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Environmental Processes
How persistent and bioavailable are oxygenated Deepwater Horizon oil transformation products? Christoph Aeppli, Robert F. Swarthout, Gregory W O'Neil, Samuel D Katz, Deedar Nabi, Collin P. Ward, Robert K. Nelson, Charles M. Sharpless, and Christopher M. Reddy Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01001 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018
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Abstract
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About half of the surface oil floating on the Gulf of Mexico in the aftermath of the 2010 Deepwater
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Horizon spill was transformed into oxygenated hydrocarbons (OxHC) within days to weeks. These OxHC
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persist for years in oil/sand aggregates in nearshore and beach environments, and there is concern that
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these aggregates might represent a long-term source of toxic compounds. However, because this OxHC
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fraction is a continuum of transformation products that are not well chemically characterized, it is not
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included in current oil spill fate and effect models. This challenges an accurate environmental risk
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assessment of weathered oil. Here, we used molecular and bulk analytical techniques to constrain the
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chemical composition and environmental fate of weathered oil samples collected on the sea surface and
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beaches of the Gulf of Mexico. We found that approx. 50% of the weathering-related disappearance of
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saturated and aromatic compounds in these samples was compensated by increase in OxHC. Furthermore,
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we identified and quantified a suite of oxygenated aliphatic compounds that are more water soluble and
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less hydrophobic than its presumed precursors, but only represents 13±3% oxygen content
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on a mass basis), had an average elemental C:O ratio of 5:1, and was newly formed during oil Page 2 of 25 ACS Paragon Plus Environment
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weathering.1 On a molecular level, oxygenated species with 1 to 8 oxygen atoms per molecule were
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detected in weathered oil as well as in artificially irradiated oil but were absent in the source oil.2, 3, 7
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The OxHC fraction is a large pool of petroleum-derived products that are currently not included in oil
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spill fate and effect models,8, 9 oil spill budget estimations,10, 11 or decisions about aerial dispersant
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application.12 It has been estimated that 10% of the approximately 4.1 million barrels (5.2 × 108 kg) of
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liquid oil that was released from the Macondo Well during the DWH oil spill reached the sea surface and
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formed an oil slick.13-15 Given that > 50% of the slick oil residue was transformed into OxHC within days
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to weeks.1, 4 at least 3 × 1010 g of OxHC were produced in the early phase of the DWH oil spill, and likely
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deposited on beaches or nearshore environments. Because the OxHC fraction is very complex,3 as well as
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mainly outside the analytical window of traditional, gas chromatography-based oil spill techniques,16 this
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pool of oil-derived compounds has not been quantified and is not included in oil spill budget estimations
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of the DWH oil spill.10
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The large amount of this uncharacterized OxHC fraction challenges current hydrocarbon-based
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approaches for risk or damage assessment, and poses questions about its fate and potential effects in the
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environment: Are these OxHC hydrophobic and recalcitrant compounds that will not be released into the
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water phase and therefore pose a minimal threat to aquatic organisms? Alternatively, can they partition
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into the water phase and therefore potentially pose a risk to organisms? The fact that the operationally-
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defined OxHC fraction persisted at least six years in oil/sand aggregates (referred to here as “sand
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patties”)4, 5 would suggest that this fraction was not chemically or biologically degraded over this period.
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In contrast, the highly oxygenated nature of OxHC1, 3, 17 would suggest that OxHC compounds can be
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relatively water-soluble and bioavailable, which could make them prone to biodegradation. The factors
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controlling this apparent contradiction between recalcitrance and biodegradability are poorly constrained.
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The goal of this study was to constrain the environmental fate and effect of OxHC better. To this end,
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we chemically characterize this fraction in the Macondo Well oil (MWO) residues as they weathered
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between 2010 and 2017. We based our study on the premise that physico-chemical properties of the
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identified structure can be determined based on the molecular compositions of oil residues, which then
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allows evaluating their environmental fate. We employed a strategy based on three methods for this study:
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First, using analytical methods that are based on bulk level–rather than molecular level–allowed for a
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comprehensive characterization of oil residues. Thin layer chromatography coupled to flame ionization
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detection (TLC-FID) and Fourier-transform infrared spectroscopy (FT-IR) have proven successful to this
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end.1, 5 Second, chemical modification of oil residues made certain polar functional groups GC amenable,
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allowing for the identification and quantification of OxHC components while maintaining the advantages
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of GC-based methods of quantification and of structural identification. Such chemical modification
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reactions—so-called derivatization reactions—are well-known for a multitude of functional groups,18 and
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allow taking advantage of the high chromatographic resolution and the ability to produce quantitative
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results that GC offers. Third, the potential release of OxHC was investigated in dissolution experiments as
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well as estimated based on physico-chemical properties of identified compounds.
