The impact of the Deepwater Horizon blowout on historic shipwreck-associated sediment microbiomes in the northern Gulf of Mexico

More than 2,000 historic shipwrecks spanning 500 years of history, rest on the Gulf of Mexico seafloor. Shipwrecks serve as artificial reefs and hotspots of biodiversity by providing hard substrate, something rare in deep ocean regions. The Deepwater Horizon (DWH) spill discharged crude oil into the deep Gulf. Because of physical, biological, and chemical interactions, DWH oil was deposited on the seafloor, where historic shipwrecks are present. This study examined sediment microbiomes at seven historic shipwrecks. Steel-hulled, World War II-era shipwrecks and wooden-hulled, 19th century shipwrecks within and outside of the surface oiled area and subsurface plume were examined. Analysis of 16S rRNA sequence libraries, sediment radiocarbon age data, sedimentation rates, and hydrocarbons revealed that the German U-boat U-166 and the wooden-hulled sailing vessel known as the Mardi Gras Wreck, both in the Mississippi Canyon leasing area, were exposed to deposited oil during a rapid sedimentation event. Impacts to shipwreck microbiomes included a significant increase in Piscirickettsiaceae-related sequences in surface sediments, and reduced biodiversity relative to unimpacted sites. This study is the first to address the impact of the spill on shipwreck-associated microbiomes, and to explore how shipwrecks themselves influence microbiome diversity in the deep sea.

Microbiome composition. On average, 7,594 and 34,429 sequences with median length of 442 base pairs were obtained per sample for bacteria and archaea respectively ( Table 2). Despite a lower sequence count, operational taxonomic units (OTU) count and Shannon diversity was greater for bacteria than archaea. The Chao 1 reveals that bacteria and archaea were under-sequenced by 30% and 21% respectively (Supplementary Table S1). The Good's coverage index was calculated to evaluate the impact of sample size on results. The index averaged 0.81 and 0.93 for bacteria and archaea, respectively, indicating excellent coverage of archaeal communities, but under-sequencing of bacterial singletons. Bacterial Shannon diversity did not differ in surface and bottom sediments collected within 2 m of shipwrecks. However, in samples away from shipwrecks, diversity was generally lower near the surface of cores (Table 2). Archaeal diversity was lower in surface vs. deep sediments both near and away from shipwrecks. Shannon diversity was higher in samples collected at the surface, near shipwrecks, compared to away from shipwrecks, except at the Mardi Gras Wreck. Analysis of similarity (ANOSIM) revealed that microbiomes near and away from shipwrecks differed significantly (p ≤ 0.05) at moderately impacted and reference sites, but not at heavily impacted sites ( Table 2).
The Delta-, Gamma-and Alpha-proteobacteria-were highly abundant at all sites (Figs 3 and S1). Deltaproteobacteria averaged 24% of sequences (range 17-26%), and increased slightly with depth. Gammaproteobacteria averaged 12% of sequences, (range 0.9-33%) and were most abundant in surface sediments. The Alphaproteobacteria were third most abundant, averaging 8% of all sequences (range 2-20%), with highest abundances in surface sediments at moderately and heavily impacted sites. The Dehalococcoidetes and Phycisphaerae averaged 5% of sequences (range 0-27% and 2-27% respectively), and generally increased with depth. Sediments at reference sites had the largest percentage of Dehalococcoidetes. Sediments at U-166 and the Mardi Gras Wreck had the largest percentage of Phycisphaerae.
Although Deltaproteobacteria were the most abundant class, one Gammaproteobacteria phylotype related to the Piscirickettsiaceae family accounted for up to 16% of sequences in surface sediments (0-5 cm below seafloor or cmbsf), and was uniformly abundant in the upper 4-6 cmbsf of cores collected proximate to the Viosca Knoll Wreck, U-166, the Mica Wreck, and the Mardi Gras Wreck (Fig. 4). The second most abundant phylotype was a member of the candidate order MSBL9 of the Phycisphaerae. Members of this candidate order may be involved in breakdown of complex polysaccharides 15 .
