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a Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701
b Department of Agronomy and Soils, Auburn University, Auburn, AL 36849
* Corresponding author (gsheng{at}uark.edu).
Received for publication July 10, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: HPLC, high performance liquid chromatography • PBS, phosphate buffer solution
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Pesticide sorption and desorption in soil are dependent on the composition and properties of the soil. Soil organic matter and clay minerals are often considered the two components responsible for pesticide sorption (Sheng et al., 2001). However, sorptive properties of a soil are often altered by agricultural practices. As a method of land clearing, post-harvest burning of crop residues is practiced in many agricultural regions worldwide. The particulate matter (ash) resulting from burning is incorporated directly into soil by tillage. The ash usually contains high-surface-area carbonaceous materials due to combustive carbonization of the residue, and can effectively sorb applied pesticides. Hilton and Yuen (1963) reported the enhanced sorption of substituted ureas and s-triazines in Hawaiian soil that received the ash from burning of sugarcane trash and the retained sorptivity of the soil following hydrogen peroxide treatment. The observations were ascribed to sugarcane ash, the carbon fraction of which was believed to be resistant to hydrogen peroxide oxidation, although no direct evidence was provided. Yang and Sheng (2003a)(2003b) recently reported the high sorptivity of ash derived from the burning of wheat and rice residues. The ash was found to be 400 to 2500 times more effective than a silt loam soil in sorbing diuron from water. Furthermore, evidence showed that the carbon fraction of the wheat ash was primarily responsible for the high sorptivity of the ash in a fashion similar to that of activated carbon. Yang and Sheng (2003a) estimated that each burning of wheat residue would result in an ash content of up to 0.1% (by weight) in a soil of furrow slice (approximately 15 cm deep) and the actual content may be much higher due to repeated annual burnings and subsequent ash accumulation in soil.
Biodegradation of organic contaminants in the presence of activated carbon is reduced due to high adsorption that effectively decreases the solution-phase concentration of contaminants and inability of microbes to utilize adsorbed contaminants directly (Guerin and Boyd, 1997). It is not clear whether the presence of ash in soil has a similar effect on pesticide biodegradation. In this study, benzonitrile and an ash resulting from the burning of wheat residue were used to test the effect of ash on biodegradation. Benzonitrile is a common structural moiety of some widely used pesticides (Nawaz et al., 1992), and wheat residue is the most frequently burned crop residue in the United States. Our objectives were to (i) evaluate the influence of wheat ash on adsorption and biodegradation of benzonitrile in soil and (ii) gain further understanding of the potential effect of agricultural practices on the environmental fate of pesticides in agricultural soils.
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MATERIALS AND METHODS |
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The bacterium was characterized by standard microbiological methods (Holt et al., 1994). The bacterium showed 0.5- x 0.2-µm rods, which were single, in pairs or short chains, and the bacterium was Gram positive and showed no spores in 48 h. The white colonies grew well on nutrient agar. The bacterium was identified with fatty acid profile (service provided by Microbial Identification, Inc., Newark, DE) as a Nocardia species with a similarity index of 0.689, and was able to use benzonitrile as the sole carbon and energy source for growth.
To obtain inocula for biodegradation measurements, cells grown in the mineral salts medium were harvested in the early stationary phase (19 h) by centrifugation (RCF = 11700 x g) for 15 min, washed twice with 0.02 M sterile phosphate buffer solution (PBS, pH 7) to remove residual benzonitrile, and resuspended in the same buffer solution before inoculation. The cell suspension was kept at room temperature (approximately 25°C) and used within 3 h.
Sorbents and Extracts
A Stuttgart silt loam (fine, smectitic, thermic Albaquultic Hapludalfs) was collected from a rice field in Stuttgart, Arkansas. The soil had 2.1% organic matter, a mechanical composition of 17.1% sand, 60.4% silt, and 22.5% clay, and a CEC of 8.5 cmol kg–1 with a pH of 6.1. The soil was air-dried, ground, and sieved through a 1-mm sieve. The soil had no history of crop residue burns, and was presumed to have minimal crop-residue-derived ash.
Wheat ash was collected by burning dry wheat straw (1 kg) on a 1- x 1-m stainless steel plate under natural conditions (Yang and Sheng, 2003a). Elemental analysis showed that the ash contained 12.9% elemental C, 1.4% carbonate C, 19.5% Si, 20.9% K, 3.4% Ca, and other trace elements. The specific surface area of the ash was 10.1 m2 g–1 as measured by N2 adsorption (service provided by QuantaChrome Instruments, Boynton Beach, FL) and a pH of 10.2.
