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Polar metabolites resulting from petroleum biodegradation are measured in groundwater samples as TPHd unless a silica gel cleanup (SGC) is used on the sample extract to isolate hydrocarbons. Even though the metabolites can be the vast majority of the dissolved organics present in groundwater, SGC has been inconsistently applied because of regulatory concern about the nature and toxicity of the metabolites. A two-step approach was used to identify polar compounds that were measured as TPHd in groundwater extracts at five sites with biodegrading fuel sources. First, gas chromatography with mass spectrometry (GC-MS) was used to identify and quantify 57 individual target polar metabolites. Only one of these compounds—dodecanoic acid, which has low potential human toxicity—was detected. Second, nontargeted analysis was used to identify as many polar metabolites as possible using both GC-MS and GC×GC-MS. The nontargeted analysis revealed that the mixture of polar metabolites identified in groundwater source areas at these five sites is composed of approximately equal average percentages of organic acids, alcohols and ketones, with few phenols and aldehydes. The mixture identified in downgradient areas at these five sites is dominated by acids, with fewer alcohols, far fewer ketones, and very few aldehydes and phenols. A ranking system consistent with systems used by USEPA and the United Nations was developed for evaluating the potential chronic oral toxicity to humans of the different classes of identified polar metabolites. The vast majority of the identified polar metabolites have a “Low” toxicity profile, and the mixture of identified polar metabolites present in groundwater extracts at these five sites is unlikely to present a significant risk to human health.
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- Results and Discussion
Total petroleum hydrocarbons (TPH) has long been a routinely required groundwater monitoring parameter at petroleum release sites. For the semi-volatile (or extractable) fraction, a common analytical approach is to use USEPA Method 3510C for solvent extraction, followed by USEPA Method 8015B/C or equivalent (gas chromatography with flame ionization detection [GC-FID]) for quantitation. The purpose of the analysis, called either TPH as diesel (TPHd) or diesel-range organics (DRO), is to measure C10 to C28 hydrocarbons dissolved in the groundwater. The TPHd concentration is then usually compared to hydrocarbon-based regulatory criteria. However, neither Method 3510C nor 8015B/C is specific for hydrocarbons, and the TPHd analysis actually measures all extractable organics within the prescribed boiling-point range (170 to 430 °C in the case of Method 8015B/C).
In order to compare sample TPHd results to hydrocarbon-based water quality objectives, a silica gel cleanup (SGC) must be applied to the sample extract prior to analysis. This was documented in a study of 21 sites (Zemo and Foote 2003), where the application of SGC to the sample extract prior to analysis for TPHd showed that the majority of samples with elevated concentrations of TPHd in groundwater at sites with biodegrading petroleum sources were composed almost entirely of dissolved polar, nonhydrocarbon compounds and not dissolved diesel-range hydrocarbons. The polar compounds were typically found in groundwater directly within the source area and downgradient from biodegraded petroleum; therefore, it was concluded that they were most likely biodegradation metabolites. This finding was confirmed by Lundegard and Sweeney (2004), Haddad et al. (2007), and Lang et al. (2009). These studies showed that the concentration of polar metabolites, quantified as TPHd, typically ranged from 100s to 10,000s micrograms per liter (µg/L), with a maximum of about 100,000 µg/L.
In addition to presumed biodegradation metabolites, other studies have demonstrated that nonhydrocarbons measured as TPHd may include natural organics, laboratory or sample equipment artifacts (e.g., phthalates), or nonpetroleum chemicals (Zemo et al. 1995; Uhler et al. 1998). Zemo and Foote (2003) recommended the routine use of SGC for TPHd analysis to facilitate comparison of the concentration of the hydrocarbons in the sample to hydrocarbon-based regulatory criteria. However, over the last decade SGC has been applied inconsistently because of regulatory concern about the nature and toxicity of the polar metabolites. This regulatory approach can result in expensive and potentially unnecessary additional investigation or remediation, or protracted site closures.
