D C Values of Polycyclic Aromatic Hydrocarbons Collected from Two

Chemical Geology 152Ž. 1998 43±58

d13C values of polycyclic aromatic hydrocarbons collected from two creosote-contaminated sites

Beth Trust Hammer a,), Cheryl A. Kelley a,1, Richard B. Coffin b,1, Luis A. Cifuentes c,2, James G. Mueller d,3 a National Research Council, cro U.S. EnÕironmental Protection Agency, Gulf Ecology DiÕision, 1 Sabine Island DriÕe, Gulf Breeze, FL 32561, USA b U.S. EnÕironmental Protection Agency, Gulf Ecology DiÕision, 1 Sabine Island DriÕe, Gulf Breeze, FL 32561, USA c Department of Oceanography, Texas A&M UniÕersity, College Station, TX 77843, USA d SBP Technologies, 1 Sabine Island DriÕe, Gulf Breeze, FL 32561, USA

Abstract

Groundwaters were sampled on two dates from several wells at each of two creosote-contaminated waste sites in Florida. Polycyclic aromatic hydrocarbonsŽ. PAHs were extracted from the groundwaters, and their individual concentrations were measured by gas chromatographyrflame ionization detectionŽ. GCrFID . The d13C values of the PAHs were then determined by gas chromatographyrion trap mass spectrometryrisotope ratio mass spectrometryŽ. GCrITMSrIRMS . At the American Creosote WorksŽ. ACW in Pensacola, concentrations of PAHs were found to decrease by over four orders-of-magnitude, both with increasing depth and with increasing distance from the most contaminated area. At a wood-preserving facility in Gainesville, concentrations were also found to decrease with increasing distance from the most contaminated area. At the ACW site, d13C values of individual PAHs ranged from y20.09½ to y32.94½, although the majority of compounds fell in a tighter range between y22.66½ and y25.31½. The d13C values of over 75% of the PAHs remained constant across all wells, both with migration of the contaminant plume and over a 3-month time period. The compounds that showed the highest variability among the wells were anthracene; the heterocyclic compounds thianaphthene, dibenzothiophene, and carbazole; and the lighter PAHs naphthalene, biphenyl, and 2-methylnaphthalene. Variability of these compounds is likely the result of variations in d13C values among different creosotes added to the sites over many years. The other compounds measured were conserved across the wells and would serve as good tracers of a contaminant plume in bioremediation settings. At the Gainesville site, d13C values of individual PAHs ranged from y18.87½ to y27.05½, with 70% of the values falling between y22.06½ and y24.53½. This range is very similar to the values for PAHs at the ACW site. Comparing d13C values of specific PAHs between the two creosote-contaminated sites, 12 of 16 compounds agreed within 1.0½. This indicates that, although there are a few compounds that may be variable across different creosotes, there may be a suite of d13C values that is conserved across PAHs of creosote origin. These characteristic PAHs could be used to

) Corresponding author. 10444 Park Tree Place, Glen Allen, VA 23060, USA. Fax: q1 804 733 9637. 1 Current address. Dept. of Geological Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA. fax: q1 573 882 5458; e-mail: [email protected] 2 Fax: q1 409 862 3172; e-mail: [email protected] 3 Dames & Moore, Inc., Rolling Meadows, IL 60008, USA. E-mail: [email protected]

0009-2541r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S0009-2541Ž. 98 00095-3 44 B.T. Hammer et al.rChemical Geology 152() 1998 43±58 determine whether or not creosote is contributing to the PAH contamination at a site. In addition, the compounds that are variable between different creosotes could be used as tracers of individual creosotes at polluted sites and to differentiate between possible creosote sources. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: d13C; Polycyclic aromatic hydrocarbon; Creosote-contaminated site

