Impact of Photooxidation and Biodegradation on the Fate of Oil Spilled

Article

Impact of Photooxidation and Biodegradation on the Fate of Oil Spilled During the Deepwater Horizon Incident: Advanced Stages of Weathering Brian Harriman, Phoebe Zito, David C Podgorski, Matthew A Tarr, and Joseph M Suflita Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017 Downloaded from http://pubs.acs.org on June 3, 2017

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Environmental Science & Technology

Title:

Impact of Photooxidation and Biodegradation on the Fate of Oil Spilled During the Deepwater Horizon Incident: Advanced Stages of Weathering

Authors:

Brian H. Harrimana,b, Phoebe Zitoc, David C. Podgorskic,d, Matthew A. Tarre and Joseph M. Suflitaa,b*

Affiliations:

a

Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019 b Institute for Energy and the Environment, University of Oklahoma, Norman, OK 73019 c National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310-3706 d Department of Earth, Ocean and Atmospheric Science, Florida State University, Tallahassee, FL 32306 e Department of Chemistry, University of New Orleans, New Orleans, LA 70148

Corresponding author:

*University of Oklahoma, Department of Microbiology and Plant Biology, 770 Van Vleet Oval, Norman, Oklahoma, 73019, USA; Phone: +1(405) 325-3771; Email: [email protected].

Coauthor emails:

BHH - [email protected], DCP [email protected], PZ - [email protected], MAT - [email protected], JMS – [email protected]

Key words: Deepwater Horizon, oil spill, sand patty, photooxidation, biodegradation

Abstract While the biogeochemical forces influencing the weathering of spilled oil have been

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investigated for decades, the environmental fate and effects of ‘oxyhydrocarbons’ in sand patties

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deposited on beaches are not well known. We collected sand patties deposited in the swash zone

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on Gulf of Mexico beaches following the Deepwater Horizon oil spill. When sand patties were

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exposed to simulated sunlight, a larger concentration of dissolved organic carbon was leached

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into seawater than the corresponding dark controls. This result was consistent with the general

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ease of movement of seawater through the sand patties as shown with a 35SO42- radiotracer. 1 ACS Paragon Plus Environment


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Ultrahigh-resolution mass spectrometry, as well as optical measurements revealed that the

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chemical composition of dissolved organic matter (DOM) leached from the sand patties under

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dark and irradiated conditions were substantially different, but neither had a significant

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inhibitory influence on the endogenous rate of aerobic or anaerobic microbial respiratory

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activity. Rather, the dissolved organic photooxidation products stimulated significantly more

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microbial O2 consumption (113 ± 4 µM) than either the dark (78 ± 2 µM) controls or the

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endogenous (38 µM ± 4) forms of DOM. The changes in the DOM quality and quantity were

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consistent with biodegradation as an explanation for the differences. These results confirm that

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sand patties undergo a gradual dissolution of DOM in both the dark and in the light, but

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photooxidation accelerates the production of water-soluble polar organic compounds that are

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relatively more amenable to aerobic biodegradation. As such, these processes represent

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previously unrecognized advanced weathering stages that are important in the ultimate

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transformation of spilled crude oil.

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Introduction On April 20, 2010, the blowout of the Deepwater Horizon (DWH) drilling platform

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resulted in the deaths of 11 workers and injury to another 17 individuals. Over the next 87 d, the

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wellhead spewed 4.2-4.9 million barrels of light, sweet crude oil from deep below the surface of

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the Gulf of Mexico (GoM), making it the largest accidental spill in history.1–3

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Numerous studies examined the fate and impact of oil within the plume4–8, on the surface

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of the Gulf 5,9,10, buried in sediments (either directly or as marine snow) 6,10,11, in marshes 12-15,

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and on beaches.10,13,15–21 The environmental fate of the oil was influenced by many weathering

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processes22–27 that resulted in a chemical fingerprint that differed substantively from the crude oil

2 ACS Paragon Plus Environment

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released at the wellhead. Specifically, the transformation of the oil resulted in the formation of a

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group of partially oxidized organic compounds collectively termed ‘oxyhydrocarbons.’22 The

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formation of the oxyhydrocarbons was coincident with a decrease in the fraction of n-alkanes

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and aromatic compounds in the residue. The oxyhydrocarbons are most obviously found in sand

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patties that wash up in the swash zone on northern GoM beaches. Sand patties are thought to be

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highly weathered oil deposits that combine with sediment particulates and migrate along the

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seafloor after a spill and eventually reach coastal beaches.16,19,22 The oxyhydrocarbons constitute

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over 50% of the organic compounds in sand patties and of the residuum on ‘oiled’ rocks.22,23

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Sand patties are likely exposed to both anaerobic conditions during their migration along

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the seafloor and aerobic settings once they reach the beachfront. It is possible that the patties

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themselves may develop an O2 gradient within pores where diffusion of this potential electron

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acceptor may be restricted. Once on beaches, sand patties are exposed to direct sunlight during

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the day and are constantly wetted from the action of the surf. Sunlight results in oil weathering

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by photooxidation that likely further transforms the constituents in sand patties.26 Continuous

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exposure to weathering processes results in the loss of saturated and aromatic compounds in sand

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patties and the formation of oxygenated resins and polar components that are difficult to resolve

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by standard chromatographic methodologies.22,25,28 While it is known that irradiation and

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biodegradation transform crude oil components, the long-term fate of many polar molecules

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resulting from the aforementioned combination of oil weathering processes is far from clear.

