Impact of Groundwater Level on BTEX Concentrations in Trees

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Plants as Bio-Indicators of Subsurface Conditions: Impact of Groundwater Level on BTEX 1

Concentrations in Trees 2

3

Jordan Wilson, Rachel Bartz, Matt Limmer* and Joel Burken 4

Department of Civil, Architectural and Environmental Engineering, Missouri University of 5

Science and Technology, Rolla, MO, USA. 6

7

*Corresponding author: malqn3@mst.edu 8

201 Butler-Carlton Hall 9

1401 N. Pine Street 10

Rolla, MO 65409 11

Cell: 419.276.5358 12

Fax: 573.341.7217 13

Impact of Groundwater Level on BTEX Concentrations in Trees

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ABSTRACT 14

Numerous studies have demonstrated trees’ ability to extract and translocate moderately 15

hydrophobic contaminants, and sampling trees for compounds such as BTEX can help delineate 16

plumes in the field. However, when BTEX is detected in the groundwater, detection in nearby 17

trees is not as reliable an indicator of subsurface contamination as other compounds such as 18

chlorinated solvents. Aerobic rhizospheric and bulk soil degradation is a potential explanation 19

for the observed variability of BTEX in trees as compared to groundwater concentrations. The 20

goal of this study was to determine the effect of groundwater level on BTEX concentrations in 21

tree tissue. The central hypothesis was increased vadose zone thickness promotes biodegradation 22

of BTEX leading to lower BTEX concentrations in overlying trees. Storage methods for tree core 23

samples were also investigated as a possible reason for tree cores revealing lower than expected 24

BTEX levels in some sampling efforts. The water level hypothesis was supported in a 25

greenhouse study, where water table level was found to significantly affect tree BTEX 26

concentrations, indicating that the influx of oxygen coupled with the presence of the tree 27

facilitates aerobic biodegradation of BTEX in the vadose zone. 28

KEY TERMS 29

Phytoscreening, Phytoforensics, SPME, Biodegradation 30

Impact of Groundwater Level on BTEX Concentrations in Trees

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INTRODUCTION 31

Petroleum-based fuels are and have been used globally as transportation has developed, 32

resulting in numerous contaminated sites (Squillace et al. 1996). Of particular concern are 33

benzene, toluene, ethylbenzene and xylenes (BTEX) in the groundwater, as benzene is a known 34

carcinogen, regulated at 5 μg/L in US drinking water (EPA 2009; NTP 2005). These moderately 35

hydrophobic organics can be long-lived in the environment as they slowly dissolve, generating 36

expansive plumes in the groundwater. Phytoremediation represents a sustainable, cost effective 37

solution for removing shallow BTEX contamination as plants, such as poplars and willows, have 38

been shown to remove petroleum hydrocarbons from soil and groundwater, often through 39

stimulation of the microbial community or uptake and translocation (Barac et al. 2009; Cook et 40

al. 2010; El-Gendy et al. 2009). 41

Trees can also be indicators of certain contaminants in the soil and groundwater, a 42

practice termed phytoforensics (Burken et al. 2011). Chlorinated solvents have been detected in 43

trees at numerous field sites at concentrations related to subsurface concentrations (e.g. 44

Schumacher et al. 2004). As BTEX have similar chemical properties, such as hydrophobicity, 45

trees can likely indicate the presence of BTEX in groundwater (i.e., phytoscreening) (Burken and 46

Schnoor 1998; Collins and Finnegan 2010). However, in several cases when high concentrations 47

of BTEX were found in shallow groundwater systems, no measureable BTEX concentrations 48

were found in nearby tree tissues (Sorek et al. 2007; Weishaar et al. 2009). 49

In considering fate and persistence of chlorinated solvents and BTEX, the rapid 50

biodegradation potential of BTEX compounds under aerobic conditions is a substantial 51

difference between the two contaminant types. The aromatic ring is a stable structure, making 52

degradation difficult under unfavorable redox conditions (i.e. anaerobic). While BTEX have 53

Impact of Groundwater Level on BTEX Concentrations in Trees

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been degraded under mixed aerobic and denitrifying conditions (Borden et al. 1997; Hubert et al. 54

1999; Ma and Love 2001) and anaerobic conditions (Lovley 1997, 2001), anaerobic degradation 55

is generally considered slower than aerobic degradation, particularly at field sites (Barbaro et al. 56