1. Introduction solved complex mixture that is inherent in many environmental samplesŽ. O'Malley et al., 1994 . In a Although a few isotopic investigations have been recent modification, an ion trap mass spectrometer conducted on individual compounds or classes of Ž.ITMS has been placed in-line with the GCrIRMS, compounds isolated from bulk samplesŽ e.g., Parker, creating a GCrITMSrIRMS. Rather than having an 1964; Des Marais et al., 1980; Macko et al., 1990; isotope ratio mass spectrometer as the sole detector, Hare et al., 1991. , stable isotope studies historically in the new GCrITMSrIRMS system, the column involve the combustion of whole organisms or tis- effluent is split between the ITMS, where compound sues. Bulk measurements may effectively mask a identification and verification of purity are obtained, great deal of information contained at the molecular and the IRMS, where isotopic composition is mea- level. Matthews and HayesŽ. 1978 were the first to sured. This configuration allows the investigator, in interface a gas chromatograph to an isotope ratio the same sample acquisition, to verify the identity of mass spectrometer via a 7508C cupric oxide-packed a specific compound and to determine if there are combustion furnace. It has been only recently, how- any coeluting compounds contributing to a peak ever, that this instrumentation has become commer- which otherwise appears to be well-resolved. This cially available. The development of so-called gas capability is especially helpful in the analysis of chromatographrcombustionrisotope ratio mass complex environmental samples such as those found spectrometersŽ. GCrCrIRMS or GCrIRMS now in contaminated areas. provides the ability for researchers to determine the The PAH contamination constitutes one of the isotope ratios of individual molecules within various world's most widespread environmental problems. In classes of compoundsŽ e.g., Freeman et al., 1990; many cases, in situ bioremediation represents the Hayes et al., 1990; Rieley et al., 1991; Silfer et al., only viable remedial solution. A challenge in the 1991; Goodman and Brenna, 1992; O'Malley et al., bioremediation of contaminated field sites is estab- 1994; CoBabe and Pratt, 1995. . lishing that organisms are actually effecting the The capability of the GCrIRMS system permits cleanup of the siteŽ U.S. Environmental Protection the resolution and determination of d13C values of Agency, 1994. , because decreases in contaminant complex mixtures of hydrocarbon pollutants, such as levels may only be due to transport of the pollutants polycyclic aromatic hydrocarbonsŽ. PAHs . The d13C to a new location or to the partitioning of the pollu- values of individual PAHs have been measured in tants into an immobile phase. A potential tool for such samples as roadsweeps, car soot, fireplace soot, proving that microorganisms are indeed remediating and sewageŽ. O'Malley et al., 1994 . These different contaminated sites is stable carbon isotope analysis. PAH sources were found to have distinctive suites of It has been well-established that consumers have d13C values which could serve as indicators of their close carbon isotopic similarities to their diets presence in contaminated environments, thereby al- ŽParker, 1964; DeNiro and Epstein, 1978; Fry et al., lowing for source apportionment. There are limita- 1978; Fry and Sherr, 1984. . Furthermore, the use of tions, however, to the GCrIRMS technique. The stable isotope analyses for tracing carbon sources accuracy and precision of d13C values obtained by into bacterial biomass has been successful in aque- GCrIRMS for individual compounds are affected ous environmentsŽ. Coffin et al., 1989, 1990 . In mainly by the degree of resolution of coeluting bioremediation settings, if bacteria are consuming compounds and the nature and extent of the unre- one or more pollutant compounds, their biomass B.T. Hammer et al.rChemical Geology 152() 1998 43±58 45 should have a carbon isotopic signal that is similar to previouslyŽ Middaugh et al., 1991; Mueller et al., that of the pollutantŽ. s . Similarly, the CO2 they 1991. . The sampling wells were located along a respire should also have the same carbon isotopic transect leading away from the most contaminated signalŽ. Trust et al., 1995 . By measuring the d13C area. ACW Wells 340, 360 and 380 were situated values of individual PAHs, background indigenous immediately adjacent to the most contaminated area carbon sources, bacterial biomass, and respired CO2 , at depths of 40 ftŽ.Ž. 12.2 m , 60 ft 18.3 m and 77 ft the flux of contaminant carbon through bacterial Ž.23.5 m , respectively. Wells 480 and 400 were biomass can be traced. Also, the percentage of con- located about 125 m downgradient of the contami- taminant vs. indigenous carbon assimilated by the nated area at depths of 80 ftŽ. 24.4 m and 100 ft bacteria can be determined. Thus, the use of natural Ž.30.5 m , respectively. Well 760 was the farthest abundance stable isotopes may be a powerful tool in downgradient, located approximately 275 m from the developing bioremediation strategies and evaluating most contaminated area at a depth of 64 ftŽ. 19.5 m . their efficacies. See site locations in Middaugh et al.Ž. 1991 for In order to determine the usefulness of stable further description. carbon isotopes in bioremediation programs where Groundwater samples also were collected from the flow of contaminant carbon into bacterial biomass the Cabot CarbonrKoppers wood-preserving facility and respired CO2 needs to be verified, it first needs in Gainesville, FL on February 6, 1995 and on May to be established that the d13C values of a specific 31, 1995. Like the ACW site, this site is heavily contaminant source are conserved over time periods impacted by coal tar. Since the mid-1920s, the for- relevant to bioremedial effortsŽ a few months to a mer Cabot Carbon operated a 34-acreŽ 0.14 km2 . few years. and with migration of the contaminant pine tar and charcoal generation facility, both of plume through a groundwater aquifer. Here, we re- which are now discontinued. During the same time, port the d13C values of PAHs extracted from ground- the Kopper's Industries plant occupied approxi- waters collected at two creosote-contaminated sites. mately 90 acresŽ 0.36 km2 . as a wood treatment We provide evidence that the d13C values of a large facility. Historically, the facility used creosote, number of PAHs are conserved over time and with pentachlorophenol, and chromated copper arsenate migration through a groundwater aquifer. Further- Ž.CCA to preserve wood utility poles and timbers. more, d13C values of individual PAHs were found to The facility continues to operate, but using only be similar between two sites, indicating that there CCA on site. Multiple process and storage areas may be a suite of d13C values, i.e., an `isotopic associated with past facility operations have been fingerprint', that is characteristic of PAHs in cre- identified as potential sources of organic wood-pre- osote. If this isotopic fingerprint of d13C values is serving constituents. Specifically, the former north found to be unique to PAHs of creosote origin, then lagoon areaŽ which has since been filled and lev- by measuring d13C values of PAHs found in plumes elled. has been identified as a potential source of where the source is unknown, it may be possible to creosote constituents, with free product being de- determine whether the contamination is from a cre- tectedŽ. Fig. 1 . As such, a UVB groundwater circula- osote or closely related source. tion systemŽ Borchert and Sick, 1992; Herrling et al., 1993; U.S. Patent Office, 1992a,b. and 12 monitoring wells were installed in January 1995 as part of a 2. Materials and methods project to determine the effectiveness of in situ bioremediation for cleaning up PAHs. Samples were 2.1. Sampling collected from two depthsŽ 3.7 m and 6.1 m below ground surface. at each of the 12 monitoring wells. Groundwater samples were obtained on June 8, Wells 1, 2, 3, 10, 11, and 12 were located on a 1994 and September 9, 1994 from the American transectŽ. Transect 1 leading away from the most Creosote WorksŽ. ACW in Pensacola, FL. This site contaminated area. Wells 4 through 9 were located in is an abandoned wood-preserving facility that was in another transectŽ. Transect 2 perpendicular to the operation from 1902 to 1981 and has been described first transect, crossing between Wells 3 and 10. After 46 B.T. Hammer et al.rChemical Geology 152() 1998 43±58

