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Jun 14, 2018 - Marine Clam, Mactra veneriformis, Chronically Exposed to Oil-. Suspended Particulate Matter Aggregates. Junsung Noh,. †. Hosang Kim,...
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Ecotoxicology and Human Environmental Health

Bioaccumulation of Polycyclic Aromatic Hydrocarbons (PAHs) by the Marine Clam, Mactra veneriformis, Chronically Exposed to Oil-suspended Particulate Matter Aggregates Junsung Noh, Hosang Kim, Changkeun Lee, Seo Joon Yoon, Seungoh Chu, Bong-Oh Kwon, Jongseong Ryu, Jae-Jin Kim, Hanbyul Lee, Un Hyuk Yim, John P. Giesy, and Jong Seong Khim Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06692 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

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Bioaccumulation of Polycyclic Aromatic Hydrocarbons (PAHs) by the Marine

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Clam, Mactra veneriformis, Chronically Exposed to Oil-suspended Particulate

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Matter Aggregates

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Junsung Noh,a Hosang Kim,a Changkeun Lee,a Seo Joon Yoon,a Seungoh Chu,a Bong-Oh Kwon,a

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Jongseong Ryu,b Jae-Jin Kim,c Hanbyul Lee,c Un Hyuk Yim,d John P. Giesy,e,f,g,h and Jong Seong Khima,*

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a

School of Earth and Environmental Sciences & Research Institute of Oceanography, Seoul National University, Seoul,

Republic of Korea b

Department of Marine Biotechnology, Anyang University, Ganghwa-gun, Incheon, Republic of Korea

c

Division of Environmental Science & Ecological Engineering, Korea University, Seoul, Republic of Korea

d

Oil and POPs Research Group, Korea Institute of Ocean Science and Technology, Geoje, Republic of Korea

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Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon,

Saskatchewan, Canada. f

Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA

g

School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China

h

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University,

Nanjing, People’s Republic of China

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Word counts

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4712 words (Abstract (197) + Text) + 2 Large Figures and 3 Small Figures (words 2100 equivalent)

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= Total 6812 words equivalent

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* Corresponding author.

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Address: School of Earth and Environmental Sciences & Research Institute of Oceanography, Seoul

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National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea.

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Tel.: +82 2 880 6750; fax: +82 2 872 0311.E-mail address: [email protected] (J.S. Khim). 1

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ABSTRACT: Dispersion and biodegradation of petroleum hydrocarbons are significantly enhanced

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by formation of oil-suspended particulate matter aggregates (OSAs), but little is known about their

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adverse effects on benthic invertebrates or microbes. In this study, we investigated: (1)

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bioaccumulation of polycyclic aromatic hydrocarbons (PAHs) by the marine bivalve, Mactra

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veneriformis and (2) changes in composition and relative abundances of microbes, during 50-d of

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OSAs feeding experiment. Total concentrations of PAHs increased more rapidly during the first week

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of exposure, peaked at Day 30, then gradually declined to the end of experiment. While

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bioaccumulation of PAHs by clams varied among the 20 target compounds, two major groups of

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PAHs were identified by the cluster analysis. One group including 3-methylphenanthrene, 1,6-

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dimethylphenanthrene, 1,2,6,9-tetramethylphenanthrene, and benzo[a]anthracene showed a fairly

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constant rate of accumulation, while the second group including 2-methyldibenzothiophene, 2,4-

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dimethyldibenzothiophene, 2,4,7-trimethyldibenzothiophene, 3-methylchrysene, 6-ethylchrysene,

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and 1,3,6-trimethylchrysene exhibited a bell-shaped pattern. Bioaccumulation of PAHs by clams was

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dependent on changes in abundance of Gammaproteobacteria, indicating active degradations of

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hydrocarbons by selected species. Six key species included: Porticoccus litoralis, Porticoccus

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hydrocarbonoclasticus, Cycloclasticus spirillensus, Alcanivorax borkumensis, Alcanivorax dieselolei,

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and Alkalimarinus sediminis. These results are the first to demonstrate interactions of OSAs and

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macrofauna/microbe in oil cleanup operations.

