A simple solid phase assay for the detection of 2,4-D in soil

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Abstract

Contaminated soils are usually characterized using chemical analyses. However, these do not assess the bioavailability of pollutants, a factor which may be important in estimating the risks associated with contamination. Thus there is a need to support chemical analyses with information on biological effects to determine the potential risks a pollutant may pose in the soil. Although bacterial bioreporters have been used to detect the presence of contaminants in soils, in general these studies have been carried out in slurries or soil extracts rather than soil itself. The following study presents the development of a simple solid-phase bioassay for the direct detection of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) in soil using Ralstonia eutropha JMP 134-32, a luxCDABE-based 2,4-D whole cell bioreporter. The bioreporter was spotted onto glass microfibre filter discs that allowed its retrieval and analysis after exposure to 2,4-D amended soils. These disc-fixed cells responded in a concentration dependent manner to 2,4-D in solution (0–25 mg/L) and in spiked soil (0–50 mg/kg). The influence of environmental factors on bioavailability was demonstrated in soil with a low moisture content which prevented 2,4-D-induced bioluminescence but which did not affect bioluminescence from already induced cells. This rapid and low cost bioassay provides a proof of concept demonstrating that retrievable disk-fixed cells can be induced in soil, thus providing a measure of solid-phase bioavailability. This method overcomes some of the limitations associated with the inoculation and monitoring of bioreporters directly in soil. Additionally, this simple system should be amenable to use with other bioreporters.

Introduction

The characterization of organic chemical contamination in polluted soils traditionally relies on destructive extraction with solvents followed by sensitive chemical analyses (Shaw et al., 1999). Although these processes are useful for quantification of pollutants and are mandated by government regulation, they do not assess the actual bioavailability of pollutants (Kelsey et al., 1997), which may be important in quantifying the risk a contaminant poses.

In an effort to overcome the limitations of traditional chemical analyses, biological endpoints are increasingly being used to characterize waste site contamination and to determine the need for remedial action (Dorn et al., 1998, Dorn et al., 2003). In addition to the use of plants and invertebrates, there are many generalized microbial endpoints amenable to the survey of soil contamination including microbial biomass, viability, survival, respiration, and enzyme activity (Palmer et al., 1998). Although the measurement of these broad responses can be useful in determining general effects, they are time consuming and not amenable to high throughput analyses. Due to their rapid response time, relative ease of handling, and high sensitivity, bacterial bioreporters such as Microtox™ have also been used as biological indicators to complement chemical analyses (Salanitro et al., 1997). The versatility and sensitivity of bacterial bioreporters contribute to their attractiveness as powerful tools for monitoring pollutants in the environment (Leveau and Lindow, 2002).

Bacterial bioreporters based on microbial bioluminescence have been shown to respond to low concentrations of bioavailable pollutants in freshwater, sewage sludge and other complex systems (McGrath et al., 1999). The Microtox™ assay (Microbics Corporation, 1992), which has become widely accepted, relies upon a naturally bioluminescent marine bacterium, Vibrio fischeri. This assay measures the decrease of bioluminescence as the environment becomes more toxic to V. fischeri. Although useful, Microtox™ has disadvantages, such as, narrow pH range and an optimal salinity tolerance that is not representative of fresh water or most soils. This is due in part to the fact that V. fischeri is a marine bacterium which suggests that it may be less appropriate for soil testing (Lajoie et al., 2002).

While bioreporters expressing the lux operon from constitutive or density related promoters seem to provide a reliable measure of generalized toxicity, they are nonspecific and cannot yield detailed information about the nature of the toxicant or the mode of intoxication. Several studies have reported on the development of such constitutive lux-based bioreporters although few have reported on their use in soil (Palmer et al., 1998, Kohler et al., 2000, Lajoie et al., 2002). While widely used in aquatic toxicology, the use of lux-based bioreporters for assessing the toxicity of terrestrial contaminants has been largely limited to soil extracts or slurries (King et al., 1990, Dorn et al., 1998, Hay et al., 2000, Lajoie et al., 2002, Acheson et al., 2004). Although this offers a significant advance over methods that rely on the analysis of solvent extracts, the relevance of the concentrations detected to the bioavailability of pollutants under normal soil conditions remains unknown. Direct application of bioreporters to soils is problematic on several fronts (Ripp et al., 2000) including analyte toxicity, as well as variability in pH, temperature, concentrations of non-inducing nutrients, luminescence quenching by soil particles, and competition from other bacteria. Concerns have also been expressed over the release of genetically modified organisms into the environment (Ripp et al., 2000, Dorn et al., 2003).

