Recalcitrance and Degradation of Petroleum Biomarkers upon

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Recalcitrance and Degradation of Petroleum Biomarkers upon Abiotic and Biotic Natural Weathering of Deepwater Horizon Oil Christoph Aeppli,*,†,∥ Robert K. Nelson,† Jagoš R. Radović,†,‡ Catherine A. Carmichael,† David L. Valentine,§ and Christopher M. Reddy† †

Department of Marine Chemistry & Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, United States ‡ Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona, 18, Barcelona 08034, Barcelona, Spain § Department of Earth Science and Marine Science Institute, University of California, Santa Barbara, California 93106, United States S Supporting Information *

ABSTRACT: Petroleum biomarkers such as hopanoids, steranes, and triaromatic steroids (TAS) are commonly used to investigate the source and fate of petroleum hydrocarbons in the environment based on the premise that these compounds are resistant to biotic and abiotic degradation. To test the validity of this premise in the context of the Deepwater Horizon disaster, we investigated changes to these biomarkers as induced by natural weathering of crude oil discharged from the Macondo Well (MW). For surface slicks collected from May to June in 2010, and other oiled samples collected on beaches in the northern Gulf of Mexico from July 2010 until August 2012, hopanoids with up to 31 carbons as well as steranes and diasteranes were not systematically affected by weathering processes. In contrast, TAS and C32- to C35-homohopanes were depleted in all samples relative to 17α(H),21β(H)-hopane (C30-hopane). Compared to MW oil, C35homohopanes and TAS were depleted by 18 ± 10% and 36 ± 20%, respectively, in surface slicks collected from May to June 2010, and by 37 ± 9% and 67 ± 10%, respectively, in samples collected along beaches from April 2011 through August 2012. Based on patterns of relative losses of individual compounds, we hypothesize biodegradation and photooxidation as main degradation processes for homohopanes and TAS, respectively. This study highlights that (i) TAS and homohopanes can be degraded within several years following an oil spill, (ii) the use of homohopanes and TAS for oil spill forensics must account for degradation, and (iii) these compounds provide a window to parse biodegradation and photooxidation during advanced stages of oil weathering.

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Most oil spill fingerprinting relies on the fidelity and variability of hopanoids, steranes and TAS (Figure 1), which are present in crude oils and some of their refined products.1 The use of these petroleum biomarker compounds for oil spill forensics is an extension of petroleum geochemistry.12 These compounds are generally assumed to be more recalcitrant toward microbial and abiotic degradation than other saturated (such as n-alkanes, branched alkanes, and isoprenoids) and aromatic compounds (benzene, alkylated congeners of benzene, and polycyclic aromatic hydrocarbons, PAHs).12,13 In laboratory studies, 17α(H),21β(H)-hopane (I in Figure 1; referred to as “C30-hopane” hereafter) has proven to be remarkably recalcitrant against biodegradation2,3 and photooxidation.14 However, biodegradation of C30-hopane and other biomarkers was achieved under aggressive laboratory conditions using aerobic enrichment cultures.15−17 Degradation of

INTRODUCTION Petroleum biomarkers (i.e., molecular fossils) are commonly used to define the identity of spilled oil1 and further serve as recalcitrant tracers for weathering studies.2,3 For example, these compounds have previously been used to study the fate of oil residues in the northern Gulf of Mexico,4−7 following the explosion of the Deepwater Horizon (DWH) oil rig on April 20, 2010, that led to a release of 6.8 ± 1.7 × 108 kg of petroleum hydrocarbons from the Macondo Well (MW).8,9 While soluble and volatile hydrocarbons were dissolved in the water column or evaporated to the atmosphere, it has been estimated that approximately 10% of the leaked mass formed surface oil slicks that eventually oiled beaches.9 This amount corresponds to roughly 70,000 t (approximately 500,000 barrels), mostly petroleum hydrocarbons larger than n-pentadecane (n-C15), and is more than twice the estimated release from the 1989 Exxon Valdez oil spill.10 Given the magnitude of release and the extent of oiled shoreline, reliable fingerprinting methods are clearly needed to distinguish MW oil from other crude oil sources such as natural seeps11 or accidental releases associated with oil production, transportation, and usage. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 6726

