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Published online 31 May 2006
Published in J Environ Qual 35:992-1000 (2006)
DOI: 10.2134/jeq2005.0271
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Bioremediation and Biodegradation

Soil Amendments, Plant Age, and Intercropping Impact p,p'-DDE Bioavailability to Cucurbita pepo

Jason C. Whitea,*, Zakia D. Parrisha, Martin P. N. Gentb, William Iannucci-Bergerc, Brian D. Eitzerc, Mehmet Isleyenc and MaryJane Incorvia Mattinac

a Department of Soil and Water, Connecticut Agricultural Experiment Station (CAES), 123 Huntington Street, New Haven, CT 06504
b Department of Forestry and Horticulture, Connecticut Agricultural Experiment Station (CAES), 123 Huntington Street, New Haven, CT 06504
c Department of Analytical Chemistry, Connecticut Agricultural Experiment Station (CAES), 123 Huntington Street, New Haven, CT 06504

* Corresponding author (Jason.White{at}po.state.ct.us)

Received for publication July 13, 2005.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Field experiments were conducted to optimize the phytoextraction of weathered p,p'-DDE (p,p'-dichlorodiphenyldichloroethylene) by Cucurbita subspecies. The effects of two soil amendments, mycorrhizae or a biosurfactant, on p,p'-DDE accumulation was determined. Also, p,p'-DDE uptake was assessed during plant growth (12, 26, 38, and 62 d), and cultivars that accumulate weathered p,p'-DDE were intercropped with cultivars known not to have that ability. Cucurbita pepo L. ssp. pepo accumulated large amounts of the contaminant, having stem bioconcentration factors, amounts of p,p'-DDE translocated, and contaminant phytoextraction that were 14, 9.9, and 5.0 times greater than C. pepo L. ssp. ovifera (L.) D.S. Decker, respectively. During 62 d, the stem BCF (bioconcentration factor) for p,p'-DDE in subspecies pepo remained constant and the total amount of contaminant accumulated was correlated with plant biomass (r2 = 0.86). For subspecies ovifera, the stem BCF was highest at 12 d (1.5) but decreased to 0.39 by 62 d, and p,p'-DDE removal was not correlated with plant biomass. Mycorrhizal inoculation increased p,p'-DDE accumulation by both subspecies by an average 4.4 times. For subspecies pepo, mycorrhizae increased the percentage of contaminant extracted from 0.72 to 2.1%. Biosurfactant amendment also enhanced contaminant accumulation by both subspecies, although treatment reduced subspecies ovifera biomass by 60%. The biosurfactant had no effect on the biomass of subspecies pepo, increased the average contaminant concentration by 3.6-fold, and doubled the overall amount of p,p'-DDE removed from the soil. Soil amendments that enhance the mobility of weathered persistent organic pollutants will significantly increase the amount of contaminant phytoextraction by Cucurbita pepo.

Abbreviations: BCF, bioconcentration factor • POP, persistent organic pollutant • TF, translocation factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
PERSISTENT organic pollutants (POPs) are of environmental concern due to their recalcitrance in soils and sediments, global distribution, and toxicity (Ritter et al., 1995; Wania and Mackay, 1996). Since many POPs are produced synthetically, few natural enzyme systems exist to facilitate their degradation (Nash and Woolson, 1967; Mattina et al., 1999). These pollutants are hydrophobic, with the potential to bioaccumulate in fatty body tissues and biomagnify through food webs (Fraser et al., 2002; Kelly and Gobas, 2001). Due to their properties, POPs bind strongly to organic matter and can become sequestered within the soil matrix. As sequestration occurs, POP bioavailability can decline sharply, dramatically limiting remedial options (Alexander, 2000).

Phytoremediation uses the inherent physiological abilities of plants to decrease pollutants in soil by mechanisms operating within the rhizosphere or by contaminant uptake into the plant (Schnoor, 2002). Organic contaminants can be transformed in the rhizosphere by plant-released enzymes or by the microbial community that develops under the influence of plant root exudates (Schnoor, 2002). Plant uptake of organic compounds is presumed to be a function of their water solubility. Moderately soluble pollutants (log of the octanol–water partition coefficient or log KOW values 1.0–3.5) are the most likely to cross the root membrane and can be accumulated, transported, transformed, or transpired by the plant.