1992; Patterson et al. 1993). 57

Poplar trees have been shown to promote BTEX degrader populations in the rhizosphere 58

and to act as a hydraulic pump, lowering the natural water table during periods of high 59

transpiration. This depression of the water table draws oxygen to the subsurface, further 60

stimulating microbial populations (Weishaar et al. 2009). While Weishaar et al. hypothesized 61

that this rise and fall of the water table was directly related to the proliferation of the BTEX 62

degrading bacteria, none have isolated this variable and statistically shown the influence of water 63

level on contaminant removal. Such a finding offers a probable explanation why BTEX are less 64

frequently encountered in trees growing above contaminated groundwater, as biological 65

oxidation may occur prior to uptake and translocation. 66

Measuring BTEX concentrations in plant tissues requires sensitive methods, as xylem 67

concentrations are reduced from groundwater concentrations. Solid-phase microextraction 68

(SPME) is a solvent-less extraction technique capable of quantifying VOCs such as BTEX in 69

complex matrices (Lord and Pawliszyn 2000; Zhang and Pawliszyn 1993). SPME has been 70

employed to measure a range of organics, including environmental contaminants, in plant 71

materials (Legind et al. 2007; Limmer et al. 2011; Ouyang et al. 2011). For these moderately 72

hydrophobic contaminants, low detection limits can be reached by headspace sampling (Legind 73

et al. 2007), particularly when using composite fibers (Almeida and Boas 2004; Popp and 74

Paschke 1997). 75

Impact of Groundwater Level on BTEX Concentrations in Trees

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This paper describes an investigation into the influence of water level on BTEX 76

concentrations in trees. SPME methods were developed to test the hypothesis that lower 77

groundwater levels can decrease BTEX concentrations in trees due to facilitation of aerobic 78

degradation in the rhizosphere and bulk soil. 79

MATERIALS AND METHODS: 80

Chemicals 81

Benzene (99%) was obtained from Acros Organics (Fairlawn, New Jersey). Toluene 82

(HPLC grade), ethylbenzene (Certified grade), xylenes (Certified ACS grade), DCM (Pesticide 83

grade) and sodium azide (Purified) were obtained from Fisher Scientific (Fairlawn, New Jersey). 84

The Bushnell-Haas broth contained 0.2 g/L MgSO4, 0.02 g/L CaCl2, 1 g/L KPO4, 1 g/L 85

(NH4)2HPO4, 1 g/L KNO3 and 0.05 g/L FeCl3 (Difco Laboratories, Detroit, MI). 86

Analytics 87

Solid-phase microextraction (SPME) was performed using an 85-μm 88

carboxen/polydimethylsiloxane (CAR/PDMS) composite fiber (Supelco, Bellefonte, PA). The 89

SPME fiber was desorbed for 5 minutes in an Agilent 7890A GC at 290°C. A range of 90

headspace extraction times were investigated to determine optimal extraction. The total runtime 91

for a five-minute extraction was 15 minutes using a CombiPAL SPME auto sampler (CTC 92

Analytics, Switzerland). The oven, initially held at 40°C, ramped at 20°C/min to 160°C, which 93

was held for 4 minutes. Chromatography was accomplished using an HP-5 5% phenyl methyl 94

siloxane column with dimensions of 30 m x 320 µm x 0.25 µm. Flame ionization detection (FID) 95

was used to quantify the mass of BTEX. 96

Impact of Groundwater Level on BTEX Concentrations in Trees

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Method detection limits (MDL) were estimated using seven replicates of BTEX spiked 97

DI water (EPA 1986). Water was used as tree tissue heterogeneities hinder quantification for this 98

passive sampling method. 99

Reactor Set-Up 100

One-liter reactors were filled with 280 g of sand (3.8 cm deep) and topped with 220 g of 101

commercial potting soil (11.4 cm deep). 72 hybrid poplar cuttings (P. deltoids x P. nigra, clone 102

DN34) were planted into 36 reactors, resulting in two plants per reactor. All cuttings, which were 103

1-2 years old, were approximately 40 cm long and were planted 8.5 cm from the bottom of the 104

reactor. 105

To dose the trees with BTEX, water was siphoned from a feed bottle to the reactor using 106

a fluorinated ethylene propylene (FEP) feed tube (see Figure 1). The 250-mL amber glass feed 107

bottles were capped with polytetrafluoroethylene (PTFE) septa-lined lids. All reactors were 108

covered with aluminum foil to prevent algal growth and light from reaching the roots. 109