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Hydrocarbons are known to inhibit and can be potentially toxic to individual

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microorganisms.29 However, we sought to assess the impact of hydrocarbon-laden sand patties

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and associated organic matter on the overall functioning of marine microbial communities. To

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this end, the endogenous rate of electron acceptor utilization was used as an integrated measure



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of the baseline functioning of the active microflora in marine samples. The overall rate of both

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O2 consumption and sulfide formation in the presence and absence of sand patty carbon was

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evaluated. Additionally, we examined the combined effects of photooxidation and

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biodegradation on the environmental fate of the oil-derived components. We found that the

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DOM leached from sand patties is a complex mixture of constituents that is more oxidized after

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exposure to sunlight than that released in the dark. The leached DOM is amenable to aerobic

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biodegradation under both conditions, but the photosolubilzed components are relatively more

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easily metabolized by the resident microflora. Collectively, these processes represent the near

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final stages in the mineralization of the contaminating oil.

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Materials and Methods

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Sample Collection, Biomarker Analysis and Anion Analysis

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Sand patties were collected from the swash zone of beaches in Gulf Shores and Fort Morgan, Alabama (30.24° N x 87.74° W and 30.22° N x 88.01° W, respectively) on January 20,

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2014 and March 18, 2014. The initial sand patty collection was conducted with Dr. Christoph

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Aeppli, who subsequently analyzed a subset of the specimens for the presence of conserved

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hopanoid, sterane, and diasterane biomarkers. Based on this analysis, the sand patties were

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clearly derived from the oil spilt during the Deepwater Horizon incident.23 Sand patties were

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placed in clean sealed glass containers, cooled with ice packs, and shipped overnight to the

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laboratory where they were stored at -800C until use. Sediment from just below the water

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surface on the same beaches was collected using a wide mouth neoprene jar to scoop material

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from a depth of 5-10 cm. The jars were nearly filled, topped off with seawater to eliminate

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headspace, sealed, and also shipped with ice packs to the laboratory, where they were stored at


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40C prior to use. Anion analysis (Cl-, NO3- and SO4-2) of the aqueous samples was performed

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with a Dionex Series 3000 Ion Chromatography System as previously described (Thermo Fisher,

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Waltham, MA).30 The sulfate analysis was used for assessing the rates of sulfate reduction using

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a radiotracer method (below).

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Impact of Sand Patties on GoM Microbial Communities

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The rate of O2 consumption during aerobic heterotrophic respiration was measured with a

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10-channel Micro-Oxymax Respirometer outfitted with an electrochemical O2 sensor (Columbus

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Instruments, Columbus, OH). Incubations were conducted at room temperature in 250 mL

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bottles containing 10 g of sediment inoculum and 10 mL of filter-sterilized seawater (0.3 µm,

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GF-75, Advantec, Dublin, CA). The headspace was automatically purged every 4 h and analyzed

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for O2. The incubations were conducted in triplicate and results averaged for each experimental

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condition.

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To assess the impact of sand patty organic matter on anaerobic respiration, we employed SO4-2 as a radiotracer and measured its conversion to 35S-sulfide using a previously described

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35

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procedure.30 Briefly, in 120 mL serum bottles under a headspace of N2:CO2 (80:20), 10 mL of

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seawater bubbled with N2 was combined with varying amounts of sediment and macerated sand

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patty material to total 10 g. Then, 100 µL of a 5 µCi 35SO4-2 stock solution was added to each of

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the bottles to reach an initial dose of 50 nCi tracer. The bottles were then incubated in the dark

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without agitation for 7 d at room temperature. After incubation, 4 mL Cr(II)Cl and 4 mL HCl

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were added to volatilize biogenically produced H235S which was subsequently trapped in a zinc

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acetate solution. Radioactivity was measured with a scintillation counter (Triathler LSC, Hidex,

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Turku, Finland) by removing a 1 mL portion of the trap and adding it to 5 mL of Ultima Gold

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Liquid Scintillation Cocktail (Sigma Aldrich, St Louis, MO). The rates of sulfate reduction in



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sand patty-amended incubations were compared to positive control incubations that received

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lactate (20 mM), a substrate-unamended control with only endogenous substrates and an

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autoclaved negative control (without sand patty amendment) using an ANOVA with Bonferroni

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correction.

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Seawater Penetration in Sand Patties Since sand patties contain a variety of hydrophobic compounds,16,19,23,25 these structures

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might resist the penetration of seawater to their interiors. To test this prospect, several sand

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patties were placed on the surface of sediment that was overlain with seawater in a standard petri

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dish. The sand patties were irregular in shape, but measured approximately 35 x 45 mm, and

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tapered from 5 mm at their narrowest point to 15 mm at the thickest. Approximately 5 µCi of

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35

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temperature under a headspace of N2:CO2 (80:20). The sand patties were recovered from the

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petri dish and dissected into approximately 1.5 mm segments with a clean razor blade for each

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cut. The resulting sections were then laid flat and the distribution of the radioactivity was

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directly imaged using a Packard Instant Imager (Packard Instruments, Meriden, CT). Total β-

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emissions from 35S-SO4 and heat maps were generated using the associated imaging software.