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investigate systematically abiotic and/or biotic natural degradation of biomarker compounds in MW oil. The aim of this study was therefore to assess the recalcitrance and degradation of biomarker compounds in MW oil that has undergone natural weathering processes including dissolution, evaporation, biodegradation, and photooxidation. In order to span a large spectrum of weathering, we revisited previously described MW oil, surface slicks, oil-soaked sand, and oilcovered rock samples4,5,8 and added newer samples collected up to 28 months after the Deepwater Horizon disaster. We used comprehensive two-dimensional comprehensive gas chromatography (GC×GC) for biomarker analysis.26,27 As noted in recent studies, GC×GC is superior to one-dimensional gas chromatography with mass spectral detection (GC-MS) in terms of analytical resolution, selectivity, and sensitivity28 and allows for accurate quantification of biomarkers in a complex mixture of petroleum hydrocarbons.29 Using this technique, we found that hopanoids consisting of 27 to 31 carbon atoms as well as steranes and diasteranes were recalcitrant toward weathering. However, we observed substantial losses of homohopanes with more than 31 carbon atoms as well as of TAS, over 28 months of weathering.

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MATERIALS AND METHODS Samples. A total of 50 oiled samples collected in the Gulf of Mexico during April 2010 through August 2012 were analyzed. This sample set comprises MW oil collected from the well on June 21, 2010,8 nine surface slicks and oiled floating DWH debris samples collected on the sea surface during the spill,4,5 and samples washed on beaches (31 oil-soaked sand samples or “sand patties”, six oil samples scraped off rocks or “rock scrapings”, and four sand-free asphalt pieces), some of which were previously described.5 Sand patties, rock scrapings, and asphalt pieces were extracted in dichloromethane (DCM) and methanol (90/10 v/v). All samples were dissolved in DCM or DCM/methanol at concentrations of 10−50 mg mL−1 for analysis. See Table S3 of the SI for sampling dates and locations. Beside MW oil and field samples, five reference crude oils were analyzed: (i) a surrogate for MW oil provided by BP, produced from the Marlin Platform on Viosca Knoll lease block 915 (VK915),30 located 60 km NE of the MW; (ii) the EPAAPI reference WP#681 Southern Louisiana Sweet crude (SLSC);31 (iii) crude oil from Eugene Island lease block 330 (EI330), located 340 km W of the MW; (iv) the petroleum standard reference material (SRM) 1582 from the National Institute of Standards and Technology (NIST), which is a Wilmington crude from the Monterey formation; and (v) cargo oil from the Exxon Valdez (EVC). Comprehensive Two-Dimensional Gas Chromatography-FID (GC×GC-FID). We used GC×GC-FID methods described in detail previously.5,32 Briefly, 1 μL of each sample in DCM or DCM/methanol (90/10) was injected in a GC×GC-FID system with a dual stage cryogenic modulator (Leco, Saint Joseph, MI), equipped with a Restek Rtx-1 firstdimension column (60 m, 0.25 mm ID, 0.25 μm film thickness) and a SGE BPX-50 s-dimension column (1.5 m, 0.10 mm ID, 0.10 μm film thickness). The inlet temperature was held at 300 °C. The injection mode was splitless, and the carrier gas was H2 at a constant flow rate of 1.00 mL min−1. The first oven was programed isothermal at 40 °C for 10 min, 40 to 340 °C at 1.25 °C min−1 (held for 5 min). The second oven was programmed

Figure 1. Structures of targeted petroleum biomarkers: (a) hopanoids, (b) steranes and diasteranes, and (c) triaromatic steroids (TAS). See Table S1 for names, abbreviations, and structures of all compounds discussed in this study. The straight alkyl chain of homohopanes and the phenanthrene backbone of TAS compounds (colored in blue) are likely targets of biodegradation and photooxidation, respectively.

