Biological Effects of Hydrocarbon Degradation Intermediates: Is the Total Petroleum Hydrocarbon Analytical Method Adequate for Risk Assessment?

In crude oil contaminant plumes, the dissolved organic carbon (DOC) is mainly hydrocarbon degradation intermediates only partly quantified by the diesel range total petroleum hydrocarbon (TPHd) method. To understand potential biological effects of degradation intermediates, we tested three fractions of DOC: (1) solid-phase extract (HLB); (2) dichloromethane (DCM-total) extract used in TPHd; and (3) DCM extract with hydrocarbons isolated by silica gel cleanup (DCM-SGC). Bioactivity of extracts from five wells spanning a range of DOC was tested using an in vitro multiplex reporter system that evaluates modulation of the activity of 46 transcription factors; extracts were evaluated at concentrations equivalent to the well water samples. The aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) transcription factors showed the greatest upregulation, with HLB exceeding DCM-total, and no upregulation in the hydrocarbon fraction (DCM-SGC). The HLB extracts were further studied with HepG2 chemically activated luciferase expression (CALUX) in vitro assays at nine concentrations ranging from 40 to 0.01 times the well water concentrations. Responses decreased with distance from the source but were still present at two wells without detectable hydrocarbons. Thus, our in vitro assay results indicate that risks associated with degradation intermediates of hydrocarbons in groundwater will be underestimated when protocols that remove these chemicals are employed.

Toxicity Assessment of Groundwater Contaminated by Petroleum Hydrocarbons at a Well-Characterized, Aged, Crude Oil Release Site.

Management of petroleum-impacted waters by monitored natural attenuation requires an understanding of the toxicology of both the original compounds released and the transformation products formed during natural breakdown. Here, we report data from a groundwater plume consisting of a mixture of crude oil compounds and transformation products in an effort to bridge the gap between groundwater quality information and potential biological effects of human exposures. Groundwater samples were characterized for redox processes, concentrations of nonvolatile dissolved organic carbon (NVDOC) and total petroleum hydrocarbons in the diesel range, as well as for activation of human nuclear receptors (hNR) and toxicologically relevant transcriptional pathways. Results show upregulation of several biological pathways, including peroxisome proliferator-activated receptor gamma and alpha, estrogen receptor alpha, and pregnane X receptor (PXR) with higher levels of hNR activity observed in more contaminated samples. Our study of affected groundwater contaminated by a crude-oil release 39 years ago shows these types of waters may have the potential to cause adverse impacts on development, endocrine, and liver functioning in exposed populations. Additionally, positive trends in activation of some of the molecular targets (e.g., PXR) with increasing NVDOC concentrations (including polar transformation products) demonstrate the importance of improving our understanding of the toxicity associated with the unknown transformation products present in hydrocarbon-impacted waters. Our results begin to provide insight into the potential toxicity of petroleum-impacted waters, which is particularly timely given the ubiquitous nature of waters impacted by petroleum contamination not only recently but also in the past and the need to protect drinking-water quality.

Examining Natural Attenuation and Acute Toxicity of Petroleum-Derived Dissolved Organic Matter with Optical Spectroscopy.

Groundwater samples containing petroleum-derived dissolved organic matter (DOMHC) originating from the north oil body within the National Crude Oil Spill Fate and Natural Attenuation Research Site near Bemidji, MN, USA were analyzed by optical spectroscopic techniques (i.e., absorbance and fluorescence) to assess relationships that can be used to examine natural attenuation and toxicity of DOMHC in contaminated groundwater. A strong correlation between the concentration of dissolved organic carbon (DOC) and absorbance at 254 nm ( a254) along a transect of the DOMHC plume indicates that a254 can be used to quantitatively assess natural attenuation of DOMHC. Fluorescence components, identified by parallel factor (PARAFAC) analysis, show that the composition of the DOMHC beneath and adjacent to the oil body is dominated by aliphatic, low O/C compounds ("protein-like" fluorescence) and that the composition gradually evolves to aromatic, high O/C compounds ("humic-/fulvic-like" fluorescence) as a function of distance downgradient from the oil body. Finally, a direct, positive correlation between optical properties and Microtox acute toxicity assays demonstrates the utility of these combined techniques in assessing the spatial and temporal natural attenuation and toxicity of the DOMHC in petroleum-impacted groundwater systems.