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Generation of Sand Patty-Derived DOM

SO4-2 was added to the seawater and the mixture was gently agitated at 45 rpm for 7 d at room

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Approximately 12-15 g of sand patty material was crumbled into 100 mL of filter

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sterilized seawater and then irradiated in borosilicate glass jacketed beakers with quartz lids for 3

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h or 12 h (equivalent to 0.75 or 3 days of average northern GoM sunlight, respectively) using a

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solar simulator (Atlas CPS+, Mount Prospect, IL) as previously described.26 After each

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irradiation increment, seawater with associated DOM was then removed, the beaker refilled with

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fresh sterile seawater, and irradiated for another time increment. The irradiation and water


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replacement was repeated for up to 84 h. Sand patties incubated in sterile seawater and kept in

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the dark served as controls. Samples were stored in pre-cleaned glass containers at -80 °C until

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use.

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Biodegradation of Sand Patty DOM

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The biodegradation of sand patty-derived and endogenous DOM was compared using

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GoM sediment (10 g) as an inoculum. Evidence for biodegradation included an assessment of

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the rate of electron acceptor utilization using the aforementioned respirometer. The endogenous

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control incubation received the inoculum and 10 mL of sterile seawater. In other replicates, the

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seawater was replaced with the same volume of sand patty-derived DOM from either the

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irradiation procedure or dark control as described above. The O2 utilization rates in the

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incubations were compared to each other and to a sterile negative control containing seawater

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and sediment that was autoclaved (20 min). All data are reported as the average of triplicate

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room temperature incubations. In addition to O2 uptake, the change in the quality of the DOM as

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a result of biodegradation was also assessed by sampling at the beginning (T0) and end (TFinal) of

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the incubation using the procedures described below.

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Dissolved Organic Carbon and Optical Measurements

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Sample DOM concentration was determined by the high temperature catalytic oxidation

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using a Shimadzu TOC-LCPH analyzer (Shimadzu Corp., Japan).32 Each sample was acidified to

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pH 2 and sparged for 5 min at 75 mL min-1 with either ultra-pure air or ultra-pure O2 to remove

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inorganic carbon prior to the measurement. The mean value of three to five 25 µL replicate

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samples is reported. The coefficient of variance (precision) was < 2% for replicate

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determinations. Chromophoric dissolved organic matter (CDOM) leached from the sand patties



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after irradiation and dark control treatments were initially characterized using a scanning

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spectrophotometer (from 200 – 600 nm), but routinely monitored at 254 nm.

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The pH of each sample was adjusted to 8 for optical measurements.33-35 Absorbance and

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fluorescence spectra were collected with an Aqualog® fluorometer (Horiba Scientific, Kyoto,

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Japan) in a 10 mm quartz cell at 20 °C. Sealed water cell blanks were analyzed initially to test

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instrument stability using the Raman peak of water at excitation 350 nm and emission 340-420

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nm. Excitation and absorbance scans were collected from 240 to 800 nm at 5 nm increments

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with an integration time of 0.5 s. Emission spectra were collected every 5 nm from 245 to 800

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nm with a charge-coupled device at 1.64 nm resolution. All samples were diluted to an

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absorbance of 0.1 at 254 nm with MilliQ water to reduce inner-filter effects.36 Excitation-

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emission matrix fluorescence intensities were Milli-Q water blank-subtracted, corrected for

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Rayleigh and Raman scattering and instrument bias in excitation and emission prior to correction

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for any inner filter effects.37 Fluorescence intensity was normalized to quinine sulfate units as

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previously described.38

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Mass Spectrometry

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Dissolved organic matter was obtained by the solid-phase extraction described by

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Dittmar et al.39 Briefly, each sample was passed through a precombusted 0.27 µm glass-fiber

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filter and acidified to pH 2 prior to loading onto a Bond Elut PPL (Agilent Technologies)

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stationary phase cartridge. Each sample was then desalted with pH 2 MilliQ water and eluted

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with methanol at a final concentration of 100 µgC mL-1. The extracts were stored in the dark at

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4 °C in pre-combusted glass vials until analysis by negative-ion electrospray ionization coupled

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with a custom-built Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS)

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equipped with a 9.4 tesla superconducting magnet (National High Magnetic Field Laboratory


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(NHMFL), Florida State University, Tallahassee).40,41 Each mass spectrum was internally

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calibrated with a “walking” calibration equation followed by molecular formula assignment with

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internally developed software provided by the NHMFL.42 The standard deviation for the number

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of assigned formulas of mass spectra from triplicate biodegradation experiments was ±7%.

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Results

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Toxicity Screening

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Toxicity evaluations often employ a single organism to assess the potential impact of a

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toxicant.43 Such evaluations are subject to many interpretational constraints when extrapolating

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the information to other organisms or to larger community effects. It is arguably more

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environmentally relevant to examine the response to perturbations by monitoring overall

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community respiration. In marine habitats, the two most quantitatively important electron

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acceptors available to the requisite microbial communities are O2 and sulfate.44 Aerobic

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microbial community respiration was monitored as the endogenous rate of O2 utilization, while

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anaerobic respiration was measured as a rate of sulfide formation (Table 1).

