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Understanding the dissipation rates of chemicals in unsaturated and saturated zones of subsurface
soils will help determine if reductions of concentrations to acceptable levels will occur. Chemical
properties and microbial biomass and activity were determined for the surface (0-15 cm), lower root
(50-105 cm), and vadose (175-220 cm) zones in a Huntington silty clay loam (Fluventic Hapludoll)
collected from an agricultural field near Piketon, OH. The rates of sorption, mineralization, and
transformation (formation of bound residues and metabolites) of atrazine were determined. Microbial
activity was estimated from the mineralization of 14C-benzoate. We observed decreased levels of
nutrients (total organic carbon, N, and P) and microbial biomass with depth, while activity as measured
with benzoate metabolism was higher in the vadose zone than in either the surface or the root zones.
Sorption coefficients (Kf) declined from 8.17 in the surface to 3.31 in the vadose zone. Sorption was
positively correlated with organic C content. Rates of atrazine mineralization and bound residues
formation were, respectively, 12-2.3-fold lower in the vadose than in the surface soil. Estimated
half-lives of atrazine ranged from 77 to 101 days in the surface soil, but increased to over 900 days
in the subsurface soils. The decreased dissipation of atrazine with increasing depth in the profile is
the result of decreased microbial activity toward atrazine, measured either as total biomass or as
populations of atrazine-degrading microorganisms. The combination of reduced dissipation and low
sorption indicates that there is potential for atrazine movement in the subsurface soils.
KEYWORDS: Microbial biomass and activity; sorption; subsurface microbiology; mineralization; kinetics
Table 1. Mean Physical and Chemical Properties of a Huntington Silty Clay Loam near Piketon, OH
nitrogen phosphorus
mineralb TKN total available
depth (cm) sand (%) silt (%) clay (%) pH TOC (%) (mg N kg-1) (mg N kg-1) (mg P kg-1) (mg P kg-1)
0−15 16.0a 46.0 38.0 6.9 2.1 18.3 1980.0 291.0 2.3
(5.3) (2.0) (4.0) (0.3) (0.1) (2.4) (108.2) (22.5) (0.4)
50−105 15.3 46.7 38.0 7.0 0.8 7.4 1056.7 186.3 0.8
(3.1) (3.1) (0.0) (0.2) (0.02) (0.4) (56.9) (0.6) (0.2)
175−220 11.3 53.3 35.3 7.5 0.5 6.0 746.3 173.0 1.1
(3.1) (3.1) (1.2) (0.2) (0.05) (0.9) (56.1) (10.1) (0.2)
a Values are means over the three sites with the standard deviations beneath the means in parentheses. b Sum of nitrate, nitrite, and ammonia forms of N.
(MSEA). The MSEA lies adjacent to the Scioto River, near Piketon, 1 M NaOH, capped, and incubated for 50 days. The shell vials were
OH. Three field locations at the MSEA field station were chosen within then removed, scintillation cocktail was added, and the 14CO2 was
a field cropped to a corn, soybean, and wheat-hairy vetch rotation. Three trapped by the NaOH determined by LSC. The vials were scored as
soil cores (220 depth × 10 cm), 2 m apart from each other, were (+) when the evolved 14CO2 exceeded 2 times the background and
collected using a truck-mounted GSRP-S (The Giddings Machine Co., (-) otherwise.
Fort Collins, CO) hydraulic soil coring unit. The cores were divided Atrazine Sorption. Atrazine sorption in the soils collected from
into sections that constitute the three depths used: surface zone (0- the different field locations and depths was determined by batch
15 cm), lower root zone (50-105 cm), and vadose zone (175-220 isotherms. Two replicates of 3 g of air-dried soil were equilibrated for
cm). To control contamination, the exterior 1 cm of each section was 48 h in 20 mL of 0.01 M CaCl2 containing a mixture of nonlabeled
removed. The sections were placed in sterile plastic bags and transported and 14C-ring-labeled atrazine (67 Bq 14C-activity per tube) at total
to the Purdue Soil Microbiology Laboratory where the soil from each concentrations of 1.4, 7, 14, and 70 µM. After centrifugation (10 000
section was mixed, passed through a 4-mm sieve, and stored at 4 °C rpm for 15 min), a 1 mL aliquot was removed from the supernatant
for no longer than 2 months. Biomass extractions (see below) were for LSC. Values of Kf were calculated from fitting the Freundlich
started within 1 h of core collection; determination of microbial activity, sorption equation: x/m ) Kf CeN, where x/m is the concentration (µmol
MPN measurement of atrazine degraders, and atrazine mineralization kg-1) of the herbicide sorbed to the soil, Ce is the equilibrium
were begun within 2 months of soil collection. concentration (µmol L-1) in solution, and Kf and N are the sorption
Table 1 shows the physical and chemical characterization of the capacity and the sorption intensity empirical indexes, respectively.
materials collected at the different depths. The particle size was analyzed Atrazine Mineralization. Atrazine mineralization and transformation
by the pipet method, pH in H2O (1:1 soil/solution), total organic carbon were determined using atrazine applied at 0.1 and 1.0 mg kg-1 soil.
