Enhanced degradation of a mixture of three herbicides in the rhizosphere of a herbicide-tolerant plant

Chemosphere,Vol. 28, No. g, pp. 1551-1557, 1994

Pergamon 0045-6535(94)E0076-6

Elsevier Science Ltd Printed in Great Britain. All rights reserved 0045-6535/94 $7.00+0.00

ENHANCED DEGRADATION OF A MIXTURE OF THREE HERBICIDES IN THE RHIZOSPHERE OF A HERBICIDE-TOLERANT PLANT

T. A. Anderson*, E. L. Kruger, and J. R. Coats Pesticide Toxicology Laboratory, Department of Entomology, Iowa State University, Ames, Iowa 50011-3140 (Received in Germany 11 August 1993; accepted 29 November 1993)

ABSTRACT The rhizosphere of herbicide-tolerant plants may be an important component in biologically remediating pesticide-contaminated soils. A pesticide-contaminated site at an agrochemical dealership in Iowa was characterized, and soil from the site was brought to the laboratory for degradation experiments. Three major herbicides were identified in the soils by gas chromatographyatrazine, metolachlor, and trifluralin. Although concentrations of these chemicals were as high as 2 to 3 times field application rates, herbicide-tolerant plants were found growing in the contaminated soil. Initial numbers of microorganisms were determined in rhizosphere soil from Kochia sp. and in edaphosphere (nonvegetated) soil. The rhizosphere soil had an order of magnitude higher microbial numbers (4.2 x 10s) compared with the edaphosphere soil (3.5 x 104.) A degradation experiment that did not incorporate vegetation was carded out by using sterile control soil, Kochia sp. rhizosphere soil, and edaphosphere soil spiked with a mixture of atrazine, metolachlor, and trifluralin at levels typical of point-source spills. Significantly (p _<0.10) enhanced degradation was observed in the rhizosphere soil after 14-d incubations. Microorganisms in nonvegetated soil also showed the ability to degrade the three compounds, but not to the extent of the rhizosphere soil. Some abiotic degradation occurred for all three herbicides. The results of these preliminary experiments suggest that the rhizosphere of certain plant species may be important for facilitating microbial degradation of pesticide wastes in soils and beneficial for remediating pesticide-contaminated sites.

*To whom correspondence should be sent.

INTRODUCTION The use of microorganisms for remediating contaminated environments is currently an area of intense interest [1]. Recent successes utilizing the natural metabolic capabilities of bacteria and fungi to cleanup soil, sediment, and water have been instrumental in encouraging this interest [2,3,4]. In addition, the relative cost-effectiveness of bioremediation compared with other remediation technologies has spurred research to identify situations for which remediation using microorganisms can be appropriate. Although use of the term bioremediation is relatively new, actual biological

