Stable Carbon Isotope Evidence of Biodegradation Zonation in a

Environ. Sci. Technol. 1996, 30, 1120-1128

Stable Carbon Isotope Evidence of Biodegradation Zonation in a Shallow Jet-Fuel Contaminated Aquifer JAMES E. LANDMEYER,* DON A. VROBLESKY, AND FRANCIS H. CHAPELLE U.S. Geological SurveysWater Resources Division, Stephenson Center, Suite 129, 720 Gracern Road, Columbia, South Carolina 29210-7651

δ13C values in dissolved inorganic carbon (DIC) ranged from -28 to +11.9‰ in a sandy, noncarbonate shallow aquifer contaminated with jet-fuel petroleum hydrocarbons. This range was observed over a 4-year study in shallow and deep monitoring wells and comprised δ13C values representative of the aerobic and anaerobic microbial biodegradation of 13C-depleted jet fuel (δ13C ∼ -27‰). The δ13C DIC values were found to be influenced by the extent of rainwater infiltration of dissolved oxygen or sulfate or, conversely, by the absence of recharge, lack of dissolved oxygen or sulfate input to the aquifer, and the ensuing methanogenic conditions. After some recharge events delivered dissolved oxygen or sulfate to the shallow part of the aquifer, low to medium DIC δ13C values were measured, and reflected biodegradation of 13C-depleted jet fuel under aerobic (δ13C DIC ∼ -26‰) or sulfate-reducing (δ13C DIC ∼ -18‰) conditions; the deeper part of the aquifer isolated from recharge was methanogenic and had higher δ13C DIC values. Conversely, when rainfall was absent and dissolved oxygen and sulfate concentrations were low in the aquifer, higher DIC δ13C values were measured in both shallow and deep contaminated groundwater (δ13C DIC up to +11.9‰) where H2 concentrations indicated that the predominant terminal electronaccepting process was methanogenesis. The highest δ13C values (+2.6 to +11.9‰) were from contaminated groundwater that contained no dissolved oxygen and little sulfate, CH4 concentrations up to 1985 µmol/L, and acetate concentrations exceeding 12 000 µmol/L. These results suggest that stable carbon isotopes in DIC can be used to indicate the zonation of 13C-depleted hydrocarbon biodegradation processes under the influence of hydrologically controlled electron-acceptor availability.

Introduction It is commonly observed that the microbial degradation of petroleum hydrocarbons in groundwater occurs under a variety of terminal electron-accepting conditions (1-4). It is also known that the spatial distribution of terminal electron-accepting processes (TEAPs) in a contaminated aquifer is not constant but rather that TEAP conditions change over time (5, 6). One factor that can cause TEAP conditions to change is the availability of potential electron acceptors in the aquifer. This availability of potential electron acceptors, such as dissolved oxygen, nitrate, or sulfate, to an aquifer can be influenced by man-made groundwater recharge (engineered bioremediation) or natural recharge (intrinsic bioremediation). Because the TEAPs often determine the fate of groundwater contaminants (5), the purpose of this paper is to show that measurements of stable carbon isotopes (δ13C) in dissolved inorganic carbon (DIC) in a contaminated aquifer can reveal that TEAP conditions are influenced by the availability of electron acceptors in natural groundwater recharge. This can be accomplished because some TEAP conditions produce characteristic δ13C values in DIC. Microbial oxidation of 13C-depleted petroleum hydrocarbons in groundwater containing dissolved oxygen (DO) produces carbon dioxide with a carbon isotope value similar to the isotopic value of the hydrocarbon being degraded. This indicates that little fractionation of carbon isotopes occurs during the degradation of 13C-depleted petroleum hydrocarbons with oxygen as the TEAP. For example, Aggarwal and Hinchee (7) reported δ13C values from soilgas CO2 near -30‰ above shallow, aerobic groundwater contaminated by JP-4 and JP-5 jet fuels; this value is about 4‰ lower than the petroleum-hydrocarbon isotopic value (jet fuel δ13C ∼ -26‰). In an organic compound contaminated aquifer, Suchomel et al. (8) observed low δ13C values in soil-gas CO2 above the contaminant source similar to the 13C-depleted δ13C values of the organic compounds undergoing aerobic biodegradation. At a crude-oil spill site near Bemidji, MN, researchers noted that aerobic crudeoil biodegradation produced δ13C values in DIC near -28‰, indicating the oxidation of 13C-depleted (δ13C ∼-26‰) petroleum compounds (9). Van de Velde et al. (10) used stable carbon isotopes to monitor bioremediation progress at two sites and reported the close agreement of CO2 δ13C values with the δ13C values of fuel oil and petroleum hydrocarbons under aerobic conditions. Such observations of 13C-depleted δ13C values clearly demonstrate that the stable isotopic composition of soil-gas CO2 or DIC can indicate the biodegradation of petroleum hydrocarbons under aerobic conditions. It is widely observed, however, that aquifers contaminated with petroleum hydrocarbons quickly become anaerobic due to the depletion of dissolved oxygen by microbial activity (11). Moreover, petroleum hydrocarbons continue to be degraded under different TEAP conditions, such as sulfate or CO2 reduction. At the Bemidji site previously mentioned, Baedecker et al. (9) reported that as groundwater in the crude-oil plume became progressively more * Corresponding author telephone: (803) 750-6128; Fax: (803) 7506181; e-mail address: [email protected].