Feed bottles were attached at two different heights and filled at two differing frequencies 110

to allow four water level treatments. With the feed bottle positioned high on the reactor, the 111

water level essentially eliminated the vadose zone in the reactor, while a low feed bottle 112

maximized vadose zone thickness. At both high and low feed bottle positions, two different 113

water level temporal conditions were maintained: steady and fluctuating. The steady condition 114

was maintained such that the feed bottle remained nearly full by filling on a daily basis, while the 115

fluctuating condition allowed the feed bottle to nearly empty before refilling. With this 116

configuration, four distinct water table treatments were created: a high and high-fluctuating 117

water level in the high feed bottle height and low and low-fluctuating water level in the low feed 118

bottle height. These treatments are shown in Figure 1, including anticipated water levels over 119

Impact of Groundwater Level on BTEX Concentrations in Trees

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time. The high-steady and low-steady water levels were 0.7 cm and 8.2 cm below ground surface 120

(bgs) respectively. The high-fluctuating and low-fluctuating water levels varied from 0.7 to 7.7 121

cm bgs and from 8.2 to 15.2 cm bgs respectively. 122

BTEX contaminated water was prepared by mixing each constituent individually with 123

water to produce a saturated solution. From the saturated solutions, composite solutions were 124

produced at 2 mg/L and 20 mg/L of each BTEX constituent. 50-mL glass syringes were used to 125

deliver BTEX contaminated water to the feed bottles. Reactors were checked daily and solution 126

was added as needed to maintain the required level. For the fluctuating treatments, the feed 127

bottles were initially filled to the high or low water mark where appropriate and allowed to drain 128

to the bottom of the feed bottle before refilling. 129

BTEX concentration and water level were each assigned such that for any combination of 130

variables there were three replicates (e.g., three reactors with a high concentration, high bottle 131

height and fluctuating water). Reactors were randomly placed in a greenhouse fume hood and 132

dosed for 5 months prior to harvesting. 133

Soil Degradation 134

To evaluate the ability of soil microorganisms to degrade BTEX, samples were taken 135

from the reactors and spiked with BTEX. 10 g of soil were removed approximately 2 cm from 136

the top of the reactor and 2 cm above the sand/soil boundary layer and placed into 250-mL 137

amber glass bottles. These samples were from one high-steady reactor and another that had a 138

low-steady water level. Two soil samples were taken from one reactor that was not spiked during 139

dosing and used as negative controls. One of the negative controls was spiked with sodium azide 140

(10 g/L) to be used as a killed control. For each soil reactor, 100 mL of a Bushnell-Haas broth 141

mineral solution void of a carbon source, 164 µg of benzene, 216 µg of toluene, 253 µg of 142

Impact of Groundwater Level on BTEX Concentrations in Trees

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ethylbenzene and 277 µg of a mixture of o-, m- and p-xylenes were added to the amber glass 143

bottles and sealed with a PTFE-lined mininert cap. 310 µg of dichloromethane (DCM) was 144

added as an internal standard (IS). Concentrations of BTEX were normalized by the IS using 145

equation 1: 146

(1) 147

Where: 148

is the calculated concentration of an individual analyte 149

is the normalized concentration of an individual analyte 150

is the peak area of the IS 151

is the average peak area of the IS over the experiment 152

153

Immediately after preparing the soil reactor, each reactor was vigorously shaken for 154

approximately one minute in an attempt to expedite equilibrium. Reactor headspace was 155

analyzed by SPME-GC as described above, with the exception that the reactor was sampled 156

manually. The headspace was then sampled approximately every two hours with the reactors 157

shaken at 40 rpm between testing. After 38 hours, sampling was ceased after noticeable 158

degradation was observed. 159

Tree Sampling 160

At predetermined times, each cutting’s trunk were excised and sectioned into four 161

samples for analysis, which were later averaged for statistical analysis. The cuttings were 162

sectioned into 5-cm long pieces and sliced in half, which were each then quickly placed into 20-163

mL glass screw-top vials sealed with Teflon septa. The other half of the tree was used for a 164

Impact of Groundwater Level on BTEX Concentrations in Trees

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degradation experiment not described here. The samples were allowed to equilibrate in the vials 165

for 24 hours and then analyzed via SPME-GC-FID as described above. 166

Differences in sample mass were corrected to obtain an accurate measure of xylem 167

