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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:
From October 12, 2009 to April 27, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.4100 (Aerobic Soil Metabolism)
Deviations:
no
GLP compliance:
yes
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
USDA (US Department of Agriculture)
Soil no.:
#1
Soil type:
sandy loam
% Clay:
9
% Silt:
15
% Sand:
76
% Org. C:
4
pH:
6.4
CEC:
13.5 meq/100 g soil d.w.
Bulk density (g/cm³):
1.02
Soil no.:
#2
Soil type:
sandy loam
% Clay:
19
% Silt:
14
% Sand:
67
% Org. C:
3.2
pH:
7
CEC:
17.2 meq/100 g soil d.w.
Bulk density (g/cm³):
1.02
Soil no.:
#3
Soil type:
clay
% Clay:
41
% Silt:
16
% Sand:
43
% Org. C:
1.5
pH:
8.2
CEC:
37 meq/100 g soil d.w.
Bulk density (g/cm³):
1.17
Details on soil characteristics:
Three test soils were ordered from Agvise Laboratories (Northwood, North Dakota). The soils were sandy loam, designated RMN-PF, from Grand Forks, North Dakota; a sandy loam, designated MSL-PF, from Grand Forks, North Dakota; and a clay, designated MT-CL-PF, from Valley County, Montana. The bulk soil was sieved through a 2.0 mm screen to remove gross debris. Once the soil was received, the bulk sample was stored in the dark in an incubator set at 4 ± 2°C in a container with free access to air until used. Soils were characterized by USDA and FAO classification with regard to texture (percent sand, percent silt, percent clay), pH (in 1:1 soil:water ratio and in 1N KCl), percent organic matter, cation exchange capacity, maximum water holding capacity and bulk density.

Soil moisture content: moisture content for the three test soils was determined prior to preparation of the test systems.

Absence of radioactivity in the soil: triplicate subsamples of the untreated soil were combusted and analyzed by liquid scintillation counting (LSC) to confirm the absence of radioactivity in the soil.

Microbial activity of the soil: the microbial biomass of the soil, was determined using a chloroform fumigation method. One sample per test system was taken for chloroform fumigation analysis prior to incubation, one sample during incubation and one sample at the final sampling time. Additionally, microbiological activity of the three soil systems was determined by total plate count analysis prior to treatment and after the final sampling time. Total colony forming units (of bacterial and fungal) per gram of soil (cfu/g) were determined by plate count analysis of dilute soil solutions.
Soil No.:
#1
Initial conc.:
1 other: μg/g
Soil No.:
#2
Initial conc.:
1 other: μg/g
Soil No.:
#3
Initial conc.:
1 other: μg/g
Parameter followed for biodegradation estimation:
CO2 evolution
Soil No.:
#1
Temp.:
20 ± 2°C
Humidity:
17.10 %
Microbial biomass:
249 ug/g dry basis
Soil No.:
#2
Temp.:
20 ± 2°C
Humidity:
13.91 %
Microbial biomass:
212 ug/g dry basis
Soil No.:
#3
Temp.:
20 ± 2°C
Humidity:
7.44 %
Microbial biomass:
223.3 ug/g dry basis
Details on experimental conditions:
Pretest System
- 1 M NaOH solution to remove carbon dioxide from the air.
Water bottle to humidify the air flow.

Test System
Soil samples connected in series fitted with appropriate connectors.

Post-Test System
- Volatile organic trap containing ethylene glycol (100 mL)
2 traps containing 1 M NaOH solution to trap 14CO2 (100 mL each).


Preparation of the Test Samples

The test vessels used to contain the soil consisted of 250-mL, wide-mouth, glass bottles. Soil was added to the test vessels to a total thickness of ca. 2.3-3.1 cm, approximately 50- g dry weight, based on the oven-dry weight of the soil. The exact weight of soil for each sample was documented in the study records. The soil moisture in the test samples was adjusted to 50% maximum water holding capacity (MWHC) using HPLC grade water. Bottles were capped with rubber stoppers each fitted with two glass tubes for inlet and outlet of air flow.