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INTRODUCTION Spills of oil associated with transportation in marine environments, oilfield drilling, petroleum

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refining, oil storage and waste management, can have consequences for aquatic ecosystems,

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particularly to subtidal and intertidal habitats1. Crude oil and oil-derivatives not only contain toxic

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components, such as polycyclic aromatic hydrocarbons (PAHs), but can also be spread widely and

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persist and be accumulated into food chains. Detrimental effects of oil spills on marine organisms

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and their habitats over an extended period can be significant 2,3,4. For example, the Hebei Spirit Oil

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Spill (HSOS) occurred 10 km off the coast of Taean, Korea in 2007 released approximately 10,800

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tonnes of crude oil into the sea and eventually damaged entire marine ecosystems5,6. Recovery of the

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marine environment from this oil spill took >five years to return to baseline conditions of water and

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sediment quality7. To date, it is still debatable whether recovery of the benthic community in the area

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is complete.

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PAHs are omnipresent contaminants in marine environments. In fact, pyrogenic PAHs are

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commonly found in marine organisms. Petrogenic PAHs, which originate from crude oil, contain a

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wide range of alkylated derivatives. Thus, it is not surprising that marine organisms exposed to oil

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spills contain significant concentrations of both PAHs and alkylated PAHs4,8,9. Lower molecular

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mass (LMM) PAHs, such as naphthalene and its alkylated groups dominate the PAHs of Iranian

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heavy crude (IHC) oil, followed by the another group of LMM PAHs including dibenzothiophene,

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phenanthrene, and fluorene, and then by relatively minor components representing higher molecular

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mass (HMM) PAHs, such as chrysene and other HMM PAHs10. Bioaccumulation and adverse effects

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of LMM PAHs on marine organisms are well known (see SI-Table S1). HMM PAHs are particularly

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hazardous because they have greater toxic potencies and are also carcinogenic to humans and

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wildlife and often degrade quite slowly. 3

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PAHs are transformed primarily by microbes, which play an essential role in degrading spilled

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oils and/or residues. In fact, a number of bacterial taxa can decompose a wide variety of

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hydrocarbons and those taxa can reproduce rapidly in oiled environments11–15. Thus, microbial

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communities could be used to effectively and economically bio-remediate environments

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contaminated by oil16.

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Suspended particulate matters (SPM) have been recognized as vectors for transport of oil from

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one environment to another and to affect accumulation by marine organisms. Oil and a variety of

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SPMs bind together in seawater, to form oil-SPM aggregate (OSA), which is known to affect

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dispersions of spilled oil and degradation of petroleum hydrocarbons17–20. In addition to being called

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OSAs, previous studies have referred to these aggregates of oil and SPM as oil-mineral aggregates,

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or oil-particle aggregates21–25. In areas where SPMs are prevalent, OSAs form naturally21. Due to

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their lesser viscosities, OSAs are not as adhesive to seashore habitats as is crude oil. This is because

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oil droplets originating from spills usually become coated with fine, non-sticky organic particles as

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oil disperses. This type of aggregation enhances biodegradation because aggregates significantly

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increase oil-water contact areas, which maximizes accessibility of hydrocarbon-degrading bacteria

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and fungi to oil26,27,28. Furthermore, by being nearly neutrally buoyant, OSAs can be easily dispersed

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away from the oil-contaminated sites by tides and/or currents29. Although characteristics of OSAs

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formed under various environmental conditions have been well studied23,30-32, there have been few

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studies examining bioaccumulation and/or biodegradation of oil in the form of the OSAs, by

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microbial communities.

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In the present study, a laboratory mesocosm experiment was used to simulate a shallow,

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subtidal environment contaminated by chronic exposure to OSAs (Figure 1A). Since this shellfish is

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commercially important worldwide and the very species prefer to inhabit in muddy tidal flat33 which 4

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has greater potential in OSAs formation compared to that in sandy areas, when exposed to the oil

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contaminated environment, the filter-feeding bivalve, clam Mactra veneriformis was studied.

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Concentrations of PAHs in exposed animals, microbial communities, and other parameters were

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monitored for 50 d (Figure 1B) because the spilled oil could last >30 days5,6 and possibly OSAs

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could be formed and persist for longer periods of time. Specific objectives were to: (1) evaluate

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bioaccumulation of PAHs into soft tissues of M. veneriformis chronically exposed to OSAs in the

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water column; (2) examine compound-specific patterns of bioaccumulation in clams targeting parent

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PAHs and alkylated PAHs; (3) identify specific constituents of microbial communities dominating an

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OSA-contaminated environment, and (4) assess how bioaccumulation of PAHs into clams influences

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microbial-mediated biodegradation.