Ash-amended soil (1.0% ash) was prepared by mixing wheat ash with soil in a ratio of 1:99 (w/w), and the soil, ash, and ash-amended soil were used as sorbents in this study. Before use, all sorbents were sterilized by 
-irradiation (5 Mrad from a 60Co source) in 150-mL polypropylene bottles, and the sealed bottles were maintained at room temperature.
Separate extracts were prepared from 280.30 g soil, 283.13 g ash-amended soil, or 2.83 g ash suspended in 700 mL of PBS and shaken on a mechanical shaker for 24 h. The liquid to solid ratio was the same as that used in the biodegradation measurements. The supernatants obtained after centrifugation were filtered through two layers of Whatman (Maidstone, UK) no. 5 filter paper. The extracts were autoclaved and stored at room temperature until use.
Sorption and Desorption
Sorption isotherms for benzonitrile were obtained by a batch equilibration technique. A predetermined weight of each sterile sorbent (2.8 g of soil, 0.6 g of ash-amended soil, and 0.005 g of ash) and 8 mL of PBS containing various amounts of benzonitrile were combined in a series of 30-mL glass centrifuge tubes with Teflon-coated caps. Initial benzonitrile concentrations ranged from 0 to 4.25 mg L–1. Following 24 h of rotation at room temperature, all tubes were centrifuged for 15 min at RCF = 6120 x g, and supernatants were analyzed for benzonitrile concentration by high performance liquid chromatography (HPLC). The amounts of benzonitrile sorbed were calculated by the difference between the initial and final concentrations. Previous tests indicated that sorption reached apparent equilibrium within 18 h, and loss of benzonitrile in the processes other than sorption was negligible. All measurements were made in duplicate and usually varied less than 5%; the average data were reported.
Desorption kinetics were measured in a series of glass centrifuge tubes containing sterile soil and ash in the same amounts as used in the sorption experiments and 8 mL of PBS with an initial benzonitrile concentration of 3.19 mg L–1. Following 24 h of equilibration, all tubes were centrifuged for 15 min at RCF = 6120 x g to separate the liquid and sorbent. The supernatants (6 mL for soil and 7 mL for ash) were removed; the benzonitrile concentrations in the supernatants were measured by HPLC. Benzonitrile-free PBS in the same amount as removed was added to the tubes to bring the volume back to the original; the contents in the tubes were resuspended immediately. At 2, 5, 20, 60, and 120 min, duplicate tubes were centrifuged and the supernatants were analyzed for benzonitrile concentrations.
Benzonitrile was analyzed by HPLC using a Phenomenex (Torrance, CA) Prodigy C18 column and a Hitachi (Tokyo, Japan) Model L-7450A diode array detector. The mobile phase was a mixture of acetonitrile and water in a ratio of 45:55 (v/v) at a flow rate of 1.75 mL min–1, and the sample injection volume was 100 µL.
Biodegradation
For biodegradation experiments, 7 mL of PBS plus 1 mL of 8.5 mg L–1 benzonitrile solution was equilibrated with soil (2.803 g), wheat ash (0.02830 g), or ash-amended soil (2.8313 g) in Teflon-sealed 30-mL glass centrifuge tubes at room temperature for 24 h to complete benzonitrile sorption. Mixing of PBS and benzonitrile solution resulted in a solution with an initial benzonitrile concentration of 1.06 mg L–1. Sterility of the sorbent slurries was checked by plating 0.1 mL of the suspension on nutrient agar plates. Controls containing 7 mL of extracts and 1 mL of 8.5 mg L–1 benzonitrile solution but no sorbents were also prepared. Each tube was inoculated with 0.1 mL of the bacteria cell suspension (initial cell density of approximately 3.6 x 108 colony-forming units [CFU] mL–1, resulting in a final cell density of approximately 4.5 x 106 CFU mL–1 in the tube) and incubated at room temperature. Sterile-sorbent slurries and extract controls without inoculation were also prepared to monitor recovery and abiotic losses of benzonitrile during the entire period. At degradation times ranging from 10 min to 33 h, the degradation was stopped by adding 0.2 mL of 0.5% Ag2SO4 solution. Termination of benzonitrile degradation following Ag2SO4 addition was verified previously in our laboratory. The tubes were rotated for another 60 min to let benzonitrile equilibrate between solid and liquid phases and centrifuged. One milliliter of supernatant was then sampled to measure benzonitrile concentrations. The supernatant (5.8 mL for soil and ash-amended soil, 6 mL for ash) was subsequently removed, and sorbent was extracted with 10 mL of acetonitrile to analyze sorbed-phase concentrations. Extraction recoveries were 85, 98, and 95% for soil, ash, and ash-amended soil, respectively. Benzonitrile concentrations were adjusted for the recoveries. All experiments were prepared in duplicate and performed at room temperature.