Sequences classified to the Dehalococcoidaceae, which are ubiquitous in marine sediments 16 , were third most abundant in the dataset. Their abundance was driven by prevalence in the lower half of cores from reference sites (Fig. 3). The most abundant Deltaproteobacteria was affiliated with the sulfate-reducing Desulfococcus genus. This phylotype was elevated towards the base of the core collected 2 m from the Mardi Gras Wreck, where it accounted for 12% of sequences. The uncultivated order NB1-j also contributed to the abundance of Deltaproteobacteria. NB1-j sequences averaged 6% of sequences in the upper 6 cmbsf at U-166 and the Mica Wreck (Fig. 4). NB1-j sequences have also been observed in high abundance in sediment and coral floc collected in the immediate vicinity of U-166 in December 2010 17 .
The archaea libraries were dominated by fewer OTUs, and the top 60 accounted for nearly 100% of sequences in samples (Fig. 4). The Thaumarchaeota were dominant in surface sediments at all sites, and sharply declined with depth (Figs 3 and S2). Members of this class accounted for 53% (range 0.7-99%) of all archaeal sequences. The Marine Crenarchaeal Group (MCG) was the second most abundant class (25%; range 0.1-83%). The MCG were most abundant at Halo, and generally increased with sediment depth. The Marine Benthic Group B (MGCB) averaged 10% of sequences (range 0-47%), and increased sharply in abundance below 5 cmbsf at most sites. The Figure 1. Study sites, and concentrations of seafloor hopane, PAH 50 , and dioctyl sodium sulfosuccinate (DOSS), a marker compound for Corexit 9500 residue. Grid data from NRDA sediment (top 10 cmbsf) collection efforts between 2010 and 2014. Data downloaded from the NOAA DIVER explorer (https://www. diver.orr.noaa.gov). Grids and maps were created using NRDA data and BOEM site coordinates in Surfer v.14 (Golden Software).
The majority of Thaumarchaeota were affiliated with a phylotype related to the ubiquitous 18 marine Nitrosopumilus, which averaged 38% of all archaeal sequences proximate to and away from shipwrecks. Sequences of this genus were observed within the DWH subsurface plume; however, they are not associated with hydrocarbon degradation. These were uniformly abundant in the upper 6 cmbsf at the Viosca Knoll Wreck, U-166, and the Mica Wreck (Fig. 4), and accounted for >78% of the microbiome at the surface (0-2 cmbsf) of all moderately and heavily impacted sites. At the Mardi Gras Wreck, Nitrosopumilus accounted for 94% of all archaeal sequences at the sediment-water interface. An unidentified MCG was second-most abundant, averaging 17% of all sequences (range 0-45%). This phylotype was most abundant towards the base of Halo and Ewing Bank Wreck cores. Two additional Thaumarchaeota phylotypes, affiliated with the Cenarchaeaceae, averaged 10% and 4% of sample sequences. Neither of these could be resolved beyond the family level. The more abundant of these was found at all sites, although were minimally abundant at the Mardi Gras Wreck. There were no clear trends with depth for either phylotype.
Bacteria at Halo formed distinct clusters in non-metric multidimensional scaling (NMDS) plots of Bray-Curtis similarity data for sequence types and abundance (Fig. 5a). These sub-divided according to whether samples were collected proximate to (2 m) or away from the shipwreck (similarity profile testing, SIMPROF, p = 0.05). The majority of samples from U-166 and the Mardi Gras Wreck created two closely ordinated groups, regardless of proximity to the shipwrecks. Bacterial microbiomes were somewhat organized by depth. There was a cluster of samples from all sites collected at 0-5 cmbsf (Fig. 5a). Within this cluster were samples from U-166 down to 6 cmbsf. The similarity profile permutation test (SIMPROF) was used to identify groupings of samples at a 0.05    Fig. 5a and SIMPROF results, not shown). Archaea were less organized by site, with the exception of Halo, and more organized by depth (Fig. 5b). The NMDS included three large groups of samples: 0-8, 8-16, and 16-20+ cmbsf. At 70% similarity, there was no distinct grouping of samples according to site type (reference, moderately, or heavily impacted). However, SIMPROF revealed distinct clusters for Halo and the Ewing Bank Wreck (p = 0.05), and U-166, Mardi Gras Wreck, and Anona.
ANOSIM on the upper 10 cmbsf of all cores revealed that Halo bacterial microbiomes differed highly significantly from other shipwrecks (Table 3). This was generally the case for archaea at Halo. Bacterial microbiomes at the Ewing Bank Wreck and U-166, and bacterial and archaeal microbiomes at the Mardi Gras were distinct from all sites except Anona.