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Sulfate reduction assays are shown in Table 1. The lactate-amended positive control

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reduced more sulfate than the endogenous control, confirming that the resident microflora

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included anaerobes that could respond to the introduction of a labile carbon source. Community

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respiration increased with sand patty addition and the most statistically significant amount of

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reduced sulfide evident in incubations receiving the largest amendment. Thus, sand patties are

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not inherently detrimental to the native sulfate-reducing microflora. Rather, the increased rate of

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sulfate reduction suggests that sediment microbes are capable of utilizing some sand patty

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components as electron donors.



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Similarly, O2 respiration was used as an indicator of the impact of sand patties on aerobic

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microbial communities (Table 1). If sand patties were inhibitory, a decrease in the O2 respiration

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rate would be evident upon sand patty addition. However, when amended with 1-2 g of sand

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patty material, the O2 respiration rate remained unaltered. Like the anaerobic incubations, larger

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sand patty amendments (5 g) significantly stimulated aerobic microbial community respiration.

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Thus, sand patties did not impact the GoM microflora negatively, but some associated

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components likely stimulated microbial metabolism.

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Seawater Penetration in Sand Patties

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Sand patties were incubated for 7 d in seawater containing 35SO4-2 and subsequently

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dissected and analyzed. The autoradiographic images show that the water-soluble tracer

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penetrated to the interior of the structures (Figure S1). The amount of radioactivity in various

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subsections of the sand patty was roughly equivalent. However, the distribution of the label was

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more even in one distal end of the sand patty, presumably reflecting variations in thickness.

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Ignoring the surface radioactivity, the tracer that reached to the interior of the sand patty was

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largely normally distributed in all subsections (only representative data shown in Figure S1).

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However, several subsections had local hot spots of accumulated radioactivity for unknown

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reasons. Collectively, these findings argue that seawater was readily able to infiltrate sand

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patties and that the chemical nature of these structures does not represent a substantive barrier to

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water penetration.

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Photogeneration of Sand Patty-Derived DOM

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Sand patty material in sterile seawater was exposed to simulated sunlight to examine the

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impact of irradiation on the weathering of these residues. The absorption characteristics of

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CDOM photo-solubilized by this procedure were measured from 200 – 600 nm and compared


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with dark controls and the seawater alone. Routine monitoring of A254 revealed an increase in

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CDOM leached from sand patties in both the light and dark (Figure S2). The increase in A254 is

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noteworthy since this value correlates with DOC concentrations.33 However, more CDOM was

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produced upon irradiation, particularly over the first 12 h. After the initial 12 h of irradiation, the

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release of CDOM decreased to a slower, but linear rate. The irradiated and dark control samples

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showed similar behavior, except the rate of increase in A254 was faster for the irradiated samples

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in both initial and subsequent time periods. The CDOM continued to leach from even the dark

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samples relative to seawater throughout the 84 h experiment. These findings indicate that sand

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patties represent a source of CDOM to the GoM and that more organic matter is released upon

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exposure to sunlight.

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Biodegradation of Sand Patty-Derived DOM

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The sediment GoM inoculum was used to determine if the indigenous microflora were

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capable of metabolizing DOM leached from the sand patties (Fig. 1). The aerobic toxicity

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screening revealed that at least some components in the whole sand patty were amenable to

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biodegradation (Table 1). A similar rate of O2 consumption was observed when the DOM from

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the dark controls were similarly assayed (Fig. 1). We presume that the same or similar suite of

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seawater-soluble DOM components leached from the whole sand patties and served as electron

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donors for the resident aerobic microflora. Since seawater could readily penetrate the sand

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patties (Fig. S1), the DOM components in both the dark controls and whole sand patty treatments

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were likely comparable.

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When photo-solubilized DOM from sand patties was used as an amendment, the rate of

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O2 consumption increased relative to the dark DOM treatment or the endogenous respiration

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level (Fig. 1). The endogenous microflora respired 38 ± 4 µM O2 with natural organic matter



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serving as electron donors, while 64 ± 2 and 67 ± 2 µM O2 were utilized when the same

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inoculum used whole sand patty organic matter or the DOM from dark controls, respectively.

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However, when photo-solubilized DOM from sand patties was similarly assayed, a total of 114 ±

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4 µM O2 was utilized. These findings attest to the increased susceptibility of the irradiated DOC

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to aerobic biodegradation.

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Evaluation of Photooxidized and Biologically Transformed DOM

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Optical Properties: The transformations of DOM leached from sand patties under

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irradiated and dark conditions and the impact of biodegradation were analyzed by FT-ICR MS as

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well as absorbance and fluorescence measurements.40,41,43 The latter procedures are typically

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applied to soil, plant or algal organic matter, but were used here to interpret changes in sand

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patty-derived DOM following phototransformation and biodegradation in a comparable manner.