biomarker compounds was also observed in several long-term field studies (an overview is given in Figure S1 of the Supporting Information, SI). For example, eight years after an oil spill experiment in a mangrove soil on Guadeloupe (Lesser Antilles), total steranes and hopanes were depleted >25%.18 Similar observations were made 20 years after an Arctic oil spill experiment,19,20 22 years after the Arrow spill on the Nova Scotia coast,21 24 years after the Metula spill in the Straight of Magellan,22 and in oiled soil samples from oil production areas17 and from natural seeps.23 Most of the above-mentioned studies were conducted at latitudes higher than the Gulf of Mexico, and it is of interest to know how the higher average temperatures and intense solar irradiation affected biomarker degradation of MW oil. We previously reported extensive degradation of saturated and aromatic compounds of MW oil within 18 months after the spill in oiled samples collected on beaches, with n-alkanes, isoprenoids, and two-ring PAHs being completely removed in most samples.5,24 Furthermore, we observed TAS depletion in a limited data set of oil-covered rock samples,25 and lower amounts of steranes relative to hopanoids in samples recovered from deep-sea corals.7 This alteration of biomarker compounds in relatively short time scales (months to years) prompted us to 6727

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Figure 2. (a) GC×GC-FID chromatograms of MW oil and (b) sand patty B105 collected August 2012 in Gulf Shores, AL. Two biomarker regions (with hopanoids, (dia)steranes, and TAS), as well as PAH elution regions are depicted. Many compounds originally present in MW oil have been removed, but the hopanoid and (dia)sterane region remained largely unaffected. An enlargement of the hopanoid and sterane region with select annotated peaks is given (see Table S1 of the SI for compound names).

as follows: isothermal at 45 °C for 10 min, 45 to 355 °C at 1.29 °C min−1 (held for 5 min). The modulation period was 15 s. Biomarker compounds were identified based on established elution order27,28 and using authentic standards obtained from Sigma-Aldrich, Chiron, and NIST SRM 2266 (Table S2 of the SI). In addition, GC×GC coupled to time-of-flight mass spectrometry (GC×GC-TOFMS, Leco, Saint Joseph, MI; operated according to ref 33) was used to confirm the identity of compounds based on their mass spectra. See Figure 2 and Figure S2 of the SI for sample GC×GC-FID chromatograms. GC×GC-FID peaks were manually integrated using the ChromaTOF software (Leco). We targeted 17 hopanoids, 13 steranes and diasteranes, and eight TAS indicated in Figure S2 and Table S1 of the SI. We also included several PAHs (phenanthrene, C1−C3 phenanthrenes/anthracenes, chrysene, C1−C3 chrysene/benz[a]anthracenes), alkanes (n-heptadecane; n-C17, n-octadecane; n-C18), and isoprenoids (pristane and phytane) in our analyses. Quality control included a linearity test of the FID responses by injecting the NIST SRM 2266 at five concentrations within our operating range. We obtained a linear calibration curve for the seven certified biomarkers present in SRM 2266 (R2 > 0.999; Table S2 of the SI). Furthermore, all seven compounds had identical response factors (i.e., signal per mass unit biomarker injected), with a relative standard deviation (RSD) of 50% for most sand patties and rock scrapings.

remarkably different sterane/hopanoid ratios. Note that it might be counterintuitive to compare GC×GC-FID (rather than GC×GC-TOFMS) to GC-MS. However, due to the superior chromatographic resolution of GC×GC, FID detection is sufficient and better suited for biomarker analysis than TOFMS detection, due to the high sensitivity and the constant response factor of the FID. Relative Enrichment of Biomarker Compounds During Weathering. We investigated weathering of MW oil on the sea surface as well as on beaches. Natural weathering changed the composition of the spilled oil, as shown in the GC×GC chromatograms of MW oil and a representative oilsoaked sand sample collected on the beach of Gulf Shores, AL more than two years after the onset of the spill (Figure 2). Two robust trends are apparent by visual comparison. First, the oilsoaked sand sample had lost volatile compounds (i.e., having early GC fist-dimension retention times) due to evaporation, including all compounds with less than 15 carbons.5 Similarly, the more polar compounds (i.e., having late GC seconddimension retention times) were lost to a greater extent than less polar ones due to preferential disolution.39 For example, we observed a preferential depletion of phenanthrene relative to alkylated (and less polar) phenanthrenes.5 Second, weathering led to a depletion of specific compound classes, including nalkanes. In contrast, the (dia)steranes and hopanoids qualitatively appear to be much less affected by weathering, representing a relative enrichment of the biomarker compounds in the weathered oil samples. Persistence of Hopanoids and Steranes. To investigate quantitatively the persistence of biomarker compounds, we correlated biomarkers in the surface slicks, sand patties, and rock scrapings (Table S3) with their degree of oil weathering. As a proxy for weathering, we used the relative amount of OxHC. We previously reported that this operationally defined oil fraction was a suitable indicator for gauging weathering of MW oil.5