Fig. 1. Locations of sampling wells at the Cabot CarbonrKoppers facility in Gainesville, FL. the initial groundwater sampling on February 6, 1995, 2.2. Analysis the groundwater circulation system was started on February 16, 1995, so that it had run for approxi- The PAHs were extracted from groundwaters ac- mately 15 weeks before the second sampling on May cording to EPA's SW-846 Method 3510AŽ Sep- 31, 1995. aratory Funnel Liquid±Liquid Extraction. . The ex- At the Pensacola site, groundwater samples for traction solvent was methylene chloride. All extracts stable isotope analysis were pumped from the wells were dried over anhydrous sodium sulfate and con- through 1r4 in. i.d. Teflonw tubing into glass bottles centrated to a final volume of 1 ml by gently evapo- fitted with Teflon-lined caps. 4 At the Gainesville rating the sample with N gas. Concentrations of site, samples were pumped through 1r4 in. i.d. 2 PAHs were analysed on a Hewlett-Packard 5890 Gas stainless steel tubing. The wells were purged by Chromatograph using flame ionization detection pumping a volume of water equivalent to three Ž.GCrFID . The GC was equipped with an HP-5 well-volumes before collecting approximately 500 columnŽ 25 m, 0.32 mm i.d., 0.17 mm film thick- ml for analysis. Samples were preserved with NaOH ness. . The carrier gas was helium-maintained at a Ž.pHs10 and kept refrigerated at 48C until they flow rate of 1 mlrmin. The injector temperature was could be extracted for analyses of concentrations and 2908C, and the detector temperature was 3158C. The carbon isotope ratios of PAHs. initial column oven temperature was 508C, which was maintained for 2 min. The temperature was then ramped to 1008C at a rate of 258Crmin, and then to 4 r Mention of trade names or commercial products does not a final temperature of 3108C at a rate of 58C min, constitute endorsement or recommendation for use. where it was held for 6 min. Concentrations of PAHs B.T. Hammer et al.rChemical Geology 152() 1998 43±58 47

were calculated using an external calibration curve 8508C for 2 h to generate CO2 gas, which was generated from standards of known concentration isolated cryogenically on a vacuum line. The carbon run under the same conditions. Analyses of random isotope content of the CO2 was measured on a samples in triplicate revealed a precision of "0.1 Finnigan MAT 252 isotope ratio mass spectrometer mgrl for all the PAHs measured in this study. having a precision better than "0.1½ for replicate The d13C values of the PAHs extracted from the analyses of a combusted sample. The individual groundwater samples were measured using a novel PAHs were then dissolved into methylene chloride to GCrITMSrIRMS system. This system consists of make a standard solution containing all 10 PAHs. three parts:Ž. 1 a Varian Star 3400 CX gas chromato- This mixture was injected into the GCrITMSrIRMS graph,Ž. 2 a Finnigan MAT Magnum ion trap mass at varying concentrations to yield major ion beam spectrometerŽ.Ž. ITMS , and 3 a Finnigan MAT Delta Ž.i.e., mass 44 signals ranging from 0.1 to 8.1 V on S isotope ratio mass spectrometerŽ. IRMS . The GC the IRMS. The 13C value of each PAH was deter- was equipped with a Hewlett-Packard Ultra 2 col- mined 54 times over a period of several months. umnŽ. 50 m, 0.32 mm i.d., 0.52 mm film thickness . Carrier gas conditions and GC temperatures were the same as for the GCrFID. As each compound eluted 3. Results from the GC column, 10% of the effluent was car- ried through a 2908C heated transfer line into the ion 3.1. Precision and accuracy of the GCrITMSrIRMS trap mass spectrometer for confirmation of com- system pound identification and purity. The remaining 90% of the effluent passed through a 9408C combustion The d13C values of the 10 standard PAHs as furnace packed with cupric oxide, platinum and determined by GCrITMSrIRMS are compared to nickel, where carbon constituents were converted those produced by the conventional IRMS method in quantitatively to CO2 gas. Co-generated water vapor Fig. 2. Although it had lower precision than the was removed via a Nafionw elimination trap, and interfering nitrogen oxides were reduced to N2 gas via a 6008C reduction column. The purified CO2 was swept into the isotope ratio mass spectrometer, where its carbon isotope composition was measured. All isotope data are reported using the conventional d13C notation:

13 s r y = d C½PDBŽ. Ž.R sampleR std 1 1000, 13 r12 where Rsampleand R std are the C C isotope ratios corresponding to the sample and the conventional PeeDee BelemniteŽ. PDB carbonate standard, respectivelyŽ. Craig, 1957 . In order to evaluate the precision and accuracy of the GCrITMSrIRMS for determining d13C values of PAHs, the d13C values of 10 individual PAHs were first measured in either duplicate or triplicate Fig. 2. Comparison of d13C values of standard PAHs measured by using conventional isotope ratio mass spectrometry. conventional IRMS and GCrITMSrIRMS. Error bars represent Following the method described by MackoŽ. 1981 , "1 standard deviation. Horizontal error bars for the conventional individual PAHs were placed into pre-combusted IRMS values are -0.1½; therefore, they are hidden by the quartz tubes. Copper wireŽ. 5 g and CuO Ž 2.5 g . symbol at each point. Compounds listed from left to right on the x-axis are phenanthrene, pyrene, fluoranthene, anthraquinone, an- were added to the samples, and the tubes were thracene, fluorene, acenaphthene, 2,6-dimethylnaphthalene, 2,3-di- evacuated and sealed. The tubes were heated to methylnaphthalene, and acenaphthylene. 48 B.T. Hammer et al.rChemical Geology 152() 1998 43±58 conventional IRMS method, as indicated by the error wells. Moving downstream from the most contami- bars Ž."1 standard deviation , the GCrITMSrIRMS nated area, concentrations of individual PAHs again method had a high degree of accuracy. The values were over four orders-of-magnitude less than at the produced by this technique for PAHs were not sig- source area, measuring from undetectable levels to a nificantly different from those produced by the con- maximum of 1.2 mgrl at Well 480 and a maximum ventional IRMS method Ž.p-0.05, Student's t-test . of 0.7 mgrl at Well 760. Concentrations of individ- The d13C values of the PAHs, as determined by ual PAHs at the previous June 1994 sampling were GCrITMSrIRMS, agreed within 1.1½ of the val- comparable to those of the September 1994 sam- ues produced by the conventional IRMS method. In pling. On this date, PAH concentrations at Well 400, fact, for eight out of the 10 PAHs, the two methods which was not sampled in September, measured produced d13C values that agreed within 0.4½ or from undetectable levels to a maximum of only 31 better of each other. The good agreement of the mgrl. GCrITMSrIRMS method with the conventional Although concentrations of individual PAHs de- IRMS method instills a high degree of confidence in creased dramatically across the wells, the d13C val- this technique. This is especially exciting because the ues of the majority of PAHs did not vary greatly GCrITMSrIRMS method, like regular GCrIRMS, from one well to the next on either sampling date. can be used on much smaller sample sizes than the The d13C values of the individual compounds mea- conventional IRMS method. The conventional IRMS sured at the ACW site in both June and September method typically requires a sample size on the order 1994 are reported in Table 1. Values for the different of 10 mmol of carbon, whereas the GCrIRMS meth- compounds varied from y20.09½ for acenaphthy- ods can be performed on nanomolar quantities of lene to y32.94½ for dibenzothiophene. The major- material. Furthermore, the accuracy of the ity of the valuesŽ. 100 out of 130 measurements , GCrITMSrIRMS method proves that there is no however, fell in a tighter range between y22.66½ fractionation of carbon isotopes as the sample is split and y25.31½. between the two mass spectrometers for analysis. Comparing the d13C values of any one compound across the three `300' wells, only thianaphthene, 3.2. Concentrations and d13C Õalues of PAHS at the dibenzothiophene, and anthracene varied by more ACW site than 1.3½ on both sampling dates. Of these three compounds, only anthracene was statistically differ- Concentrations of 21 PAHs extracted from ent Ž.p-0.05, Student's t-test for both dates. The groundwaters collected at the ACW site both on June remaining 12 compounds for which d13C values 8, 1994 and September 9, 1994 were found to de- could be measured at two or more wells did not vary crease with increasing distance from the known to a significant degree. Although four compounds source area. Fig. 3 shows a plot of the PAH concen- Ž.the dimethylnaphthalenes, carbazole and biphenyl trations measured by GCrFID for groundwaters col- varied across the `300' wells by more than 1.3½ on lected on September 9, 1994. Well 340, the well one of the two dates, each compound varied much closest to the source, had the highest concentrations less Ž.-1.0½ on the other date Ž 2,6-dimethylnaph- of PAHs, ranging from 1079 to 45,790 mgrlŽ 1.1 to thalene varied the greatest on the September 1994 45.8 grl. for individual compounds. The compound sampling date, whereas the other three compounds found in the largest concentration was phenanthrene, varied the greatest on the June 1994 sampling date. . followed by naphthalene. Wells 360 and 380, located These results indicate that d13C values of the major- immediately adjacent to Well 340 but at increasingly ity of PAHs are conserved with depth, i.e., vertical greater depth, had concentrations of individual PAHs migration through an aquifer. that were over four orders-of magnitude less than at Results also indicate that d13C values of PAHs Well 340. Concentrations for individual PAHs at are conserved as they move horizontally through the Well 360 ranged from 0.1 to 6.3 mgrl and from 0.1 aquifer via groundwater flow. In a comparison of the to 4.9 mgrl at Well 380. Phenanthrene and naphtha- wells moving away from the source area, biphenyl lene were again the most abundant PAHs in these and 1-methylnaphthalene had d13C values that dif- B.T. Hammer et al.rChemical Geology 152() 1998 43±58 49