Evidence of Coupled Carbon and Iron Cycling at a Hydrocarbon-Contaminated Site from Time Lapse Magnetic Susceptibility.

Conventional characterization and monitoring of hydrocarbon (HC) pollution is often expensive and time-consuming. Magnetic susceptibility (MS) has been proposed as an inexpensive, long-term monitoring proxy of the degradation of HC. We acquired repeated down hole MS logging data in boreholes at a HC-contaminated field research site in Bemidji, MN, USA. The MS data were analyzed in conjunction with redox conditions and iron availability within the source zone to better assess whether MS can serve as a proxy for monitoring HC contamination in unconsolidated sediments. The MS response at the site diminished during the sampling period, which was found to coincide with depletion of solid phase iron in the source zone. Previous geochemical observations and modeling at the site suggest that the most likely cause of the decrease in MS is the transformation of magnetite to siderite, coupled with the exhaustion of ferrihydrite. Although the temporal MS response at this site gives valuable field-scale evidence for changing conditions of iron cycling and stability of iron minerals it does not provide a simple proxy for long-term monitoring of biodegradation of hydrocarbons in the smear zone.

Impacts of an ethanol-blended fuel release on groundwater and fate of produced methane: Simulation of field observations.

In a field experiment at Vandenberg Air Force Base (VAFB) designed to mimic the impact of a small-volume release of E10 (10% ethanol and 90% conventional gasoline), two plumes were created by injecting extracted groundwater spiked with benzene, toluene, and o-xylene, abbreviated BToX (No-Ethanol Lane) and BToX plus ethanol (With-Ethanol Lane) for 283 days. We developed a reactive transport model to understand processes controlling the fate of ethanol and BToX. The model was calibrated to the extensive field dataset and accounted for concentrations of sulfate, iron, acetate, and methane along with iron-reducing bacteria, sulfate-reducing bacteria, fermentative bacteria, and methanogenic archaea. The benzene plume was about 4.5 times longer in the With-Ethanol Lane than in the No-Ethanol Lane. Matching this different behavior in the two lanes required inhibiting benzene degradation in the presence of ethanol. Inclusion of iron reduction with negligible growth of iron-reducers was required to reproduce the observed constant degradation rate of benzene. Modeling suggested that vertical dispersion and diffusion of sulfate from an adjacent aquitard were important sources of sulfate in the aquifer. Matching of methane data required incorporating initial fermentation of ethanol to acetate, methane loss by outgassing, and methane oxidation coupled to sulfate and iron reduction. Simulation of microbial growth using dual Monod kinetics, and including inhibition by more favorable electron acceptors, generally resulted in reasonable yields for microbial growth of 0.01-0.05.

Photochemical mobilization of dissolved hydrocarbon oxidation products from petroleum contaminated soil into a shallow aquifer activate human nuclear receptors.

Elevated non-volatile dissolved organic carbon (NVDOC) concentrations in groundwater (GW) monitoring wells under oil-contaminated hydrophobic soils originating from a pipeline rupture at the National Crude Oil Spill & Natural Attenuation Research Site near Bemidji, MN are documented. We hypothesized the elevated NVDOC is comprised of water-soluble photooxidation products transported from the surface to the aquifer. We use field and laboratory samples in combination with complementary analytical methods to test this hypothesis and determine the biological response to these products. Observations from optical spectroscopy and ultrahigh-resolution mass spectrometry reveal a significant correlation between the chemical composition of NVDOC leached from photochemically weathered soils and GW monitoring wells with high NVDOC concentrations measured in the aquifer beneath the contaminated soil. Conversely, the chemical composition from the uncontaminated soil photoleachate matches the NVDOC observed in the uncontaminated wells. Contaminated GW and photodissolution leachates from contaminated soil activated biological targets indicative of xenobiotic metabolism and exhibited potential for adverse effects. Newly formed hydrocarbon oxidation products (HOPs) from fresh oil could be distinguished from those downgradient. This study illustrates another pathway for dissolved HOPs to infiltrate GW and potentially affect human health and the environment.

Hydrocarbons to carboxyl-rich alicyclic molecules: A continuum model to describe biodegradation of petroleum-derived dissolved organic matter in contaminated groundwater plumes.