as the MS fragmentation pattern is highly specific for individual compounds and is dependent on the condition of the ionization source.35 Furthermore, GC-MS biomarker analysis is frequently performed semiquantitatively, i.e., by calculating ratios of mass fragments without external standards for each compounds.35 Consequently, biomarker ratios determined by GC-MS do not accurately reflect the relative concentrations of the compounds in the sample and are more prone to vary among different laboratories. As example, this issue arose with Ts/Tm in these samples. While Geomark and our GC-MS measurement resulted in Ts/Tm of 1.1 for MW oil, Mulabagal et al. recently reported a ratio of 0.9 (also using GC-MS).36 Similarly, biomarker analysis by an analytical laboratory using GC/MS without external TAS standards led to an overestimation of TAS concentrations by a factor of eight, as compared to our GC×GC-FID results (data not shown). In contrast, our approach using GC×GC-FID should be more consistent across different users. While we argue that GC×GC-FID provides advantages over GC-MS we are not implying that GC-MS is inaccurate; when standardized protocols are followed for GCMS analysis of biomarker ratios (such as the “Nordtest method”37), round-robin test laboratories routinely determined consistent results.38 Third, because GC×GC separates compounds according to two distinct physical properties, the elution space of compounds on the GC×GC plane provides information about their physical properties or structures. For example, the unknown compound that coelutes with Tm in the first dimension elutes very close to C28ααα(S) in GC×GC. Therefore, the unknown compound is expected to have a similar structure or physical properties to this C28-sterane. To compare biomarker results of GC×GC-FID and GC-MS, we performed GC-MS analysis of hopanoids and (dia)steranes for 24 field samples (Table S5 of the SI). For ratios of hopanoids, we observed similar biomarker ratios with GC-MS and GC×GC-FID. In contrast, the two methods produce 6730

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When the C30-hopane normalized compounds (eq 1) are compared to OxHC, their loss due to weathering can be visualized (Figure 3). For hopanoids containing 27 to 31 carbon atoms, such as Tm (C27H46) or HH (C31H54), no significant loss with weathering was observed for all investigated surface slicks, sand patties, and rock scrapings (Figure 3a-c). Consequently, no weathering-dependent change in Ts/Tm was observed either (Figure S5). Interestingly, such a weatheringdependent Ts/Tm change has previously been observed on longer time scales, e.g., 24 years after an oil spill in the Strait of Magellan,22 eight years after an artificial oil spill in a Mangrove soil,18 and in laboratory experiments.15,16 Similar to hopanoids ≤ C31, no trend in loss with increasing weathering was observed for steranes and diasteranes (Figure 3e-f). We also did not observe preferential degradation of C27 > C28 > C29 steranes in the investigated samples (Figure S5). In contrast to our study, a susceptibility of steranes over hopanoids as well as a preferential degradation of C27 steranes over C29 steranes has been observed on time scales of 8 to 24 years (see Figure S1 of the SI for an overview of past studies).18,19,21,22 Biodegradation of Homohopanes. In contrast to the persistence of Ts and Tm relative to H, we found preferential losses of homohopanes > C31, such as 3HH (C33H58) or 5HH (C35H62), relative to C30-hopane (Figure 3d-e). We calculated an average 5HH loss of 18 ± 10%, 35 ± 8%, and 47 ± 9% relative to the MW oil for surface slicks, sand patties, and rock scrapings, respectively (Table S3). In contrast, C30-hopanenormalized HH or 2HH did not change significantly. This preferential degradation of homohopanes with longer alkyl chains is consistent with observations from field studies of salt marshes (on the Strait of Magellan) and mangroves (on Guadeloupe) and has been associated with biodegradation.18,22 In line with biodegradation, we also observed a preferential degradation of the R vs the S epimer of 3HH, 4HH, and 5HH for heavily degraded samples (i.e., with OxHCtot > 60%; Figure S5).18,22 In summary, hopanoids ≤ C31, steranes, and diasteranes present in MW oil appeared to be recalcitrant toward weathering processes in the Gulf of Mexico on the time scale of two years, whereas homohopanes >C31 were degraded. The degradation patterns point to microbial activity as being responsible for this degradation. Using hopanoids ≤ C31, steranes, and diasteranes is therefore suitable for fingerprinting weathered MW oil. Photooxidation of Triaromatic Steranes. We found that TAS were less stable than C30-hopane (Figure 3g-h). An average loss of TAS-C28 of 36 ± 20%, 65 ± 9%, and 76 ± 12% was calculated using eq 1 for surface slicks, sand patty, and rock scrapings, respectively (Table S3). We observed that all investigated TAS were lost to remarkably similar degrees with no preferential removal of individual congeners (Figure 4). For example, the C20/C28 TAS ratios for MW oil and surface slick samples were similar (1.6 ± 0.2 and 1.5 ± 0.2, respectively) despite the latter having traveled vertically through the 1.5 km water column and more than 200 km horizontally on the water surface. The variable degree of alkylation of the alkyl side chain of TASs (Figure 1) leads to a range in their molecular weight (260−372 g mol−1), vapor pressure (1.6 × 10−5 to 6.8 × 10−9 Pa), octanol−water partitioning coefficient (KOW = 1.0 × 107 to 1.8 × 1010), and aqueous solubility (0.001 to 3.6 μg L−1; see Table S4 of the SI). Therefore, physical processes such as evaporation, sorption, or