BTEX concentration. This correction is derived from a simple mass balance described elsewhere 168

(Limmer et al. 2011; Ma and Burken 2002), using dry wood:BTEX partitioning coefficients 169

described in the literature (MacKay and Gschwend 2000; Trapp et al. 2001). Minimal correction 170

was required, as most samples met negligible depletion criteria (Legind et al. 2007). 171

Sample Storage 172

Field application of BTEX phytoscreening requires proper sample storage, to reduce 173

analyte losses prior to analysis. Eleven trees were grown and dosed with 20 mg/L BTEX as in 174

the above experiments, with the exception that laurel leaf willows (Salix pentandra) were grown 175

for one month prior to harvesting. Several different preservation techniques were employed to 176

reduce BTEX losses, including acidification with 0.1 M hydrochloric acid (HCl) or 0.1 M 177

sulfuric acid (H2SO4). Ethylene glycol was also used to limit microbial activity. For each liquid 178

preservative, 1 mL was added to a 20-mL vial prior to adding the tree sample. Calcium sulfate 179

desiccant was also added to minimize water bioavailability. Eight grams of desiccant were added 180

to each vial, which could capture 0.5 g of water, assuming 6% water storage by mass. 181

Temperature control included refrigeration at 5ºC for the duration of the experiment, freezer 182

storage at -20ºC for one day, and freezer storage at -20ºC for the duration of the experiment 183

(positive control). No preservative was considered the negative control. 184

Each tree was sectioned into eight pieces of similar size and randomly assigned one of 185

the preservatives. Sections of the tree were typically 2 cm in length and included only the 186

previous year’s growth except in one case where new growth was used. Samples were analyzed 187

Impact of Groundwater Level on BTEX Concentrations in Trees

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by SPME-GC as described above after storage for 6 days. Frozen or refrigerated samples were 188

allowed to warm to room temperature for 12 hours prior to analysis. Concentrations were 189

normalized by the positive control and analyzed via 1-way ANOVA using SAS (SAS Institute, 190

Cary, NC). 191

RESULTS: 192

Analytics 193

The SPME method resulted in rapid and sensitive detection of BTEX. With 194

approximately 10 mL of headspace, a 5-minute extraction time was sufficient for equilibration of 195

the system (See Figure 2). MDLs for benzene, toluene, ethylbenzene and xylenes were measured 196

at 1.2 ng/L, 4.3 ng/L, 1.3 ng/L and 0.89 ng/L respectively, which are similar to results obtained 197

using other composite fibers (Almeida et al. 2004). 198

Tree Sampling 199

Trees dosed at the low concentration (2 mg/L) exhibited the hypothesized trend where 200

lower water levels led to decreased BTEX concentrations in the trees (see Figure 3). This trend 201

indicates reactors with larger aerobic zones (i.e., low bottle, fluctuating level) likely underwent 202

greater biodegradation of the BTEX compounds. However, the measured concentrations were 203

near the MDL, which prevented accurate estimation of variance and proper statistical analysis. 204

Trees dosed at 20 mg/L BTEX showed a similar trend to that of the 2 mg/L dose, as 205

reactors with larger aerobic zones exhibited lower BTEX concentrations in tree tissues (see 206

Figure 4). This trend was observed for all four BTEX constituents. For statistical analysis, tree 207

concentrations were log-transformed to ensure equal variance. 1-way ANOVA showed water 208

level in the reactor significantly affected BTEX concentrations in trees (all p<0.05). The high-209

steady water level was shown to be statistically different from the low-steady water level for all 210

Impact of Groundwater Level on BTEX Concentrations in Trees

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BTEX constituents using Tukey-adjusted multiple comparisons in SAS (p<0.05) (see Figure 4 211

for details). Trends observed with the 2 mg/L reactors agree with the trends observed with the 20 212

mg/L reactors, where increasing aerobic zone thickness likely led to more degradation of BTEX 213

in the soil, subsequently lessening the amount of BTEX measured in planta. While the statistical 214

analysis was not powerful enough to separate out each treatment, the analysis provides clear 215

evidence towards the observation that increasing aerobic zone thickness reduced in planta 216

concentrations. 217

Soil Degradation 218

In addition to increased biodegradation in aerobic reactors, other phenomena could lower 219