Polar compounds naturally present in crude oil (nitrogen-, sulfur-, or oxygen-containing heteromolecules) are largely eliminated in the refining processes used to produce gasoline, jet fuel, and diesel because they are deleterious to fuel performance (Hamilton and Falkiner 2003; Strauss 2003; Westbrook and LeCren 2003). Therefore, the water-soluble fraction of these fresh unbiodegraded fuels typically contains few or virtually no oxygen-containing polar compounds, except for chemicals such as oxygenates purposely added to the fuel. The presence of a high proportion of oxygen-containing polar compounds other than additives at fuel release sites is direct evidence that biodegradation is occurring (Barcelona et al. 1995; Beller et al. 1995; Beller 2002). The oxidative biodegradation of petroleum hydrocarbons has been studied extensively, and intrinsic and enhanced biodegradation are widely accepted remediation methods for petroleum releases (Wiedemeier et al. 1995; USEPA 1999). Both aerobic and anaerobic biodegradation processes involve transformation of the hydrocarbon molecules by sequential oxidative reactions, ultimately producing small organic acids, which are transformed to carbon dioxide and water (Atlas 1981; Dragun 1988; Cozzarelli et al. 1994; Barcelona et al. 1995; Beller et al. 1995). Intermediate steps result in the formation and subsequent biodegradation of oxygen-containing polar compounds (metabolites) that can be categorized by chemical structure into five families: acids/esters, alcohols, phenols (from aromatic hydrocarbons only), aldehydes, and ketones (Healy et al. 1980; Harayama et al. 1999; Griebler et al. 2004; Young and Phelps 2005; Chakraborty and Coates 2005; Callaghan et al. 2006; Geig et al. 2009). These five families can be further subdivided into structural classes (normal and branched, cyclic, aromatic, bicyclic and polycyclic aromatic) based on precursor hydrocarbon structures, which results in a total of 22 structural classes for the potential polar metabolites as shown in Table 1. Individual metabolites are transient. Because fuels are mixtures of hundreds of individual hydrocarbons, thousands of individual transient polar metabolites are possible.
Table 1. Structural Classes of Polar Metabolites from Biodegradation of Fuels and Expected Chronic Oral Toxicity to Humans
|Polar Family||Specific Structural Class||Expected Chronic Oral Toxicity to Humans|
|Alcohols (and diols)||Alkyl alcohols||Low|
|Bicyclic alkyl alcohols||Low|
|Polycyclic aromatic alcohols||Low to Moderate|
|Acids (and esters)||Alkyl acids||Low|
|Bicyclic alkyl acids||Low|
|Polycyclic aromatic acids||Low to Moderate|
|Ketones||Alkyl ketones||Low to Moderate|
|Bicyclic alkyl ketones||Low|
|Aromatic ketones||Low to Moderate|
|Polycyclic aromatic ketones||Low to Moderate|
|Aldehydes||Alkyl aldehydes||Low to Moderate|
|Cycloalkyl aldehydes||Low to Moderate|
|Bicyclic alkyl aldehydes||Low to Moderate|
|Aromatic aldehydes||Low to Moderate|
|Polycyclic aromatic aldehydes||Low to Moderate|
Identifying polar metabolites produced by biodegrading fuels in environmental samples using traditional analytical techniques is challenging because of the large number of potential compounds that can be present at low concentrations, that is, µg/L. Most work on metabolites has focused on identifying organic acids in groundwater using derivatization procedures followed by GC (Barcelona et al. 1995; Cozzarelli et al. 1995; Beller 2002; Martus and Puttmann 2003; Alumbaugh et al. 2004; McKelvie et al. 2005). Using GC-MS, Langbehn and Steinhart (1995) identified acids and ketones in soil affected by biodegrading diesel. Recently, the availability of two-dimensional separation techniques, such as two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-MS), has increased the ability to characterize an increased number of compounds. GC × GC performs complimentary separations in two dimensions simultaneously and results in a greater sensitivity than traditional one-dimensional GC (Marriott et al. 2012; Ryan and Marriott 2003). Mao et al. (2009) identified acids/esters, alcohols, phenols, aldehydes, and ketones in a laboratory-generated leachate of diesel-containing soil undergoing aerobic biodegradation using high-performance liquid chromatography followed by two-dimensional gas chromatography with flame ionization detection (HPLC-GC × GC-FID) and GC × GC-MS.