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The DOM concentration, slope ratio, humification index, and freshness index were

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determined for each sample at the beginning (T0) and end (TFinal) of the biodegradation

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experiment (Figure 2). The amount of sand patty DOC after exposure to simulated sunlight

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increased relative to the dark controls; an indication that photooxidation enhanced the formation

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of water-soluble organic components from the weathered oil residuum as previously noted.26 The

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fact that the DOC concentration was significantly reduced at the end of the incubation for dark

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and irradiated samples (67% and 56%, respectively) suggests that the leached material was also

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amenable to biodegradation (Fig. 2A).

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The amount of DOC in the endogenous incubation (no sand patty material) at either T0 or

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TFinal was only about a third and a fifth of that initially formed in the dark and irradiated samples,

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respectively. The lack of a substantive change at such low DOC concentrations suggests that the

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determination is likely too insensitive an indicator when losses due to aerobic respiration are


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possibly offset by increases in microbial growth (Fig. 2A). The DOC concentration in the sterile

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incubation increased over the endogenous control following the intense heating during

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sterilization. The release of DOC from marine sediments following heating is not without

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precedence.47 However, such extreme heating has no substantive environmental relevance in

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coastal marine sediments and is included here for comparative purposes.

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The composition of CDOM at the beginning and end of the biodegradation experiment

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were further characterized by the changes in spectral properties as previously detailed.45 The

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spectral slope was calculated by applying a nonlinear fit of an exponential function to an

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absorbance spectrum in the range of 275-295 nm (Fig. 2B) and 350-400 nm (Fig. 2C) and the

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spectral slope ratio (SR) was calculated as the ratio of S275-295 to S350-400 (Fig 2D).48 The three

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parameters were shown to correlate with the molecular weight, aromaticity and source of DOM

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and relative changes in these values can be attributed to photochemical and microbial

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degradation processes.48 Basically, the increase in the slope ratio from 1.4 to 1.6 in the dark and

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irradiated samples, respectively indicates that more lower molecular weight constituents were

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photosolubilized prior to the biodegradation experiment (Fig. 2D). Figure 2 also shows that

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mean S275-295 and SR values increased for the DOM produced from sand patty-amended

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incubations relative to the endogenous or sterile controls, particularly upon exposure to sunlight.

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Relatively steep S275-295 and high SR are related to a decrease in DOM molecular weight and

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aromaticity.48-51 The compounds leached from the sand patties were utilized by the resident

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microflora during the course of the biodegradation experiment as evidenced by an increase in

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S350-400 and decrease in SR (Fig. 2C and 2D). The significant decrease in SR after biodegradation

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was a result of a corresponding increase in S350-400 while S275-295 remained constant. This



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decrease in SR was likely a result of microbial metabolism of labile components and/or selective

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preservation of relatively large aromatic compounds.

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Microbial transformation of low molecular weight organic compounds into relatively

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condensed, high molecular weight macromolecules is often assessed through the humification

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index. Humification results in a red shift in emission spectra that corresponds to a decrease in

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the H to C ratio. The humification index is calculated by dividing the area under Em. 435-480 by

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the peak area 300-435 nm + 435-480 nm, with excitation at 254 nm.36 The humification index of

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the DOM, was nearly equivalent at the beginning of the biodegradation experiment in both the

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endogenous and sterile incubations (Fig 2E). The DOM humification index decreased from 1.5

323

in dark samples to 1.0 in irradiated incubations. This result suggests that the DOM

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photosolubilized from sand patties had a higher H to C ratio relative to material leached in the

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dark and was presumably more bioavailable.

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humification index rose in all incubations except the sterile control. Presumably, the more labile

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DOM constituents were preferentially metabolized by the GoM microflora resulting in a

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relatively higher humification index in each case. The increase in the TFinal humification index

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for the sand patty-derived DOM (83%) leached in the dark was higher than the comparable

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measure for DOM leached in the presence of sunlight (62%). This difference may reflect an

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increase in the quantity of relatively aliphatic, small, bioavailable DOM formed after the sand

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patties were exposed to sunlight. An increase in the pool of labile DOM may result in a slower

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rate of change in the humification index values relative to a small pool that was rapidly depleted

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by microbes. The DOM at TFinal in the endogenous incubations had a higher humification index

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than either of the sand patty incubations. However, the humification index in the sterile

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incubation decreased with time, probably reflecting an unknown abiotic change in DOM quality.

At the end of the biodegradation experiment, the


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Freshness index (β:α) describes the ratio of “fresh-like” (aliphatic) to “humic-like”

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(aromatic) DOM.48 The value is obtained from the emission intensity at 380 nm divided by the

339

Emmax between 420 and 435 nm, with excitation at 310 nm.52 Relatively high β:α values are

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indicative of labile, bioavailable DOM.45 Microbial utilization of the labile DOM pool results in

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a decrease in β:α as the material is converted to more persistent DOM. Thus, the increase from

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1.1 in dark samples to 1.4 in irradiated samples indicates an increase in smaller, less conjugated

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compounds due to photooxidation prior to microbial treatment (Fig. 2F). Similarly, the freshness

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index of the endogenous DOM at the beginning of the experiment (0.9) was lowest (more

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recalcitrant) relative to either the dark or irradiated samples; another indication that the DOM

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produced in sand patty-amended incubations was chemically distinct. At the end of the

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biodegradation experiment (TFinal; Fig 2F), an increasing trend in the DOM freshness index is

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apparent when comparing the endogenous, dark and irradiated samples. However in each case,

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the index decreased relative to the T0 determinations. This result suggests that the conjugated

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DOM is at least partially amenable to microbial decay, resulting in the residual pool of organic

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matter increasing in relative recalcitrance.