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This article not subject to U.S. copyright. Published 1996 by the American Chemical Society.

FIGURE 1. Site location and altitude of the water table in the surficial aquifer in April 1991 (modified from ref 5).

reducing, DIC previously depleted in 13C became more enriched with 13C, and δ13C values up to -4.05‰ were measured. This shift toward higher δ13C values under anaerobic conditions occurred because of an increase in the methanogenic biodegradation of hydrocarbons and subsequent fractionation of fuel carbon isotopes into 13Cdepleted CH4 and 13C-enriched CO2 (12). It is clear then that characteristic δ13C values are produced during petroleum-hydrocarbon degradation under oxygen-reducing and methanogenic conditions. Because the distribution of such redox end members in a contaminated aquifer is influenced by the availability of electron acceptors, and this availability is influenced by groundwater recharge, the site hydrology plays a major role in determining biodegradation zonation. It seems reasonable to suggest, therefore, that measurements of stable carbon isotopes in DIC can be used to show that TEAP zonation is influenced, in part, by the hydrologically controlled availability of electron acceptors to an aquifer.

Methods Study Area. This study was performed between 1991 and 1995 at a petroleum tank-farm site in Hanahan, near Charleston, SC (Figure 1). The hydrocarbon-release history and surficial geology have been discussed previously (5). To summarize, 83 000 gal of JP-4 jet fuel spilled in 1975 from an underground pipe near tank 1 (Figure 2) into a shallow water-table (10-ft below land surface, bls) aquifer of unconsolidated Pleistocene alluvial sands and isolated clay lenses. Aquifer contamination near tank 3 (Figure 2) resulted from a combination of undetected leaks and tankbottom cleaning processes. This site enables the meaningful interpretation of DIC δ13C values, TEAPs, and hydrologically controlled electron-acceptor availability because of extensive water-chemistry, water-level, and rainfall measurements that have been collected quarterly since December 1990. Water-chemistry data includes groundwater H2 concentrations useful for TEAP determination in the aquifer at the time of sampling (5, 6, 13).

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FIGURE 2. Distribution of benzene, toluene, ethylbenzene, and total xylene (BTEX) in ground water during June 18-July 1, 1991 (one sampling) (modified from ref 5).