The purpose of this study is to characterize the chemical structure and potential toxicity of polar metabolites measured as TPHd in groundwater samples at five fuel-impacted sites. A two-step analytical approach was used, in which groundwater samples were analyzed quantitatively for a target list of potential individual polar metabolites for which toxicity information and analytical standards were available using traditional GC-MS analyses, and additional polar compounds were tentatively identified using nontargeted (Hoh et al. 2012) GC-MS and GC × GC-MS techniques. The potential human toxicity of the mixtures of identified polar metabolites was then assessed. Due to the transient nature of the polar metabolites and uncertainty associated with specific isomeric identification, the nontargeted investigation focused on the five families and 22 structural classes presented in Table 1, rather than on individual compounds.
Results and Discussion
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- Results and Discussion
Results for the polar compounds that are potential metabolites, which were the vast majority of the polar TICs in these samples, are presented and discussed herein. Other polar compounds that were infrequently identified and that are not potential metabolites (chlorinated compounds, benzothiophenes, plasticizers, pesticides) are not presented or discussed. These nonmetabolites were not a significant component of these samples either in terms of numbers of TICs or detector response (peak area), with the exception of the plasticizers in a few samples. Except for the benzothiophenes, which were very infrequently identified, the nonmetabolites have no relationship to the presence of the residual fuel at these sites and thus would have no role in risk management associated with the residual fuel.
Detailed analytical results for the commercial lab are shown in Table 4. The GC × GC-MS analytical results and toxicity evaluation results are summarized in Tables 5 and 6. Esters, which are reversibly formed from an acid and an alcohol, were classified with the acids for this study.
Table 4. Results of Commercial Lab Analyses
Table 5. Summary of GC×GC-MS Results: Average per Well % of TICs by Polar Families and by Expected Human Chronic Oral Toxicity
|Site||Avg TPHd wo/w SGC||Total # TICs||Polar Chemical Families||Expected Chronic Oral Toxicity|
|Acids||Alcohols||Phenols||Ketones||Aldehydes||Low||Low to Moderate||Moderate|
|Total Results—All Samples and All Sites|
|Average % all sites|| || ||46||25||2||22||4||84||14||1|
|Source Zone Samples|
|Site 1||2800 p / 1100 p||173||36||30||2||28||3||78||20||2|
|Site 2||27,000 p / 20,000 p||173||25||31||3||32||8||72||25||3|
|Site 3||1600 / 200||80||43||23||0||28||6||80||20||0|
|Site 4||3100 / <100||153||31||29||0||32||8||86||14||0|
|Site 5||6800/830 nc||737||13||26||8||45||7||65||26||8|
|Average % source area|| || ||30||28||3||33||6||76||21||3|
|Site 1||840 / <100||34||70||13||2||13||2||88||12||0|
|Site 2||800 / <100||40||66||32||0||2||0||98||2||0|
|Site 4||340 / <100||28||71||16||8||6||0||98||2||0|
|Site 5||1400 / 270 nc||35||65||23||0||12||0||97||3||0|
|Average % downgradient area|| || ||68||21||2||8||1||95||5||0|
Based on the results for the natural attenuation parameters (Table 4), all wells were within the zone of biodegradation. The redox conditions were generally anaerobic and varied from nitrate-reducing to methanogenic, depending on the site and the relative position of each well with respect to the source area.
All TPHd results (Table 4), including those with SGC, were within the laboratory's acceptable control ranges. The TPHd concentrations for each groundwater sample were similar to previous monitoring events. The TPHd concentrations without SGC (representing all organics extracted by Method 3510C and with boiling points between 170 and 430 °C) for samples containing only dissolved organics ranged from 1000 to 8100 µg/L in source-area samples, and from 98 to 1700 µg/L in downgradient samples. A review of the chromatograms revealed that 4 of the 13 source-area samples contained a nondissolved product component (Site 2 MW-6, Site 1 MW-5A, Site 1 MW-100/5A duplicate, Site 1 MW-26A). Nondissolved product is characterized by a chromatogram with a distinctive fuel pattern (dominated by an unresolved complex mixture [UCM] in the appropriate carbon range in the case of middle distillates) and not by the individual hydrocarbon peaks that correspond to the water-soluble fraction of fuels (primarily the C14 and smaller aromatics and very small aliphatics). The inclusion of a nondissolved component was an artifact of sheen or petroleum-impacted soil particles (turbidity) in the samples caused by the act of sampling. These four samples had TPHd concentrations ranging from 2000 to 27,000 µg/L.
