taken after dosing of the flasks. HPLC-grade water was added after dosing to obtain 75% of field moisture capacity at 0.33 bar. Duplicate flasks for each time period were sealed and placed in a dark incubator. Each set of duplicate flasks for each sample interval were continuously purged with humidified air under negative pressure with the air being passed through a series of volatile collection traps. Traps consisted of two 1 N potassium hydroxide in methanol and one ethylene glycol trap in series. Duplicate flasks were sampled following incubation for 0 and 4 hours, and 1, 2, 4, 7, 14, 21, 45. 93 and 150 days. Incubator temperatures throughout the study ranged from 22.0 - 26.0°C with a mean of 24.9 ± 0.6°C (mean ± standard deviation). At sampling, soil was removed from the flasks and extracted with chloroform (CHCI3) followed by extraction with chIoroform:methanol (CHCl3:CH3OH, 1:1, v:v). Quantitative analysis of soil extracts was determined by reverse-phase high performance liquid chromatography (HPLC). Degradate identity was confirmed by thin layer chromatography (TLC).

Degradation of ferbam was rapid with formation of carbon disulfide (CS2), carbon dioxide (CO2) and tetramethylthiuram disulfide (TMTD, "thiuram") as the major degradates. CS2 and CO2 were trapped as volatile products in the methanolic KOH trap solutions. Following 150 days of incubation, volatile products (I4C-CS2 and I4C-CO2) comprised 61.9% of the applied radiocarbon. 14C-CO2 was the dominant product comprising approximately 90% of the volatile radiocarbon versus approximately 10% 14CS2- The primary product extracted from the soil with either CHCI3 or CHC^CHsOH was TMTD. No unknown products were observed at >10% of the applied radiocarbon activity. Soil bound residue, not extractable with CHCI3 or CHCl3:CH30H shake extractions, increased during the course of the 150-day incubation. Bound residues reached a maximum of approximately 49.0% (mean of replicates) of the applied dose at Day 7, then decreased to 26.3% by Day 150 (mean of replicates).

Material balance was quantitative throughout the study with 96.5 ± 2.7% (mean ± standard deviation) of the applied radiocarbon recovered.

Ferbam degraded too rapidly to permit meaningful assessment of the degradation rate. The half-life of TMTD was calculated to be 4.9 days.

Supporting Information: Daly, 1987a

An aerobic soil metabolism similar or equilvalent to EPA OPPTS 835.4100 (Aerobic Soil Metabolism) study with 14C-Ferbam is being conducted under dark conditions on sandy loam soil. The study soil was initially treated with 14C-Ferbam primary stock solution to achieve a nominal concentration rate of 10 ug/g. The concentration of 14C-Ferbam in the test system immediately after dosing was analyzed to be 9.59 ug/g.

On days 0, 1, 3, 7, 14 and 1, 2, 3, 4, 6, 9 and 12 months after dosing and aerobic incubation, soil sample were analyzed by combustion radioanalysis to determine the level of 14C-activity remaining on the soil and training solutions were analyzed by liquid scintillation counting for 14C-volatiles. The KOH trapping solutions were analyzed for CS2 by gas chromatography.

An aerobic half-life of 6.60 days was estimated for 14C-Ferbam based on the total residues in the soil for the first seven days of the study.

The mean 14C-mass balance accountability after 12 months was 93.2%.

Supporting Information: Coody, 1998

This study was conducted to provide data for the anaerobic metabolism requirement, as specified in U.S. EPA Pesticide Assessment Guidelines, Subdivision N, Series 162-2 and 162 -3. The study design follows guidance given in Subdivision N: 162-3 Anaerobic Aquatic Metabolism with the exception that terrestrial field soil and HPLC-grade water will be used rather than pond sediment and water. The 162-3 guideline was selected to allow for a longer study period to more fully quantify the anaerobic process.

Ferbam was insufficiently stable in either reverse- or normal-phase high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) systems to allow these methods for determination of purity of the test substance. As a result of this instability, the purity of [14C]ferbam was determined in a chloroform-^ solution by proton nuclear magnetic resonance spectroscopy (NMR). Purity of [13C]ferbam in chloroform-d3 (deuterated) solution was likewise determined by NMR.