Figure 4. Calculated losses of TAS-C21, TAS-C27(R), and TAS-C28(R) vs losses of TAS-C28(S). Shown are MW oil (yellow circles), surface slicks (blue circles), sand patties (black circles), and rock scrapings (red circles), the 1-to-1-line (solid black line), and the linear regression (blue line, including slope and R2). Despite differences in aqueous solubility and vapor pressure of 3 orders of magnitude (see Table S3 of the SI) and different stereochemistry, the various TAS compounds are similarly depleted relative to C30-hopane. This suggests that photooxidation, rather than evaporation, dissolution, or biodegradation, is responsible for their degradation.

dissolution are expected to affect different TAS compounds to different degrees. For example, evaporation of TAS was hypothesized to be the main driver for preferential disappearance of TAS-C20 and TAS-C21 relative to the larger TAS compounds at an arid spill site in Egypt.40 This is in contrast to our study, where the behavioral similarities of all TASs suggest that dissolution or evaporation were not the main processes responsible for the observed TAS depletion. Beside physical processes, biodegradation might be responsible for TAS removal, similar to the above-described biodegradation of homohopanes. In a previous study, TAS biodegradation has been reported under oxic laboratory conditions.17 TAS-C20 and TAS-C26 (S) were degraded during a 20-week incubation experiment, while the C27 and C28 TAS compounds were recalcitrant. In the same study, a preferential depletion of TAS-C20 and TAS-C26 (S) was observed in soil field samples from a retired oil production area in Ecuador.17 This preferential depletion was attributed to biodegradation. In contrast to these findings, our results showed no such preferential loss of lower-molecular weight TAS relative to TAS-C27 and TAS-C28 (Figure 4). This suggests that biodegradation is not the main driver of TAS degradation in our samples. Finally, this leaves photooxidation as a likely process for TAS degradation. Intuitively, the condensed aromatic backbone of TAS compounds makes them good candidates for photooxidation, similar to phenanthrene or other petroleum PAHs (Figure 1).41,42 Comparing TAS losses to (alkylated) 6731