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RESULTS AND DISCUSSION |
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High sorption of benzonitrile by wheat ash alone suggested that the presence of ash in soil may substantially enhance the sorptivity of the soil. With 1% wheat ash in soil, the ash-amended soil was approximately 10 times more effective than the ash-free soil in sorbing benzonitrile (Fig. 1). Assuming that soil and ash independently contributed to the overall sorption, calculations indicate that approximately 90% of the total benzonitrile was sorbed by the 1% ash. The soil, which was 99% of the sorbent mass, sorbed only about 10% of the benzonitrile. The sorptive capacity of the ash was decreased by approximately 55% when the ash was mixed with the soil. The reduction in benzonitrile sorption is presumed to result from the competitive adsorption of various soluble organic molecules in the soil (i.e., dissolved organic carbon such as fulvic acids and carbohydrates) (Yang and Sheng, 2003b). A competitive mechanism has been proposed to account for the decrease of isotherm nonlinearity in the presence of cosolutes in soils containing high-surface-area carbonaceous material (Chiou and Kile, 1998). Despite competitive sorption, the presence of wheat ash in the soil substantially enhanced benzonitrile sorption.
Desorption of benzonitrile from the soil and wheat ash alone was expressed in percent of the total sorbed benzonitrile desorbed at the time of measurement (Fig. 2) . Although the percent of benzonitrile desorbed was similar for ash and soil, desorption kinetics in the ash differed from that in the soil. Desorption from the ash was clearly slower than that from the soil. The Student's t test at the 95% level of confidence was used to compare the individual desorption values. Desorption from the soil was significant within the first 20 min; however, further desorption from the soil beyond 20 min was not significant. Desorption from the ash was significant within the first 60 min. Slower desorption from the ash than from the soil may be due to the surface adsorption of benzonitrile on the ash carbon, which is expected to be stronger as compared with partitioning into soil organic matter. We did not measure the desorption of benzonitrile from the ash-amended soil. It was expected to resemble that from the ash alone.
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As sorption decreased the solution-phase concentration of benzonitrile, decreased bioaccessibility would also slow the degradation of benzonitrile. The degradation of benzonitrile in soil slurry did not have an acclimation period and was completed at approximately 500 min (Fig. 3). Degradation in the soil slurry was slower than that in PBS and soil extract, as shown by the smaller slope of the soil slurry degradation curve compared with that with PBS and soil extract. Thus, sorption of benzonitrile by soil reduced the biodegradation rate. The HPLC analysis showed that the initial equilibrium concentration of benzonitrile in the soil slurry solution was 0.632 mg L–1, or about 40% lower than that in PBS and soil extract. Using the time for complete degradation in soil slurry (500 min), calculation shows that soil sorption reduced the biodegradation by 44%.
Biodegradation of benzonitrile in the slurries of ash and ash-amended soil was substantially reduced as compared with soil slurry (Fig. 3). An acclimation period again contributed to the slower overall biodegradation. Due to high sorption by the ash, the initial concentrations of benzonitrile in the solutions of ash slurry and ash-amended soil slurry were reduced to 3.5 x 10–3 and 3.7 x 10–2 mg L–1, respectively. Higher concentration of benzonitrile in the solution of ash-amended soil slurry was attributed to competitive sorption of dissolved organic carbon that reduced the sorptive capacity of the ash, as described earlier. With low initial solution-phase concentrations, only 20% of benzonitrile in ash slurry and 44% in ash-amended soil slurry were degraded at 2000 min.
As desorption is often a prerequisite for biodegradation, lower degradation of benzonitrile in the ash and ash-amended soil slurries may have been partially due to the differences in desorption rate in soil and ash (Fig. 2). That is, slower desorption may also have contributed to the lower degradation of benzonitrile in the ash and ash-amended soil slurries. We do not know if the soil bacterium was able to utilize ash-sorbed benzonitrile directly. While direct access to soil-sorbed organic contaminants has been reported (Park et al., 2001, 2002; Feng et al., 2000; Guerin and Boyd, 1992), such a process with granular activated carbon was not observed (Guerin and Boyd, 1997).
It has been reported that benzonitrile can be utilized by bacteria as a carbon and nitrogen source and converted to benzoic acid and ammonia (Rezende et al., 2000; Dhillon and Shivaraman, 1999; Masunaga et al., 1995; Nawaz et al., 1992). In our study, we simply measured the dissipation of benzonitrile over time, and thus did not provide mechanistic information about the degradation pathway. However, our results clearly showed that the presence of the ash resulting from the burning of wheat residue substantially reduced the biodegradation of benzonitrile. The reduction apparently resulted from a combination of the acclimation of bacterium, the high sorption by wheat ash, and the slow desorption from the ash.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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-acceptors. Soil Sci. Soc. Am. J. 62:369–378.Related articles in JEQ:
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