Preparation of the Sterile Test Samples

The test vessels used to contain the soil consisted of 250-mL wide-mouth, glass bottles with foam stoppers. Soil was added to the test vessels to a total thickness of ca. 2.3-3.1 cm, approximately 50-g dry weight, based on the oven-dry weight of the soil. The samples were sterilized by autoclaving once a day for 3 days prior to the treatment of the test substance. The soil moisture in the test samples was adjusted to 50% MWHC using sterile HPLC grade water.

Test Systems

A total of three aerobic test systems were prepared using the RMN-PF (Nr. 1), MSL-PF (Nr. 2) and MTCL-PF (Nr. 3) soils incubated at 20 ± 2 °C in the dark. During acclimation and incubation the aerobic test systems were maintained under aerobic conditions by passing carbon dioxidefree humidified air over the soil samples using a vacuum pump. Each test system consisted of test vessels connected in series followed by volatile traps. Air was passed first through a sodium hydroxide volatile trap (100 mL) followed by a glass washer bottle containing 100 mL HPLC water and then through the test vessels. For the acclimation period, each test system was followed by a gas washer containing HPLC grade water. For the incubation period, each test system was followed by one volatile trap containing 100 mL ethylene glycol and 2 traps containing 100 mL 1 M NaOH solution to trap 14CO2.

For the sterile test systems for all three soils, following application of the test material, the sample containers were stoppered with foam plugs and placed in a flow through desiccator attached to a vacuum pump. The stream of air entered the system through a sterile filter (Millex-FG, Vent Filter Unit, Hydrophobic Fluoropore, PTFE Membrane, Millipore Corporation, Billerica, MA) followed by a gas washer bottle containing 1 M NaOH then a second gas washer bottle, containing water, to humidify the air flow. The carbon dioxide-free, humidified air then passed through the desiccator and exited into an ethylene glycol trap (100 mL) followed by two 1 M NaOH traps (100 mL each). The sterile soil samples were incubated at 20 ± 2 °C in the dark. Prior to test substance application, the test systems were allowed to equilibrate in the dark at 20 ± 2 °C under aerobic conditions for 26 days. The water content of individual samples was monitored. Samples were periodically removed from the environmental chamber and the soil moisture level adjusted to 50% MWHC as necessary. Incubation under study conditions allowed the establishment of equilibrium and the microbial population to acclimate under the aerobic incubation conditions.


TEST SYSTEM ANALYSIS

Soil Extraction and Analysis
For each test soil sample, the soil was transferred to a plastic centrifuge bottle. Extraction solvent, 100 mL of 80:20 acetonitrile:0.1 M NH4OH (v:v), was added. Samples were sonicated for 10 minutes then placed on a wrist-action shaker for 1 hour. Samples were centrifuged to settle the solids. The supernatant was decanted into a graduated cylinder, the volume recorded and the extract was analyzed by LSC in triplicate. Samples were extracted a total of five times.

For each sample, all five extracts were pooled and aliquots analyzed by LSC. A subsample of each of the pooled extracts was centrifuged. Aliquots of the resulting supernatant were analyzed by LSC to monitor any loss of radioactivity. An aliquot of the supernatant was evaporated to dryness under a nitrogen stream then redissolved in 250 μL 25:75 acetonitrile:0.1 M NH4OH (v:v). Aliquots of the concentrate were analyzed by LSC to determine 14C recovery. The reconstituted residues were then centrifuged to remove any particulates; the supernatant was analyzed by LSC to determine 14C recovery. Aliquots of the concentrates, along with MON 51803 reference standard, were analyzed by HPLC as described in the High Performance Liquid Chromatography section.

ANALYSIS OF THE PES (POST EXTRACTION SOLIDS)
The solids remaining after extraction of soil were air dried in a flow hood. The weight of each dried PES sample was then obtained. The dried PES was homogenized using a mortar and pestle. Triplicate aliquots (approximately 0.25-0.5 g each) were combusted to determine the amount of radioactivity remaining in the PES samples.

Further extractions, designated “Exhaustive Extraction,” and bound residue analyses were performed on the unextracted radioactivity remaining in the Day 7 and Day 90 for the RMN-PF and MSL-PF soil samples and Days 3, 7 and 90 for the MT-CL-PF soil samples.