352

Mass Spectrometry: Van Krevelen plots of the mass spectral data (Figure S3) show a

353

vast array of molecular formulas representing DOM with a diverse range of oxygen to carbon

354

(O/C) and hydrogen to carbon (H/C) ratios leached from the sand patties under both dark and

355

irradiated conditions. The van Krevelen plots of DOM leached in the dark represent 17,737 and

356

16,582 molecular formulas before and after the biodegradation experiment, respectively (Fig S3).

357

The comparable numbers of molecular formulas for the irradiated samples were 17,280 and

358

14,795, respectively. The overlap in van Krevelen compositional space does not allow for ready

359

comparison of the qualitative (or quantitative) nature of the leached DOM before and after



360

biodegradation. More revealing are van Krevelen difference plots that remove molecular

361

formulas that were common to and persisted over the course of the biodegradation experiment,

362

as common molecular formulas account for 88% of the DOM leached in the dark, and 86% of

363

the DOM leached in the light (Figure 3). Thus, Fig. 3A and 3C emphasize the 2,627 and 3,560

364

molecular formulas altered during the biodegradation experiment using the DOM leached to the

365

seawater under dark and irradiated conditions, respectively. Similarly, Fig. 3B and 3D highlight

366

the 1,433 and 1,039 chemical transformation products that were newly formed during the course

367

of the biodegradation experiment.

368

Prior to biodegradation, the DOM in the irradiated samples had more molecular formulas

369

and more diverse chemical constituents that exhibited a wider range of both H/C and O/C ratios

370

(Figs 3A and 3C). Molecular formulas with low H/C tend to be removed through photochemical

371

processes, while microbial transformation activities remove features with high H/C.46 In both

372

cases, the general trend is for the transformation products to be shifted toward the center of the

373

plot and relatively high O/C values.

374

However, there is only a 24% degree of similarity between Figure 3A and 3C. Thus, the

375

DOM leached from the sand patty into seawater in the dark and the light is fundamentally

376

different in molecular composition. The greater tendency toward formulas with higher O/C and

377

lower H/C ratios in the irradiated samples indicates that photooxidation of aromatic compounds

378

in the sand patties provides a mechanism for solubilization of the material into seawater. This

379

observation is in agreement with previous reports.26

380

The relative differences in the DOM formed in both the dark and irradiated samples are

381

also evident at the end of the biodegradation experiment (Fig. 3). A greater number and

382

diversity of molecular formulas were formed in incubations receiving dark DOM (Fig 3B&D).


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383

These metabolic features exhibited a wide range in both the H/C and O/C ratios, but a greater

384

tendency for the production of less oxidized and more saturated constituents. In contrast, the

385

constituents formed in the experiment amended with irradiated DOM were less diverse and

386

relatively more oxidized. Presumably the fewer and more oxidized constituents reflect that fact

387

that more DOM was in a relatively more advanced state of decomposition and/or completely

388

mineralized. These findings are consistent with the O2 respiration data and further suggest that

389

the irradiated DOM was more amenable to biodegradation and complete mineralization.

390 391 392

Discussion Sand patties deposited on northern GoM beaches have been reported as a residual form of

393

the Deepwater Horizon oil.16,19,22,23 Aeppli and colleagues noted the apparent recalcitrance of

394

sand patties and characterized their chemical composition.22 They reported that >50% of the

395

mass in these structures is not hydrocarbon at all, but oxygenated hydrocarbon-derived

396

molecules produced through a combination of weathering processes. We investigated the

397

environmental fate and impact of these residual oil structures and explored the susceptibility of

398

sand patties to the most likely forms of advanced decomposition – photoxidation and

399

biodegradation.

400

Even though we retrieved sand patty samples from beaches, previous work demonstrated

401

that such residual oil components could be buried in sediments where anaerobic conditions often

402

prevail.53-56 The potential impact of sand patties on the resident microflora was assessed under

403

the predominant electron accepting conditions in marine coastal areas. Whole sand patty

404

amendments did not have a negative impact on the endogenous rate of either aerobic or

405

anaerobic metabolism (Table 1). In fact, with increasing amendment, whole sand patties



406

stimulated respiratory processes, confirming that at least some of the chemical constituents

407

represented suitable electron donors for the indigenous microflora. Such findings have two

408

important implications. First, the subsequent biodegradation experiments were unlikely to be

409

predisposed to failure due to the inhibitory nature of the constituent chemicals in sand patties.

410

Secondly, given the lack of impact on the rate of microbial community respiration, toxicological

411

concerns associated with residual oil deposition should probably be targeted at other trophic

412

levels.

413

The presumed hydrophobic nature of sand patties was also evaluated with a seawater-

414

soluble radiotracer. We found that 35SO4-2 readily penetrated into the interior of the sand patties.

