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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:
08 March 2001 to 04 July 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: US EPA Pesticide Assessment Guidelines, Subdivision N, Environmental Fate, 162-1: Aerobic Soil Metabolism Studies (1982)
GLP compliance:
yes
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
USDA (US Department of Agriculture)
Year:
2001
Soil no.:
#1
Soil type:
sandy loam
pH:
6.5
Details on soil characteristics:
Sandy Loam, pH 6.5, % OM 1.48, soil moisture content 8 %
Soil No.:
#1
Initial conc.:
>= 31.4 - <= 31.6 mg/kg soil d.w.
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
Soil No.:
#1
Temp.:
20
Details on experimental conditions:
Approximately 120 g soil per vessel. Duplicate anaylis at time 0, 1, 2, 3, 4, 5, 6, 8, 24, 96, 168 and 288 hours after treatment. Treated and non-treated biomass samples analysed for microbial activity at time 0, 24 and 288 hours after treatment.
Soil No.:
#1
DT50:
2 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r2=0.9837
Transformation products:
no
Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
yes
Details on results:
PROPERTIES OF THE TEST SYSTEM
Characterisation and Moisture Content:
- Results from the A & L Great Lakes Laboratories, Inc. characterisation show that the soil obtained from Watsonville, CA is a Sandy Loam, with a pH of 6.5 and organic matter content of 1.48 %. No other pesticides had been applied to the area where the soil was collected since 1998. Pesticides from the same class as the test material had not been applied over the past five years.
- The soil moisture content was determined to be 8.0 % (%RSD of 0.067). Since the water holding capacity of the soil at 75 % of 1/3 Bar is 7.95 % (10.60x0.75 = 7.95 ), no additional water was added to the soil during the test system preparation process. At 16 days after the test systems were prepared (8 days following fortification), two of the extra samples were measured for moisture loss. A 0.3 % moisture difference was determined for both samples showing that moisture had remained stable and that no additional moisture adjustment were necessary.

Temperature
- Temperatures for the test systems stored in the environmental chamber ranged from 19.3 to 20.1 °C during the pre-incubation period. After fortification, the sample temperatures ranged from 19.3 to 21.6 °C. Temperatures were above 21.0 °C for a total of 90 minutes of the definitive study starting on the sixth day after fortification. Since this slight temperature deviation occurred for only a short period of the study and after > 90 % of the test material had degraded and dissipated from the test system, it will not have a significant impact on the outcome of the study.

Microbial Activity
- Viable bacterial and fungal populations were present prior to and after fortification with the test material. No significant quantitative differences between the bacterial or fungal counts of the treated and untreated samples within a selected interval were observed. Qualitative differences were seen in the size of the fungal colonies, with slower apparent growth in the treated samples.

MATERIAL BALANCE
The total applied radioactivity was determined from the theoretical amount of the test material applied to each test vessel. The material balance for individual samples was determined by summing the recovered radioactivity from the soil column and volatile traps and comparing these values to the theoretical amount applied. The material balance for individual samples ranged from 91.2 to 101.7 %, with an average value of 95.0 %. Overall, these results indicate that acceptable material balance was maintained (between 90 and 110 %), allowing for conclusions to be drawn regarding chemical degradation in the test system.


DISTRIBUTION OF RADIOACTIVE RESIDUES

Distribution within the Extraction and Trapping Systems:
- The reaction of the test material with the tripropylamine in the 2 % tripropylamine in DMSO traps produced [14C]methyltripropylarnrnonium ion, with the ( 14C]methyl group of iodomethane being transferred to tripropylamine. This quaternary amine was then ion-paired with 1-heptanesulfonic acid so that it could be retained and analysed on a reversed phase HPLC system. lodomethane was determined to be the only component of the 2% tripropylamine in DMSO traps by comparing the chromatograms of samples to a standard of iodomethane that was directly reacted with 2 % tripropylamine in DMSO. The chromatograms from the actual samples and from the iodomethane standard both had a small quantity of diffuse radioactivity eluting within the same early region of the chromatograms. This diffuse radioactivity was collected and analysed using HPLC system 1. It was determined that this diffuse area was one major peak that did not match the retention time of any reference standards. Since this diffuse radioactivity is a side product of the reaction of iodomethane with the 2 % tripropylamine in DMSO solution, and not a metabolite, its identity was not explored further. Each of the 2 % tripropylamine in DMSO traps analysed by HPLC had lower or equivalent proportions of the diffuse radioactivity discussed above, the methyltripropylammonium ion pair, and no other peaks. The total recovered radioactivity in each of the 2% tripropylamine in DMSO traps was therefore found to have originated solely from iodomethane and will be counted as iodomethane when determining the percent of applied radioactivity as each compound (or component).
- Barium chloride precipitation was performed on all 1N NaOH traps containing >0.25 % of the total applied radioactivity. The volatile 1N NaOH trap for sample Mel-ASM-5 precipitated as well. Results show that 68.10 to 87 .68 % of the radioactivity in the samples analysed precipitated out of the solution. The average amount of radioactivity precipitated from the samples was approximately 80.12 % with a standard deviation of 4.7 %. This average value was applied to all samples < 0.25 % of the applied radioactivity in the NaOH traps. Overall, the radioactivity remaining in solution accounted for < 0.3 % of the total applied radioactivity in any given sample and will be classified as "unknown volatiles" along with the radioactivity present in the coconut charcoal traps.
Distribution of the Individual Components within the Total System:
- The percent of applied radioactivity as iodomethane within the soil column decreased from an average value of 95.3 % at time 0 to 0.0 % by 7 days (168 hours). Iodomethane within the volatile traps correspondingly increased to an average value of 96.5 % by 7 days. Residues remaining on the soil after the heated nitrogen purge of the soil column increased from an average of 0.3 % at time 0 to 1.1 % by hour 3. After hour 3, the residues remained relatively constant, ranging from average values of 1.1 to 1.2 % for the duration of the study. Levels of carbon dioxide and other unknown volatiles gradually increased from average values of 0.2 to 1.1 % and 0.0 to 2.6 %, respectively, by 12 days. The radioactivity listed as other unknown volatiles is a combination of the radioactivity that remained in solution in the 1N NaOH traps after barium chloride precipitation and the radioactivity collected on the coconut charcoal traps. Since the levels of radioactivity on the soil residues and those listed as "other unknown volatiles" were less than 1.4 and 3.0 % of the applied radioactivity, respectively, no further characterisation was performed.