For many POPs, their resistance to biodegradation, hydrophobicity, and low bioavailability in soil suggest that the impact of vegetation would be negligible. This is supported by data from our laboratory, which indicate that plant species may vary in their ability to access POPs such as p,p'-DDE and chlordane, but generally accumulate minimal amounts (Mattina et al., 2000; White, 2000; White et al., 2005). Work by Hülster et al. (1994), however, first suggested that certain subspecies of Cucurbita pepo can effectively accumulate weathered dioxins from soil, a result substantiated in our lab. We have shown that p,p'-DDE and chlordane concentrations in the root and stem tissues of Cucurbita pepo ssp. pepo are frequently an order of magnitude greater than other plant species (Mattina et al., 2002; White, 2001, 2002; White et al., 2003a; Mattina et al., 2004). We speculate that root exudation of low molecular weight organic acids by this Cucurbita alters contaminant bioavailability in the soil and results in enhanced uptake (White et al., 2003a); however, similar to other plants, we expect that POP availability in soil still limits uptake by C. pepo ssp. pepo.

This study sought to maximize the remedial potential of C. pepo ssp. pepo by investigating the impact of soil amendments, plant developmental stage, and cropping strategy on p,p'-DDE uptake. Two subspecies of C. pepo were chosen because they differ in their ability to accumulate this POP, with C. pepo ssp. pepo taking up significantly greater quantities than C. pepo ssp. ovifera (White et al., 2003a). For ease of discussion and analysis, cultivars within these subspecies were grouped and referred to as accumulators and nonaccumulators. Soil was amended with a mycorrhizal inoculant or a biosurfactant. Both are thought to impact soil structure or sequestration and could, therefore, change p,p'-DDE bioavailability (Meharg and Cairney, 2000; Zhang and Miller, 1992; White et al., 1998). As root exudation (and the effect on bioavailability) can vary at different stages of growth, the uptake and translocation of weathered p,p'-DDE was assessed in plants 12, 26, 38, and 62 d after germination, as well as in plants that were prevented from fruiting. Lastly, we assessed the effect of intercropping cucurbits known to effectively accumulate weathered POPs with those that do not.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Experimental Field Plot
Five cultivar varieties of Cucurbita pepo ssp. pepo (‘Black Beauty’, ‘Raven’, ‘Goldrush’, ‘Costata Romanesco’, and ‘Connecticut Field’) and four of Cucurbita pepo ssp. ovifera (‘Seneca’, ‘Sunray’, ‘Patty Pan’, and ‘Zephyr’) were acquired from Johnny's Selected Seeds (Albion, ME) or Seedway (Hall, NY). All subspecies pepo cultivars are zucchini, except for Connecticut Field, which is a pumpkin. All ovifera cultivars are non-zucchini summer squash. Experimental plots were established at the Connecticut Agricultural Experiment Station's Lockwood Farm (Hamden, CT) in areas contaminated with weathered p,p'-DDE residues. The soil is designated as a fine sandy loam (56% sand, 36% silt, 8% clay) with a pH of 6.7 and an organic C content of 1.4%. To minimize contaminant volatilization, limit the growth of unwanted vegetation, and aid in water retention, the experimental plot was covered with 1400 m2 of black polyethylene sheeting. Before planting, 30-cm2 squares were excised from the plastic at 3.0-m intervals (in all directions). Each 30-cm2 square served as a single replicate mound.

Approximately 100 seeds of each cultivar were germinated and, after 3 d at room temperature, the seedlings were planted in the field. To deter local fauna, seedlings were protected with row covers for the first 2 wk of growth. With one exception, each treatment consisted of duplicate mounds of individual cultivars, with each mound containing four plants. For testing the effect of plant development on p,p'-DDE uptake, replication was included at several stages of growth (as described below). Plants were weeded and watered as necessary. Fruits (of approximately 200 g wet weight) were harvested throughout the season, except for pumpkin, where fruit was collected only at the end of the experimental period. All plants (except those used to test earlier developmental stages) were grown for 62 d and then destructively harvested in August. There were eight treatments (four growth stages, biosurfactant amendment, fungal amendment, no fruit, and intercropping) that were distributed randomly across the 1400-m2 experimental plot.

Soil Extraction
Soil cores (2.5-cm diameter, 6- to 10-cm depth) were collected from each treatment before planting and were extracted as described previously (White et al., 2003a). Eight separate soil cores were collected from each set of duplicate mounds, four per replicate. Briefly, soils (3.0 g) from each treatment were air dried, sieved, and extracted with hexanes at 70°C for 5 h. The moisture content was determined separately by placing a 3.0-g portion of soil in an oven at 100°C for 24 h. A 1-mL aliquot of the supernatant was passed through a glass microfiber filter (0.2 µm, Laboratory Science, Sparks, NV) and collected in a chromatography vial. The average p,p'-DDE content in the soil of each set of duplicate mounds for all cultivar and treatment combinations was determined. Postharvest soils were not analyzed as part of this study.