415

The penetration of seawater through sand patties has important implications for the

416

transformation of the constituent chemicals. Seawater is rich with nutrients, microbes and a

417

variety of potential electron acceptors and donors that may be carried to the interior of sand

418

patties.44 Conversely, metabolic end products and partially transformed organic molecules can

419

readily be leached from the sand patty interiors. Thus, sand patties likely represent a suitable

420

habitat for the enrichment and proliferation of microorganisms with the ability to metabolize the

421

oil-derived organic matter. Moreover, the relocation of potentially photosolubilized organic

422

material or microbial transformation products from the interior of sand patties to the surrounding

423

environment would likely be greatly facilitated by the penetration of seawater through these

424

structures.

425

Irradiated and dark sand patty material that partitioned to seawater showed increased

426

absorbance between 250-280 nm – the adsorption spectrum of aromatic ring structures - relative

427

to the endogenous organic matter (Figure S4), indicating that the resulting DOM is likely oil-

428

derived. Similarly, the quantity of DOM in both the irradiated and dark samples exceeded the


Page 18 of 34

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429

endogenous DOM levels (Fig 2A). The general absorbance features in this range of wavelengths

430

of the sand patty-derived DOM were reminiscent of other reports of DOM emanating from the

431

weathering of the Deepwater Horizon oil.57 Most notably, the quantitative increase in aromatic

432

DOM constituents formed as a result of natural weathering processes seems to be a generalizing

433

feature.26

434

The aerobic biodegradability of sand patty-derived DOM was examined by comparing

435

the rate of O2 consumption in sediment samples amended with dark or irradiated DOM, whole

436

sand patty material or only endogenous organic matter. The rate of O2 consumption {and thus

437

the amount of biodegradable organic matter} was lowest in samples amended with endogenous

438

organic matter, intermediate with dark DOM or whole sand patty material, and highest with

439

irradiated samples (Fig. 1). Presumably, the potential electron donors emanating from the whole

440

sand patty and the dark DOM are somewhat comparable as they gave overlapping rates of O2

441

consumption. The significant increase in rate of O2 consumption observed with the irradiated

442

DOM suggests that this material is more susceptible to aerobic decay processes. There is also no

443

doubt that both the quality (Fig S2) and quantity (Fig 2) of the DOM in this sample were far

444

different from either the dark or endogenous DOM. Thus, a comparison of the changes in DOM

445

both before and after the biodegradation experiment was conducted.

446

There were clear qualitative differences in DOM at the start of the biodegradation

447

experiment between the sand patty-derived organic matter formed by the dark and irradiation

448

procedures. The van Krevelen difference plots (Fig. 3) revealed that both sources of DOM

449

represented complex molecular mixtures. These plots confirm that there were a greater number

450

of molecular features in the DOM produced through irradiation than the corresponding dark

451

samples. In addition, the DOM formed by irradiation tended to have lower H/C and higher O/C,



452

confirming the more oxidized nature of the starting substrates for microbial attack.

453

Following the biodegradation experiment, the major chemical difference in DOM

454

produced in the presence of artificial sunlight is the removal of an abundant group of molecular

455

features with an O/C value of ~0.4 and H/C of ~1.25. A plausible explanation is that

456

photochemical processes result in the disaggregation of relatively small, but highly alkylated

457

aromatic compounds (1-2 ring) that are then readily available to the indigenous microflora. This

458

contention is supported by the relatively high slope ratio, low humification index, and high

459

freshness index observed for the molecular features produced at the end of the experiment (Fig.

460

2). In contrast, the lower spectral slope and freshness index as well as the higher humification

461

index collectively argue that the dark DOM, at both T0 and TFinal, is more aromatic than the

462

comparable organic matter formed in the presence of sunlight. The reduced aromaticity likely

463

accounts for the greater susceptibility of the irradiated DOM to aerobic biodegradation.

464

The dark and irradiated DOM exhibited a classical biodegradation trend of the removal of

465

predominately high H/C molecular features and production of others with relatively low H/C

466

(Fig 3). The molecular features that were manifest at the end of the biodegradation experiment

467

exhibited higher O/C, suggesting that more oxidized intermediates or end products were formed.

468

These results corroborate the increase in the humification index, as well as a decrease in both the

469

freshness index and the slope ratio associated with the biodegradation of endogenous, dark and

470

irradiated DOM. Finally, a comparable set of experiments designed to evaluate the anaerobic

471

biodegradation of the sand patty-derived DOM suggests that this organic material tends to be

472

recalcitrant under sulfate reducing conditions (data not shown).

473 474

The long-term effects of the Deepwater Horizon oil spill are still under scientific scrutiny. Numerous studies examined the fate of the spilled oil and its transformation by a variety of


Page 20 of 34

Page 21 of 34


475

weathering processes. This study found that both sunlight and aerobic microbial metabolism

476

further transform oil-derived sand patties and represent major advanced weathering processes.