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Material and Methods
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Samples, Sample Extraction, and Chemical Modification. Table S1 in the Supporting Information (SI)
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lists the oil samples used in this study. A standard reference material (SRM) No. 2779, which is a
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Macondo Well source oil, was obtained from the National Institute of Standards and Technology (NIST
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SRM2779). A surrogate oil (“BP Surrogate Oil”) that is similar to the Macondo Well source oil was
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obtained from BP. Slick oils, collected from the sea surface in 2010, were collected by skimming vessels
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during the DWH oil spill response and obtained from BP and NOAA (sample S018 and S019) or were
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collected by us as described previously.1 Slick samples were binned into “early slick samples” for
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samples with estimated residence times on the sea surface of 5 days. Oil/sand aggregates (“sand patties”)
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were collected on beaches of the Gulf of Mexico in 2011 and 2012 as described previously.1, 6 Additional
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sand patties were collected on beaches at Fort Morgan, AL and Gulf Shores, AL in 2014, 2016, and 2017
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(Table S1 in SI). The samples were extracted according to literature using dichloromethane:methanol (8:2
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v:v; sand patties) or dichloromethane (slick samples).1 A set of samples was subjected to chemical
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modification reactions described in the Section S-II in the SI (silylation, acetylation, transesterification,
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methoximation, reduction).
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Dissolution Experiments. For short-term dissolution experiments (18 h), water accommodated fractions
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(WAFs) were created with artificial seawater and extracted oil residues as described in the SI. For long-
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term dissolution experiments (up to 312 h), intact sand patties were incubated in artificial seawater on a
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shaker table as described in Section S-I of the SI.
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Analytical Methods. The amount of saturated, aromatic, and oxygenated hydrocarbon fractions in
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extracts was quantified by TLC-FID as described previously.1 Briefly, sample extracts were spotted on
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the base of a silica-gel sintered glass rods and developed sequentially in hexane, toluene and
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dichloromethane:methanol (97:3). The rods were then analyzed on a TLC-FID analyzer, and four distinct
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peaks were observed (saturated, aromatic, OxHC1 and OxHC2 fractions). For this study, we combined the
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two OxHC fractions (i.e., OxHC = OxHC1 + OxHC2). Hopane-normalization of all areas was performed
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following an approach described previously1 by determining the ratio of 17α(H), 21β(H)-hopane (referred
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to as “hopane” throughout this manuscript) relative to the combined saturated and aromatic fractions from
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comprehensive two-dimensional gas chromatography analysis (GC×GC-FID; see next paragraph). This
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normalization was performed since TLC-FID only provides the relative abundance of saturated, aromatic,
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and OxHC fraction, and would not allow detecting changes if, e.g., all fractions were degraded by the
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same amount. In contrast, such a disappearance could be detected by normalizing TLC-FID data to
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hopane. This native petroleum biomarker compound has been found to be degraded to less than 5% over
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four years in samples with hopane concentrations > 0.75 µg g-1 (oil/sand aggregates typically contain >10
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µg hopane per g sample).19 After hopane-normalization, TLC-FID data were corrected for method-
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associated loss of compounds with boiling points < n-C15 according to a previously described procedure
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(see Table S2 in SI).1
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GCxGC-FID analyses were performed according to methods described in the SI. The cumulative
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signal intensity under the GC×GC-FID chromatogram in the range of n-pentadecane (n-C15) through n-
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nonatriacontane (n-C39) in the first dimension and from 1.5 sec to 10 seconds in the 2nd dimension was
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determined in MATLAB using raw chromatograms extracted from the ChromaTOF software (Leco, Saint
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Joseph, MI). To correct for column bleeding and detector offset, a blank chromatogram was subtracted
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(blank injection of dichloromethane). This blank-corrected total GC×GC-FID chromatogram was
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normalized by the height of the hopane peak. We chose n-C15 as a lower limit because compounds with
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lower boiling points are lost during TLC-FID analysis.20 Compounds with higher boiling points than n-
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C39 amounted to approx. 15% in MW source oil (see Table S3 in SI). Therefore, this hopane-
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normalization approach will lead to a conservative estimation (i.e., slight underestimation) of the
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oxygenated fraction.