Fig. 3. Concentrations of PAHs in groundwaters collected from ACW Wells on September 9, 1994. The panelsŽ. A through E are in order of increasing distance from the source of contamination. Error bars represent the standard deviation of triplicate samples. Note the changes in scale on the concentrations axes. The PAH compounds are listed in the order that they elute from the GC column. fered the most between the wells on the September Well 760, measuring y27.48½. This value for 9, 1994 sampling date. Whereas biphenyl had d13C biphenyl at Well 760 was statistically different from values of y20.65 to y21.16½ at the `300' wells, all three `300' wells Ž.p-0.01, Student's t-test . this compound was depleted in 13C downstream at Similarly, 1-methylnaphthalene measured y23.48 to 50 B.T. Hammer et al.rChemical Geology 152() 1998 43±58 25.71 23.05 23.62 24.01 26.89 24.96 24.82 23.05 23.71 30.00 23.94 23.77 23.41 21.50 23.80 23.53 22.73 25.55 23.66 23.94 24.45 y y y y y y y y y y y y y y y y y y y y y ab 94 24.70 22.69 23.56 23.88 26.89 23.79 22.47 23.50 30.82 24.15 24.14 23.49 22.90 25.27 23.27 22.60 25.51 23.72 24.44 24.37 r y y y y y y y y y y y y y y y y y y y y 94 9 26.72 23.41 23.68 24.11 24.82 ± 26.51 23.63 23.98 28.78 23.69 23.31 23.31 20.09 22.81 23.79 22.85 25.59 23.58 23.32 24.55 r y y y y y y y y y y y y y y y y y y y y 94 6 24.26 23.25 23.96 23.36 22.97 22.90 27.48 28.63 23.49 23.57 24.45 r y y y y y y y y y y y 94 9 r 94 6 23.20 22.14 23.45 27.04 23.15 26.59 23.31 23.47 23.56 r y y y y y y y y y 94 9 r 94 6 Ž. r 94 9 25.04 ± ± 23.44 22.53 22.66 22.52 23.71 ± 31.27 21.76 21.50 24.45 r y y y y y y y y y y 94 6 23.20 ± ± ± 23.50 26.89 ± ± ± ± 23.43 ± 23.42 ± ± ± 23.89 28.91 ± ± ± 23.99 22.88 23.48 21.59 ± ± ± 21.16 24.80 23.46 26.93 24.35 r 94. y y y y y y y y y y y y y y y y r 94 9 23.96 23.69 25.77 23.83 24.20 30.76 23.98 23.51 23.53 21.50 ± ± ± ± 24.24 23.77 24.01 23.97 23.01 24.29 r y y y y y y y y y y y y y y y y 94 6 24.70 ± ± ± ± ± 22.69 ± ± ± ± ± 23.91 24.37 24.26 20.66 24.27 32.94 25.30 23.31 23.73 25.58 23.95 21.12 23.48 24.11 24.00 24.41 r y y y y y y y y y y y y y y y y y y 94 9 23.43 23.58 25.61 22.82 23.88 24.35 23.42 23.47 24.60 23.22 21.19 23.61 24.60 24.77 24.73 94 and All Well average 9 r r y y y y y y y y y y y y y y y ±±±±±± ± ± ±±±± ±±±±±± ± 23.57 23.78 23.32 23.95 30.61 ± 24.05 24.09 24.10 24.96 24.26 20.65 24.04 94 6 24.24 24.21 25.07 r y y y y y y y y y y y y y y y Ž. C½ 94 9 26.72 ± ± 23.41 ± ± 23.65 24.11 24.82 28.16 ± 24.24 24.41 26.79 23.88 23.65 23.72 20.09 22.34 23.91 22.72 23.48 24.00 24.01 24.72 r 13 y y y y y y y y y y y y y y y y y y y Well 3406 Well 360 Well 380 Well 400 Well 480 Well 760 All Well average Total average y d average of all the wells on that date. s average of All Well average 6 s C values of PAHs extracted from groundwaters collected at the American Creosote Works ACW in June and September 1994 13 Ž. d Total average All Well average Chrysene Benz a anthracene Pyrene Fluoranthene 2-Methylanthracene Anthraquinone ± Carbazole Anthracene Phenanthrene Dibenzothiophene Fluorene Dibenzofuran Acenaphthene Acenaphthylene 2,3-dimethylnaphthalene 2,6-Dimethylnaphthalene Biphenyl 1-methylnaphthalene 2-Methylnaphthalene Thianaphthene The Table 1 Naphthalene a b Compound B.T. Hammer et al.rChemical Geology 152() 1998 43±58 51 y24.80½ at the `300' wells on September 9, 1994 d13C values of the individual PAHs changed over the but was a more negative y28.63½ at Well 760 on 3-month period of time between samplings, the `All the same date. However, the value for 1-methylnaph- Well' averages for each compound were compared thalene at Well 760 was only significantly different statistically between the two dates. Of all the com- from the value at Well 360 Ž p-0.05, Student's pounds, only carbazole differed between the two t-test. . Besides biphenyl, only three other compounds dates Ž.p-0.05, Student's t-test . differed significantly between the `300' wells and either Well 480 or 760 Ž.p-0.05, Student's t-test . 