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MATERIALS AND METHODS

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Preparation of OSAs. OSAs were prepared by previously described methods25, with a slight

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modification (see details in SI-Table S3). In brief, OSAs were prepared using 1 L (1000 g) of filtered

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seawater in a glass carboy, with 0.6 g of IHC oil, and 0.2 g of ground and sieved sediment (Φ 6% loss on ignition).

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This enriched, dried sediment was mixed with seawater, and then IHC was added by use of pre-

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calibrated pipettes. A reciprocating shaker (set up at 170 rpm) in a temperature-controlled room (18 ±

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0.5 oC) was used to simulate mixing of the crude oil, sediment, and seawater. After 24 hours, the

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carboy was allowed to stabilize for about 5 minutes, and then 700 ml of a mixture of OSAs and

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seawater was extracted using a faucet located at the bottom. The mixture was used for feeding

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materials of 300 mL and 600 mL, respectively, in OSAlow and OSAhigh treatments (Figure 1).

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Experimental Design. Individuals of the test organism, M. veneriformis (mollusks) were collected

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in June of 2016, from intertidal mudflat off the west coast of Korea (36.095ºN, 126.613ºE). After

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collection clams were immediately (within 4 h) transported to laboratory aquariums where they were

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acclimated to lab conditions for one month. For each OSA feeding experiment (described below), 40

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clams (3.5 ± 0.5 mm shell length) were placed in experimental aquariums (40 × 40 × 30 cm), which

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were filled with sandy sediment (3 cm deep) and seawater (40 L). Clams were acclimated in

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aquariums for 2 d before the beginning of experiments. Seawater was filtered through 0.7-µm glass-

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fiber filters. Movement of water inside aquariums was maintained by use of a continuous-flow pipe

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attached to an air pump. During the experiment, salinity was maintained at 34 (± 1.0) psu. To

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minimize the influence of organic carbon from exogenous sources, a sediment composed of cleaned

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and dried sandy loam (95% of the population in samples, except dried sediment.

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In particular, Proteobacteria and Bacteroidetes represented >92% of the microbial populations, while

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in the source sample (dried sediment) represented 5% of

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members, were identified in all three temporal sampling times of the experiment, including

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representatives of Bacteroidetes (unresolved taxa) and Proteobacteria (Alphaproteobacteria and

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Gammaproteobacteria) (Figure 4B’). The Shannon-Weaver index of the core communities ranged

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from 2.5 to 3.8, which suggested that the number of species and evenness indicated relatively more 12

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diverse bacterial communities in each class compared with that of other classes in phyla.

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Gammaproteobacteria was the most prevalent class in all samples. It was abundant in the OSA of all

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feeding tanks and the Shannon-Weaver index of Gammaproteobacteria increased over the

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experimental period (Figure 4B’).

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Two of dominant genera (>10%) in initial source sediment were Hydrogenophaga and

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Panacagrimonas, however, the dominant genera in treatments with OSA indicated changes during

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experimental periods (Figure 5A). Communities of Proteobacteria bloomed in the beginning of

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experiment (at Day 1), representing over 85% dominated by anaerobic and halophilic species, then

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the proportion of Proteobacteria sharply declined until Day 14 (Figure 5B). After Day 14, there was

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still a large proportion of Proteobacteria which predominated, with relative abundance of ca. 70% to

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the total. The sum of Proteobacteria and Bacteroidetes were maintained collectively at greater than

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90% in relative abundance during the experiment.

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Comparisons of three phylogenetic representations at Day 1, Day 30, and Day 50 showed that

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specific genera of Gammaproteobacteria were remarkably changed and more abundant in the OSA

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treatments compared to Control. The corresponding genera include Pseudomonas, Methylophaga,

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Rheinheimera, Porticoccus, Cyclosticus, Methylophaga, and Alcanivorax (Figure 5A). At species

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level, nine Gammaproteobacteria taxa were found to be abundant in the OSA treatments, including

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Porticoccus litoralis, Pseudomonas guineae, Pseudomonas taeanensis, Porticoccus

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hydrocarbonoclasticus, Cycloclasticus spirillensus, Thioprofundum lithotrophicum, Alcanivorax