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treatment of wastes has been occurring for decades, especially in the areas of waste water treatment and composting. The rhizosphere is a zone immediately surrounding the plant root [5]. The numbers of microorganisms in the rhizosphere are typically an order of magnitude higher than in edaphosphere (nonvegetated) soil. In addition, the rhizosphere contains a more diverse, active, and synergistic microbial community than edaphosphere soil. These phenomena often result in greater rates of microbial degradation of organic compounds in the root zone compared with the edaphosphere [6-11]. The variety of plants and chemicals for which this has been observed and the remediation processes associated with the rhizosphere were recently reviewed [12-14]. The results of these studies suggest that vegetation potentially could be managed to facilitate biological remediation of waste sites by enhancing microbial degradation. In soils contaminated with pesticides, such as those at agrochemical dealerships, a unique problem exists because of the presence of herbicides-chemicals designed to prohibit the growth of vegetation. However, herbicide-tolerant plants can survive at these sites and are ideal candidates for testing whether vegetation can be used to enhance microbial degradation of pesticide wastes. In this paper, we report on preliminary work with a pesticide-contaminated site in Iowa. MATERIALS AND METHODS Site Characterization and Sample Collection. An appropriate study site was selected on the basis of several criteria, including accessibility, type of contamination, imminent hazard, and availability of background information. The site selected is an agrochemicals dealership in Iowa plagued by herbicide contamination in the groundwater since the mid-1980s. A variety of herbicides such as atrazine, metolachlor, alachlor, and trifluralin have also been detected in surface soil at the site at concentrations severalfold field application rates. In most instances, the contaminants are confined to the upper 40 cm of soil but are not homogeneously distributed. An initial vegetation survey of the site revealed several herbicide-tolerant plant species including Kochia sp., knotweed (Po/ygonum sp.), and crabgrass (Digitaria sp.). The presence of these plants was consistent with previous observations in the literature of their resistance to certain herbicide classes, as well as with the types of chemicals present in the soil. We collected rhizosphere soil from Kochia sp. and edaphosphere soil from the site for use in degradation studies. Soils were transferred to sterile WhirI-Pak® bags, sealed, and stored on ice for transport to Iowa State University. The soils were sieved (2.4 mm) and stored in the dark at 4 °C. A composite soil sample was analyzed for percent organic matter, pH, cation exchange capacity, and particle size distribution. Microbial Plate Counts. Estimates of microbial numbers (colony-forming units per gram of soil) in Kochia sp. rhizosphere soil and edaphosphere soil were made by the spread plate technique on trypticase soy agar (BBL Microbiology Systems, Cockeysville, MD). Serial dilutions were made in sterile, distilled water. Petri plates were incubated in the dark at 23 °C. Colonies were counted on day 3. In addition to the initial plate counts, determinations of colony-forming units were also conducted on pesticide-treated soils at the conclusion of the degradation experiment.

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Microbial Degradation. The purpose of the degradation experiment was to test the influence of rhizosphere soil on microbial degradation of a mixture of pesticides. The degradation of three representative chemicals found at the site, atrazine, metolachlor, and trifluralin, was tested by monitoring removal of parent compound from rhizosphere soil, edaphosphere soil, and sterile control soil (autoclaved for 1 h on three consecutive days). Although chemical concentrations in all soils were near or exceeding field application rates, the soils were spiked with additional amounts of the three chemicals to increase the concentrations to levels typical of point-source spills (> 10 ppm). Samples were incubated in an environmental chamber with a 14 h photopedod. Diurnal temperatures were 30/24 °C. Chemical Analysis. Soils were extracted by mechanical agitation with ethyl acetate for 2 hours to remove the chemicals of interest. Spike-recovery tests were used to confirm the adequacy of this extraction method. The % degradation values were corrected for chemical extraction efficiency based on the results of these tests. The residual water in the soil extracts was removed with anhydrous sodium sulfate, and the extracts were concentrated by rotary evaporation. Concentrated extracts were analyzed using a GC9A gas chromatograph (Shimadzu Corporation, Kyoto, Japan) with a flame thermionic detector. Chromatographic conditions were: column, 10% DC 200:2% OV 225 (2.0 mm x 1.8 m); carder gas, N2 (35 mL/min); injector temperature, 250 °C; column temperature, 230 °C; detector temperature, 250 °C. Chemical concentrations were determined by using calibration curves constructed with pesticide standards. The significance of concentration differences in sterile, edaphosphere, and Kochia rhizosphere soil on day 14 was tested by ANOVA. RESULTS Physicochemical properties of the composite soil sample are shown in Table 1. Soils were a sandy loam texture (pH 7.5) with 2.2% organic matter. The high sand content of the soil probably has contributed to the presence of herbicides in groundwater below the site; although, clay lenses have undoubtedly retarded some contaminant movement. Although concentrations of individual herbicide residues at specific areas of the site were occasionally quite high (>10 ppm), the wastes were not homogeneously distributed in the soil. Thus, herbicide concentrations in the composite soil sample composed of contaminated and uncontaminated soils were, in some instances, lower than field application rates. Although the soil was quite sandy, there seems to be adequate organic matter to support microbial growth as indicated by the plate count data. Rhizosphere soils from Kochia sp. had 4.2 x 105 colony-forming units per gram of soil, whereas edaphosphere soils had 3.5 x 104 colony-forming units per gram, an R/S ratio of 12. At the conclusion of the degradation experiments (14 days), colonyforming units per gram of soil had increased in both the rhizosphere (1.3 x 108) and edaphosphere soil (1.8 x 106), indicative of growth on the herbicide mixture and/or removal of competition by the toxic effects of the herbicides. Sterile control soils remained sterile throughout the experiment. Degradation tests with sterile soil, edaphosphere soil, and rhizosphere soil collected from the root zone of Kochia sp. indicated a significant (p _<0.10) enhanced microbial degradation of trifluralin,