Monitoring Wells. δ13C values were measured in groundwater from 40 PVC monitoring wells screened across and below the water-table surface. Near tank 1 (Figure 2), shallow wells 27A-30A and 2 are screened between 6 and 17 ft bls; deep wells 27B-30B are screened between 15 and 22 ft bls. Near tank 3 (Figure 2), shallow wells 12A, 31A34A, 40A, and 41A are screened between 4 and 15 ft bls; deep wells 12, 31B-34B, 40B, and 41B are screened between 15 and 30 ft bls. Outside of the site boundary in the neighborhood, wells 5, 6, 8, and 9 (Figure 1) are screened between 2 and 12 ft bls and wells 4, 5A, and 22 are screened between 5 and 25 ft bls. δ13C Sampling and Analysis. Analysis of groundwater DIC for δ13C used two standard methods: (1) direct DIC precipitation with SrCl2 or BaCl2, and (2) bubble stripping. Analyses for δ13C utilizing the two methods were performed by the Marine Biological Laboratory, Ecosystems Center, Stable Isotope Laboratory, Woods Hole, MA. Direct DIC precipitation with SrCl2 or BaCl2 involved groundwater sample collection using a bottom-discharge bailer and transfer into a 1-L glass bottle (glass was used to minimize the loss or entry of CO2 during transfer). DIC was precipitated as SrCO3 or BaCO3 by raising the sample pH upon addition of 50 mL of SrCl2-NH4OH (ammoniacal strontium chloride is prepared by dissolving 500 g of granular strontium chloride hexahydrate in a 2.5-L bottle of ammonium hydroxide) or after the addition of 50 mL of BaCl2-NaOH. The glass bottle was sealed with a Teflonlined screw cap. Following complete precipitation of SrCO3 or BaCO3 (usually within 48 h), the samples were filtered using 1-2 µm glass filters. The recovered filtrate was then oven dried at 100 °C for 4 h. At the lab, the precipitate was reacted in vacuo with 85% phosphoric acid to produce CO2 for analysis with a Finnigan MAT 251 isotope ratio mass spectrometer (IRMS). SrCl2 precipitation was used during sampling in June and October 1991 and BaCl2 precipitation was used in January 1992. The bubble stripping method used was based on that of Deuser and Hunt (14). Groundwater samples were collected in 250-mL glass bottles, fixed with 1 mL of

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saturated HgCl2, and sealed with a Teflon-lined screw cap. Samples were acidified on a vacuum line to produce CO2 and bubbled with N2 gas, and the released CO2 was analyzed using IRMS. This method was used to collect samples in February 1994 and July 1995. δ13C determinations were also performed on carbonateshell material and jet-fuel samples. Shell material was collected from tank-basin floors and from a marl zone present in the water-table aquifer below the deepest screened monitoring well. These samples were analyzed for δ13C upon acidification to CO2 with 5 N H2SO4 for 24 h. Weathered jet fuel was collected from free-product wells near tanks 1 and 3 (Figure 2), and fresh jet fuel was acquired from the Defense Fuel Supply Center. Both samples were combusted at 590 °C for 3 min with 1 g of fine-ground CuO, and the captured CO2 was analyzed by IRMS. The stable isotope composition (δx) of a groundwater, shell material, or jet-fuel sample is expressed in per mil (‰, parts per thousand) deviations from the PDB standard as shown in the following equation:

δx ) [(13C/12C(sample) 13

C/12C(standard))/13C/12C(standard)] × 1000 (1)

Because different analytical methods were used to determine δ13C values over the duration of the study, the SrCl2 direct-precipitation method was compared to the bubble stripping method on replicate samples to evaluate the potential for method-induced changes to occur in the reported δ13C values. Although SrCl2 precipitation is a more common procedure in stable carbon isotopes than either BaCl2 precipitation (15) or bubble stripping, there was little to no isotopic differences for δ13C using the two methods on duplicate samples (Table 1). Groundwater Chemistry. Groundwater sample collection and analyses for benzene, toluene, ethylbenzene, and total xylenes (BTEX), H2, Fe(II), SO42-, CH4, DO, and acetate concentrations followed that previously specified in Vroblesky and Chapelle (5). BTEX samples were collected in 40-mL glass vials using a bottom-discharge bailer,

