Environ. Sci. Techno/. 1991, 25, 1178-1180
(6) Harrison, R. M.; Johnston, W. R. In Pollutant Transport
and Fate in Ecosystems; Coughtrey, P. J., Martin, M. H.,
Unsworth, M. H., Eds.; Blackwell: Oxford, 1987. (7) Harrison, R. M.; Chirgawi, M. B. Sci. Total Environ. 1989, 83, 13. (8) Harrison, R. M.; Chirgawi, M. B. Sci. Total Environ. 1989, 83, 47. (9) Tjell, J. C.; Hovmand, M. F.; Mosbaek, H. Nature 1979, 280, 425. (10) Nriagu, J. 0. Nature 1979, 279, 409. (11) Jones, K. C. Enuiron. Pollut., in press. (12) Nriagu, J. 0. Sci. Total Environ. 1990, 92, 13. (13) Salmon, L.; Atkins, D. H. F.; Fisher, E. M. R.; Healy, C.; Law, D. V. Sci. Total Environ. 1978, 9, 161.
(14) Alderton, D. H. M. In Historical Monitoring; Monitoring
and Assessment Research Centre, University of London: London, 1985. (15) Jones, K. C.; Stratford, J. A.; Waterhouse, K. S.; Furlong, E. T.; Giger, W.; Hites, R. A.; Schaffner, C.; Johnston, A. E. Environ. Sci. Technol. 1989, 23, 95. (16) Ministry of Agriculture, Fisheries and Food Lead in Food: Progress Report; Food Surveillance Paper 27; HMSO: London, 1989. Received for review May 15,1990. Revised manuscript received January 28,1991. Accepted January 31,1991. W e are grateful to the Agricultural and Food Research Council for financial support.
COMMUNICATIONS Monitorlng in Sltu Biodegradation of Hydrocarbons by Using Stable Carbon Isotopes Pradeep K. Aggarwal' and Robert E. Hinchee Battelle Columbus Operations, 505 Klng Avenue, Columbus, Ohio 4320 1-2693
Introduction Spilled or leaked nonhalogenated petroleum hydrocarbons in the soil can generally be metabolized by indigenous, aerobic bacteria. In situ biological degradation of hydrocarbons may be accelerated by supplying inorganic nutrients and/or oxygen. Approaches to monitoring and verifying enhanced in situ biodegradation have included measurements of changes over time in the (a) concentration of hydrocarbons, (b) temperature, (c) number of hydrocarbon-degrading microorganisms, (d) ratio of fastdegrading hydrocarbons (e.g., n-C,, or n-C18) to slowly degrading hydrocarbons (e.g., pristanes or phytanes), and (e) metabolic intermediates. Measurements of oxygen consumption over time and elevated carbon dioxide concentrations in soil gas also have been used as indicators of hydrocarbon degradation ( I ) . All of these methods have some merit; however, because of the high variability at many sites and difficulty in obtaining an accurate mass balance, it is difficult to demonstrate that the disappearance of hydrocarbons is due to biodegradation rather than volatilization, dissolution, or simply dilution. Methods based on changes in soil gas composition also are less accurate because these changes may occur by processes other than hydrocarbon degradation. An alternative approach that may help substantiate biodegradation is to measure stable carbon isotope ratios in soil gas COz. Stable carbon isotope ratio analysis is inexpensive and commercially available at many laboratories. Carbon dioxide produced by hydrocarbon degradation may be distinguished from that produced by other processes based on the carbon isotopic compositions characteristic of the source material and/or fractionation accompanying microbial metabolism (2-4). Here we demonstrate the applicability of the stable isotope technique for monitoring enhanced, aerobic biodegradation of hydrocarbons using data from three locations in the United
* Present address: Argonne National Laboratory, Environmental Research Division, Argonne, IL 60439. 1178 Envlron. Scl. Technol., Vol. 25, No. 8, 1991
States. A laboratory study is currently underway to extend this technique to estimate the amount of hydrocarbons degraded over a certain period of time. Site Description The three sites used in this study were located a t Hill Air Force Base, UT; Patuxent River Naval Air Station, MD; and Tyndall Air Force Base, FL. Climatic conditions ranged from arid (average precipitation 20 cmfyear) at Hill and temperate at Patuxent to humid, subtropical (average precipitation 100 cm/year) at Tyndall. All three sites were contaminated over several years with spilled or leaked jet fuel (JP-4 and/or JP-5). At the Hill and Tyndall sites, subsurface soils in the unsaturated zone were vented with atmospheric air, resulting in aerobic conditions in the fuel-contaminated soils, which apparently stimulated biodegradation. Aerobic conditions at the Patuxent River site were achieved by pumping approximately 4000 L of atmospheric air into the contaminated, unsaturated zone soils over a 12-h period. Soil gas samples at Hill and Tyndall were collected after several months of soil venting. A t Patuxent, sampling occurred 120 h after air injection. Sampling and Analysis Sampling of soil gas from the zone of soil venting or air injection and from nearby uncontaminated locations at each site occurred in March and April at Hill and Tyndall and in July at Patuxent. Soil gas was collected in plastic (Tedlar) bags commonly used for air sampling. These bags have ports for collecting or withdrawing a gaseous sample by use of either a pump or a needle syringe. In addition to soil gas, uncontaminated and hydrocarbon-contaminated soils from Tyndall were analyzed. Stable carbon isotopic ratios were measured by Geochron Laboratories in Cambridge, MA, using standard techniques. Carbon dioxide was separated from the soil gas by first passing through a trap at dry ice-methanol temperature to remove water and then through two traps maintained at liquid oxygen temperatures. Any hydrocarbons collected along