3.3. Concentrations and d13C Õalues of PAHs at the These were 2-methylnaphthalene, acenaphthene, and GainesÕille site phenanthrene. Only naphthalene was significantly different between Well 480 and Well 760 Ž p-0.05, Concentrations of 24 PAHs were measured in Student's t-test. . groundwaters collected from shallowŽ 3.7 m below There was one well at the ACW site that had ground surface.Ž and deep 6.1 m below ground distinct d13C values of PAHs. On the June 8, 1994 surface. ports at each of 12 monitoring wells in sampling date, d13C values for eight of the 10 com- February and May 1995. Concentrations of these pounds at Well 400 were statistically different Ž p- PAHs were summed, and the resulting `Total PAH' 0.05, Student's t-test. from at least two of the three values are plotted in Fig. 4 for both shallow and deep `300' wells measured on the same date. The only wells on the two sampling dates. two compounds that were not different between the Generally, concentrations of PAHs were higher in `300' wells and Well 400 were naphthalene and the deep ports relative to the shallow ports at any biphenyl. As mentioned above, the `300' wells did one well. For example, in February 1995, Total PAH not differ isotopically from Wells 480 or 760 for the concentration in the deep port of Well 5 was 8.9 majority of compounds at the September 1994 sam- mgrl, whereas it was only 0.2 mgrl in the shallow pling, so it is interesting that Well 400, which is port. Similarly, in May 1995, the Total PAH concen- situated close to Well 480, albeit 6.1 m deeper, was tration in the deep port of Well 5 was 6.4 mgrl, statistically different from the `300' wells at the June whereas it was 0.2 mgrl in the shallow port. Being sampling. It is possible that there was a second one of the more water-soluble constituents of cre- source of PAHs to Well 400 that did not contribute osote, naphthalene was the most abundant PAH in 17 to the contamination at the other wells. In order to of the 24 water sampling ports on both dates. Com- determine if Well 400 was unique from all wells, paring concentrations between the wells along Tran- Well 400 was also compared to Wells 480 and 760, sect 1Ž. Wells 1±3, 10±12 for the February sam- even though they were not sampled on the same pling, PAH concentrations tended to decrease with date. In this comparison, only thianapthene and 2- increasing distance from the north lagoon area. Total methylnaphthalene differed between Well 400 and PAH concentrations in the shallow wells decreased both Well 480 and Well 760. Furthermore, these two from a maximum value of 10.3 mgrl at Well 1 to a compounds were not found to differ between the minimum of 0.6 mgrl at Well 12. Similarly, Total `300' wells and Wells 480 and 760 when they all PAH concentrations in the deep wells decreased were sampled on September 9, 1994. All of these from a maximum of 12.7 mgrl at Well 3 to a information together make it difficult to conclude minimum of 1.6 mgrl at Well 12. Along Transect 2 that a secondary source of PAHs was contributing to Ž.Wells 4±9 , concentrations in the wells showed no the contamination in Well 400. regular pattern. Concentrations in the shallow ports Because the majority of PAHs for which d13C varied between 0.2 mgrl Total PAHsŽ. Well 5 and values were obtained were not statistically different 16.6 mgrl Total PAHsŽ. Well 9 , and in the deep between the wells at the ACW site, the values for ports they ranged from 1.2 mgrl Total PAHsŽ Well each individual compound were averaged across the 4.Ž. to 10.0 mgrl Total PAHs Well 6 . The lack of a wells to produce an `All Well' average for each regular pattern along Transect 2 was not unexpected, compound on both the June and September 1994 as these wells lie approximately equidistant from the sampling datesŽ. Table 1 . In order to determine if the source area. 52 B.T. Hammer et al.rChemical Geology 152() 1998 43±58