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GC×GC coupled to time of flight mass spectrometry (GC×GC-TOFMS) was performed using the
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methods described in the SI. Gas chromatography coupled to mass spectrometry (GC-MS) quantification
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(n-alkanes, PAHs, and oxygenated compounds) and analyses of DOC were performed using the method
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described in the SI. FT-IR analyses were performed according to literature.5
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Results and Discussion
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Persistence of OxHC in Weathered MWO Residue. We first investigated how the mass of the OxHC
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fraction in MWO residues changed over time. To this end, we quantified the relative amount of saturated,
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aromatic, and OxHC fractions in oil samples using TLC-FID, and normalized these data to the recalcitrant
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tracer hopane. We previously used a similar approach with samples collected in 2010 and 2011 to
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demonstrate that the OxHC fraction was increasing relative to hopane.1 Here, we now focused on the
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overall budget of oil-derived material and expanded the time series until 2017.
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The results of our TLC-FID analysis showed 79±2% depletion of the saturated and aromatic fractions
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(sand patty samples collected between 2011 and 2017 relative to MWO; Figure 1A). This extensive
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depletion, which was clearly visible in GC×GC (Figure 1B), represents the result of the various physical
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and chemical oil weathering processes. However, our TLC-FID results furthermore showed that this
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depletion of saturated and aromatic compounds was partly compensated by a concurrent increase in the
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OxHC fraction, leading to an overall oil depletion of only 42±12% (2011-2017 sand patty samples
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compared to MWO: Figure 1A).
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Three conclusions can be made from our results. First, the formation of OxHC occurred mainly in
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slick samples in the initial stage of the oil spill, when the oil was floating on the water surface. In slick
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samples, the OxHC fraction increased by more than three-fold, while the combined saturated and
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aromatic fractions were depleted 54±13%. The overall mass depletion from MWO to slick samples was
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34±6%. This number is close to the estimated fraction that evaporated from slick oil (approx. 30%),13
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suggesting that evaporation was the main mass removal process for surfaced oil. Our finding of an early
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formation of OxHC is in line with our recent report that partial photo-oxidation of oil on the sea surface
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led to extensive formation of OxHC within ten days.4 This photo-oxidation just transformed and did not
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remove oil hydrocarbons. The formation of OxHC at the early stage of oil weathering has also been
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suggested based on oil slick samples collected during the DWH oil spill17 or based on photo-oxidation
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experiments.21, 22
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Second, the mass of the saturated, aromatic, and operationally-defined OxHC fractions remained
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relatively stable after the oil slick encountered the beach and sank as oil/sand aggregates. All sand patty
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samples collected between 2011 and 2017 had a strikingly similar amount of OxHC based on TLC/FID
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(relative standard deviation of 12%; Table S2). This consistency suggests that after 2011, (i) chemical
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weathering did not lead to a noticeable move of saturated or aromatic compounds into the operationally-
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defined OxHC fraction, and (ii) no net formation or degradation of the OxHC fraction occurred. Note that
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we previously reported a significant increase of OxHC fraction between 2012 and 2014 (p n-C22) due to
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biodegradation. Even if the disappearance of n-alkanes and PAHs looks extensive on a GC chromatogram
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(Figure 1B), MWO only contains 87% of
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the investigated compounds are still larger than 5 (Figure 4B). These relatively high KOW values suggest
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that the long carbon backbone (≥ C15) is making OxHC compounds these compounds still hydrophobic
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enough, so that partitioning from the oil phase into the water phase only leads to a minimal mass
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depletion in sand patties. The KOW values are also in the range of hydrophobic PAHs such as pyrene and
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chrysene (log(KOW) = 4.9 and 5.8, respectively); these PAHs remained at a constant level in sand patties
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between 2011 and 2014.28 1,000,000
(A)
O4
WS_i / WS_n-alk 1/Kow_i / 1/Kow_n-alk O2
100,000 10,000
O5
O3
1,000
O1
100 10
10
(OH)(OOH)2
25
(=O)(OOH)2
24
(OOH)2
23
(OH)(=O)OOH
(=O)OOH
OOH
(OH)2
(=O)2
(OH)2OOH
12
(=O)OOH (OH)OOH (=O)2OOH (OH)2OOH (OH)(=O)OOH (OOH)2 (=O)(OOH)2
(=O)2OOH
nAlk =O OH (=O)2 (OH)2 OOH (=O)OOH
(OH)OOH
16 14
log(Kow)
=O
nAlk
(B)
OH
O0
1
(=O)(OOH)2 (OH)(OOH)2
8 6 4 2 15
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16
17
18
19
20
21
22
26
27
28
29
30
carbon number
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Figure 4. Estimated water solubility (WS) and octanol-water partitioning (KOW) of homologous
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series of C12 to C30 n-alkanes and corresponding oxygenated compounds containing one or
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multiple hydroxyl (OH), carbonyl (=O), and carboxyl groups (OOH). (A) WS and 1/KOW of
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compounds containing one to five oxygens (O1 to O5) increased by one to five orders of
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magnitude relative to the values of n-alkane. Given are the averages and standard deviations
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(error bars) of all 19 compounds in the considered homologous series divided by the values of
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their corresponding n-alkane. (B) The estimated log(KOW) values of homologous series of n-
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alkanes and oxygenated analogues show that >87% of the considered compounds have KOW
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values > 10,000.