DEGRADATION KINETICS
Non-linear regression analysis for iodomethane in the total sediment-water system was performed using a one-phase exponential decay with GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). The plateau constant for the equation was set to zero, since complete degradation and dissipation occurred by the end of the study. The rate constant (k) was determined to be 0.3408 (R = 0.9837) giving a DT50 of 2.0 hours and a DT90 of 6.8 hours. Since the correlation coefficient is > 0.98, there is a high level of confidence in the slope (rate constant) from the regression analysis and consequentially the calculated DT50 and DT90 values.

IDENTIFICATION OF IODOMETHANE
Confirmation of lodomethane by GC-MS:
- The identity of iodomethane was confirmed by GC-MS prior to fortification of the test vessels. The ions at 142 and 144 m/z correspond to the molecular ions (M+) of non-radiolabelled and radiolabelled iodomethane, respectively. The fragment at 127 m/z corresponds to iodide, and resulted from the loss of the methyl group (15 m/z units).
Co-Chromatography of Derivatised lodomethane:
- The major peak observed from the injection of a 2 % tripropylamine in DMSO trap from a sample was compared to the major peak found from the direct reaction of [ 14C]iodomethane standard with 2 % tripropylamine in DMSO, by co-chromatography. Results show that by co-injecting solutions from the standard and sample together that only one definitive major peak was present, indicating that the two peaks are from the same compound.
Confirmation of the Identity of the lodomethane Derivative by LC-MS:
- The identity of the major peak found in the 2% tnpropylamine in DMSO trapping solutions was determined by LC/MS using (+)ESI. The ions at 158 and 160 m/z correspond to the molecular ions of non-radiolabelled and radiolabelled methyltripropylammonium ion, respectively. The fragment ions at 116 and 114 m/z are likely from the loss of C3H6 and C3H1+H, respectively.

Exracted Iodomethane declined from 95.3 % at 0 hours to 0.8 % at 24 hours.

Volatilised Iodomethane increased from 0 % at 0 hour to 96.5 % at 168 hours.

Conclusions:
The DT50 and DT90 of the test material in the aerobic soil from Watsonville, California are 2.0 and 6.8 hours, respectively. The soil was determined to be microbially viable throughout the study. Total carbon dioxide and other "unknown compounds" gradually increased to an average of 1.1 and 2.6 % of the total applied radioactivity, respectively, by the end of the study (12 days). Soil residues increased to approximately 1.1 % of the applied radioactivity by 3 hours and then remained relatively constant for the duration of the study. Overall, the major fate of iodomethane in the aerobic test systems was volatilisation, with minor contributions from microbial processes (carbon dioxide and other volatiles) and possible direct reactions with the organic material in the soil (soil residues).
The mass spectral and HPLC co-chromatographic data confirm that the material collected in the 2 % tripropylamine in DMSO trapping solutions was the proposed derivative of iodomethane, the methyltripropylammonium ion.
Executive summary:

The biodegradation of the test material in soil was investigated in accordance with the standardised guideline EPA 162-1, under GLP conditions.