The similarity percent (SIMPER) analysis was performed on square root transformed data (upper 10 cmbsf) to reveal the OTUs driving similarity between and among surface sediments at each site. The differences in bacterial microbiomes were explained by numerous OTUs (Supplementary Table S2); archaeal differences were explained by one OTU (Supplementary Table S3). Bacterial similarity within sites ranged 61-74%, with the lowest within-group similarity at the Mardi Gras Wreck and U-166. Halo and Mardi Gras Wreck bacteria had the greatest dissimilarity (Supplementary Table S2) to other sites. Dissimilarity between Halo and other sites was driven by Dehalococcoidaceae-related OTUs. Dissimilarity of the Mardi Gras Wreck to other sites was driven by Piscirickettsiaceae-related OTUs. Archaeal within-site similarity ranged 59-74% (Supplementary Table S3). Archaea at the Mardi Gras Wreck had the greatest dissimilarity to other sites, followed by Halo. In all comparisons, either the Nitrosopumilus-related OTU, or MCG-related OTUs explained 11% or more of the dissimilarity between sites.
Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) 19 was used to predict metagenomes of bacterial taxa. The analysis was not conducted on archaea given a poor return of OTUs in closed reference OTU picking. Predicted functional genes involved in PAH and naphthalene degradation and metabolism of xenobiotics by cytochrome P450 were elevated in surface sediments at Anona, U-166 and the Mardi Gras Wreck ( Supplementary Fig. S3).

Sediment Physical Properties.
Cores from U-166 had a reddish-brown layer at the surface that extended down to ~5 cmbsf. The layer was unconsolidated, gelatinous, and stained nitrile gloves in a manner consistent with observations of MOSSFA made by others (Joye et al. 20 and Ziervogel et al. 21 ) in cores from the Mississippi Canyon leasing area.
Highest porosities were observed at the surface at Mardi Gras and Viosca Knoll Wrecks, and U-166 ( Fig. 6a). At the former two, porosity decreased through the upper 6 cmbsf, while at U-166 it remained high (~90%). Porosity at Halo was significantly reduced compared to other sites. This relatively shallow site (Table 1) experiences high current velocities as was observed during both dives in 2014, along with poor visibility (~3 m). As a result, sediments at Halo are sandy with larger grain size and lower water content.
Hydrocarbon analysis. PAH concentrations were near the limit of detection (LOD) in most cores, likely because samples were collected four years post-spill. The low concentrations could have further been driven by potential sorption of PAHs to whirl-pak bags used for sample collection (Fig. 6b). However, a peak at 6 cmbsf at Halo was detected. Total petroleum hydrocarbons (TPHs) ranged 0-17 mg/g, and were elevated at Halo, U-166,  and Mica Wreck cores various depths from surface to bottom (Fig. 6c). Halo, an oil tanker, was carrying crude oil when it sank in 1942, which explains observations in this core. U-166 and the Mica Wreck had peak TPH concentrations at mid-core. PAHs and TPHs were near the LOD at Anona. The concentration of C7-C40 alkanes ranged 0-4 μg/g in all samples (Fig. 6d). Peak concentrations of C7-C40 alkanes were observed mid-core at the Mica Wreck, U-166, and Halo, the latter having a secondary peak at the base of the core. Sediment radiocarbon natural abundance (Δ 14 C) was used to indicate if petrocarbon (presumably oil) was deposited near shipwrecks ( Table 4). The analysis assumes that surface sediment contains a more modern Δ 14 C signature than 'fossil' hydrocarbons 4 . The approach is powerful when tracking the fate of weathered oil (~petrocarbon) through the Gulf ecosystem 4 . Halo surface and deep sediments were significantly Δ 14 C depleted (Table 4)  torpedo strike have a nearly fossil petroleum Δ 14 C signature (−1000‰). This signal is most likely attributable to the crude oil that the vessel was carrying when it sank. In all sites other than U-166, age increased with depth. At U-166, there was no change in Δ 14 C with depth.
The rate of sedimentation was analyzed at Halo, the Mica Wreck, U-166, the Mardi Gras Wreck, Anona, and the Ewing Bank Wreck (Table 4 and Supplementary Fig. S4). Through the upper 10 cmbsf, the rate ranged from 0.08 to 0.63 cm year −1 . The lowest rates were observed at reference sites Halo and the Ewing Bank Wreck, and the highest at U-166. The Pb-210 xs activity profiles (Supplementary Fig. S4)

Discussion
Microbiome, physical property, and chemical data comparisons were used to evaluate the impact of the DWH spill to sediments surrounding historic shipwrecks. To rule out the influence of sediment berms formation around these high-relief structure, or the physical disruption of sediments during wrecking events, analyses were conducted in proximity to, and away from each shipwreck's lateral extent.