Plant Tissue Analysis
At harvest, unless otherwise noted, a 1.0 by 1.0 by 0.25 m volume of soil that contained the root system was excavated. The roots were carefully separated from the soil and the entire plant was washed thoroughly with water to remove attached particles. The fresh mass of plant tissues (fruit, leaf, stem, and root) was determined. These tissues were then separated by treatment, cultivar, and tissue type; the biomass was then finely chopped and extracted as described previously (White et al., 2003a). Briefly, vegetation was mixed in an explosion-proof blender (Fisher Scientific, Springfield, NJ) with 2-propanol (Ultra-Resi-Analyzed, J.T. Baker, Phillipsburg, NJ) for 30 s before the addition of petroleum ether (Ultra-Resi-Analyzed, J.T. Baker, Phillipsburg, NJ) and subsequent blending for 5 min. The extract was decanted through a funnel packed with glass wool and collected in a 500-mL glass separatory funnel with Teflon stopcock. After draining for 20 min, the petroleum ether was rinsed three consecutive times with reverse osmosis water and a saturated Na2SO4 solution. The petroleum ether was collected in a graduated cylinder containing 10 g of anhydrous Na2SO4 and allowed to sit for 1 to 2 h before transfer to a chromatography vial.

Experimental Treatments
Plant Developmental Stages
To assess the dynamics of p,p'-DDE uptake and translocation across stages of plant growth, the nine cultivars of C. pepo were harvested at four different intervals after planting: 12 , 26 , 38 (male flowers only), and 62 d (mature fruiting plants). Due to concerns about limited biomass at the 12- and 26-d harvests, the nine cultivars were grown in triplicate mounds instead of duplicate (27 mounds total). The mounds consisted of 60-cm2 holes in the plastic and contained 8 to 10 individual plants. The 38- and 62-d treatments were established as described above—duplicate 30-cm2 mounds each containing four plants. The 62-d treatments were the controls for the other experiments described below and denoted as surfactant, fungal inoculation, no fruit, and intercropping. In other words, all treatments were compared to one set of control plants: the nine cultivars grown to maturity (62 d).

To assess the relationship between fruiting and p,p'-DDE accumulation, eight of the cultivars were grown in duplicate mounds for 62 d (16 total mounds) but female flowers were removed on a daily basis to prevent fruit development. Due to the enormous biomass of pumpkins, C. pepo ssp. pepo Connecticut Field was excluded from these trials.

Intercropping
Duplicate mounds of C. pepo were established in which a single individual of subspecies ovifera was surrounded by three individuals of subspecies pepo. The following four cultivar combinations were used: three Black Beauty with one Patty Pan, three Goldrush with one Zephyr, three Costata Romanesco with one Sunburst, and three Raven with one Seneca (eight mounds total—the four combinations in duplicate). The plants were harvested at 62 d as described above; special care was taken to separate the root systems of the two subspecies of C. pepo within the individual mounds of intercropped vegetation.

Soil Amendments
Two different amendments of the soil were tested. The first was a rhamnolipid biosurfactant solution (JBR425, 25% surfactant in water) produced commercially by a mixed culture of Pseudomonas aeruginosa strains (Jeneil Biosurfactant Co., Saukville, WI). Before planting the duplicate mounds of nine C. pepo cultivars (18 total mounds, each containing four plants), the soil was amended with 1 L of a 1000 mg/L solution. After surfactant addition, the soil was mixed manually with a trowel and the seedlings were then planted. Two weeks after planting, a second surfactant amendment of 200 mL of a 100 mg/L solution was made. To facilitate surfactant mixing into the soil and rhizosphere, a 1-cm-diameter bamboo stake was used to make six 12-cm-deep cores around the plants. Plants were harvested after 62 d of growth.

The second soil amendment tested was a mycorrhizal root inoculant (BioVam) purchased from T&J Enterprises (Spokane, WA). The product contains vesicular arbuscular mycorrhizae as a primary constituent and is recommended for use to enhance the growth and survivability of a range of agricultural and nonagricultural plant species. Specifically, the material consists of endomycorrhize (40–100 spores/cm3), ectomycorrhizae (100–500 spores/cm3), two Trichoderma species (up to 10 000 cells/cm3), as well as the following bacteria (total of 20 000 cells/cm3): Arthrobacter globiformis, Bacillus subtillis, two Azobacter species, and four Pseudomonas species. At planting of the nine C. pepo cultivars (duplicate mounds for a total of 18, each with four plants), 0.6 cm3 of inoculant was placed into the soil with each individual seedling. Plants were harvested after 62 d of growth.