Relationships between dissolved organic matter (DOM) reactivity and chemical composition in a groundwater plume containing petroleum-derived DOM (DOMHC) were examined by quantitative and qualitative measurements to determine the source and chemical composition of the compounds that persist downgradient. Samples were collected from a transect down the core of the plume in the direction of groundwater flow. An exponential decrease in dissolved organic carbon concentration resulting from biodegradation along the transect correlated with a continuous shift in fluorescent DOMHC from shorter to longer wavelengths. Moreover, ultrahigh resolution mass spectrometry showed a shift from low molecular weight (MW) aliphatic, reduced compounds to high MW, unsaturated (alicyclic/aromatic), high oxygen compounds that are consistent with carboxyl-rich alicyclic molecules. The degree of condensed aromaticity increased downgradient, indicating that compounds with larger, conjugated aromatic core structures were less susceptible to biodegradation. Nuclear magnetic resonance spectroscopy showed a decrease in alkyl (particularly methyl) and an increase in aromatic/olefinic structural motifs. Collectively, data obtained from the combination of these complementary analytical techniques indicated that changes in the DOMHC composition of a groundwater plume are gradual, as relatively low molecular weight (MW), reduced, aliphatic compounds from the oil source were selectively degraded and high MW, alicyclic/aromatic, oxidized compounds persisted.

Nitrogen fluxes through unsaturated zones in five agricultural settings across the United States.

The main physical and chemical controls on nitrogen (N) fluxes between the root zone and the water table were determined for agricultural sites in California, Indiana, Maryland, Nebraska, and Washington from 2004 to 2005. Sites included irrigated and nonirrigated fields; soil textures ranging from clay to sand; crops including corn, soybeans, almonds, and pasture; and unsaturated zone thicknesses ranging from 1 to 22 m. Chemical analyses of water from lysimeters and shallow wells indicate that advective transport of nitrate is the dominant process affecting the flux of N below the root zone. Vertical profiles of (i) nitrogen species, (ii) stable isotopes of nitrogen and oxygen, and (iii) oxygen, N, and argon in unsaturated zone air and correlations between N and other agricultural chemicals indicate that reactions do not greatly affect N concentrations between the root zone and the capillary fringe. As a result, physical factors, such as N application rate, water inputs, and evapotranspiration, control the differences in concentrations among the sites. Concentrations of N in shallow lysimeters exhibit seasonal variation, whereas concentrations in lysimeters deeper than a few meters are relatively stable. Based on concentration and recharge estimates, fluxes of N through the deep unsaturated zone range from 7 to 99 kg ha(-1) yr(-1). Vertical fluxes of N in ground water are lower due to spatial and historical changes in N inputs. High N fluxes are associated with coarse sediments and high N application rates.

Limited occurrence of denitrification in four shallow aquifers in agricultural areas of the United States.

The ability of natural attenuation to mitigate agricultural nitrate contamination in recharging aquifers was investigated in four important agricultural settings in the United States. The study used laboratory analyses, field measurements, and flow and transport modeling for monitoring well transects (0.5 to 2.5 km in length) in the San Joaquin watershed, California, the Elkhorn watershed, Nebraska, the Yakima watershed, Washington, and the Chester watershed, Maryland. Ground water analyses included major ion chemistry, dissolved gases, nitrogen and oxygen stable isotopes, and estimates of recharge date. Sediment analyses included potential electron donors and stable nitrogen and carbon isotopes. Within each site and among aquifer-based medians, dissolved oxygen decreases with ground water age, and excess N(2) from denitrification increases with age. Stable isotopes and excess N(2) imply minimal denitrifying activity at the Maryland and Washington sites, partial denitrification at the California site, and total denitrification across portions of the Nebraska site. At all sites, recharging electron donor concentrations are not sufficient to account for the losses of dissolved oxygen and nitrate, implying that relict, solid phase electron donors drive redox reactions. Zero-order rates of denitrification range from 0 to 0.14 micromol N L(-1)d(-1), comparable to observations of other studies using the same methods. Many values reported in the literature are, however, orders of magnitude higher, which is attributed to a combination of method limitations and bias for selection of sites with rapid denitrification. In the shallow aquifers below these agricultural fields, denitrification is limited in extent and will require residence times of decades or longer to mitigate modern nitrate contamination.