TABLE 1 13

TABLE 2

δ C Values (in ‰) for Replicate Groundwater Samples Using Different Analytical Methods well (Figures 1 and 2)

strontium carbonate precipitation

bubble stripping evolution

34A 34B WT6

-23.7, -23.5 +11.0 -25.2, -25.1

-23.7, -23.8 +9.7 -24.0

Water-Chemistry Data from an Uncontaminated Well (WT6), Sampled on April 4, 1994, Hanahan, SC

a

FIGURE 3. (A) Groundwater level changes in wells W108 and W107 from February 28, 1991, to August 30, 1995 (modified from ref 5), and (B) rainfall at the Charleston airport.

preserved with hydrochloric acid, and capped with Teflonlined septa. Dissolved hydrogen was collected using the bubble-strip method of Chapelle and McMahon (16) and quantified using a gas chromatograph equipped with a reduction gas detector. Groundwater filtered through a 0.45-µm filter was analyzed for Fe(II) using a colorimeter. Sulfate concentrations were quantified by conductivity detection using anion-exchange chromatography. Methane was collected in sealed serum vials using a syringe with a 0.45-µm filter, and methane concentrations were quantified using flame ionization detection gas chromatography. Dissolved oxygen was determined by the Winkler titration method. Acetate concentrations were measured by ion chromatography.

Results and Discussion Groundwater samples were collected between 1991 and 1995 from uncontaminated and contaminated areas of the water-table aquifer at the site. Throughout this 4-year sampling period, monitoring wells were sampled under hydrologic conditions of varying amounts of precipitation (Figure 3B), and hence, varying amounts of groundwater recharge (Figure 3A). Sampling in October 1991 and April 1994 was during drought conditions, and sampling in January 1992 occurred after groundwater levels were slightly increased by rainfall. Sampling in June 1991 and February 1994 also followed periods of rainfall that increased groundwater levels in representative shallow wells (Figure 3A). Sampling in July 1995 was after rainfall had ended a

parameter measured

value

dissolved oxygen (mg/L) pH DIC (mg/L) Fe(II) (mg/L) magnesium (mg/L) calcium (mg/L) sulfate (mg/L) TOC (mg/L) δ13C (‰)

2.46 5.30 72.80 0.80 0.72 0.27 12.20 2.3 -25.2a -24.0b

SrCl2 direct precipitation.

b

Bubble stripping evolution.

4-month drought. These differences in precipitation, and hence groundwater recharge, can cause fluctuations to occur in the delivery of potential electron acceptors (such as dissolved oxygen and sulfate) to the aquifer (5). In turn, δ13C values characteristic of particular TEAPs are also affected by such changes in hydrologic conditions, as presented below. Uncontaminated Aquifer Conditions. Water-chemistry data was collected in April 1994 from monitoring well WT6 (Table 2; Figure 1 for location). The low amount of total organic carbon (TOC ) 2.3 mg/L), the presence of 2.46 mg/L DO, and the location hydrologically upgradient from the contamination sources at tanks 1 and 3 confirm that WT6 represents background shallow groundwater unimpacted by jet fuel. The groundwater carbon isotope signature in WT6 (-24.6‰) is similar to the 13C-depleted δ13C values that indicate actively respiring or decomposing C3 plant material in temperate climates (δ13C ) -25‰; refs 17-19)). The aerobic groundwater condition, isotopically low δ13C values, low DIC concentration, and lack of BTEX suggest that, during sampling, root respiration or plant decomposition by microorganisms was the predominant control on groundwater δ13C values at uncontaminated well WT6. The lack of significant amounts of aquifer soil organic matter (SOM) encountered during installation of WT6 precluded SOM δ13C determination. Jet-fuel related BTEX compounds were not detected in wells 3, 5, 9, 15, and 16 in June 1991 and in wells 1, 5, 5A, and 9 in July 1995 (Tables 3 and 6); the average δ13C value in groundwater from these wells was -16.4 and -12.1‰, respectively. These averages are similar to the groundwater δ13C value of -17.8‰ in well 15, being farthest downgradient from the contamination sources (Figure 1). Such δ13C values that are higher than that seen at WT6 (even though all do not contain BTEX compounds) could reflect the aerobic degradation of 13C-enriched SOM, the value that can vary by 10‰ (20). Contaminated Aquifer Conditions. Between 1991 and 1995, a wide range of δ13C values was measured (-28 to +11.9‰) in two localized areas near tanks 1 and 3 (Figure 2). This range incorporates δ13C values characteristic of aerobic and anaerobic controls on carbon flow in a petroleum-hydrocarbon contaminated aquifer and probably mixing of waters from both end members (Tables 3-6). Generally, monitoring wells with high concentrations of BTEX had higher δ13C values than wells with little or no BTEX. This bias toward higher δ13C values in contaminated