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atrazine, and metolachlor in the rhizosphere soil after 14 days (Figure 1). Because plants were absent in these tests, root uptake of the herbicides was eliminated and therefore did not obscure the degradation data. However, the absence of a living plant in the rhizosphere soil during the degradation experiments may have reduced the impact of the rhizosphere microorganisms by changing the composition of the microbial community. Nonetheless, degradation of the parent compounds was significantly accelerated in the rhizosphere soil. Surprisingly, some Kochia sp. seedlings emerged from the rhizosphere soils spiked with additional concentrations of the test chemicals, suggesting the ability of these plants to survive in soils containing high concentrations of herbicide mixtures. Chemical degradation of the herbicides also seemed to take place, as indicated by the decrease in herbicide concentrations in the sterile control soil (data not shown). DISCUSSION

Contaminated soil taken from an agrochemical dealership site in Iowa was tested to determine the potential role of rhizosphere microorganisms from herbicide-tolerant plants in bioremediation of pesticide wastes. It was hypothesized that microorganisms in the rhizosphere of these tolerant plants might be involved in enhanced microbial degradation of herbicides and possibly protecting the plant from herbicidal injury, as suggested previously [7]. Kochia sp., a plant with several herbicide-resistant biotypes, sustained microbial populations an order of magnitude greater than did edaphosphere soil. The increased microbial numbers in the rhizosphere soil was further stimulated by herbicide treatment and correlated well with the enhanced microbial degradation observed in rhizosphere soil compared with edaphosphere and sterile control soils, however, the composition of the microbial community rather than its size may be more important for herbicide degradation [6,7,8,15] We have also tested whether a commodity plant such as soybean (Glycine max) could survive in the pesticide-contaminated soil not spiked with the herbicide mixture, and if its presence could enhance biodegradation of atrazine, metolachlor, and trifluralin. Because soybean is sensitive to damage from these chemicals, we hypothesized that microbial degradation of the herbicides would not be significantly enhanced in the rhizosphere compared to edaphosphere soil. Although soybean survival in the soil was high, its presence did not seem to enhance the degradation of the chemicals (data not shown). The time that these chemicals have been present in the soil undoubtedly has affected their bioavailability (and, thus, toxicity) to soybean. This is further illustrated in that the addition of similar concentrations of chemicals to uncontaminated soil prohibited the growth of soybean seedlings. The purpose of these experiments was not to obtain information for remediating the specific site described but, rather, to attempt to understand what it is about plants at these sites, in relation to the type of chemical contamination, that enhances microbial degradation in the root zone. Evidence for enhanced microbial degradation of a variety of hazardous organic chemicals in the rhizosphere continues to accrue [13,14] suggesting that plants could be managed at contaminated sites to facilitate microbial degradation of unwanted organics. The preliminary information presented herein further illustrates the role that rhizosphere microbial communities could play in maintaining and/or remediating herbicide-contaminated soils through enhanced metabolism in the root zone. The rhizosphere contains a diverse microbial community capable of vast metabolic activities. A better

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understanding of the complex relationship between plants, microorganisms, and chemicals in the root zone could be aided by characterizing the microbial communities associated with different plant species under contaminated and uncontaminated conditions and determining the role of exudates in selection of those communities. Ultimately, such information will help provide additional management strategies that may facilitate the biological remediation of these contaminated sites. ACKNOWLEDGMENTS

The authors thank Jennifer Chaplin and Pam Rice for technical assistance. This project was partly funded by a seed grant from the Center for Health Effects of Environmental Contamination (CHEEC), University of Iowa. Journal Paper No. J-15493 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3187. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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