VERIFICATION OF 14CO2
Aliquots of the NaOH trap solutions from representative samples were assayed by LSC.
The 1 M NaOH traps from Day 7 and Day 90 from the RMN-PF and MSL-PF soil systems were analyzed for 14CO2, Day 1 and Day 90 NaOH traps were analyzed from the MT-CL-PF soil system.

METABOLITE IDENTIFICATION
The parent compound was identified based on co-chromatography of [14C]test substance in the soil extracts with the authentic reference standard. Further identification of metabolites was outside the scope of the study.
Soil No.:
#1
Remarks on result:
other: Aerobic Test Systems
Remarks:
92.7 - 111.3%
Soil No.:
#2
Remarks on result:
other: Aerobic Test Systems
Remarks:
97.6 - 108.3%
Soil No.:
#3
Remarks on result:
other: Aerobic Test Systems
Remarks:
75.6-108.2%
Soil No.:
#1
Remarks on result:
other: Sterile Aerobic Test Systems
Remarks:
102.5-107.8%
Soil No.:
#2
Remarks on result:
other: Sterile Aerobic Test Systems
Remarks:
100.8-107.3%
Soil No.:
#3
Remarks on result:
other: Sterile Aerobic Test Systems
Remarks:
104.6-106.4%
Key result
Soil No.:
#1
DT50:
8.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Key result
Soil No.:
#2
DT50:
7.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Key result
Soil No.:
#3
DT50:
2.3 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Transformation products:
not specified
Details on results:
This study demonstrated that test substance was degraded at a rapid rate under the aerobic, microbially active, soil conditions employed in this study. The major degradation product
of test substance was released as radioactive carbon dioxide. Approximately 78-90% of the applied radioactivity (AR) was detected in the 1 M NaOH volatile traps from all three soil test systems after 90 days; however, only trace amounts of radioactivity (0.1% AR or less) were detected in the ethylene glycol volatile organic traps. Analysis of soil extracts by HPLC showed that the main metabolism products consisted of polar material eluting at the solvent front. In contrast, no radioactive carbon dioxide was formed and only minimal amounts of metabolism products were formed in the corresponding sterile soil systems indicating that degradation under aerobic conditions was due to microbial activity.

Analysis of the dissipation of test substance in the three soils used in the study assuming simple first-order kinetics did not give acceptable curve fits on natural log transformed data using linear regression analysis. However, non-linear regression analysis using untransformed data based on the Gustafson-Holden model gave curves from which DT50 values of 8.6, 7.6, and 2.3 days were calculated for the RMN-PF, MSL-PF and MT-CLPF soils, respectively. The DT90 values calculated using this method were 28.5, 36.5, and 7.7 days for the RMN-PF, MSL-PF, and MT-CL-PF soils, respectively. The curve fits gave r2 values of 0.9933, 0.9785 and 0.9933 for each of the soils, respectively. The DT50 values calculated for each soil are in good agreement with those approximated from visual inspection of the plotted data. The calculated DT90 values for the RMN-PF and MT-CL-PF soils are also in good agreement with those approximated from visual inspection of the plotted data. However, the calculated DT90 value for the MSL-PF soil is actually underestimated and the plotted data appears to give a DT90 of approximately 55 days.

Based on the results of this study, it can be concluded that test substance will rapidly dissipate from natural soil systems under aerobic conditions.
Conclusions:
Under aerobic soil metabolism conditions using microbially active soils, the test substance is metabolized at a rapid rate.
Executive summary:

A study was conducted to determine the rate of degradation of [14C] radiolabeled test substance in three representative U.S. soil types at 20°C under aerobic conditions according to OECD Guideline 307 and EPA OPPTS Method 835.4100, in compliance with GLP. The [14C] test substance was applied to each soil (50.0 g dw) to achieve a final concentration of 1.0 ppm based on the soil dry weight. Each test system was maintained in the dark at 20 ± 2°C under aerobic conditions by continuously flowing air over the samples for 90 days. Sodium hydroxide (1 M NaOH) and ethylene glycol traps were included in the flow-through test systems in order to collect 14CO2 and any volatile 14C-labeled components that evolved during the study. Samples from the aerobic test system were assayed at 0, 1, 3, 7, 14, 21, 44 and 90 days after application of the test substance. At each sampling interval, the aerobic test soils were extracted with acetonitrile:0.1 M aqueous ammonia (80:20, v:v). The soil extracts were radioassayed and analyzed by HPLC for [14C]test substance and degradates. Non-extracted radioactivity in the postextracted solids (PES) was quantified by combustion analysis. The 1 M NaOH and volatile organic traps were radioassayed directly by liquid scintillation counting (LSC). Total 14C mass balance (based on the sum of the soil extractable, volatile and bound nonextracted radioactivity divided by the applied radioactivity) of all replicates ranged from 92.7 to 111.3% of the applied radioactivity (% AR) for the RMN-PF soil system, 97.6 to 108.3% AR for the MSL-PF soil system and 75.6 to 108.2% AR for the MT-CL-PF soil system at all sampling points. For the MT-CL-PF soil system, one sampling point, Day 7 replicates 807 and 808, had a mass balance of 75.6 and 77.2% AR, respectively. All other replicates of the MT-CL-PF soil system had acceptable mass balances of 90% or greater of the applied radioactivity. In the RMN-PF soil, the amount of extractable radioactivity from the soil steadily decreased over time to a minimum average of 4.6% AR at 90 days while the amount of bound nonextractable radioactivity increased to a maximum average of 14.5% AR at Day 3 then decreased to 9.1% at 90 days. The amount of 14CO2 detected significantly increased throughout the study to a maximum average of 79.1% AR at Day 90. A negligible amount of volatile organic compounds was evolved (0.1% AR) through 90 days. In the MSL-PF soil, the amount of extractable radioactivity steadily decreased over time to a minimum average of 9.1% AR at 90 days while the amount of bound nonextractable radioactivity increased to a maximum average of 30.9% AR at Day 7 then decreased to 12.3% AR at 90 days. The amount of radioactivity as 14CO2 reached a maximum level of 77.8% AR at 90 days. A negligible amount of volatile organic compounds was evolved (0.1% AR) through 90 days. In the MT-CL-PF soil, the amount of extractable radioactivity steadily decreased over time to a minimum average of 2.2% AR at 90 days while the amount of nonextractable radioactivity increased to a maximum average of 59.9% AR at 7 days then decreased to 9.8% AR at 90 days. The amount of radioactivity as 14CO2 reached a maximum level of 89.6% AR at 90 days. A negligible amount of volatile organic compounds was evolved (<0.1% AR) through 90 days. A significant portion of the parent compound was mineralized and released as radioactive carbon dioxide. Minor metabolites were also observed in the breakdown of test substance. In contrast, no radioactive carbon dioxide was formed and only minimal amounts of degradation products were formed in the corresponding sterile soil systems indicating that degradation under aerobic conditions was due to microbial activity. The DT50 (half-life) and DT90 values for dissipation of test substance in the aerobic test systems were evaluated using first-order linear regression analysis, but gave unacceptable correlation coefficients. The data were subsequently evaluated using a non-linear regression analysis based on the Gustafson-Holden model. The decline of test substance was calculated using the amounts of test substance detected in the soil extracts through Day 90 for all three soil systems. The DT50 values calculated for each soil are in good agreement with values that can be approximated from visual inspection of the plotted data. The calculated DT90 values for the RMN-PF and MT-CL-PF soils are also in good agreement with values that can be approximated the plotted data. However, the calculated DT90 value for the MSL-PF soil is actually underestimated and the plotted data appears to give a DT90 of approximately 55 days. Under aerobic soil metabolism conditions using microbially active soils, the test substance is metabolized at a rapid rate (Herczog, 2010).

Description of key information

Under aerobic soil metabolism conditions using microbially active soils, the test substance is metabolized at a rapid rate.

Key value for chemical safety assessment

Half-life in soil:
8.6 d
at the temperature of:
20 °C

Additional information