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TABLE 3

Concentrations of Dissolved Oxygen, Iron, Sulfate, Methane, Hydrogen, δ13C Values, Total BTEX, Acetate, and pH in Groundwater Monitoring Wells, Overall Site, June 1991, Hanahan, SCa well (Figures 1 and 2) 17 12 105 11A

a

DO (mg/L)

Fe2+ (mg/L)

SO42- (mg/L)

0.0 0.0 0.0

3.20 8.20 16.0

8.35 0.00 22.25

0.0

17.0

H2 (nM)

δ13C (‰)

BTEX (µg/L)

acetate (µM)

pH

Tank 3 -

3.8 9.0 6.0

-14.8 -10.5 -24.0

537.9 2030 15.0

-

5.05 5.44 5.97

11.25

Tank 1 -

1.8

-20.9

13.3

-

6.25

6.4 0.7 11. 9.2

-11.2 -28.1 -13.3 -15.2

322 408.5 ND ND

-

5.81 6.18 4.99 6.44

7.2 5.7 0.6 0.3 8.0

-17.4 -14.7 -12.6 -17.8 -23.2

178.5 44.4 ND ND ND

-

6.07 6.88 6.81 5.44 6.35

CH4 (µm/L)

7 19 3 16

0.0 0.0 0.8 0.0

10.2 26.6 ND 3.10

8.72 7.13 19.09 7.37

Background -

1 8 9 15 5

0.0 0.0 0.0 2.8 0.0

4.00 6.00 5.60 0.00 29.2

0.00 0.00 7.42 23.52 0.00

Neighborhood -

ND, not detected; -, no data collected.

TABLE 4

Concentrations of Dissolved Oxygen, Iron, Sulfate, Methane, Hydrogen, δ13C Values, Total BTEX, Acetate, and pH in Groundwater Monitoring Wells, Tank 1, October 1991, Hanahan, SCa well (Figures 1 and 2)

DO (mg/L)

Fe2+ (mg/L)

SO42- (mg/L)

CH4 (µm/L)

H2 (nM)

δ13C (‰)

BTEX (µg/L)

acetate (µM)

pH

27A 27B 28A 28B 29A 29B 30A 30B 2 JP-4 (from 27B) shell material

0.0 0.0 1.83 0.0 0.0 0.0 0.0 0.0 0.0

23.4 27.6 8.80 8.20 11.2 5.0 9.60 4.80 0.80

0.34 0.61 1.51 0.22 0.85 21.8 0.39 24.9 1.56

-

7.5 19.7 10.3 2.1 39 20.4

-0.5 +11.9 -16.1 -13.6 -14.1 -27.9 -19.5 -21.1 -19.0 -27.1 +2.1

503.0 4130 23.4 3218 3375 3.2 7590 2.0 2455

1.5 3.3 3.4 0.6 1.3 0.5 58.2 2.5 0.9

6.03 5.83 6.09 5.91 5.65 5.81 5.43 5.79 5.84

a

ND, not detected; -, no data collected.