Except for the samples with entrained nondissolved product, the TPHd chromatograms for the study samples were all dominated by a prominent UCM that was not representative of a fuel pattern or dissolved hydrocarbon pattern but is typical for complex mixtures of polar compounds at sites with biodegrading petroleum sources (see Figure 4 of Zemo and Foote 2003). Except for Site 5 (discussed below) and samples with entrained nondissolved product, TPHd concentrations for all samples but one were reduced to nondetect (<100 µg/L) after the SGC. This indicates that virtually all of the organics in groundwater and the components of the UCM at these sites are polars, and not dissolved hydrocarbons. Based on the difference between the TPHd concentration without SGC and with SGC for each sample, the percentage of the dissolved organics that were polars ranged from 84 to 100% in source-area samples and was 100% in downgradient samples. Samples with entrained nondissolved product (Site 2 MW-6, Site 1 MW-5A, Site 1 MW-26A) had less reduction in TPHd concentration after SGC because of the hydrocarbons present.
At Site 5, the TPHd concentrations were significantly reduced after SGC, but remained above 100 µg/L. The SGC was incomplete for all of the Site 5 samples, as indicated by either a capric acid recovery greater than 1% or the chromatogram pattern. The reason for the incomplete SGC at Site 5 is unclear. Even with an incomplete SGC, the percentage of dissolved polar compounds in the Site 5 samples ranged from at least 65% to at least 91%.
Targeted GC-MS Analysis
The quantitative results from Modified EPA Method 8270C for the 57 target polars (Table 4) show that, except for 11 µg/L dodecanoic acid in one sample, none of the individual compounds were detected in any of the extracts/eluates for any groundwater sample (most reporting limits were 10 µg/L). The LCS/LCSD recoveries and the Method 8270C surrogate recoveries were generally within the laboratory's acceptable range.
Dodecanoic acid (also known as lauric acid CASRN 143-07-7) is a C12 saturated fatty acid. Based on the toxicity ranking system, this compound and its structural class (alkyl acid) are of Low toxicity. It is equally important to note that included among the 57 target polars were 12 alkyl phenols, representing the relatively more toxic polar metabolites (as shown on Figure 1), none of which were detected in any sample at a reporting limit of 10 µg/L.
Nontargeted GC-MS Analysis
Combining the GC-MS Library Search results for the DCM extracts and methanol eluates for all wells at each site, the number of polar metabolite TICs for each site ranged from 4 (Site 2) to 27 (Site 5) (Table 4). The number of unique polar metabolite TICs in a single well ranged from 1 to 8. The three Site 5 source area samples had the largest number of TICs and included organic acids, ketones, phenols, and one aldehyde. Organic acids were the only compounds tentatively identified in other samples, except for one phenol in Site 2 MW-6. Most of the organic acids were identified as “unknown carboxylic acid”; “naphthalene carboxylic acid” was identified in four samples.
Nontargeted GC×GC-MS Analysis
Because of the uncertainty associated with MS library matching, inability to distinguish among potential isomers, and lack of standard-based confirmation, specific individual TICs are not discussed in detail here but rather are reported by family and structural class. TIC concentrations could not be calculated because standards were not available, which prevented the generation of calibration curves; however, based on the 28 standards that were run, it was determined that the LOQ for a majority of the identified compounds is in the range of 1 to 5 µg/L. The GC × GC also detected several of the 57 target polar metabolites that were not detected by the traditional GC at detection limits of 10 µg/L, further suggesting that the target polars identified by the GC × GC were present at single digit µg/L levels. A complete list of unique TICs from this study and additional details about the GC × GC-MS analysis are presented in Mohler et al. (in press). DOI: 10.1021/es401706m.