plant concentrations of BTEX in aerobic environments, such as higher volatilization rates from 220

the thick vadose zone reactors or increased root permeability in saturated reactors. To ensure that 221

the variations in tree BTEX concentrations were accurately reflecting biodegradation, soil 222

degradation rates were investigated for a subset of the samples. Two samples from a control, 223

low-steady and high-steady were tested. These soil samples were taken from near the bottom and 224

top of the reactor. 225

Figure 5 provides evidence that the differences in BTEX concentration observed in the 226

tree resulted from differing soil biodegradation rates. The soil from the lower portion of the low-227

steady reactor had the highest rate of BTEX degradation while also being the first sample to 228

resume degradation of BTEX. Rapid degradation was expected given the sample was near the 229

water table of a highly aerobic reactor. The soil from the upper portion of the high-steady and 230

low-steady reactor showed similar rates of degradation. The smallest degradation rate was 231

observed from the lower portion of the high-steady reactor. As expected, there was not 232

considerable degradation of BTEX in either control over the sampling period. 233

Impact of Groundwater Level on BTEX Concentrations in Trees

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Degradation rates were as expected, as samples from reactors with greater saturated 234

zones exhibited less degradation. Rates were particularly slow in the ‘high-steady’ reactor’s 235

lower sample, where aerobes were expected to be lacking. The higher degradation rates of the 236

‘low-steady’ reactor samples and the ‘high-steady’ reactor’s upper sample demonstrates the 237

presence of BTEX-degrading aerobes, as these samples were not consistently below the water 238

table. The relatively slow degradation from the largely aerobic low-steady upper sample might 239

be explained by the sample’s dry location, as a lack of moisture would inhibit bacterial growth. 240

An alternate explanation may be that limited BTEX was available, given the aerobic degradation 241

observed deeper in the reactor. 242

Sample Storage 243

Analysis of variance showed preservative method significantly affected measured BTEX 244

concentrations six days post-harvest (p<0.0001). Average concentrations normalized by the 245

positive control are plotted in Figure 6. Ethylene glycol and desiccant worked poorly as 246

preservatives, likely due to partitioning of BTEX to the fluid (ethylene glycol) and exhaustion of 247

the minimized headspace (desiccant). Sulfuric acid was most successful, with the 95% 248

confidence interval including unity for each BTEX constituent. Over this relatively short storage 249

period, acidification and refrigeration were not significantly different from the control in most 250

cases, but trends generally indicate that these preservatives reduced losses of BTEX during 251

storage. 252

CONCLUSIONS 253

The soil degradation rates agree with the observations from the tree concentration data: 254

lower water tables increased aerobic biodegradation of BTEX, likely decreasing the BTEX 255

concentrations in trees. This finding has several important implications to the field of 256

Impact of Groundwater Level on BTEX Concentrations in Trees

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phytoremediation. First, from a remediation perspective, the amount of BTEX transpired is likely 257

related to the redox conditions where the plants are rooted. Phytoremediation systems can 258

therefore be designed to promote biodegradation. The subsurface conditions may change 259

seasonally with the elevation of groundwater levels, resulting in varying seasonal contaminant 260

fates. The ability of trees to lower the water table during periods of high transpiration can result 261

in additional subsurface mineralization of BTEX in mature plots. From a phytoforensics 262

perspective, these findings imply limits in the applicability of phytoscreening at BTEX 263

contaminated sites. Phytoscreening will likely be less reliable at sites with substantial vadose 264

zones, where mineralization of BTEX occurs prior to translocation of the contaminant in the tree. 265

Subsurface conditions should be considered in phytoforensic approaches for BTEX sites. Sample 266

storage for BTEX should also be considered if analysis it not performed rapidly, with the 267

optimized storage method determined to be acidification. 268

ACKNOWLEDGEMENTS 269

This work was funded in part by the Missouri S&T Opportunities for Undergraduate 270

Research Experiences (OURE) with support for Ms. Bartz and Mr. Wilson. The authors would 271

like to thank Mikhil Shetty for helping to dose and maintain the plants and Jeff Weishaar for his 272

initial research. We thank Dr. David Tsao and BP for assistance and funding of initial research as 273

well as Honglan Shi for making the analytics possible. This research was also supported by the 274

National Science Foundation through a graduate research fellowship to Matt Limmer. 275

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