Fig. 4. Contour plots of Total PAH concentrations in groundwaters collected from shallowŽ. 3.7 m and deep Ž. 6.1 m ports at the Gainesville site on February 6, 1995 and May 31, 1995.

Trends in concentrations along the two transects to have decreased from the February 1995 levels in were generally the same in May 1995 as in February 18 sampling ports, whereas they had increased in the 1995. In May 1995, PAH concentrations were found remaining six wells. For example, Total PAH con- B.T. Hammer et al.rChemical Geology 152() 1998 43±58 53 s 23.25 23.25 y y average of s 23.26 23.26 y y dibenzofuran; Phen s 22.65 ± ± 22.33 ± ± 22.52 ± ± 22.81 ± ± 22.26 21.87 22.64 ± ± 21.31 ± ± 22.30 ± ± 22.08 ± ± 24.83 ± ± 22.90 ± ± y y y y y y y y y y y y 24.46 23.42 23.53 23.76 24.80 25.79 ± ± ± 24.43 25.94 ± ± ± 26.11 ± ± ± 25.16 26.55 25.32 25.60 26.27 y y y y y y y y y y y y y y acenaphthene; DiBF s 23.48 23.15 ± ± ± ± 22.09 22.80 22.63 ± ± ± ± 22.67 ± ± ± ± 22.21 22.68 23.72 22.13 ± ± ± ± 22.51 23.15 23.10 22.84 23.32 ± ± ± ± 22.05 22.82 22.96 23.14 ± ± ± ± 22.91 y y y y y y y y y y y y y y y y y y y y All Well average for February 1995; Total average 23.54 23.84 22.37 22.70 23.20 22.91 22.59 22.93 23.01 22.85 22.89±±±±± 22.79 23.40 23.18 22.64±±±±± 23.07 23.37 24.41 ± ± 21.89 22.74 23.55 23.63 22.76 s y y y y y y y y y y y y y y y y y y y y y y y 95 acenaphthylene; Acen r s 23.26 23.06 22.82 23.38 22.39 22.71 22.31 23.06 22.42 22.45 24.09 23.24 22.99 22.47 22.18 22.87 23.67 23.34 21.80 22.87 23.08 23.86 22.26 y y y y y y y y y y y y y y y y y y y y y y y 23.55 24.53 22.48 22.95 22.48 23.33 22.38 23.03 22.82 22.76 22.43 22.88 24.58 23.17 21.74 23.17 23.44 24.43 22.06 22.77 25.35 23.08 y y y y y y y y y y y y y y y y y y y y y y dimethylnaphthalene; Acy s 20.35 21.64 20.99 21.78 22.50 21.49 22.10 y y y y y y y All Well average for May 1995; AWA 2 s 95 18.87 ± 21.04 ± 20.64 20.51 20.97 19.93 21.19 21.08 21.83 ± 23.02 r y y y y y y y y y y Koppers site in Gainesville, FL on May 31, 1995 r methylnaphthalene; DMN s 21.74 ± ± 20.05 23.56 19.18 ± ± 21.58 ± ± 21.42 22.70 21.06 23.47 25.09 ± ± 22.82 21.93 21.92 ± ± 22.36 23.36 20.91 ± ± ± y y y y y y y y y y y y y y y y pyrene; AWA 5 s 22.24 21.60 22.85±±± 22.42±±± 22.39 22.24 20.07±±± 21.94 22.51 22.99±±± 21.88 21.22 21.97 21.33 21.93 23.68 21.08±±± y y y y y y y y y y y y y y y y y thianaphthene; MN s fluoranthene; Pyr s 24.12 25.15 24.49 23.84 ± 23.83 ± 24.84 24.36 24.68 24.22 24.99 25.83 24.61 23.04 24.36 24.87 23.47 y y y y y y y y y y y y y y y y 23.19 24.03 23.24 23.16 23.06 23.29 24.41 23.45 23.76 23.73 23.36 23.32 22.73 23.17 20.78 ± 23.45 23.19 22.54 ± carbazole; Fla naphthalene; Thia y y y y y y y y y y y y y y y y y y s s 26.37 ± ± ± 23.52 27.05 26.52 25.82 26.98 26.36 25.45 23.67 ± ± 25.80 25.00 26.52 26.83 25.02 25.90 25.47 26.97 25.77 25.73 y y y y y y y y y y y y y y y y y y y 95. r anthracene; Carb Ž. s ±±±±±± ± ± C½ 23.36 24.10 ± 23.72 24.17 24.34 23.82 23.79 23.95 24.03 23.63 24.27 24.19 23.94 23.27 23.78 23.08 23.88 23.72 23.55 13 y y y y y y y y y y y y y y y y y d y Naph Thia 2-MN 1-MN Biph 2,6-DMN 2,3-DMN Acyy Acen DiBF Fluor Phen Anth Carb Fla Pyr 95 and AWA 2 95 95 r C values of PAHs extracted from groundwaters collected at the Cabot Carbon r r 13 d 4-Deep -hlo±±±±± 4-Shallow±±±±±±± 3-Deep 3-Shallow 11-Shallow 10-Deep 10-Shallow 7-Deep 2-Deep Total average 7-Shallow ± AWA 2 2-Shallow 6-Deep AWA 5 6-Shallow 1-Deep 12-Deep 12-Shallow±±±±± 5-Deep Table 2 The Well 1-Shallow 5-Shallow 11-Deep Abbreviations arephenanthrene; as Anth follows:AWA Naph 5 54 B.T. Hammer et al.rChemical Geology 152() 1998 43±58 centration was 10.1 mgrl in the deep port of Well 6 cases, phenanthrene differed in 24%, and 1-methyl- in February, but it was only 3.3 mgrl in May. In naphthalene differed in 35%. contrast, Total PAH concentration in the deep port of Because the majority of d13C values for the PAHs Well 9 measured 6.1 mgrl in February, and it at the Gainesville site were not significantly different increased to 10.6 mgrl by May. The reason for the between the different wells, an `All Well' average variability in concentrations between sampling dates for each compound was calculated by averaging the is unknown, but it perhaps can be attributed partly to values for each wellŽ. shallow and deep . An `All the start-up of the groundwater circulation system on Well' average was calculated for both the February February 16, 1995, which increased groundwater 1995 and May 1995 samplingsŽ. Table 2 . Comparing flow in the area of the monitoring wells. the d13C values of the compounds between the two At the Gainesville site, d13C values of the various dates, only naphthalene, thianaphthene, and 2- PAHs ranged from y18.87½ to y27.05½, with methylnaphthalene differed statistically Ž p-0.05, 70% of the values falling between y22.06½ and Student's t-test. . y24.53½Ž. Table 2 . Similar to the ACW site, d13C values did not vary greatly between the wells, even 3.4. Comparison of d13C Õalues of PAHs at the ACW when concentrations ranged from fairly contami- and GainesÕille sites natedŽ. Wells 1, 2, and 3 to only trace amounts Ž Well 12. . In a statistical comparison of individual com- The d13C values of PAHs at the Gainesville site, pounds between different wellsŽ e.g., naphthalene at averaged over all wells and sampling timesŽ Table 2, Well 1 shallow vs. naphthalene at Well 10 deep. , the last row. , are plotted along with the corresponding d13C values of all the PAHs were found to be values for the ACW site in Fig. 5. Twelve of the 16 significantly different in 35% or less of the compar- compounds agreed within 1.0½ for the two sites. isons Ž.p-0.05, Student's t-test . For example, an- There were five compounds which were statistically thracene only differed between wells in 5% of the different between the sites Ž p-0.05, Student's t-