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borkumensis, Alcanivorax dieselolei, and Alkalimarinus sediminis (Figure 5C). In general,

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Pseudomonas was found to be the predominant genus, particularly peaked during early exposure

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(until Day 7 or 14) among treatments. However, its relative abundance gradually decreased over the

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last period of time. Alternatively, the relative abundance of Porticoccus increased consistently, 13

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ultimately to become the most prevalent taxa, representing over 20% of the total microbial

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abundances (Figure 5C). Relative abundances of Porticoccus and Pseudomonas seemed to indicate a

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competitive interaction, which might be influenced by temporal changes in PAHs concentrations

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and/or compositions apparently related to the degree of PAHs degradation.

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DISCUSSION Relative compositions of PAHs in OSAs were a function of the process used to prepare OSAs.

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Naphthalene (Nap), 1-methylnaphthalene (C1-Nap), 2-methylnaphthalene (C2-Nap), 1,4,5-

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trimethylnaphthalene (C3-Nap), 1,2,5,6-tetramethylnaphthalene (C4-Nap), fluorene (Flu), 9-

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methylfluorene (C1-Flu), 1,7-dimethylfluorene (C2-Flu), 9-n-propylfluorene (C3-Flu), Phe, C1 to

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C4-Phe in the OSAs of this study, had relative compositions of PAHs as did weathered oil in situ

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sediments, while relative proportions of other PAHs, including Dbt, C1 to C3-Dbt, Chr, and C1 to

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C3-Chr resembled that of IHC, supporting lesser weathering at the beginning stage of oil exposure in

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the environment10 (SI-Figure S5). Characteristics that typically occur after weathering of HMM

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PAHs were not observed during preparation of OSAs. Shaking was performed to form OSAs, thus,

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the weathering of Nap, Flu, Phe, and their alky derivatives proceeded as the input of energy and

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microbial activities were experienced during formation of OSAs.

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Fecal pellets were produced in tanks in which clams were fed, such as OSAhigh, which in turn

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confirmed that M. veneriformis fed on OSAs. In order to evaluate bio-assimilation of oil components

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in OSAs, carbon and nitrogen isotopic ratios in soft tissue of M. veneriformis were measured (see

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Supporting Information), however, there was no evidence for assimilation of oil-derived materials

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during the experiments (SI-Figure S6). Results of several previous studies indicated that some

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marine mollusks possess detoxification enzymes, especially with cytochrome P450 (CYP 450) 14

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catalyzed enzymatic reactions such as by benzo[a]pyrene-hydroxylase, ethoxycoumarin O-

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deethylase, and N,N-dimethylaniline N-demethylase, which can biotransform PAHs44,45,46.

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Nevertheless, due to high hydrophobicity in stable lipid-rich tissues, bivalves usually have been

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reported to be a media as retaining xenobiotic contaminants47.

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In general, the major route of exposure for LMM PAHs is via water, while GMM PAHs

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generally accumulate via an intake of particulate materials from sediments48,49. In this study, LMM

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PAHs were more abundant than HMM PAHs in OSAs. The concentration of Nap fluctuated among

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LMM PAHs, of which accumulation was seemingly controlled by osmotic uptake, such that slight

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variations in rates of filtration and elimination by M. veneriformis resulted in differences in rates of

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accumulation. This is because relatively greater hydrophilicity (i.e., the log octanol-water partition

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coefficient, log Kow) of Nap was less (3.3) than those of other targeted PAHs (SI-Table S4). Among

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alkylated PAHs during 30 days, bioaccumulation was proportional to log Kow of PAHs, such that

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PAHs with greater log Kow were more accumulated by clams (SI-Figure S7). The TPAH in Group I

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gradually increased until the end of 50-d experiment without any inflections, with C2-Phe accounting

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for >75% of the total concentration of PAHs. In spite of the regular and lasting intake of OSAs, the

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TPAH in Group II included not only LMM PAHs (C1- to C3-Dbt) but also HMM PAHs such as

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alkylated chrysene (C1- to C3-Chr) reported to exhibit greater log Kow (refer to SI-Table S4).

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Therefore, PAHs of Group II did not follow predictions based on log Kow, although substituted,

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chrysene compounds have been reported to exhibit greater log Kow (Chr = 5.81, C1-Chr = 6.42, C2-

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Chr = 6.88, C3-Chr = 7.44) (refer to SI-Table S4, references given in table only).