Relative contributions of microbial and infrastructure heat at a crude oil-contaminated site.

Biodegradation of contaminants can increase the temperature in the subsurface due to heat generated from exothermic reactions, making temperature observations a potentially low-cost approach for determining microbial activity. For this technique to gain more widespread acceptance, it is necessary to better understand all the factors affecting the measured temperatures. Biodegradation has been occurring at a crude oil-contaminated site near Bemidji, Minnesota for 39 years, creating a quasi-steady-state plume of contaminants and degradation products. A model of subsurface heat generation and transport helps elucidate the contribution of microbial and infrastructure heating to observed temperature increases at this site. We created a steady-state, two-dimensional, heat transport model using previous-published parameter values for physical, chemical and biodegradation properties. Simulated temperature distributions closely match the observed average annual temperatures measured in the contaminated area at the site within less than 0.2 °C in the unsaturated zone and 0.4 °C in the saturated zone. The model results confirm that the observed subsurface heat from microbial activity is due primarily to methane oxidation in the unsaturated zone resulting in a 3.6 °C increase in average annual temperature. Another important source of subsurface heat is from the active, crude-oil pipelines crossing the site. The pipelines impact temperatures for a distance of 200 m and contribute half the heat. Model results show that not accounting for the heat from the pipelines leads to overestimating the degradation rates by a factor of 1.7, demonstrating the importance of identifying and quantifying all heat sources. The model results also highlighted a zone where previously unknown microbial activity is occurring at the site.

Weathering of Oil in a Surficial Aquifer.

The composition of crude oil in a surficial aquifer was determined in two locations at the Bemidji, MN, spill site. The abundances of 71 individual hydrocarbons varied within 16 locations sampled. Little depletion of these hydrocarbons (relative to the pipeline oil) occurred in the first 10 years after the spill, whereas losses of 25% to 85% of the total measured hydrocarbons occurred after 30 years. The C6-30 n-alkanes, toluene, and o-xylene were the most depleted hydrocarbons. Some hydrocarbons, such as the n-C10-24 cyclohexanes, tri- and tetra- methylbenzenes, acyclic isoprenoids, and naphthalenes were the least depleted. Benzene was detected at every sampling location 30 years after the spill. Degradation of the oil led to increases in the percent organic carbon and in the δ 13 C of the oil. Another method of determining hydrocarbon loss was by normalizing the total measured hydrocarbon concentrations to that of the most conservative analytes. This method indicated that the total measured hydrocarbons were depleted by 47% to 77% and loss of the oil mass over 30 years was 18% to 31%. Differences in hydrocarbon depletion were related to the depth of the oil in the aquifer, local topography, amount of recharge reaching the oil, availability of electron acceptors, and the presence of less permeable soils above the oil. The results from this study indicate that once crude oil has been in the subsurface for a number of years there is no longer a "starting oil concentration" that can be used to understand processes that affect its fate and the transport of hydrocarbons in groundwater.

Crude Oil Metabolites in Groundwater at Two Spill Sites.

Two groundwater plumes in north central Minnesota with residual crude oil sources have 20 to 50 mg/L of nonvolatile dissolved organic carbon (NVDOC). These values are over 10 times higher than benzene and two to three times higher than Diesel Range Organics in the same wells. On the basis of previous work, most of the NVDOC consists of partial transformation products from the crude oil. Monitoring data from 1988 to 2015 at one of the sites located near Bemidji, MN show that the plume of metabolites is expanding toward a lakeshore located 335 m from the source zone. Other mass balance studies of the site have demonstrated that the plume expansion is driven by the combined effect of continued presence of the residual crude oil source and depletion of the electron accepting capacity of solid phase iron oxide and hydroxides on the aquifer sediments. These plumes of metabolites are not covered by regulatory monitoring and reporting requirements in Minnesota and other states. Yet, a review of toxicology studies indicates that polar metabolites of crude oil may pose a risk to aquatic and mammalian species. Together the results suggest that at sites where residual sources are present, monitoring of NVDOC may be warranted to evaluate the fates of plumes of hydrocarbon transformation products.

A simple method for calculating growth rates of petroleum hydrocarbon plumes.