zones implies that anaerobic TEAPs, such as Fe(III) and sulfate reduction (21) and methanogenesis (12), are degrading 13C-depleted jet-fuel hydrocarbons to 13C-enriched CO2. However, because nonbiological processes such as (1) interaction with atmospheric CO2 during recharge events or (2) dissolution of carbonate minerals can also result in higher groundwater DIC δ13C values, the extent these processes affect δ13C values needs to be evaluated before the measured higher δ13C values can be attributed primarily to TEAP zonation influenced by recharge-delivered electron acceptors. Atmospheric CO2 is characterized by δ13C values near -8.5‰ (17). This relatively heavy (compared to 13Cdepleted hydrocarbons) isotopic signature could raise δ13C values of groundwater if this CO2 source was a significant contributor to DIC in the aquifer (22, 23) during recharge. However, because uncontaminated shallow groundwater from WT6 has a δ13C value around -25‰, it is assumed that an atmospheric source of CO2 has little role in the high δ13C values observed in some shallow groundwater at the site. Dissolution of 13C-enriched carbonate shell minerals (δ13C ∼0‰) can also increase the groundwater DIC δ13C

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value to those characteristic of anaerobic microbial processes (24). Two potential sources of carbonate-shell material are present at the site. One source is carbonateshell material forming the tank-basin floors (tank 1 shell material was +2.1‰; Table 4). Precipitation can infiltrate through this material and recharge shallow, contaminated groundwater with δ13C values of dissolving calcite. A second source of carbonate-shell material is a marl zone below the contaminated sand aquifer and the monitoring well with the deepest screened interval (45-50 ft bls); δ13C ) +1.0‰ (Table 5). The extent of calcite dissolution at the site and its affect on δ13C values can be estimated from calcium concentrations. The dissolution of carbonate minerals in groundwater can be expressed as

CaCO3 + H2O + CO2 f Ca2+ + 2HCO3-

(2)

Assuming limited atmospheric interaction with groundwater suggested by the low δ13C value at WT6 (Table 2), CaCO3 dissolution should lead to an increase in Ca2+ and 13C concentrations. However, it appears that carbonate dissolution does not play a major role in controlling the

TABLE 5

Concentrations of Dissolved Oxygen, Iron, Sulfate, Methane, Hydrogen, δ13C Values, Total BTEX, Acetate, and pH in Groundwater Monitoring Wells, Tank 3, 1992 and 1994, Hanahan, SCa well (Figures 1 and 2)

DO (mg/L) Fe2+ (mg/L) SO42- (mg/L) CH4 (µm/L) H2 (nM) δ13C (‰) BTEX (µg/L) acetate (µM)

31A 31B 32A 32B 33A 33B 34A 34B

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

6.60 24.00 8.20 4.40 3.60 4.20 5.60 32.0

34A

0.5

6.00

34B

0.0

WT6

2.5

January 1992 30.12 1.76 2.72 1.20 14.87 1.04 25.39 2.60

1266 1985 1379 1638 2015 1011 2388

5.4 36.1 6.6 14.9 7.6 12.0 7.4 15.0

-20.2 +2.6 +4.5 -11.1 -12.7 -2.8 -15.4 -8.7

353 2435 3138 1224 1425 1363 323.5 2205

-

-23.6b -23.8c +11.0b +9.2c

540

-25.2b -24.0c +1.0

ND

February 1994 29.23 123.5

28.8

0.40

1692

April 1994 12.20

0.80

30.7*

-

ND

shell material (from marl zone beneath water-table aquifer) fresh jet fuel weathered jet fuel a

2400

pH

12 700 114 51.2 15.7 15.6 18.1 7910

5.15 5.40 5.35 5.11 5.54 4.98 5.40 4.92

-

5.63

1116

5.46

-

4.93

-26.6 -27.8

ND, not detected; -, no data collected; *, average 1994 H2 concentration.

b

SrCl2 precipitation. c Bubble stripping.