At Halo and the Ewing Bank and Viosca Knoll Wrecks, bacterial and archaeal microbiomes were significantly different proximate to vs. away from the shipwrecks. This was not the case at the heavily impacted sites (Mica Wreck, U-166, and Mardi Gras Wreck). Shannon diversity was generally higher in surface sediments near the shipwrecks as compared to away from them, with the exception of the Mardi Gras Wreck. Shipwrecks are described as islands of biodiversity owing to the higher alpha diversity of macrobiota on shipwrecks compared to the surrounding environment. However, shipwreck microbiomes have never been evaluated. This study is the first to evaluate the effect of shipwreck proximity on microbiome diversity in deep-sea sediments, albeit with coarse resolution. The results of the ANOSIM and alpha diversity analyses indicate that an island effect may be imparted on some sediment microbiomes as a result of proximity to shipwrecks. However, at the spill impacted sites Mica, U-166 and Mardi Gras, this effect was not evident, either due to the greater depth of these sites, or possibly the presence of spill residues. The area that U-166 and the Mardi Gras Wreck are located in has been intensely sampled from 2010 to 2014 1,6 . Impacts to sediment and coral microbiomes in this location have been reported by other research groups 8,17,22,23 . Our study found similarities with these prior works. First, domination of surface sediments by unidentified sequences associated with the Thiotrichales, specifically, the Piscirickettsiaceae, was observed. Two studies of DWH spill-impacted benthic 17 and pelagic communities 18 noted significant enrichment of Thiotrichales. At U-166 and the Mardi Gras Wreck, Piscirickettsiaceae-related sequences accounted for 12-16% of all sequences in surface sediments. At these locations, Gammaproteobacteria, commonly observed in deep marine sediment, averaged 30% of bacterial sequences at the surface of cores nearest the shipwrecks. Yang et al. 23 observed Gammaproteobacteria to average 23% of sequences in surface sediments prior to the arrival of DWH spill contaminants on the seafloor within 4 km of the Macondo well. In Yang's study, the percent of Gammaproteobacteria rose to 33%, post-spill in line with observations in the present study. This finding suggests that the spill impact on surface sediments was still evident four years later. The PICRUSt prediction data also indicate an increase in KEGG pathways involved in PAH metabolism in bacterial communities suggesting impacts on function at sites within the spill's footprint.
The most abundant Deltaproteobacteria at the Mardi Gras Wreck was affiliated with the sulfate-reducing Desulfococcus. Members of this genus were among the top-ranked phylotypes in metagenomes created for samples less than 3 km from the wellhead in 2010 22 , and Desulfobacteraceae were also elevated in 16S rDNA profiles of spill-contaminated sediment samples in the vicinity of the Mardi Gras Wreck in October 2010 23 . Desulfococcus sequences and sequences affiliated with the NB1-j were also in elevated at sites collected near the wellhead in this study and a previous work 17 .
Prior studies have addressed water column archaea in context with the DWH spill and have noted high abundances of Thaumarchaeota 24,25 . These prior works showed that their presence and abundance was consistent in samples collected within and outside of the DWH subsurface plume; accordingly, they are thought to have no role in hydrocarbon degradation. Nitrosopumilus maritimus-affiliated sequences in those studies were only slightly depressed when amended with oil in laboratory experiments, further suggesting that spill-related contaminants may have no significant impact on these water column archaea. However, Yergeau 26 noted enrichment of Thaumarchaeota affiliated with the Nitrosopumilus genus in deep-water samples located within the subsurface plume. Nitrosopumilus-related sequences were the most abundant archaea at all sites in the current study, suggesting that they are a poor marker for spill contamination in sediments. However, we note their significant enrichment in surface sediments at the Mardi Gras Wreck relative to other locations.