(TOC) by the Leco total carbon analyzer, after removal of inorganic Two replicates of 25 g of soil (oven-dry basis) were treated with the
carbon with a 10% phosphoric acid solution. Total N and total P were two concentrations of 14C-atrazine (1682 Bq of 14C-activity per flask),
determined by acid digestion followed by analysis of soluble N and P and the water content was adjusted to -33 kPa equivalent and incubated
by colorimetric flow analysis with a Technicon Auto Analyzer under the same conditions as the benzoate. Atrazine mineralization was
(Technicon Inc., New York, NY). Mineral N and available P were estimated from 14CO2 evolution over a period of 205 days. The vials
extracted with 2 M KCl and analyzed with the Technicon Auto with the NaOH trapping solution were exchanged weekly for the first
Analyzer. month and biweekly thereafter.
Microbiological Analysis. Soil microbial biomass was estimated Atrazine Transformation. To determine atrazine transformation,
from phospholipids-PO4 content of the soils following the extraction jars from the mineralization experiment were sacrificed at seven
of microbial phospholipids (18). Biomass extraction was done on five different times over a period of 205 days. After removal of the NaOH
replicates of approximately 5 g of soil and was initiated within 1 h of vials, the jars were stored frozen (-20 °C) until extraction. Frozen
core collection. samples were thawed, and the soils were immediately extracted with a
Soil microbial activity was estimated from the mineralization of 14C- 4:1 (v/v) methanol:water solution at a 3:1 solvent:soil ratio, by shaking
benzoate. Three replicates of 25 g of soil (oven-dry equivalent) were for 2 h, sitting overnight, and then shaking for another 30 min. The
treated with an aqueous solution of analytical grade benzoate, 99% soils were then centrifuged (5000 rpm for 30 min), the supernatant
purity, fortified with 14C-ring-labeled benzoate (specific activity of 4.9 was removed, and the soils were reextracted with the methanol:water
× 108 Bq mmol-1 ) (Sigma Chemicals, St. Louis, MO). A concentration solution by shaking for 1 h. After centrifugation, the extracts were
of 1.0 mg kg-1 soil with 420 Bq of 14C-activity was applied per flask. combined, and 1 mL was sampled to measure the total extractable 14C.
Samples were adjusted with sterile water to a water content equivalent The methanol was evaporated with a rotary evaporator (50 °C), and
to -33 kPa. A vial containing 10 mL of 0.5 M NaOH as a CO2 trapping the aqueous phase was centrifuged (10000 rpm for 15 min) to remove
solution was placed inside the incubation jar, and the jars were incubated suspended sediments. After acidification with 1 mL of 30% HCl,
at 22 °C. The NaOH solution was exchanged periodically, and 1 mL atrazine and metabolites were separated from the suspension by
of the solution was mixed with 15 mL of scintillation cocktail (Ecolume, partitioning three times with 50 mL of chloroform. The solvent extracts
ICN Biochemicals Inc., Costa Mesa, CA). The 14C activity was were combined and evaporated in a rotary evaporator (30 °C). The
measured in a liquid scintillation counter (LSC) (Tri-Carb 1600 TR, extracts were resuspended in 5 mL of acetonitrile, filtered through 0.45
Packard Instruments, Downers Grove, IL). µm nylon membrane filters, and concentrated to a final volume of 2
Atrazine degrading microorganisms were enumerated by the 14C- mL under a stream of nitrogen. The samples were stored at -20 °C
most probable number method (3, 19). The culture medium consisted until analysis by high performance liquid chromatography (HPLC). The
of a basal salt solution, 100-fold diluted nutrient broth, sterile trace methanol-water extractions removed approximately 95% of added 14C
elements, and atrazine. The final concentration of atrazine was 1.0 mg atrazine, but recovery after the evaporation and filtration steps declined
L-1 (nonlabeled analytical grade atrazine, 99% purity) (ChemService, to 55%. This loss represents the formation of nonextractable residues,
West Chester, PA), plus 14C-ring-labeled atrazine (specific activity of polar metabolites, and nonpolar metabolites in samples treated with
4.3 × 108 Bq mmol-1) (Sigma Chemicals, St. Louis, MO). 14C-activity atrazine and frozen immediately after treatment and losses in extraction
of 84 Bq was used per vial. Serial dilutions of the soils were prepared process. The data showing atrazine remaining over time are corrected
to remove the bacteria from soil particles, and aliquots of the dilutions for this recovery.
and the medium were transferred to shell vials in five replicate sets. Atrazine and the formation of the metabolites deethylatrazine (DEA),
Each shell vial was placed into a scintillation vial containing 1 mL of deisopropylatrazine (DIA), and dealkylatrazine (DEDIA) was deter-
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