Chemical Analysis
The p,p'-DDE content in the soil or tissue extracts was determined on a Agilent (Avondale, PA) 6890 gas chromatograph (GC) with a 63Ni microelectron capture detector. The column (30 m by 0.53 mm by 0.5 µm) contained a SPB-1 film (Supelco, Bellefonte, PA) and the GC program was 175°C initial temperature ramped at 3.5°C/min to 225°C, then ramped at 25°C/min to 250°C with a hold time of 4.71 min. The total run time was 20 min. A 2-µL splitless injection was used, and the injection port was maintained at 250°C. The carrier gas was He, and the makeup gas was 5% CH4 in Ar at 60 mL/min. The electron capture detector was maintained at 325°C. The retention time of p,p'-DDE was 10.3 min.

Crystalline p,p'-DDE was acquired from the USEPA National Pesticide Standard Repository (Fort Meade, MD) and portions were transferred to petroleum ether (vegetation) or hexane (soil). The p,p'-DDE solution was diluted to prepare calibration standards at 10, 25, 50, 100, 150, 250, and 500 ng/mL, and the p,p'-DDE concentration in the tissue or soil extracts was quantified using external standard calibration.

Statistical Analysis
The soil surrounding each cultivar of each treatment was extracted in quadruplicate. Triplicate vegetation extractions were conducted on the tissues of each cultivar within each treatment. Average tissue p,p'-DDE concentrations were determined for the 62-d-old plants within the two subspecies. Then across the seven other treatments, the following indices were analyzed statistically by an ANOVA followed by a Dunn's multiple comparison test blocked on cultivar or subspecies: root bioconcentration factor, stem bioconcentration factor, translocation factor, biomass, and percentage of p,p'-DDE phytoextracted. The definitions of these parameters can be found below.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The treatments used in this series of experiments allowed us to first establish a baseline for the uptake and translocation of a persistent soil pollutant—p,p'-DDE—by cultivars of C. pepo, and then to evaluate the optimization of this system by assessing the impact of soil amendments or alteration of the cropping strategy on contaminant accumulation by the plants.

Soils
The p,p'-DDE content of the soil ranged from 68 (intercropped Costata Romanesco with Sunburst) to 630 µg/kg (62-d Raven) (Table 1). The concentrations are indicative of historical contamination as a function of routine DDT [1'-(2,2,2-trichloroethylidene)bis(4-chlorobenzene)] use until 1970 and the levels are consistent with previous studies in this area of the farm (White, 2001, 2002; White et al., 2003a).


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  		Table 1. Concentrations of p,p'-DDE in the soils of all cultivar and treatment combinations. Values represent the average of four replicates with the standard deviation in parentheses.

 
Vegetation
With the exception of certain Patty Pan tissues (noted below), all tissues of all cultivars contained measurable quantities of p,p'-DDE. In most cases, the concentration of contaminant was highest in the roots, followed by decreasing amounts in the stems and leaves. Direct comparison of contaminant levels in C. pepo tissues under the various treatment regimes is confounded by the different soil p,p'-DDE concentrations. In previous work with a soil containing weathered chlordane at 0.37 to 4.6 mg/kg, contaminant content in the plant increased linearly with increasing soil concentrations (Mattina et al., 2004a). Given this point and the fact that direct comparison of the p,p'-DDE concentrations in the plants is impossible due to variable soil content, the calculation of bioconcentration factors is necessary. Bioconcentration factors were determined for each tissue under each treatment by calculating the dry-weight ratio of p,p'-DDE concentration in a plant tissue to that in the associated soil. Bioconcentration factors (roots or stems) are of interest because this parameter permits a direct comparison of the abilities of different plants to accumulate weathered POPs relative to soil content. In addition, the effect of treatments on three other parameters will be discussed. The first is total plant biomass. The second is translocation factor (TF), defined as the ratio of p,p'-DDE concentration in the stem to that in the roots. The TF is of interest because, from a practical point of view, a successful candidate for contaminant phytoextraction in the field must transfer much of the pollutant from the roots to the aerial tissues. Last, the amount of p,p'-DDE phytoextracted is calculated as the absolute mass of contaminant transferred from the soil to the plant. This requires an estimate of the soil volume at destructive harvest, converted to mass with a measured soil density of 1.14. For all treatments except the 12- and 26-d plants in the growth cycle treatment, the soil volume was 1.0 by 1.0 by 0.25 m, yielding a soil mass of 290 kg. For the 12- and 26-d harvests, the soil masses used were 3.3 kg (0.15 by 0.25 by 0.08 m) and 7.2 kg (0.15 by 0.25 by 0.08 m), respectively. A second exception is C. pepo ssp. pepo Connecticut Field, which is a pumpkin and has greater biomass. Here, a volume of 1.5 by 1.5 by 0.25 m was used, yielding a soil mass of 652 kg. With the known plant biomass, tissue concentrations of p,p'-DDE, soil contaminant concentration, and estimated soil mass, the percentage of p,p'-DDE phytoextracted by the different cultivars can be calculated. Contaminant phytoextraction percentage is perhaps the parameter of most interest because it conveys the actual amount of POP removal that is possible and provides the most realistic assessment of phytoremedial potential in the field.