TABLE 6

Concentrations of Dissolved Oxygen, Iron, Sulfate, Methane, Hydrogen, δ13C Values, Total BTEX, Acetate, and pH in Groundwater Monitoring Wells, Tank 3 and Vicinity, July 1995, Hanahan, SCa well (Figures 1 and 2)

DO (mg/L)

Fe2+ (mg/L)

SO42- (mg/L)

31A 31B 32A 32B 33A 33B 34A 34B 40A 40B 41A 41B 12A 12

0.0 0.0 3.5 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0

4.3 22.0 ND 3.4 1.8 ND 2.7 25 3.0 21 1.0 12 1.4 11

9.35 0.11 139.1 0.57 49.4 0.7 22.6 0.31 15.6 0.48 81.63 0.86 21.25 0.27

Tank 3 396.7 1183 ND 671.8 66 911 122.4 1708 414.8 1615 114.7 675.2 224.4 598.4

2

0.0

-

1.42

Tank 1 163.2

1 4 5 5A 6 8 9 22 a

0.0 0.0 0.0 0.0 0.0 0.0 0.28 0.0

15.6 21.6 22.2 12 3.0

0.51 0.15 0.04 0.04 0.65

CH4 (µm/L)

Neighborhood 414.8 632.4 1137 153 282.9

H2 (nM)

δ13C (‰)

BTEX (µg/L)

acetate (µM)

pH

2 4.4 4.9 0.24 7.6 0.28 9.7 1.8 9.0 0.65 3.7 2.3 9.2

-15.7 +8.9 -3.5 -2.2 -5.7 -1.4 +4.2 -14.3 +5.2 -8.4 +7.7 -14.4 -5.5

37.7 1580 ND 839 194.7 1450 ND 1910 209.7 1870 130.3 2320 75.49 1956

ND 45.6 ND ND ND 4.7 ND 22.3 2.7 107.8 ND 3.7 ND ND

5.28 6.07 7.57 5.16 6.99 5.08 5.35 5.69 5.15 5.69 7.24 4.94 6.74 5.58

-12.5

1089

ND

5.8

-14.5 -14.3 -14.2 -5.5 -13.2 -13.8 -14.1 -12.5

ND 553.6 ND ND 149.3 32.5 ND 1325

ND ND ND ND ND ND

5.98 5.98 6.1 6.84 5.66 7.03 6.89 5.88

14 1.9 1.2 6.3 8.8 9.1 5.0 0.6 5.5

ND, not detected; -, no data collected.

13C-enriched

stable carbon isotope composition observed at the site overall (Figure 4A) or near tank 1 (Figure 4B). For example, the highest δ13C value measured near tank 1 (+11.9‰) was in a well that contained less than 6 mg/L calcium. However, a calcium concentration of 80 mg/L and a DIC δ13C value of +2.6‰ in groundwater near tank

3 in 1992 (well 31B) suggest possible infiltration of DIC from calcite dissolution (Figure 4C). Resampling wells near tank 3 in 1995, however, suggests that carbonate dissolution is not a major factor in producing the 13C-enriched stable carbon isotope composition observed because the δ13C value in 31B increased from +2.6‰ to +8.9‰ while the

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A

B

C

D

A

B

FIGURE 4. Calcium concentration (mg/L) and δ13C (‰) in groundwater for (A) June 1991 at the overall site, (B) October 1991 near tank 1, (C) January 1992 near tank 3, and (D) July 1995 near tank 3.