The physical property data and geochemical data provide evidence that U-166 and the Mardi Gras Wreck experienced hydrocarbon contamination as a result of the DWH spill. These sites had surface porosities of ~90%, which exceeds other locations in this study, and average porosities for Mississippi Canyon 1 . Ziervogel and colleagues noted elevated sediment porosity with greater proximity to the Macondo well in their study 21 . Porosity can be altered through physical manipulation of sediment, or through deposition of highly porous (96-99%) MOSSFA 5 . Surface porosities in this study at U-166 and the Mardi Gras Wreck were elevated proximate to and away from these shipwrecks indicating that lower surface porosity cannot be attributed to the wrecking events.  Table 3. Analysis of Similarities (ANOSIM). Analysis conducted on samples collected above 10 cmbsf *indicates significant difference (p < 0.05). **Indicates highly significant difference (p < 0.001). This was important to delineate at U-166 as the wrecking event resulted in a 3-m impact crater that the main wreckage rests in (Fig. 7a). Notably, samples for this study were not collected near the main U-166 wreckage to avoid what is ostensibly a sediment trap, and instead samples were collected at the bow section disarticulated from the main wreckage, ~150 m to the northwest (Fig. 7b).
The PAH data for this study are inconclusive in terms of linking the observed hydrocarbons to residual DWH spill contaminants. TPH and long-chain n-alkane data offer evidence of contamination at U-166, but were unavailable for the Mardi Gras Wreck. However, the radiocarbon and Pb-210 xs activity data for this study provide compelling evidence for a sedimentation event at both locations. Riverine organic matter (OM) Δ 14 C ranges from −86 to −223‰ 4 . Surface sediments in Mississippi Canyon ordinarily reflect the signature of new OM and are Δ 14 C enriched. With increasing depth, samples should become more Δ 14 C depleted. These are guides that must take into account location relative to the Mississippi River plume source of OM. However, the profile shape observed at U-166, specifically the deviation from an expected Δ 14 C enriched signature at surface and depleted  28 observed Δ 14 C depleted surface sediments in cores from DeSoto canyon, collected in December 2010. The Δ 14 C in the upper 1 cm of that study was nearly the same as at 15 cmbsf, however, Δ 14 C enrichment was observed through 1-5 cmbsf.
In the current study, evidence for a sedimentation event is also found in the modeled sedimentation rates provided by the Pb-210 xs data. A 2004 study 29 in the Mississippi Canyon leasing area revealed that sedimentation rates were 0.09 cm/year. This rate is consistent with rates in other coastal areas at similar depths 27 , and the average for the past 100 years in DeSoto Canyon, north east of the study area 28 . Sedimentation rates at Halo and the Ewing Bank Wreck are consistent with prior observations 29 . A normal decay pattern for Pb-210 xs (decreasing with depth) was evident for all sites except U-166, as was Δ 14 C attenuation with depth at all sites except U-166. The Pb-210 xs profile at U-166 immediately declined and then spiked at 6 cmbsf, nearly equivalent to the surface value of Pb-210 xs . A sedimentation rate at this location of 0.63 cm/year is seven times higher than expected, based on pre-spill estimates 29 . Brooks et al. 28 used short-lived radioisotopes (i.e., 7 Be and 234 Th), to compare sediment accumulation across a time series from 2010 to 2012. The study revealed a brief but rapid depositional event in DeSoto Canyon that occurred as a consequence of MOSSFA sedimentation during and immediately following the DWH spill. Taken together with the Δ 14 C profile at U-166, it is evident that a similar rapid sedimentation event occurred at U-166, and buried younger sediments. Pb analysis on cores collected during the July 2014 cruise to determine sedimentation rate. Previous studies support our finding of a rapid sedimentation event following the DWH spill 28 . However, sediment dynamics in the Mississippi Canyon leasing area is complicated by lateral shelf-slope transport and seafloor currents. Shipboard Acoustic Doppler Current Profiler was only able to resolve currents to ~700 m, and thus, no data is available for U-166. Seafloor currents can be disrupted by high-relief structures, although, sampling occurred where no berm formation was evident. Currents below 1000 m can exceed critical shear stress (0.1 to 0.4 Pa) that would induce incipient motion and facilitate lateral transport 30,31 . However, vane sheer strength at U-166 ranged from 0.42 to 0.65 Pa in the upper 6 cmbsf, proximate to and away from the shipwrecks, thus, lateral transport of sediment would not provide sufficient sediment entrainment to account for what amounts to massive deposits (~5 to 6 cm) at U-166.