Developmental Stages
The uptake and translocation of weathered p,p'-DDE across the lifespan of C. pepo is shown in Table 2. At 12 and 26 d, the plants were growing vegetatively (nonflowering); the 38-d harvest coincided with the flowering stage, but all flowers were male and no fruit were being produced. The 62-d old plants had reached maturity and the data acquired at this stage agrees with previous findings where C. pepo ssp. pepo demonstrated significantly greater stem BCFs, TFs, and contaminant removal percentages than did C. pepo ssp. ovifera (White et al., 2003a). Table 2 shows the various tissue BCFs and other parameters of interest for the two subspecies. For the subspecies ovifera cultivars, the highest BCFs for all tissues were observed at 12 d, with values declining at later stages. This trend also held for the subspecies pepo roots and leaves, although the stem BCFs for these cultivars remained relatively constant throughout the entire growth period. Both subspecies of C. pepo achieved their highest TF at 38 d: 0.12 for subspecies ovifera and 1.1 for subspecies pepo. Obviously, the biomass of all cultivars increased significantly during the four sampling period but for the subspecies pepo cultivars, the total amount of contaminant removed was correlated with the log of plant biomass (r2 = 0.86) whereas no correlation existed for subspecies ovifera (r2 = 0.04). These findings agree with Wang et al. (2004) where for subspecies pepo, the magnitude of contaminant removal was a direct function of total plant biomass.


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  		Table 2. Bioconcentration factors (BCFs) and the percentage phytoextracted of weathered DDE by Cucurbita pepo ssp. pepo and ssp. ovifera at 12, 26, 38, and 62 d of growth. Mean plant biomass is for all cultivars within each subspecies. Values in parentheses are standard deviations.

 
In addition to the harvests at different growth periods, replicate mounds of the eight cultivars were also grown for 62 d but were physically prevented from fruiting. Although the fruit compartment comprised 21 to 59% of the total plant biomass for the control plants, the biomass of plants that were not allowed to fruit was unchanged at 96% that of their fruiting counterparts (averaged across all cultivars). In addition, the root BCFs of both subspecies for p,p'-DDE were unaffected by the lack of fruit. Alternatively, the stem BCFs of the subspecies pepo cultivars was significantly reduced (14%) while the TFs were unaffected by the lack of fruit; however, the stem BCF of the subspecies ovifera cultivars was more than doubled, resulting in a 2.7-fold increase in translocation when no fruit were present. For both groups of cucurbits, the leaf BCFs increased dramatically: 6.6-fold for subspecies ovifera and 14- fold for subspecies pepo. Lastly, largely driven by the increases in tissue BCFs, the overall amount of contaminant extracted was significantly increased by 41% for subspecies pepo and by 2.6 times for subspecies ovifera when plants bore no fruit.

We have previously hypothesized that unique patterns of low-molecular-weight organic acid exudation as a nutrient acquisition strategy are in part responsible for the ability of C. pepo ssp. pepo to accumulate large quantities of weathered persistent organic pollutants from soil (White et al., 2003a, 2003b; Mattina et al., 2004). Low-molecular-weight organic acids are known to impact the soil nanostructure through chelation of soil cations, resulting in soil matrix deconstruction followed by the anticipated increased availability of nutrients and unanticipated increased availability of previously sequestered organic pollutants (White et al., 2003b; Yang et al., 2001; White and Kottler, 2002). In hydroponic experiments inducing P depletion, C. pepo ssp. pepo exuded greater amounts of citric acid than did subspecies ovifera, and this enhanced exudation pattern correlated well with the accumulation of p,p'-DDE and nutrients from soil (Gent et al., 2005). Wang et al. (2004) observed that the concentrations of four organic acids were greater in the rhizosphere of C. pepo ssp. pepo and Cucumis sativus L. (cucumber) grown under dense and presumably nutrient-stressed conditions than of those from the field, although this translated to greater p,p'-DDE accumulation only for Cucumis sativus. In addition, White et al. (2003a) observed that 10 cultivars of subspecies pepo had significantly higher amounts of p,p'-DDE, P, and several other nutrients in their tissues than did 11 cultivars of subspecies ovifera.