Consumption of aquifer Fe(III) during biodegradation of ground water contaminants may result in expansion of a contaminant plume, changing the outlook for monitored natural attenuation. Data from two research sites contaminated with petroleum hydrocarbons show that toluene and xylenes degrade under methanogenic conditions, but the benzene and ethylbenzene plumes grow as aquifer Fe(III) supplies are depleted. By considering a one-dimensional reaction front in a constant unidirectional flow field, it is possible to derive a simple expression for the growth rate of a benzene plume. The method balances the mass flux of benzene with the Fe(III) content of the aquifer, assuming that the biodegradation reaction is instantaneous. The resulting expression shows that the benzene front migration is retarded relative to the ground water velocity by a factor that depends on the concentrations of hydrocarbon and bioavailable Fe(III). The method provides good agreement with benzene plumes at a crude oil study site in Minnesota and a gasoline site in South Carolina. Compared to the South Carolina site, the Minnesota site has 25% higher benzene flux but eight times the Fe(III), leading to about one-sixth the expansion rate. Although it was developed for benzene, toluene, ethylbenzene, and xylenes, the growth-rate estimation method may have applications to contaminant plumes from other persistent contaminant sources.

Relating subsurface temperature changes to microbial activity at a crude oil-contaminated site.

Crude oil at a spill site near Bemidji, Minnesota has been undergoing aerobic and anaerobic biodegradation for over 30 years, creating a 150-200 m plume of primary and secondary contaminants. Microbial degradation generates heat that should be measurable under the right conditions. To measure this heat, thermistors were installed in wells in the saturated zone and in water-filled monitoring tubes in the unsaturated zone. In the saturated zone, a thermal groundwater plume originates near the residual oil body with temperatures ranging from 2.9°C above background near the oil to 1.2°C down gradient. Temperatures in the unsaturated zone above the oil body were up to 2.7°C more than background temperatures. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. Enthalpy calculations and observations demonstrate that the temperature increases primarily result from aerobic methane oxidation in the unsaturated zone above the oil. Methane oxidation rates at the site independently estimated from surface CO2 efflux data are comparable to rates estimated from the observed temperature increases. The results indicate that temperature may be useful as a low-cost measure of activity but care is required to account for the correct heat-generating reactions, other heat sources and the effects of focused recharge.

Inverse modeling of BTEX dissolution and biodegradation at the Bemidji, MN crude-oil spill site.

The U.S. Geological Survey (USGS) solute transport and biodegradation code BIOMOC was used in conjunction with the USGS universal inverse modeling code UCODE to quantify field-scale hydrocarbon dissolution and biodegradation at the USGS Toxic Substances Hydrology Program crude-oil spill research site located near Bemidji, MN. This inverse modeling effort used the extensive historical data compiled at the Bemidji site from 1986 to 1997 and incorporated a multicomponent transport and biodegradation model. Inverse modeling was successful when coupled transport and degradation processes were incorporated into the model and a single dissolution rate coefficient was used for all BTEX components. Assuming a stationary oil body, we simulated benzene, toluene, ethylbenzene, m,p-xylene, and o-xylene (BTEX) concentrations in the oil and ground water, respectively, as well as dissolved oxygen. Dissolution from the oil phase and aerobic and anaerobic degradation processes were represented. The parameters estimated were the recharge rate, hydraulic conductivity, dissolution rate coefficient, individual first-order BTEX anaerobic degradation rates, and transverse dispersivity. Results were similar for simulations obtained using several alternative conceptual models of the hydrologic system and biodegradation processes. The dissolved BTEX concentration data were not sufficient to discriminate between these conceptual models. The calibrated simulations reproduced the general large-scale evolution of the plume, but did not reproduce the observed small-scale spatial and temporal variability in concentrations. The estimated anaerobic biodegradation rates for toluene and o-xylene were greater than the dissolution rate coefficient. However, the estimated anaerobic biodegradation rates for benzene, ethylbenzene, and m,p-xylene were less than the dissolution rate coefficient. The calibrated model was used to determine the BTEX mass balance in the oil body and groundwater plume. Dissolution from the oil body was greatest for compounds with large effective solubilities (benzene) and with large degradation rates (toluene and o-xylene). Anaerobic degradation removed 77% of the BTEX that dissolved into the water phase and aerobic degradation removed 17%. Although goodness-of-fit measures for the alternative conceptual models were not significantly different, predictions made with the models were quite variable.