477

Hayworth et al.19 detailed a conceptual model where sand patties washed onto beaches may be

478

degraded and shrink in size until they become non-recoverable. Our study helps provide a

479

mechanistic basis for how sand patties might undergo this reduction in mass. Thus, sand patties

480

get deposited onto GoM beaches and are exposed to sunlight during daylight hours and wave

481

action throughout the day. The sand patties then represent a source of DOM to the surrounding

482

environment as seawater readily penetrates these structures. Upon exposure to sunlight and

483

seawater, a complex suite of oxidized organic material is photosolubilized from the sand patties.

484

Under dark conditions, a different suite of complex molecular features is leached from the sand

485

patties. In either the dark or the light, the water-soluble DOM is not inhibitory to the resident

486

microflora and at least partially amenable to aerobic biodegradation processes. However, the

487

photooxidized DOM is quantitatively more important than the dark DOM and represents a better

488

source of electron donors supporting aerobic microbial respiration. These experiments illustrate

489

the importance of sunlight in controlling the fate of highly weathered oil residues as well as the

490

complex interplay between photochemical and biological transformation processes, resulting in

491

the transformation and solubilization of oil-derived compounds.

492 493

Supporting Information. Method detail for photogeneration of sand patty-derived DOM;

494

Figure illustrating the transport of radioactively labeled sulfate in seawater to the interior of a

495

sand patty; Figure showing absorbance at 254 nm of the DOM from irradiated and non-irradiated

496

sand patties as a function of time; Regular and subtracted van Krevelen plots associated with the

497

aerobic biodegradation experiment; Absorbance scans of DOM in seawater leached from sand

498

patty material following 12h and 24h exposures to either irradiated or dark control conditions.

499 21 ACS Paragon Plus Environment


500

Acknowledgements

501

This research was made possible by a grant from the Gulf of Mexico Research Initiative and in

502

part by the National Science Foundation (DMR-1157490), State of Florida and the FSU Future

503

Fuels Institute. The authors wish to thank Dr. Christoph Aeppli, Bigelow Laboratory for Ocean

504

Sciences for willing assistance with sand patty sampling and biomarker analysis, Drs. Amy

505

Callaghan and Jamie Johnson Duffner, University of Oklahoma, for early access to information

506

on the microbial communities in sand patties, and Dr. Robert G.M. Spencer, FSU Department of

507

Earth, Ocean and Atmospheric Science for access to analytical instrumentation. Data are

508

publicly available through the Gulf of Mexico Research Initiative Information & Data

509

Cooperative [GRIIDC] at https://data.gulfresearchinitiative.org).

510


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List of Figures Table 1. Impact of sand patties on the rate of microbial community respiration in seawatersediment incubations. O2 consumption and sulfide production were monitored in 10 mL incubations over 3 d and 7 d, respectively. The positive and negative controls contained only seawater and sediment. Figure 1. Oxygen consumption over the course of the 68 h biodegradation experiment. Standard deviations for triplicate measurements are indicated. All incubations contain 10 g GoM sediment and 10 ml of: filter-sterilized GoM seawater (Endogenous; ∆); DOM from the nonirradiated control material leached from sand patties (Dark, ◇); filter-sterilized GoM seawater and 1 g of crumbled whole sand patty material (Sand Patty, O); DOM leached from irradiated sand patties (Irradiated, ☐ ); and GoM seawater, but the entire incubation was autoclaved prior to the start of the experiment (Sterile, ◇). Figure 2. Dissolved organic carbon concentration and optical indices before (T0) and after (TFinal) the aerobic biodegradation experiment. Bar charts were generated by measuring DOC (A) and fluorescence indices for Spectral Slopes (S275-285) (B), and (S350-400) (C), Slope Ratio (SR) (D), Humification Index (HIX) (E), and Freshness Index (β:α) (F). Standard deviations are indicated for triplicate endogenous, dark, and irradiated incubations, and sterile samples represent single replicates. Figure 3. Subtracted van Krevelen plots associated with the aerobic biodegradation experiment. Plots were generated by removing molecular formulas common to both T0 and TFinal, thereby revealing only those molecular formulas present prior to biodegradation experiment (A and C) as well as those newly formed at the end of the incubation (B and D).



Figure 1 338x190mm (72 x 72 DPI)

ACS Paragon Plus Environment

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TOC Art 338x190mm (180 x 180 DPI)



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Table 1. The impact of sand patties on the rate of microbial community respiration in room temperature GoM seawater-sediment incubations. Sulfide production was monitored in 10 mL incubations over 7 d while oxygen consumption was measured in 10 mL incubations over 3 d. The positive and negative controls contained only seawater and sediment. Sand Patty Amendment (g) nM S • day-1 • g-1 µM O2 • day-1 • g-1 0 (endogenous) 1 2 2.5 5 7.5 10

38.9 ± 0.4 -a 57 ± 14 53 ± 10 80 ± 7 * 99 ± 30 *

1.76 ± 0.08 2.1 ± 0.4 2.1 ± 0.5 2.7 ± .3 * -

Positive Control b

65 ± 9 *

-

Negative Control c 3.3 ± 0.3 * 0.7 ± 0.2 * *significant difference from the endogenous rate of respiration based on ANOVA with Bonferroni correction. a -: not determined b Positive Control: Lactate (20 mM) c Negative Control: Autoclaved (20 min)


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