The GC × GC-MS analysis resulted in a greatly increased number of polar metabolite TICs for each site, ranging from 80 (Site 3) to 772 (Site 5). The number of unique polar metabolite TICs in a single well ranged from 5 to 310. The greatly increased number of TICs for the GC × GC-MS as compared to the GC-MS confirms that two-dimensional chromatography is necessary to resolve individual compounds in these complex mixtures of polar metabolites. The highest number of polar metabolite TICs (and highest concentration of polars measured as TPHd) was present in source-area samples at each site, with significantly fewer TICs (and very low concentrations of polars as TPHd) in downgradient samples at each site.
Summaries of the average percentage of TICs in each of the polar metabolite families for each site, and separately for the source and downgradient areas at each site, are shown in Table 5. For this study, average percentage calculations are always based on the percentage for each individual well at each site, and not on the total number of TICs for the site as a whole, so that results for the downgradient samples (with fewer TICs) are weighted equally with source-area samples. The average TPHd concentration without and with SGC for each area at each site is also shown in Table 5 to provide context regarding the “bulk” concentration of polar compounds in each area.
The site-wide results for all five sites were similar and indicate that an average of 46% of the polar metabolite TICs were acids/esters, 25% were alcohols, 2% were phenols, 22% were ketones, and 4% were aldehydes (Table 5, Figure 2). All five sites also showed that there is a marked difference in the distribution of the identified polar metabolite families between source-area and downgradient samples at each site. In source-area samples, the acids/esters (average of 30% of the polar metabolite TICs), alcohols (28%), and ketones (33%) are approximately equally distributed, with far fewer phenols (3%, including alkyl phenols) or aldehydes (6%). In downgradient samples, the acids/esters dominate (average of 68% of the polar metabolite TICs), with fewer alcohols (21%), and far fewer ketones (8%), phenols (2%, all as phenol and no alkyl phenols), and aldehydes (1%). Although the GC × GC results are not quantitative, the relative response factors show that it is reasonable to compare trends for each family and class within samples in the C8 to C12 carbon range.
Figure 2. Summary of GC×GC-MS results for polar families: distributions overall and in source and downgradient areas.
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A summary of the average percentage of identified structural classes in the source and downgradient areas for all five sites combined is shown in Table 6. All 22 expected structural classes were tentatively identified. The most frequently identified structure was alkyl acids/esters, which averaged about 21% of the polar metabolite TICs in the source area and about 56% of the polar metabolite TICs in the downgradient-area samples. The results show that, when identified, the more complex bicyclic and polycyclic aromatic structures are primarily in source-area samples, as would be expected due to their proximity to the residual hydrocarbon source, with less complex structures predominating in downgradient samples. Alkyl phenols were identified in only five samples, all of which were in source areas and two of which contained a nondissolved product component.
Table 6. GC×GC-MS Results—Avg% for Each Structural Class in Source and Downgradient Areas
|Polar Family||Specific Structural Class||Expected Chronic Oral Toxicity to Humans||Source Area Samples||Downgradient Samples|
|Avg % per Sample||Avg % per Sample|
|Alcohols (and diols)||Alkyl alcohols||Low||13||11|
| ||Cycloalkyl alcohols||Low||10||5|
| ||Bicyclic alkyl alcohols||Low||2||6|
| ||Aromatic alcohols||Low||3||<1|
| ||Polycyclic aromatic alcohols||Low to Moderate||<1||0|
|Acids (and esters)||Alkyl acids||Low||21||56|
| ||Cycloalkyl acids||Low||2||<1|
| ||Bicyclic alkyl acids||Low||2||0|
| ||Aromatic acids||Low||3||12|
| ||Polycyclic aromatic acids||Low to Moderate||<1||0|
|Ketones||Alkyl ketones||Low to Moderate||6||3|
| ||Cycloalkyl ketones||Low||9||2|
| ||Bicyclic alkyl ketones||Low||9||2|
| ||Aromatic ketones||Low to Moderate||8||<1|
| ||Polycyclic aromatic ketones||Low to Moderate||<1||0|
|Aldehydes||Alkyl aldehydes||Low to Moderate||4||<1|
| ||Cycloalkyl aldehydes||Low to Moderate||<1||0|
| ||Bicyclic alkyl aldehydes||Low to Moderate||<1||0|
| ||Aromatic aldehydes||Low to Moderate||2||0|
| ||Polycyclic aromatic aldehydes||Low to Moderate||<1||0|
Because the relative response factors for each polar metabolite family were reasonably similar, the GC × GC-MS results were also evaluated by reviewing each sample to determine the polar families that were represented in the top 5, 10, and/or 20 peaks by peak area response. This showed that for each sample the acids and/or alcohols were typically a higher percentage of the highest peak area response than their percentage based on the number of TICs. Conversely, the ketones, aldehydes, and phenols were typically a smaller percentage of the highest peak area response than their percentage based on the number of TICs. This means that the results as presented in this paper based on numbers of TICs may underestimate the proportion of acids and/or alcohols, and may overestimate the proportion of ketones, aldehydes, and phenols, actually present in each sample.