An aerobic soil metabolism study was conducted with the test material at a concentration of 31 µg/g in soil from Watsonville, California, in the dark at 20 °C. This concentration of the test material approximates the single maximum field use rate (263 kg a.i./ha) calculated for a 55.9-cm (22-inch) soil layer, based on a bulk density of 1.5 g/cm^3. Test vessels consisted of glass columns approximately 30 cm in length with an internal diameter of 2.5 cm. Both ends of the columns were sealed with screw-top PTFE lined silicone septum (Kontes Glass Company, Vineland, NJ). Individual columns were filled to a depth of approximately 25 cm, and pre-incubated for 8 days before fortification. The soil moisture content was assessed to be 8.0 %, which is approximately 75 % of 1/3 Bar. Each test vessel was fortified with the appropriate quantity of treatment solution by inverting the column and applying the fortification solution to the bottom of the soil through the septum. Each column was then re-inverted and placed upright with a humidified air intake and output entering the top of the vessel through the septum. A series of traps collected the volatile 14C-labelled materials exiting each test vessel. Duplicate test systems were extracted and analysed at time 0 and 1, 2, 3, 4, 5, 6, 8, 24, 96, 168, 288 hours. Microbial evaluations were performed using untreated soil at 3 days prior to fortification, and at time 0, 24, and 288 hours. Microbial evaluations were also performed using treated soil at time 0, 24 and 288 hours after treatment. Both the untreated and treated soil were viable, with similar population sizes.

The DT50 and DT90 of the test material in the aerobic soil from Watsonville, California are 2.0 and 6.8 hours, respectively. The soil was determined to be microbially viable throughout the study. Total carbon dioxide and other "unknown compounds" gradually increased to an average of 1.1 and 2.6 % of the total applied radioactivity, respectively, by the end of the study (12 days). Soil residues increased to approximately 1.1 % of the applied radioactivity by 3 hours and then remained relatively constant for the duration of the study. Overall, the major fate of iodomethane in the aerobic test systems was volatilisation, with minor contributions from microbial processes (carbon dioxide and other volatiles) and possible direct reactions with the organic material in the soil (soil residues).

The mass spectral and HPLC co-chromatographic data confirm that the material collected in the 2 % tripropylamine in DMSO trapping solutions was the proposed derivative of iodomethane, the methyltripropylammonium ion.

Description of key information

The DT50 and DT90 of the test material in the aerobic soil from Watsonville, California are 2.0 and 6.8 hours, respectively. The soil was determined to be microbially viable throughout the study. Total carbon dioxide and other "unknown compounds" gradually increased to an average of 1.1 and 2.6 % of the total applied radioactivity, respectively, by the end of the study (12 days). Soil residues increased to approximately 1.1 % of the applied radioactivity by 3 hours and then remained relatively constant for the duration of the study. Overall, the major fate of iodomethane in the aerobic test systems was volatilisation, with minor contributions from microbial processes (carbon dioxide and other volatiles) and possible direct reactions with the organic material in the soil (soil residues).

The mass spectral and HPLC co-chromatographic data confirm that the material collected in the 2 % tripropylamine in DMSO trapping solutions was the proposed derivative of iodomethane, the methyltripropylammonium ion.

Key value for chemical safety assessment

Half-life in soil:
2 h
at the temperature of:
20 °C

Additional information

The biodegradation of the test material in soil was investigated in accordance with the standardised guideline EPA 162-1, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

An aerobic soil metabolism study was conducted with the test material at a concentration of 31 µg/g in soil from Watsonville, California, in the dark at 20 °C. This concentration of the test material approximates the single maximum field use rate (263 kg a.i./ha) calculated for a 55.9-cm (22-inch) soil layer, based on a bulk density of 1.5 g/cm^3. Test vessels consisted of glass columns approximately 30 cm in length with an internal diameter of 2.5 cm. Both ends of the columns were sealed with screw-top PTFE lined silicone septum (Kontes Glass Company, Vineland, NJ). Individual columns were filled to a depth of approximately 25 cm, and pre-incubated for 8 days before fortification. The soil moisture content was assessed to be 8.0 %, which is approximately 75 % of 1/3 Bar. Each test vessel was fortified with the appropriate quantity of treatment solution by inverting the column and applying the fortification solution to the bottom of the soil through the septum. Each column was then re-inverted and placed upright with a humidified air intake and output entering the top of the vessel through the septum. A series of traps collected the volatile 14C-labelled materials exiting each test vessel. Duplicate test systems were extracted and analysed at time 0 and 1, 2, 3, 4, 5, 6, 8, 24, 96, 168, 288 hours. Microbial evaluations were performed using untreated soil at 3 days prior to fortification, and at time 0, 24, and 288 hours. Microbial evaluations were also performed using treated soil at time 0, 24 and 288 hours after treatment. Both the untreated and treated soil were viable, with similar population sizes.

The DT50 and DT90 of the test material in the aerobic soil from Watsonville, California are 2.0 and 6.8 hours, respectively. The soil was determined to be microbially viable throughout the study. Total carbon dioxide and other "unknown compounds" gradually increased to an average of 1.1 and 2.6 % of the total applied radioactivity, respectively, by the end of the study (12 days). Soil residues increased to approximately 1.1 % of the applied radioactivity by 3 hours and then remained relatively constant for the duration of the study. Overall, the major fate of iodomethane in the aerobic test systems was volatilisation, with minor contributions from microbial processes (carbon dioxide and other volatiles) and possible direct reactions with the organic material in the soil (soil residues).

The mass spectral and HPLC co-chromatographic data confirm that the material collected in the 2 % tripropylamine in DMSO trapping solutions was the proposed derivative of iodomethane, the methyltripropylammonium ion.