0013-936X/91/0925-1178$02.50/0
0 1991 Amerlcan Chemlcal Soclety
0 From Unmntamlnated
Table 1. Stable Carbon Isotope Compositions of Gas and Soil Samples sample
Locations
depth, m
coz,
613C, L
%
(PDB)
0.3-1.0 0.3-1.0
0.5 0.5
-18.1 -18.7
d-26-1
0.3-1.5 0.3 1.5 0.3-1.5 1.5 0.9
4.5 5.8 7.1 4.7 13.0 13.0
-21.1 -24.1 -23.7 -22.7 -24.3 -24.3
0
0.3-1.0 0.3-1.0 0.3-1.0
2.7 2.7 2.7
-22.8 -23.2 -22.3
Tyndall uncontam soil gas V4-1 V4-2 contam soil gas v1 V1-1A v1-1c v2 v2-c V1-2-B soil gas from spiked air injectn study V3-1 V3-2 v3-3 uncontam soil (natural organic matter) BG-2 BG-3 contam soil (hydrocarbons + organic matter) V2-3 floating fuel (with organic matter)
1.0 1.0
-22.6 -21.8
1.0
-24.1 -24.4 (-24.2)' -26.0
fresh JP-4 Hill uncontam soil gas H-1 contam soil gas H-M H-V10
3-18
0.2
-23.6
18 3-18
9.5 0.6
-29.4 -27.4
-24.4 (-24.5)'
Patuxent uncontam soil gas P-B3
4.5
2.2
contam soil gas P-A2
4.5
6.6
P-A1
4.5
-27.6 (-27.5)" 7.5 -30.1
Duplicate analysis.
with carbon dioxide in the liquid oxygen traps were separated at liquid pentane temperatures. Soil samples were treated with hydrochloric acid to dissolve any carbonate fractions and then burned in an oxygen atmosphere to produce carbon dioxide, which was purified as discussed above. Isotopic composition of a sample (x) is reported in the conventional 6 notation as parts per thousand (per mil, 960) deviation from the PDB standard (5): =R x - R 0 x 1000 R, where R is the ratio of 6 13C/12C in the sample or the standard. The reproducibility of isotopic values is better than f0.3%0for samples with more than a few tenths of a percent carbon dioxide and approximately f l % o for samples with approximately 0.1% or less carbon dioxide. 6,
(%o)
Results and Discussion Carbon isotopic compositions of soil gas COz from the Hill, Tyndall, and Patuxent sites are given in Table I. 6 13Cvalues of soil gas COPfrom uncontaminated locations are -23.6%0 a t Hill, -24.5%0 at Patuxent, and -18.4% a t Tyndall. Soil gas C 0 2 from contaminated locations has 6 13Cvalues averaging -28.4% at Hill, -28.4% at Patuxent, and -23.3% at Tyndall. The C 0 2 concentrations in soil
\
\
5
% co,
10
15
Figure 1. Isotopic composition of soil gas COPfrom uncontaminated and hydrocarbon-contaminatedlocations. Solid lines are visual best fits for data from each of the three sites.
gas varied from 0.2 to 2.2% in uncontaminated locations and from 0.6 to 13.0% in contaminated locations. During the growing season, C02 in soil gases at uncontaminated locations is derived dominantly from plant root respiration and from decaying organic matter; in other seasons the isotopic composition of soil gas carbon dioxide is influenced by atmospheric carbon dioxide with 6 13C values of -7 to -10% (6-8). The isotopic composition of C 0 2 produced from organic matter decay is nearly the same as that of the source substance. 613C values of plant respiratory C 0 2 and natural organic matter are --25%0 in temperate climates where plants use the Calvin or C-3 cycle of carbon fixation and -12 f 5% in tropical and subtropical grasslands and deserts where vegetation uses the Hatch-Slack or C-4 pathway (9);areas with mixed C-3 and C-4 vegetation have intermediate 613C values for respiratory C 0 2 and organic matter. vegetation in Utah is dominantly of the C-3 type (7), while that near Tyndall Air Force Base is mainly of the C-4 type (9). In the temperate climate at the Patuxent site, C-3 plants are likely to be the dominant vegetation. Thus 613C values of soil gas C 0 2 from uncontaminated locations at the three sites in this study are within the range of typical values observed for plant respiratory C 0 2 from local vegetation and decaying organic matter (6-9). The carbon isotopic composition of soil gas C02 from contaminated locations analyzed in this study decreases with increasing C 0 2 concentration and becomes nearly constant at C 0 2concentrations greater than -5% (Table I, Figure 1). Highest COP concentrations are found in samples collected from contaminated locations where soil venting or air injection activities had been stopped. The slightly higher 613C values of C 0 2 in samples with lower C 0 2 content (