Conclusions
This study is the first to examine sediment microbiomes proximate to deep-sea historic shipwrecks. The purpose of this was to reveal if historic shipwrecks were impacted by the DWH spill. Close to the Macondo wellhead, there is evidence for oiling of U-166-a historically significant shipwreck, war grave, and ecological resource. Sedimentation rates at U-166 cannot be attributed to riverine sediment load or ordinary resuspension and lateral transport of seafloor sediments. The U-166 Pb-210 xs profile displayed a 5-cm layer of older sediment overtop younger material. The porosity of that interval, and Piscirickettsiaceae sequence abundance are both uniform and high. Radiocarbon data reveals surface sediments with the same approximate age as at the base of the core. It is likely that the Mardi Gras Wreck also experienced fallout of DWH contaminants, given its location and the commonalities between its microbiome and that of U-166. However, due to a lack of hydrocarbon data for the site, and a Pb-210 xs profile that indicates markedly lower sedimentation rates than at U-166, this cannot be definitively stated. The Mica Wreck also bears high microbiome similarity with U-166 and the Mardi Gras Wreck, but deviates on physical and chemical parameters. The Mica Wreck is located north of the fallout plume, and possibly narrowly avoided the DWH sedimentation event. Prior studies have identified a patchy distribution of impacts from the spill on benthic habitats and microbiomes in the area around the wellhead 1,2,6,7 .
The study also examined the influence of shipwrecks on microbiome diversity in sediments surrounding anthropogenic structures. Shipwreck proximity positively influenced diversity in surface sediments near World War II-era and 19 th century shipwrecks. Shipwreck-associated microbiomes were distinct from the surrounding environment, at four of 7 locations, indicating that some shipwrecks impart an island effect on seafloor microbiomes. However, at the Mica and Mardi Gras Wrecks, and U-166, diversity and composition were similar near and away from the sites. This may suggest the effect is not present at these deeper sites, or that spill associated effects obscure evidence for an island effect.

Materials and Methods
Sample Collection. Sediment was collected during two expeditions: PE14-15 and PE15-02 in March and July 2014, respectively, as part of the Gulf of Mexico Shipwreck Corrosion, Hydrocarbon Exposure, Microbiology, and Archaeology (GOM-SCHEMA) project (https://www.boem.gov/GOM-SCHEMA) using the Global Explorer remotely operated vehicle (GEX-ROV -Deep Sea Systems International, http://www.deepseasystems.com). Prior to sample collection, a visual survey of each shipwreck was performed to update archaeological site plans and identify suitable areas for sediment sampling that are devoid of archaeological materials.
Push core samples were obtained using a 7-function manipulator arm without damaging the archaeological sites or associated debris fields within 2 m of each shipwreck, and 100-200 m away from each shipwreck (Fig. 2). Replicate cores for various analyses were obtained and sampled according to standard techniques (See Supplementary Materials).
DNA extraction and sequencing of 16S rRNA gene amplicons. Genomic DNA was extracted using a modification of the FastDNA ™ protocol described in a previous study 32 and 16S rRNA gene amplification and sequencing was carried out according to Comeau et al. 33 at the Microbiome Resource (IMR) facility at Dalhousie University. Bioinformatics, sequence quality control, and calculation of alpha diversity statistics (Shannon Diversity, Good's Coverage) were carried out in a pipeline generated with UPARSE 34 and Quantitative Insights into Microbial Ecology (QIIME) 35 (See Supplementary Materials). Shannon Diversity was calculated for both rarefied and un-rarefied datasets. Data were also processed through PICRUSt 1.1.0 to predict metagenome functional content 19 . Sediment Physical Properties. Sediment porosity was determined from wet and dry weights at 1 cm intervals. Grain density was determined using a PentaPycnometer. Sediment accumulation rates were determined using Pb-210 xs activity measurements of sediments from the top 10 cmbsf at the Louisiana State University, Department of Geology.
Hydrocarbon Analysis. Analyses were not conducted on Mardi Gras and Viosca Knoll samples, as they were not visited during the first GOM-SCHEMA cruise in March 2014 due to weather complications. Samples were analyzed at the University of Georgia (UGA) with a protocol developed by Yi Yang (See Supplementary Materials). Sediments were sequentially extracted with dichloromethane:methanol (9:1, vol/vol) and hexane in a horn ultrasonicator. Combined extracts were reduced using a Rotovap ® , desulfurized, washed, and further reduced under nitrogen stream prior to analysis. Hydrocarbons were analyzed using a LECO Pegasus 4D GCxGC-Time of Flight Mass Spectrometer system equipped with an electron ionization source. PAH (TCL PAH mix, Supelco, Bellefonte, PA) and TPHs (BP surrogate oil) standards were used as references to quantify PAHs and TPHs concentrations in extracts.