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distinctive feature of the asphalt biomarker ratio was the much lower sterane/hopane ratio (e.g., Ratio 17: 0.2 ± 0.1) compared to the MW oil (1.0 ± 0.2). The nonmatch was also confirmed by oleanane, a compound that was detected in the asphalt samples but not in MW oil (Figure S7). Oleanane is a biomarker for flowering plants, which can be often used to date oil in a reservoir.45,46 We tested the sensitivity of the chosen biomarkers to distinguish MW oil from other crude oils. The oil VK915, which is from the Marlin platform on the VK lease block 915 (60 km NE of MW), matches MW relatively well (Table 1 and Figure S9). VK915 was found to be physically, chemically, and toxicologically similar to the MW oil and was therefore chosen by BP as a MW surrogate oil.30 Similarity in biomarker ratios of oils from deep wells in the Viosca Knoll and Mississippi Canyon Blocks have previously been reported.47 In contrast, the biomarker ratios from other crude oils that have origin in the Gulf of Mexico region, such as the SLSC (EPA standard from a shallow-water platform) and oil from Eugene Island Block 330 (EI330; 340 km W of MW), are different from MW oil. As expected, Exxon Valdez crude (EVC) and NIST SRM 1582 oils also did not match MW, as these oils have very different origins (Alaska north slope and Monterey formation, respectively). Next, we explored the use of TAS biomarker ratios to fingerprint weathered oil samples. Some TAS/TAS ratios in our samples were suitable to distinguish asphalt samples from weathered MW samples (e.g., Ratios 21 and 22; Table 1); this can be attributed to the fact that all TAS compounds were lost to a similar extent, thus preserving TAS/TAS ratios. However, ratios of TAS and C30-hopane produced false-negative results for weathered MW samples (e.g., Ratios 23 and 24; see Figure 5). This is due to the weathering-induced depletion of the TAS relative to hopanoids. The utility of TAS biomarker ratios is also limited because TAS are significantly depleted for sand patties and rock scrapings (67 ± 10% for TAS-C28), causing analytical challenges for the detection and quantification of TAS. Similar to the TAS biomarker, C30-hopane normalized 5HH (Ratio 9) can lead to false-negative results (Table 1). Nevertheless, 5HH(R)/5HH(S) (Ratio 7) was found to be suitable, since we observed only a slight preference of R over S epimer degradation (Figure S5). However, anticipating that ongoing oil weathering will accentuate degradation of homohopanes > C 31 , we caution against reliance on homohopane ratios for fingerprinting MW oil in the future. Implications. Several aspects of this study inform the use of biomarkers in oil spill forensics. First, our investigations show that most biomarkers are suitable to fingerprint MW oil samples on the investigated time frame of 28 months postspill. Even in severely weathered MW oil that was exposed to the elements in the Gulf of Mexico for more than two years, we did not see any indication for degradation of hopanoids ≤ C31, steranes, or diasteranes. In a recent study, we also found no degradation of biomarker compounds in MW oil that was trapped in a cofferdam structure on the seafloor for more than two years.29 However, it is known from studies on longer time scales (decades) that steranes20 as well as hopanoids22 may eventually become susceptible toward biodegradation (Figure S1). Also note that our study focused on surface slicks and oil samples collected from beaches (sand patties, rock scrapings). Other environments might exhibit different degradation patterns. For example, a depletion of steranes relative to

phenanthrene and chrysene reveals a similar degradation pattern of PAHs and TAS (Figure S6). Furthermore, abiotic oxidation processes were suggested to be responsible for TAS depletion in naturally weathered bitumens and crude oils from natural seeps.23 We previously demonstrated that exposing MW oil to sunlight leads to loss of C20- through C28-TAS compounds and attributed this degradation to photooxidation.25 Although TAS appear to be recalcitrant in reservoirs,12 we hypothesize that the aromatic structure makes TAS susceptible to photooxidation when exposed to sunlight. Furthermore, we suggest that the diverse physical properties of TAS make this compound class ideal to study oil photochemistry in the environment. Application: Use of Biomarker Ratios to Fingerprint MW Oil. We found that hopanoids ≤ C31, sterane, and diasterane compounds are recalcitrant toward weathering in the Gulf of Mexico on the investigated time scale. We therefore conclude that the use of diagnostic ratios of these biomarkers is valid to distinguish MW oil from dissimilar sources. We determined a set of biomarker ratios in field samples and compared it to corresponding ratios of MW oil (Figure 5).

