Bioaccumulation of Polycyclic Aromatic Hydrocarbons (PAHs) by the

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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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Environmental Science & Technology

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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

e

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

ACS Paragon Plus Environment


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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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29



635

FIGURE CAPTIONS

636

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

Page 30 of 37

637

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

638

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

639

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,

665

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

666

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

667

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

668

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

669

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

670

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

671

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

672

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

674

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

675

OSAhigh treatments.

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Bioaccumulation of Polycyclic Aromatic Hydrocarbons (PAHs) by the

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