calcium concentration decreased from 80 to 21 mg/L. Additionally, the potential calcium ion sink of cation exchange onto clay minerals probably does not play a role in the overall low calcium concentrations measured (predominantly less than 20 mg/L), due to the absence of appreciable aquifer clay minerals. Methanogenesis. Degradation of 13C-depleted organic matter by fermentative microorganisms and production of acetate for cleavage by methanogenic microorganisms produce 13C-depleted CH4 and 13C-enriched CO2 (12). Methane concentrations measured on January 1992, February 1994, and July 1995 near tank 3 in BTEX-contaminated monitoring wells positively related to δ13C values. The highest CH4 concentrations were measured in wells having the highest δ13C values and the greatest BTEX concentrations (Figure 5A,B; Tables 5 and 6). High δ13C values were also measured in three of the four deeper monitoring wells in 1992, the deeper well in 1994 (34B), and all the deeper wells in 1995 (wells identified with the suffix B on Figure 5A,B have screened intervals between 16 and 30 ft bls). Because jet fuel contains only small amounts of dissolved CH4 and no alternative source of CH4 to shallow groundwater exists at the site, concentrations of CH4 in groundwater indicate the influence of methanogenic bacteria on groundwater geochemistry. Dissolved hydrogen (H2) is an intermediate product of anaerobic metabolism, and its measurement in groundwater is an indication of the terminal electron acceptor being utilized in the degradation of petroleum hydrocarbons (8). Because methanogenesis is a relatively inefficient utilizer of H2, concentrations of H2 remain high (>5 nM). It is evident from Figure 6A,B, however, that there appears to be a range of δ13C values for H2 concentrations indicating methanogenesis. This variability can be explained in three ways. First, the half-life of H2 in groundwater is less than 1 min, and therefore H2 concentrations measured in the field reflect the predominant microbial process at the relatively short time of sampling (8); conversely, the δ13C

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FIGURE 5. CH4 concentration (µmol/L) and δ13C (‰) in shallow and deep groundwater near tank 3 in (A) January 1992 and February 1994 and (B) July 1995 [shallow wells (b), deep wells (9), February 1994 wells (2)].

A

C

B

D

FIGURE 6. H2 concentration (nM) and δ13C (‰) in shallow and deep groundwater for (A) June 1991 at the overall site, (B) October 1991 near tank 1, (C) January 1992 near tank 3, and (D) July 1995 near tank 3.

value reflects an integrated value from all the TEAPs occurring in the aquifer near the sampled well over a longer period of time. Second, the δ13C value reflects not only the

integrated microbial processes near the sampled well but also the mixing of groundwaters that may have evolved under different TEAPs and, therefore, have different δ13C values before mixing (25). Third, differences in the delivery rate of recharge-introduced electron acceptors (such as DO or SO42-) results in different amounts of time (from days to months) for microbial populations to shift TEAPs, for example, between methanogenic and sulfate-reducing conditions (5). However, the overall trend is still one of higher δ13C values with H2 concentrations above 5 nM. An additional result of methanogenesis is the accumulation of organic acids in groundwater (1, 26-28). Organic acid accumulation is an indication that organic acid production is outpacing consumption by respirative processes. Because this accumulation is most evident in methanogenic parts of the aquifer, a positive relation between concentrations of organic acids, CH4, H2, and high δ13C values would be expected. As such, in 1992 near tank 3 the deepest well (31B) had an acetate concentration of 12 700 µmol/L, the highest measured in all wells at the site, the highest H2 concentration, and a high δ13C value (Table 5). Although acetate concentrations decreased in tank 3 wells between 1992 and 1995, the highest acetate concentration measured in 1995 (107.8 µmol/L) was in deep well 40B that also had a 13C-enriched δ13C value (Table 6). In fact, the most enriched δ13C values were in deep wells that also contained measurable acetate concentrations. δ13C and Biodegradation Zonation. In January 1992, stable carbon isotope ratios from paired shallow and deep monitoring wells near tank 3 exhibited a stratification of values indicative of a zonation of anaerobic processes in the aquifer controlled by the availability of electron acceptors contained in recharge. Sampling in January 1992 followed an increase in groundwater levels caused by rainfall (Figure 3A,B). The shallow monitoring wells that received greater than 14.87 mg/L sulfate had lower δ13C values no more enriched than -12.7‰ (Table 5). Conversely, the deep monitoring wells with low sulfate concentrations (