The current investigations on p,p'-DDE accumulation across stages of the lifespan were based on studies in the literature suggesting that root exudation patterns for nutrient acquisition may vary dramatically during plant development. Rovira (1956, 1959) observed that the exudation of amino acids and carbohydrates from clover (Trifolium pratense L.), tomato (Lycopersicon esculentum Mill.), oat (Avena sativa L.), and pea (Pisum sativum L.) was significantly greater during the initial 2 wk of growth than during the second 2-wk period. More recently, Gransee and Wittenmayer (2000) noted that the overall release of C from maize (Zea mays L.) plants decreased with plant age. The findings of our study agree with these early reports in that five of the six highest root BCFs for p,p'-DDE were observed when exudation was probably intense, at the 12-d harvest. Alternatively, Aulakh et al. (2001) and Lucas-Garcia et al. (2001) both observed that among rice (Oryza sativa L.) and Lupinus cultivars, respectively, organic acid exudation peaked at flowering and decreased significantly at maturity. It is noteworthy that in our study, the highest TFs were observed during the flowering period (38-d harvest). This could be the result of a peak in root exudation (Lucas-Garcia et al., 2001) or may simply be a function of time-dependent contaminant dilution and transport out of the root system. In addition, the goal of the "no fruit" trial was to investigate the resulting effects on p,p'-DDE accumulation when the potential pattern of increased exudation present during flowering was prolonged; these effects included 41 to 260% increases in the amount of p,p'-DDE removed from the soil.

Intercropping
For intercropping studies, mounds of vegetation consisted of 4 plants: one nonaccumulator subspecies ovifera plant surrounded by three accumulating subspecies pepo plants. Because the number of plants of each subspecies differed from that present in the control mounds of vegetation (four plants), analysis of the effects of intercropping on biomass and contaminant phytoextracted percentage are not appropriate. Table 3 shows the effects of intercropping the two subspecies on tissue BCFs. Clearly, intercropping increased the tissue p,p'-DDE content of cultivars within both subspecies pepo and ovifera. The average BCFs of the two subspecies were statistically analyzed by an ANOVA with a Dunn's multiple comparison test. The p,p'-DDE BCFs in subspecies pepo tissues were increased significantly by the presence of a single individual of subspecies ovifera, ranging from a 2.0-fold increase in the roots to an 11-fold increase in the leaves. In addition, the TF of subspecies pepo cultivars was significantly increased by 1.9 times with intercropping. Similarly, the intercropped subspecies ovifera plants had significantly greater levels of p,p'-DDE in all tissues, ranging from a 1.5-fold increase in the fruit to a 7.8-fold increase in leaves. The TF of subspecies ovifera cultivars was not significantly affected by intercropping.


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  		Table 3. Tissue bioconcentration factors (BCFs) of Cucurbita pepo cultivars grown alone or intercropped with the other subspecies (one subspecies ovifera individual with three subspecies pepo individuals). Values are means with standard deviations in parentheses.

 
The literature on the effects of intercropping on contaminant remediation is not extensive, with the majority of investigations focusing on heavy metal accumulation. Given the inherent nondegradability of POPs and the hypothesized mechanism of subspecies pepo for contaminant release through root-exudate-mediated soil matrix deconstruction, however, studies focusing on heavy metals have some relevance. Xiaomei et al. (2005) observed that when co-cropped, the Zn-accumulating plant Sedum alfredii decreased the metal content of Z. mays. Whiting et al. (2001) showed that intercropping the Zn hyperaccumulator Thlaspi caerulescens J. Presl & C. Presl with T. arvense L. and Festuca rubra L. had no impact on the uptake of Zn by the co-cultivated species. Such studies have been used to suggest that metal-accumulating species could offer a protective role for other plants. Conversely, Ni et al. (2004) showed that heavy metal accumulation by Astragas sinicus L. intercropped with the Cu-accumulating species Elsholtzia splendens Nakai ex F. Maek. was significantly increased. Similarly, Gove et al. (2002) reported that T. caerulescens co-cropped with two other species (Hordeum vulgare L. and Lepidium heterophyllum Benth.) increased the Cd content of not only the intercropped nonaccumulating plants but also of the hyperaccumulating species. These findings are in direct agreement with our study, where p,p'-DDE content of all intercropped species was increased. Gove et al. (2002) suggest that the soil structure alterations and increased metal availability in the rhizosphere of the low-biomass T. caerulescens may be a useful remediation tool if intercropped with other species.