Fig. 5. Comparison of d13C values of PAHs at the ACW site with those at the Gainesville site. Values for each site are averages of all wells sampled over both sampling dates. Error bars represent "1 standard deviation. B.T. Hammer et al.rChemical Geology 152() 1998 43±58 55 test.Ž . These compounds are naphthalene differed by served along Transect 1 at the Gainesville siteŽ Table 0.42½.Ž , thianaphthene differed by 1.51½ . , 2,3-di- 2.. methylnaphthaleneŽ. differed by 3.29½ , fluorene Most bioremediation programs monitor concentra- Ž.Ždiffered by 1.01½ , and carbazole differed by tions of the contaminant compounds through time. 2.70½. . Anthracene differed by 1.75½ between the For stable isotopic analyses to be useful in such two sites, but this difference was not statistically programs, contaminant compounds need to maintain significant. their isotopic composition through time as well as during migration. Samples of groundwater obtained in June and again in September 1994 from the ACW 4. Discussion wells had similar suites of d13C values for PAHs. Likewise, groundwaters collected 3 months apart at The combined GCrITMSrIRMS system has the Gainesville site had similar isotopic compositions proven both useful and reliable in measuring the for contaminants. These results indicate that d13C stable carbon isotopic composition of individual values are maintained through time, at least over PAHs in groundwater samples. In order for d13C short periods, and that the measuring techniques values to be effective in examining the fate of these presented in this paper are reproducible and reliable. compounds in bioremediation scenarios, two basic The constancy of PAH d13C values with both criteria need to be satisfied. First, the d13C values of migration through a groundwater aquifer and time the individual PAH compounds need to be main- indicates that stable carbon isotopes could be useful tained over distance, i.e., migration, from the con- indicators of bioremediation at contaminated envi- taminated area, and secondly, they need to be main- ronments. Typically, microorganisms are only able tained through time. Once these criteria are satisfied, to degrade contaminants which are in the dissolved stable isotopic measurements of contaminant com- phase. High molecular weight compounds such as pounds could be used to fingerprint contaminant PAHs tend to have very low aqueous solubilities; sources and to trace their fate in the environment. therefore, most PAHs become adsorbed to soil parti- Furthermore, comparisons between d13C values of cles and are unavailable for biodegradation. Because contaminants, indigenous organic matter and respired there is an equilibrium between the dissolved and carbon dioxide may provide evidence that microor- adsorbed PAHs, as the dissolved PAHs are biode- ganisms are participating in the remediation of a graded or otherwise removed from the area, adsorbed polluted site and may lead to greater understanding PAHs dissolve to replenish the supplyŽ e.g., Morris of biodegradation processes. et al., 1995. . Thus, in bioremediation scenarios, Our data provide evidence that the d13C values of changes in aqueous PAH concentrations are incom- individual PAH compounds are maintained over dis- plete indicators of in situ bioremediation because tance from the source area as the contaminants mi- concentrations of PAHs in groundwaters tend not to grate both horizontally and vertically through a decrease or are highly variable, even though groundwater aquifer. At the ACW site, concentra- biodegradation is occurring. In these cases, d13C tions of PAHs decreased by over four orders-of-mag- values of bacterial biomass and respired carbon diox- nitude both in a horizontal transect from Well 340 to ide could be measured at both a contaminated envi- Well 760 and in a vertical transect from Well 340 to ronment and an uncontaminated control site. The Well 380. Concentration changes were most likely values could then be compared to those of the con- due to a combination of biodegradation and dilution. taminants andror background indigenous organic Unlike concentrations, stable carbon isotopic values matter, providing evidence that biodegradation is for the majority of PAHs, especially the higher occurring. By coupling stable isotope measurements molecular weight compounds, did not change dra- with carbon mass balances, it would be possible to matically or systematically across the wellsŽ Table estimate the amount of the contaminant that is being 1. ; therefore, it appears that neither dilution nor degraded over time. degradation significantly alters the stable isotopic Although the majority of d13C values for individ- composition of PAHs. The same results were ob- ual PAHs were consistent both with migration and 56 B.T. Hammer et al.rChemical Geology 152() 1998 43±58 time at the two creosote-contaminated sites, it should many different creosote sources were added to the be pointed out that there were a few compounds that lagoons and holding ponds at these sites. It is also were variable. At the ACW site, thianaphthene, likely that these various sources had different suites dibenzothiophene, anthracene, and biphenyl showed of d13C values associated with them which in turn the most variability with migration, and carbazole produced the differences in d13C values observed was the most variable with time. At the Gainesville between the wells for the more variable compounds. site, d13C values of the compounds were well-con- It is important to stress that even though a few of served with migration, but `All Well' averages of the compounds were variable at any one site, the naphthalene, thianaphthene, and 2-methylnaph- bulk of the compounds were not. Similarly, in a thalene were statistically different with time. comparison of the d13C values of the PAHs between Anthracene has been documented to become iso- the sitesŽ. i.e., ACW vs. Gainesville , the majority of topically enriched in 13C when it undergoes pho- compoundsŽ. 11 out of 16 were also found not to be tolytic decompositionŽ. O'Malley et al., 1994 . Per- statistically different. Furthermore, of the five com- haps, this compound also fractionates isotopically pounds that were statistically different, naphthalene, when it is subjected to other degradation mecha- thianaphthene, and carbazole have already been men- nisms in the environment. Of the other compounds tioned above as being variable at either the ACW or that were the most variable, thianaphthene, dibenzo- the Gainesville site with migration or time. These thiophene, and carbazole are heterocyclic com- results indicate that the majority of PAHs in creosote pounds. Perhaps, the sulfur or nitrogen atom imparts may have characteristic d13C values. In other words, a relative instability to these compounds which causes there may be a distinctive `isotopic fingerprint' of them to be isotopically fractionated during transport d13C values which identifies PAHs at a contaminated or degradation. Lastly, because naphthalene, 2-meth- site as being from creosote. In addition, the com- ylnaphthalene, and biphenyl are fairly easy to de- pounds that are more variable could serve to differ- grade relative to higher molecular weight PAHs entiate between two or more possible creosote Ž.Mueller et al., 1996 , they also may be subjected to sources. In future studies, the d13C values of more isotopic fractionations that do not affect the higher PAHs, both of creosote and non-creosote origin, molecular weight compounds. should be measured to see, first of all, if there is a It is not likely, however, that high molecular characteristic pattern of d13C values for creosote and weight compounds such as PAHs are isotopically if this pattern is distinct from other PAH sourcesŽ we fractionated during chemical or physical transforma- did not have enough compounds in common with tions. For example, O'Malley et al.Ž. 1994 found that O'Malley et al.Ž. 1994 to compare our creosote the d13C of naphthalene, which is the lightest com- PAHs with their PAHs of various origin. , and sec- pound analysed in this study, did not change during ondly, if there are a few individual compounds that microbial degradation, even after a 95% reduction in vary across different creosotes and thus can be used naphthalene concentration occurred. In similar stud- as tracers of specific creosotes. ies, d13C values of acenaphthene, fluorene, phenanthrene, and fluoranthene have also been found to be unaffected by microbial degradationŽ Kelley et al., 1995; Trust et al., 1995. . It is also unlikely that Acknowledgements isotopic fractionation occurs during other transforma- tions, such as the partitioning of compounds between This work was funded by the U.S. Navy under the dissolved and adsorbed phases. Department of Defense Strategic Environmental Re- A more likely explanation for the variability of search and Development Program Project no. 030. the few aforementioned PAHs is that the d13C values The help of Suzanne Lantz, Cherie Heard, Charylene of these compounds are variable across different Gatlin, Pam Hollenbeck, and Fayaz Lakhwala of creosote sources. The creosote facilities at both the SBP Technologies, Lynn Roelke at Texas A&M ACW and Gainesville sites were in operation for University, and Vicky Nelms of the Avanti Corpora- over 50 years. Over this time period, it is likely that tion is gratefully acknowledged. This work was per- B.T. Hammer et al.rChemical Geology 152() 1998 43±58 57 formed while Dr. Hammer and Dr. Kelley held Hayes, J.M., Freeman, K.H., Popp, B.N., Hoham, C.H., 1990. National Research Council±U.S. Environmental Pro- Compound-specific isotope analyses: a novel tool for recon- struction of ancient biogeochemical processes. Org. Geochem. tection Agency Research Associateships. This paper 16, 1115±1128. is contribution no. 961 of the U.S. Environmental Herrling, B., Stamm, J., Alesi, E.J., Bott-Bruening, G., Diekmann, Protection Agency, Gulf Ecology Division, Gulf S., 1993. In situ bioremediation of groundwater containing Breeze, FL. hydrocarbons, pesticides, or nitrate using vertical circulation flowsŽ.Ž. 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