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Theoretically, by exposing a much greater surface area of oil to microbial communities, the

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formation of OSAs would increase rates of degradation of oil by associated microbes, however,

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studies of this process are scarce50. Bioremediation of spilled oil by PAHs-degrading microbial 15

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communities could mitigate bioaccumulation of PAHs by benthic organisms. In our OSA-

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contaminated environment, Proteobacteria (especially Gammaproteobacteria) responded rapidly to

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greater concentrations of PAHs by increasing both abundance and diversity. This means that they

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have potential to degrade PAHs that tend to accumulate on surfaces of OSAs. One of the major

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insights provided by the results of the present study was that changes in microbial community

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composition over a 50-d period was observed in the various OSA treatments. As a result,

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bioaccumulation of PAHs by clams was influenced by several factors, including chemical properties

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and exogenous degradation such as chemical weathering and/or microbial degradation.

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Several studies, in marine environments, have described species of Gammaproteobacteria as

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key transformers of PAHs and that they respond immediately to crude oil spills, even within a day or

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so13,51–54. Results of this study generally confirmed this quick microbial response to OSAs. For

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instance, a rapid recolonization by microbial communities with some selected species was observed,

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particularly in the presence of OSAhigh. The associated microbial taxa responding most rapidly to oil-

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soaked organic-rich suspended particles included mainly the class Gammaproteobacteria

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Pseudomonas, Porticoccus, Cycloclasticus, Methylophaga, and Alcanivorax spp. Such rapid changes

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in composition in response to OSAs by microbial communities persisted for almost 2 months (50

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days in our study). This result suggests that Gammaproteobacteria could be used to control

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bioaccumulation of PAHs in marine environments.

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Similarly, increases in abundances of species of Cycloslasticus and Alcanivorax have also been

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reported in environments contaminated by petroleum hydrocarbons55–61. Pseudomonas has been

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reported to degrade Nap, Ant, Phe, and Flu in various types of terrestrial soils62–66 and is thought to

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be involved in co-metabolization of Flu67. Although Thioprofundum was considered to be mesophilic,

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anaerobic, and sulfur-oxidizing bacterial community occurred in hydrothermal areas68, the decrease 16

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of concentrations of Dbt and alkylated Dbt in clams also could be explained by the increase of

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Thioprofundum, which could have accelerated desulfurization of sulfur heterocycles. P. litoralis and

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P. hydrocarbonoclasticus have been reported to be specialized hydrocarbon-degrading bacteria living

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in association with marine phytoplankton69. In this study, by Day 50, at the end of experiment, P.

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litoralis composed about 21% of the entire bacterial composition in the OSAhigh treatment, which

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was almost 42-fold greater in relative abundance than in the Control treatment (0.6%). A. sediminis

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has been only recently isolated from marine sediment70, hence studies of biodegradation by that

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species are limited. However, A. sediminis was considered one of the hydrocarbon degraders because

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relatively great abundance was evidenced in the OSA treatments compared to that in the Control.

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Overall, bioaccumulation of PAHs into soft tissues of M. veneriformis was related to temporal

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changes of hydrocarbon-degrading bacterial compositions and abundances. Such an association

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between bioaccumulation and microbial degradation should be taken into account when handling and

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utilizing the OSAs in remediating oil spills. Of course, there might be differences in the microbial

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communities that decompose OSA depending on the type and characteristics of crude oil. In

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particular, what mechanisms are involved in degradation of PAHs by microbes is the limit of this

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study and further studies are needed.

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The primary goal of the present study was to address the role of microbial degradation

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associated with bioaccumulations of PAHs by benthic organisms. The results presented here indicate

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the importance of Porticoccus spp., which can use PAHs as a source of carbon. Some of the results

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presented here provide insight into how OSAs are related to bioaccumulation by bivalves and

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biodegradation by microbes. Based on the results of this study, we can consider ways to promote the

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formation and degradation of OSA by injecting fine sediment particles and oil-degrading bacterial

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agents in offshore oil spill accidents. To our knowledge, no other study has focused on 17

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bioaccumulation of OSAs in association with microbial communities. Thus, the present study

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appears to be the first step, in developing protocols for OSAs to remediate oil spills.