Immobilization of bioreporters onto surfaces or in gels may provide one possible solution to some of these problems. In general, immobilized bioreporters have been used to measure pollutants in the vapor phase (Ripp et al., 2003), soil solutions, chemical extracts (McGrath et al., 1999), slurries (Brandt et al., 2002), and wastewater (Lajoie et al., 2002). Although Edenborn and Brickett (2001) and others (reviewed in Wimpenny and Jones, 1988) have used immobilized bacteria to report on microbial processes in sediments and Ripp et al. (2000) monitored the persistence and activity of a naphthalene specific recombinant bioreporter inoculated directly in soil, we could not find any published studies reporting on the direct use of immobilized recombinant bioreporters in unsaturated soils.

Several groups have constructed bioreporters that maintain the lux genes under the control of promoters that respond to either specific compounds including organic pollutants and heavy metals, or that respond to generalized cell stresses such as DNA damage or oxidative stress (reviewed in Kohler et al., 2000). Hay et al. (2000) constructed a 2,4-D lux-based bioreporter (Ralstonia eutropha JMP134-32 ) by placing the lux genes under the control of the tfdDII promoter, which responds in a highly specific manner to 2,4-dichloromuconate, a metabolite of 2,4-D.

2,4-D is the third most used herbicide in the US and Canada, and it is the most commonly used herbicide worldwide (Chu et al., 2004). It owes its popularity to two main features; first, it mimics the plant growth hormone indole acetic acid, making it a plant-specific toxicant; second, it is regarded as readily biodegradable. This latter point is evidenced by the large number of microorganisms which have been reported to metabolize 2,4-D (McGowan et al., 1998). Based on these two important properties, specificity and biodegradability, it has been suggested that 2,4-D is not likely to cause harm to non-target species (Hawkins and Harwood, 2002). However the breakdown of 2,4-D is often incomplete, leaving 2,4-dichlorophenol, a toxic metabolite that can persist in the soil (Quan et al., 2004).

R. eutropha JMP134-32 was previously used to detect the presence of 2,4-D in slurries of soils that had historical contamination with 2,4-D (Hay et al., 2000). Given the solubility of 2,4-D however, it was not clear if the use of slurries might have misrepresented the actual bioavailability of 2,4-D under more realistic field conditions. While others have used nonspecific lux-based bioreporters to monitor the presence of 2,4-D-degrading bacteria in 2,4-D contaminated soils (Masson et al., 1993, Shaw et al., 1999), there is a dearth of reports detailing methods for assaying the bioavailability of 2,4-D or other contaminants (Ripp et al., 2000) in soils with moisture contents approximating those found under field conditions. In the present study we report the development of a simple solid phase bioreporter assay for the detection 2,4-D directly in soil using R. eutropha JMP134-32 cells immobilized on a retrievable support matrix.

Section snippets

Soil description

A fine textured Hudson silt loam soil was used in all soil based assays and was obtained from Cornell University, Ithaca, NY. It has a pH of 5, an organic matter content of 3.5%, and has been described previously (Richards et al., 2000).

Bioreporter-disc preparation

To ensure maximum reproducibility between experiments, 1 mL aliquots of log-phase JMP134-32 cultures where frozen with 20% glycerol at −80 °C and a single aliquot was sacrificed as an inoculum for each experiment. Briefly, a previously unopened, frozen vial of

Results

When the induced bioreporter cells were placed on glass microfiber filters they yielded bioluminescent signals lower than but similar to those obtained from free cells (Fig. 1). None of the other filters however, permitted the detection of acceptable levels of bioluminescence from preinduced cells. Filters with no cells were used as controls and showed no response above background.

Fig. 2 shows the effect that the presence of the glass microfiber filter had on the light emitted by 2,4-D induced

Discussion

The purpose of this work was to develop a simple method for directly assessing solid phase bioavailability using a known bioreporter. This was done in part to overcome limitations associated with assessing contaminant levels in soil slurries which include limited relevance to field moisture conditions, suboptimal growth conditions, and matrix opacity. The 2,4-D bioreporter chosen for this study was known to be capable of detecting 2,4-D in aqueous solutions and soil slurries (Hay et al., 2000).

Acknowledgment

The authors are grateful to Robert Murdoch for his critical reading of the manuscript.

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