Surfactant Amendment of Soil
Among the surfactant-amended cultivars, subspecies variability in contaminant accumulation agreed with previous findings (White et al., 2003a). The subspecies pepo cultivars had stem BCFs, TFs, and overall contaminant extraction percentages that were 9.2, 8.1, and 14 times greater (all statistically significant), respectively, than the subspecies ovifera plants. The effect of surfactant addition on the root and stem BCFs of individual subspecies pepo and subspecies ovifera cultivars is shown in Fig. 1 and 2. Statistical analysis of the pooled data for the respective subspecies confirms that surfactant amendment significantly increased all tissue BCFs (including leaf and fruit) for both groups but that TFs were unaffected. The biomass of subspecies ovifera cultivars was significantly reduced (60%) by surfactant addition but the overall percentage of p,p'-DDE extracted was not affected: 0.14 and 0.10% with and without treatment, respectively. The subspecies pepo biomass was not affected by the surfactant amendment, and the percentage of contaminant phytoextracted increased significantly from 0.72 to 1.3% with treatment.


Figure 1
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  		Fig. 1. Effect of biosurfactant amendment on the (A) root and (B) stem bioconcentration factors (BCFs) of Cucurbita pepo ssp. pepo cultivars. The untreated control for each cultivar is set equal to 1.0 and the surfactant treatment is normalized to the control.

 

Figure 2
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  		Fig. 2. Effect of biosurfactant amendment on the (A) root and (B) stem bioconcentration factors (BCFs) of Cucurbita pepo ssp. ovifera cultivars. The untreated control for each cultivar is set equal to 1.0 and the surfactant treatment is normalized to the control.

 
It has been known for some time that synthetically produced surface-active compounds or surfactants could enhance the water solubility of hydrophobic organic compounds (Kile and Chiou, 1989). Mechanistically, the solubility of water-insoluble compounds is enhanced as the hydrophobic molecules are encapsulated within the amphipathic micelles (Edwards et al., 1994), although there is also evidence of enhanced contaminant solubility at surfactant concentrations below the critical micelle concentration (Noordman et al., 1998). Yeom et al. (1996) reported that the desorption of polycyclic aromatic hydrocarbons from a "manufactured gas plant" soil was enhanced significantly in the presence of synthetic polyoxyethylene surfactants above the critical micelle concentration. Concern over the toxicity of synthetic surfactants to soil bacteria have limited their application, however; both Tiehm (1994) and Roch and Alexander (1995) observed significant toxicity of several synthetic surfactants to a range of pure and mixed cultures of soil bacteria. In addition, microorganisms have been shown to preferentially degrade the surfactant as a C and energy source, resulting in the need to increase amendment levels significantly to yield the desired enhanced solubilization of hydrophobic contaminants (Kim and Weber, 2005). Conversely, biologically produced compounds or biosurfactants possess many of the same physical and chemical characteristics of their synthetic counterparts but are generally nontoxic at equivalent concentrations (Makkar and Rockne, 2003). The most commonly employed biosurfactants are those used in our study, the rhamnolipids produced by Pseudomonas aeruginosa; Maier and Soberon-Chave (2000) provided a review of their isolation and applications. Zhang and Miller (1992) used a rhamnolipid biosurfactant from P. aeruginosa to enhance the solubilization of octadecane by more than four orders of magnitude. Hwang and Cutright (2002) observed much greater than anticipated biodegradation of aged phenanthrene and pyrene in soil and attributed the enhanced removal to indigenous P. aeruginosa populations producing large amounts of rhamnolipid biosurfactants during stationary growth.

The findings of our study are in general agreement with these reports in that the availability of weathered p,p'-DDE in soil under field conditions is increased in the presence of the biosurfactant, resulting in significantly greater accumulation of the contaminant by both subspecies of C. pepo. The levels of surfactant added to the soil were clearly problematic for subspecies ovifera, however, resulting in >60% reductions in total biomass. Current studies are focusing on determining the appropriate biosurfactant concentration for maximizing contaminant uptake and translocation without negatively impacting plant health.