Figure 5. Fingerprinting weathered oil samples using biomarker ratios. Box plot including all 24 biomarker ratios given in Table 1 for the surface slick, sand patty, and rock scraping samples. Given are the relative differences to the ratios of MW oil; the gray shaded area marks the ±20% analytical uncertainty, which is the determined maximal RSD for MW (see the Method section). Ratios with magenta boxes and marked with as asterisk are affected by oil weathering, limiting their utility for fingerprinting MW oil.

Thereby we used diagnostic ratios in Table 1, which are commonly used in oil fingerprinting.1 Most ratios determined for samples lie within ±20% (the maximum RSD of ratios determined for eight replicates of MW oil) of the MW oil ratios, which implies a match with MW oil. However, for ratios 8, 9, 23, and 24 most samples are outside the MW range. This is because these ratios include 3HH, 5HH, and TAS, which are susceptible to degradation. In order to validate MW as the source of the surface slicks, sand patties, and rock scrapings, we used principal component analysis (PCA).43,44 Thereby we excluded weathering-affected ratios 8, 9, 23, and 24. We included four sand-free asphalt samples collected on Grand Isle, LA, Elmer’s Island, LA, and Dauphin Island, AL in July 2011 (Figure S8), as well as five reference crude oils. As can be seen from a PCA plot (Figure S9), MW oil, surface slicks, sand patties, and rock scrapings group together, while the asphalt samples are located in a different PCA space. As can be seen in Table 1, the most 6732

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hopanoids has been hypothesized for MW oil that impacted deep-sea corrals,7 or anaerobic degradation pathways in salt marshes might lead to a different degradation pattern than reported here for surface slicks, sand patties, and rock scrapings. Second, we found that some biomarker compounds were susceptible to biodegradation and photooxidation. In the investigated sand patties and rock scrapings, we observed an average of depletion of 37 ± 9% for 5HH and 67 ± 10% for TAS. We therefore caution against the use of TAS and homohopanes > C31 for fingerprinting weathered oil in the environment. Third, the differences in biomarker degradation can be used in order to track petroleum degradation in the environment. Commonly used biodegradation indices rely on n-alkanes and isoprenoids or PAHs to assess biodegradation. As these compounds are not detectable or highly depleted in the majority of the investigated MW oil samples, it is difficult to assess the progress of biodegradation for these samples. In contrast, comparing the disappearance of more recalcitrant compounds such as homohopanes or TAS provides a window to identify and quantify active processes at advanced stages of weathering. We recently presented a similar concept based on other recalcitrant compounds (e.g., cyclic and acyclic isoprenoids).24 Finally, GC×GC-FID was an excellent tool to investigate the described dynamics of biomarkers. Although GC-MS also proved useful for differentiating MW oil from non-WM oil, the increased chromatographic resolution, the elimination of interference due to coelution, and the robust quantification by FID proved to be critical advantages of GC×GC-FID over GC-MS for this work.

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ASSOCIATED CONTENT

Supporting tables (Tables S1−S5) and figures (Figures S1− S9), as noted in the text, are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: 207 315 2567. E-mail: [email protected]. Present Address ∥

Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, P.O. Box 380, East Boothbay, ME 04544, USA. Notes

The authors declare no competing financial interest.

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REFERENCES

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Article

ACKNOWLEDGMENTS

This research was made possible in part by grants from the NSF (OCE-0960841, RAPID OCE-1043976, RAPID OCE1042097, EAR-0950600, OCE-0961725, OCE-1333148, OCE1333162) and in part by a grant from BP/the Gulf of Mexico Research Initiative (GoMRI-015) and the DEEP-C consortium. C.A. acknowledges a Swiss National Science Foundation Postdoctoral Fellowship. J.R.R. kindly acknowledges a predoctoral fellowship (JAE Predoc) from the Spanish National Council of Research (CSIC) and European Social Fund (ESF). The authors wish to thank to Molly Redmond and George Wardlaw for collecting surface slicks and Karin Lemkau and Charlotte Main for sampling assistance. 6733

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NOTE ADDED AFTER ASAP PUBLICATION The version published on June 2, 2014 needed revisions to Figure 2 and Figure S2 in the Supporting Information. The revised version was reposted on June 5, 2014.

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