Comparison of surficial CO2 efflux to other measures of subsurface crude oil degradation.

At a spill site near Bemidji, Minnesota, crude oil at the water table has been undergoing anaerobic biodegradation for over 30years. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. To compare microbial activity measurement methods from multiple locations in the oil body, surficial carbon dioxide efflux, methanogen and methanotroph concentrations, and oil degradation state were collected. Carbon dioxide effluxes over the oil body averaged more than four times those at the background site. Methanotrophic bacteria concentrations measured using pmoA were over 10(5) times higher above the oil-contaminated sediments compared with the background site. Methanogenic archaea measured using mcrA ranged from 10(5) to over 10(7) in the oil and were below detection in the background. Methanogens correlated very well with methanotroph concentrations (r=0.99), n-alkylcyclohexane losses as a proxy for degradation state (r=-0.96), and somewhat less well with carbon dioxide efflux (r=0.92). Carbon dioxide efflux similarly correlated to methanotroph concentrations (r=0.90) and n-alkylcyclohexane losses (r=-0.91).

A mass balance approach to investigating geochemical controls on secondary water quality impacts at a crude oil spill site near Bemidji, MN.

Secondary water quality impacts can result from a broad range of coupled reactions triggered by primary groundwater contaminants. Data from a crude-oil spill research site near Bemidji, MN provide an ideal test case for investigating the complex interactions controlling secondary impacts, including depleted dissolved oxygen and elevated organic carbon, inorganic carbon, CH4, Mn, Fe, and other dissolved ions. To better understand these secondary impacts, this study began with an extensive data compilation of various data types, comprising aqueous, sediment, gas, and oil phases, covering a 260m cross-sectional domain over 30years. Mass balance calculations are used to quantify pathways that control secondary components, by using the data to constrain the sources and sinks for the important redox processes. The results show that oil constituents other than BTEX (benzene, toluene, ethylbenzene, o-, m- and p-xylenes), including n-alkanes and other aromatic compounds, play significant roles in plume evolution and secondary water quality impacts. The analysis underscores previous results on the importance of non-aqueous phases. Over 99.9% of the Fe(2+) plume is attenuated by immobilization on sediments as Fe(II) and 85-95% of the carbon biodegradation products are outgassed. Gaps identified in carbon and Fe mass balances and in pH buffering mechanisms are used to formulate a new conceptual model. This new model includes direct out-gassing of CH4 and CO2 from organic carbon biodegradation, dissolution of directly produced CO2, and sorption with H(+) exchange to improve pH buffering. The identification of these mechanisms extends understanding of natural attenuation of potential secondary impacts at enhanced reductive dechlorination sites, particularly for reduced Fe plumes, produced CH4, and pH perturbations.

Crude oil at the bemidji site: 25 years of monitoring, modeling, and understanding.

The fate of hydrocarbons in the subsurface near Bemidji, Minnesota, has been investigated by a multidisciplinary group of scientists for over a quarter century. Research at Bemidji has involved extensive investigations of multiphase flow and transport, volatilization, dissolution, geochemical interactions, microbial populations, and biodegradation with the goal of providing an improved understanding of the natural processes limiting the extent of hydrocarbon contamination. A considerable volume of oil remains in the subsurface today despite 30 years of natural attenuation and 5 years of pump-and-skim remediation. Studies at Bemidji were among the first to document the importance of anaerobic biodegradation processes for hydrocarbon removal and remediation by natural attenuation. Spatial variability of hydraulic properties was observed to influence subsurface oil and water flow, vapor diffusion, and the progression of biodegradation. Pore-scale capillary pressure-saturation hysteresis and the presence of fine-grained sediments impeded oil flow, causing entrapment and relatively large residual oil saturations. Hydrocarbon attenuation and plume extent was a function of groundwater flow, compound-specific volatilization, dissolution and biodegradation rates, and availability of electron acceptors. Simulation of hydrocarbon fate and transport affirmed concepts developed from field observations, and provided estimates of field-scale reaction rates and hydrocarbon mass balance. Long-term field studies at Bemidji have illustrated that the fate of hydrocarbons evolves with time, and a snap-shot study of a hydrocarbon plume may not provide information that is of relevance to the long-term behavior of the plume during natural attenuation.