Just prior to dosing, aliquots of [14C]ferbam and [13C]ferbam solutions were transferred to foil-covered, silylated, glass 22-ml vials. Aliquots of the solutions containing 163.47 µg (23.56 uCi) of [14C]ferbam and 1,234 µg of [13C]ferbam were applied to the test system, which consisted of 20 g of sandy loam and 100 ml HPLC-grade water in each of the incubation flasks. Flasks had been previously prepared, flushed with nitrogen periodically, and amended with dextrose prior to dose day. A final ferbam concentration of 11.6 ppm in the aquatic system was based on the mean of radioassays of two diluted, dose solution aliquots taken before and after the dosing of the flasks. Duplicate flasks for each time period were sealed and placed in a dark incubator. All flasks were purged with nitrogen approximately every two weeks to evacuate the headspace of the flasks. Headspace gases were pulled through a series of volatile collection traps. Traps consisted of two methanolic KOH traps (IN KOH) and one ethylene glycol trap in series. Duplicate flasks were sampled at Day 0 and following incubation for 3, 7, 14, 21, 51, 93, 134, 182, 272 and 364 days. Incubator temperatures throughout the study ranged from 24.0 - 26.5°C with a mean of 24.9 ± 0.5°C (mean ± standard deviation).

At sampling, dissolved oxygen, Eh (mV) and pH readings were taken of each test system. The soil and water phases were separated by centrifugation. Water was decanted and analyzed, if possible (up to Day 7), by reverse-phase high performance liquid chromatography (HPLC). Soil was extracted with chloroform (CHCl3j Extract 1) followed by extraction with chloroform:methanol (CHCbMeOH, 1:1, v:v, Extract 2). Quantitative analysis of soil extracts was determined by HPLC, if possible. Degradate identity in soil Extract 1 was confirmed by thin layer chromatography (TLC) and/or proton nuclear magnetic resonance spectroscopy (NMR). CS2 identity was confirmed by gas chromatography/mass spectrometer (GC/MS) analysis.

Degradation of ferbam was immediate in the test system with formation of tetramethylthiuram disulfide (TMTD), cobalt (II) dimethyldithiocarbamate (Co-DMDTC), carbon disulfide (CS2) and carbon dioxide (CO2). CS2 and CO2 were trapped as volatile products in the methanolic KOH trap solutions. Following 364 days of incubation, volatile products (14CS2 and 14CO2) comprised 58.1% (mean of replicates) of the applied radiocarbon. 14CS2 was the dominant volatile product comprising approximately 87% of the volatile radiocarbon versus approximately 13% as 14CO2. The products extracted from the soil with either CHCI3 or CHC13:CH30H were TMTD and Co-DMDTC. TMTD was present at Day 0 at levels of 33.8% (mean of replicates) of the applied radiocarbon, declining to 8.1% of the applied radiocarbon by Day 364. Co-DMDTC was present at Day 0 at levels of 39.6% of the applied radiocarbon, increasing to 46.5% of the applied at Day 21, then decreasing to 16.8% of the applied radiocarbon by Day 364. Only one unidentified degradate was observed at a level >10% of the applied radiocarbon at any time during the study. This degradate was present at 11.7%» (mean of replicates) of the applied radiocarbon at Day 0, and was present in the water phase only. This peak had decreased to 2.8% of the applied radiocarbon by Day 7. Following Day 7, the water samples were not analyzed due to their low levels of radiocarbon (<3.0% of the applied radiocarbon). No other unknowns representing > 1.0% of the applied radiocarbon were detected throughout the study. Soil bound residue, not extractable in CHCI3 or CHCl3:CH30H shake extractions, ranged from 11.0 to 16.1% (mean of replicates) of the applied radiocarbon between Day 0 and Day 182. Day 272 and Day 364 bound residue levels had dropped to 6.6 and 5.6% (mean of replicates) of the applied radiocarbon, respectively.

First-order decay kinetics for ferbam could not be obtained in the study due to the rapid reaction in soil (no ferbam was observed in the Day 0 samples). Decay kinetics for the primary degradates, Co-DMDTC and TMTD resulted in half-lives (DT50) of 269 and 184 days for these degradates, respectively. DT90 values for Co-DMDTC and TMTD were calculated as 893 and 611 days, respectively.

Total radiocarbon recovery throughout the study (mean ± standard deviation) was 95.8 ±5.2%.

Supporting Information: Daly, 1987b

An anaerobic soil metabolism study with 14C-Ferbam has been conducted equivalent or similar to EPA OPPTS 835.4200 (Anaerobic Soil Metabolism) in sandy loam soil at 25° ±1°C in the dark. Prior to initiation of the study, soils were aerobically incubated to one half-life of the compound.

An anaerobic half-life for 14C-Ferbam was estimated based on total residues in the test sediment and water versus time. The estimated half-life was 107 days.

Volatile 14C-residues, assumed to be CS2 show a definite decrease over time under anaerobic environmental conditions.

The mean 14C-mass balance accountability was 97.4% at the end of the study.