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ASSOCIATED CONTENT

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Supporting Information

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Additional details of the methods, results, and discussions are given for 1) carbon and

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nitrogen stable isotopic ratios in soft tissue of M. veneriformis (Supporting Materials and Methods),

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2) comparison of carbon and nitrogen stable isotopic ratios in clams (Supporting Results), 3) mini-

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review of bioaccumulation of PAHs in marine organisms (Table S1), 4) baseline information of

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sediment properties (Table S2), 5) experimental conditions of preparation of the OSAs (Table S3), 6)

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list of target PAHs compounds measured by GC/MSD (Table S4), 7) GC/MSD conditions for the

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analyses of PAHs and alkylated PAHs (Table S5), 8) mean concentration of PAHs in soft tissue of

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clams under OSAs feeding experiment (Table S6), 9) result of Mann-Whitney U-test for comparison

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of replicates per treatment in total PAHs (Table S7), 10) spearman rank correlation results for

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concentrations of 20 PAHs in soft tissue of clams (Table S8), 11) result of Kolmogorov-Smirnov

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normality test for concentrations of PAHs in soft tissue of clams over the period of 50 days in OSAs

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feeding treatments (Table S9), 12) total PAHs concentration in sediment (Figure S1), 13) PAHs and

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alkylated PAHs in OSAs (Figure S2), 14) concentrations of 25 PAHs in soft tissue of clams (Figure

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S3), 15) PAHs compounds in soft tissue of clams by the bioaccumulation of OSAs feeding

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experiment (Figure S4), 16) relative compositions of PAHs and alkylated PAHs in crude oil,

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sediments contaminated by Hebei Spirit oil spill, 17) OSA made of IHC (Figure S5), 18) stable

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isotopic ratios of carbon and nitrogen in clams, OSAs, sediment, and crude oil (Figure S6), and 19)

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relationship between Log Kow and concentration of PAHs in soft tissue of clams (Figure S7). (PDF)

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ACKNOWLEDGMENTS

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This work was supported by projects entitled “Marine ecosystem-based analysis and decision-

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making support system development for marine spatial planning (20170325)” and “Integrated

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management of marine environment and ecosystems around Saemangeum (20140257)” funded by

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the Ministry of Oceans and Fisheries of Korea (MOF) granted to JSK. JPG was supported by the

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Canada Research Chair program, the 2012 "High Level Foreign Experts" (#GDT20143200016)

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program, funded by the State Administration of Foreign Experts Affairs, the P.R. China to Nanjing

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University, the Einstein Professor Program of the Chinese Academy of Sciences and a Distinguished

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Visiting Professorship in the School of Biological Sciences of the University of Hong Kong.

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Finally, we thank Ms. Ji Hyun Kim for the technical assistance for the part of experiments.

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(4) Sammarco, P. W.; Kolian, S. R.; Warby, R. A.; Bouldin, J. L.; Subra, W. A.; Porter, S. A.

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FIGURE CAPTIONS

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Figure 1. Schematic overview showing the study design of the OSAs feeding experiment. (A)

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Control and OSAs feeding treatments in constant-temperature water bath. (B) Experimental

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flow chart with information on the feeding materials and interval, water renewal, and sampling

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time. Seawater, Iranian Heavy Crude oil, and fine sediment particles (0.01%, n = 489) relative to sediment source and experimental treatments over time. A

664

phylogenetic representation of the taxonomic composition in experimental treatments (at Day 1,

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30, and 50), including Control (inner ring), OSAlow (middle ring), and OSAhigh (outer ring). The

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average relative abundance of each genus is plotted within the concentric rings, represented by

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the shaded cells, with higher relative abundance indicated by darker shades. The phylum to

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which each taxa belongs is indicated by the phylogenetic tree. Abundant taxa in Proteobacteria

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are labelled by the red outline. (B) Relative abundances of two dominant phyla, Proteobacteria

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(orange) and Bacteroidetes (blue), with total concentrations of Group II PAHs of OSAhigh

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treatments (black line) (*C1-, C2-, C3-Dbt, C1-, C2-, and C3-Chr). Acronyms for PAHs

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compounds refer to SI-Table S4). (C) The most abundant nine species (in the OSAs feeding

673

treatments) belonging to class Gammaproteobacteria. Blue lines and dots denote Control

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treatments, green lines and dots denote OSAlow treatments, and red lines and dots denote

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OSAhigh treatments.

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