Mycorrhizal Inoculation of Soil
Among the cultivars inoculated with the fungi, similar subspecies differences were observed to that of the control plants, with subspecies pepo having stem BCFs, TFs, and phytoextracted percentage values that were 9.2, 6.6, and 11 times greater, respectively (all statistically significant), than subspecies ovifera. With regard to fungal inoculation, however, the two subspecies did respond similarly, as measured by a number of parameters; the effects on the individual cultivars are shown in Table 4. The root and stem BCFs of all cultivars increased from 1.1 to 14 times with the treatment. The overall amount of contaminant extraction also increased by 2.5 times across the cultivars. Statistical analysis (ANOVA with Dunn's multiple comparison test) is reserved for pooled data for both subspecies and shows significant increases in root, stem, and leaf BCFs for both C. pepo subspecies on inoculation with fungi. The TF of subspecies ovifera was significantly increased (65%) by the mycorrhizae but the subspecies pepo TF was unaffected. Fungal inoculation reduced the total biomass of inoculated subspecies ovifera cultivars by 38% (significantly different from the control plants) but had no effect on the biomass of subspecies pepo. In spite of the decreased biomass, the percentage of contaminant removed by the subspecies ovifera cultivars was unchanged with the treatment: 0.14 and 0.19% for control and inoculated cultivars, respectively. Conversely, fungal inoculation significantly increased the magnitude of contaminant removal by subspecies pepo, with values rising from 0.72 to 2.1%, including a level of 6.0% removal by treated Connecticut Field plants.


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  		Table 4. Effect of inoculated mycorrhizal fungi on the root and stem bioconcentration factors (BCFs) and total percentage of p,p'-DDE accumulated by cultivars in two subspecies of Cucurbita pepo.

 
Mycorrhizae are fungi that symbiotically colonize plant roots, exchanging their unique ability to solubilize soil nutrients for a ready supply of C released from plant roots (Smith and Read, 1997). It is because of the metabolic diversity of the mycorrhizae, along with their ability to influence soil nanostructure and micronutrient availability through lignin degradation, that these organisms are of interest in bioremediation. Ectomycorrhizal fungi (EMF) are generally host specific and form a mycelial sheath around the roots of the plant. Much research has focused on the EMF, as well as on the unique abilities of Phanerochaete species to degrade certain organic pollutants (Meharg and Cairney, 2000; Dittman et al., 2002); however, a variety of issues, including difficulty to culture and short life span, have limited the large-scale use of these organisms in bioremediation scenarios (Dittman et al., 2002). The AMF (arbuscular mycorrhizal fungi) infect plant roots nonspecifically and emit their hyphae from the root into the soil. Oudeh et al. (2002) observed that inoculation with AMF enhanced Cd and Zn uptake by leek (Allium porrum L.) from sewage-sludge-amended soil. Jamal et al. (2002) reported similar enhanced Zn and Ni accumulation by soybean [Glycine max (L.) Merr.] and lentil (Lens culinaris Medik. ssp. culinaris) in the presence of an AMF inoculum. Given that the degradative ablities of EMF and AMF are not expected to impact the transformation of POPs, we speculate that the enhanced accumulation of p,p'-DDE observed in our study is due to increased contaminant bioavailability as a function of more intimate contact with, and alteration of, the soil nanostructure by fungal hyphae. For example, Schnabel and White (2001) reported decreased uptake of the POP aldrin from soil by willow [Salix alaxensis (Andersson) Coville] if the mycorrhizal community (EMF and AMF) was suppressed by fungicidal treatment. In our study, the fungal inoculum used contains a mixture of EMF and AMF, as well as Trichoderma and several bacterial species. Given that EMF do not form symbioses with cucurbits and that the bacterial strains present are common ones native to many soils, we speculate that the AMF or Trichoderma are the organisms of interest. Investigations elucidating the specific role of these species are currently underway. In addition, in this study, no effort was made to quantify the extent of root colonization by the inoculated organisms; however, the correlation between fungal amendment and dramatically increased accumulation of weathered p,p'-DDE is a significant and noteworthy observation.

The findings of our study indicate that p,p'-DDE accumulation by both subspecies of C. pepo was high at the seedling stage but for subspecies ovifera, the uptake potential declined dramatically during growth. For subspecies pepo, the accumulation of p,p'-DDE by these plants actually increased with growth and overall contaminant removal is correlated with total biomass. Cucurbita pepo ssp. pepo has been of significant interest due to its abilities to extract and translocate significantly greater quantities of weathered POPs than any other plant species investigated to date. It is noteworthy that, in spite of these abilities, soil amendments and cultivation practices designed to enhance the bioavailability of hydrophobic organic compounds in soil do significantly increase contaminant accumulation by C. pepo ssp. pepo. Current investigations are focusing on maximizing the accumulation potential of this plant species and assessing the potential for implementation at sites of regulatory concern in the field.


   ACKNOWLEDGMENTS
 
This work was funded partially through USEPA STAR Grant R829405 and USDA Hatch Grant CONH00772. We thank Terri Arsenault, Lydia T. Wagner, and Christine Manuck for technical assistance.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





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