A summary of the average percentage of the total number of polar metabolite TICs in each toxicity ranking (Low, Low to Moderate, and Moderate) for each site, and separately for source and downgradient areas at each site, is shown in Table 5. For all five sites combined, an average of 84% of the polar metabolite TICs are ranked as “Low” toxicity, 14% as “Low to Moderate” toxicity, and only 1% as “Moderate” toxicity. In source-area samples, the average percentages are 76% “Low” toxicity, 21% “Low to Moderate” toxicity, and 3% “Moderate” toxicity. In downgradient samples, the profile shifts toward a lower toxicity, with average percentages of 95% “Low” toxicity, 5% “Low to Moderate” toxicity, and 0% “Moderate” toxicity. The increase in the average percentage of “Low” toxicity compounds and the decrease in the “Low to Moderate” and “Moderate” toxicity compounds in the downgradient samples is due to the dominance of acids/esters, and the virtual lack of aromatic/polycyclic aromatic ketones, alkyl phenols, and aldehydes in downgradient samples (Table 6).
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- Results and Discussion
The purposes of this study were to (1) identify as well as possible the polar compounds in the DCM extracts of groundwater samples from five sites with biodegrading fuel sources that are quantified as TPHd unless a SGC is used to separate the polars from hydrocarbons, and (2) estimate the potential chronic human toxicity of the identified polar compounds. The GC × GC-MS analysis provided detail not previously available for actual groundwater samples, and documented that the vast majority of polar compounds identified in the groundwater sample extracts were oxygen-containing metabolites of biodegradation. The mixture of identified polar metabolites is composed of organic acids/esters, with variable alcohols and ketones, and very few phenols and aldehydes. The analytical results were similar among the five sites. The mixture of identified polar metabolites in the source area samples had approximately equal average percentages of organic acids/esters, alcohols, and ketones, which reflects the ongoing sequential oxidation reactions proximal to the residual hydrocarbon molecules. The mixture of identified polar metabolites in the downgradient area samples was dominated by organic acids/esters. The observed spatial trend in the relative proportions of the polar families, combined with the predominant simpler structures and decreasing bulk concentrations of polar compounds (measured as TPHd) seen in downgradient samples, documents the continued biodegradation of the polar metabolites themselves and their ultimate natural attenuation with migration away from the residual hydrocarbon in the source area. The oxidation of the various polar families to small organic acids, and their ultimate transformation to carbon dioxide and water, is consistent with known metabolic pathways.
An RfD-based toxicity ranking system that is consistent with systems used by USEPA and the United Nations was developed and applied to each of the identified polar metabolite structural classes. The results from this study show that the vast majority of the hundreds of polar metabolites that were identified using GC × GC-MS in groundwater sample extracts from these five biodegrading fuel sites are in structural classes of “Low” toxicity hazard to humans. These results indicate that the mixtures of polar metabolites identified in groundwater extracts at these five sites are unlikely to present a significant human health risk, assuming that the affected groundwater were to be consumed as drinking water.
The results from this study also show (and confirm results from previous studies) that the organics in groundwater quantified as TPHd at these five sites were primarily polar metabolites and not dissolved hydrocarbons. Therefore, a SGC is necessary if groundwater sample TPHd results are to be compared to hydrocarbon-based regulatory criteria.