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We want to stress that the presented estimates of physio-chemical properties should be viewed as very
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rough and preliminary. WSKOWWIN is useful buy simple screening tool that is based on a fragment
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contribution method to estimate KOW and on linear correlation between log(WS) and log(KOW) that
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includes functional-group specific correction terms to estimate WS.46 Before detailed fate assessments of
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OxHC compounds can be made, validations with experimental data or with more advanced computational
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methods are needed (e.g., quantum chemical based methods47).
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Dissolution Experiment to Determine Partitioning of OxHC Compounds into Water. Because of the
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high oxygen content of the OxHC fraction in weathered MWO residue (1.8 – 11.1% in surface slicks, 4.8
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– 11.9% in sand patty samples1), we expect that oil weathering products show increased water solubility
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as compared to their precursors. To test this assumption, we performed two types of dissolution
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experiments. First, we measured the dissolved organic carbon (DOC) content in WAFs that were prepared
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with extracted oil residues (1 g oil per L of seawater). We used a fresh crude oil (BP surrogate oil), a
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surface slick oil (“Juniper oil” sample S019), and a sand patty oil (composite sample B131 collected in
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2016). To have a minimal DOC background (0.5 mg L-1) we used artificial seawater. We obtained DOC
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concentrations of 11.4±1.2 mg L-1 for the crude oil WAF, 2.2±0.4 mg L-1 for the slick oil WAF, and
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7.4±0.6 mg L-1 for the sand patty WAF (Figure 3C; n=3 for each WAF). These DOC results suggest that
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even though the slick oil and sand patty samples were depleted in water-soluble PAHs (see Figure 3A),
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water-soluble compounds—presumably OxHC compounds—are still present in weathered oil.
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Furthermore, the observed increase in DOC from the slick oil to the sand patty WAF might be explained
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with increasing oxygenation in sand patties, leading to more water-soluble compounds. Although we
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filter-sterilized the artificial seawater, we did not autoclave the sand patties. Therefore, microbial growth
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is principally possible. However, the fact that the final DOC concentration remains stable after 100 h at a
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similar level two different sand patty loadings does not suggest biological activity. Furthermore, the 24-h
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WAF incubations produced DOC concentrations in the same order of magnitude as the 300 h incubation,
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which also suggests that equilibration rather than microbial growth kinetics is occurring.
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To identify these water-soluble oil-derived compounds contributing to the DOC, we extracted these
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WAFs and quantified straight-chain carboxylic acids and alcohols, along with PAHs and alkanes using
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GC-MS (Figure 3C). In the crude oil WAF, the highly soluble BTEX compounds (benzene, toluene,
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ethylbenzene, and xylenes) were dominant (1.7 mg L-1), followed by PAHs (252 µg L-1) and n-alkanoic
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acids (50 µg L-1). For the slick oil WAF, PAH concentrations were approx. 100 times lower than in the
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crude oil WAF, while the n-alkanoic acids were only a factor of two lower than in the crude oil WAF.
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For the sand patty WAF, the PAH concentrations were below the detection limit (
