Differential Toxicokinetics Determines the Sensitivity of Two Marine

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Differential toxicokinetics determines the sensitivity of two marine embryonic fish exposed to Iranian Heavy Crude Oil Jee Hyun Jung, Moonkoo Kim, Un Hyuk Yim, Sung Yong Ha, Won Joon Shim, Young Sun Chae, Hana Kim, John P. Incardona, Tiffany Linbo, and Jung-Hwan Kwon Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03729 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 17, 2015

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Environmental Science & Technology

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Differential toxicokinetics determines the sensitivity of two marine embryonic fish exposed

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to Iranian Heavy Crude Oil

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Jee-Hyun Jung†*, Moonkoo Kim†, Un Hyuk Yim† Sung Yong Ha†, Won Joon Shim†,

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Young Sun Chae , Hana Kim , John P. Incardona , Tiffany L. Linbo , Jung-Hwan Kwon§

7 8 9 10 11 12 13 14

†

†

‡

‡

†

Oil & POPs Research Group, Korea Institute Ocean Science & Technology, 41 Jangmok 1-gil Geoje, 656-834, Korea ‡ Environmental and Fisheries Science Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 2725 Montlake Blvd E, Seattle, WA 98112 USA §Division

of Environmental Science and Ecological Engineering, Korea University, Seoul 136713, Korea

15 16 17 18 19 20

*Corresponding author:

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Jee -Hyun Jung, Ph.D

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Oil & POPs Research Group, Korea Institute of Ocean Science & Technology

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391 Jangbuk-Ri, Jangmok-myon Geoje, 656-834, Korea

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Phone/Fax: +82 (55)639-8680/8689,

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E-mail: [email protected]

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Key word: Oil spills, embryo, Cardiotoxicity, Bioconcentration, PAHs 1 ACS Paragon Plus Environment


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Abstract

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Interspecific difference in the developmental toxicity of crude oil to embryonic fish allows the

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prediction of injury extent to a number of resident fish species in oil spill sites. This study

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clarifies the comparative developmental effects of Iranian Heavy crude oil (IHCO) on the

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differences of bio-uptake and toxic sensitivity between embryonic spotted sea bass (Lateolabrax

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maculates) and olive flounder (Paralichthys olivaceus). From 24 hrs after exposure to IHCO,

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several morphological defects were observed in both species of embryonic fish, including

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pericardial edema, dorsal curvature of the trunk, developmental delay and reduced finfolds. The

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severity of defects was stronger in flounder compared to sea bass. While flounder embryos

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accumulated higher embryo PAH concentrations than sea bass, the former showed significantly

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lower levels of CYP1A expression. Although bioconcentration ratios were similar between the

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two species for some PAHs, phenanthrenes and dibenzothiophenes showed selectively higher

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bioconcentration ratios in flounder, suggesting that this species has reduced metabolic capacity

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for these compounds. While consistent with a conserved cardiotoxic mechanism for petrogenic

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PAHs across diverse marine and freshwater species, these findings indicate that species-specific

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differences in toxicokinetics can be an important factor underlying species’ sensitivity to crude

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oil.

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Key word: Oil spills, embryo, Cardiotoxicity, Bioconcentration , PAHs

2 ACS Paragon Plus Environment

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■ Introduction

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Immediately after the Hebei Spirit oil spill (HSOS; 7th December, 2007), many marine

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species were found dead on rocky shores and beaches and more than 8,571 ha of land-based fish

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aquaculture facilities were directly affected by the crude oil. Hatching success rate in marine fish

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species declined to lower than 50% in the vicinity of the spill, and there has been continuing

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controversy about the impacts of residual oil

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that some resident species, in particular the greenling (Hexagrammos otakii), showed low

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abundance rates up to four years following the HSOS 3. This decrease was potentially due to an

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overlap of the timing of embryonic periods (from late November to December) of these species

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with the persistence of residual crude oil in nearshore areas. These findings prompted laboratory

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studies on the effects of crude oil from the HSOS on fish development.

1,2

. Surveys of juvenile fish populations revealed

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The effects of crude oil on fish early life history stages were first highlighted after the 1989

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Exxon Valdez oil spill contaminated nearshore spawning areas for Pacific herring (Clupea

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pallasi) and pink salmon (Oncorhynchus gorbuscha) with Alaska North Slope crude oil

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(ANSCO). Investigations carried out revealed that Pacific herring (Clupea pallasi) larvae

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hatching from adhesive demersal eggs at spawning sites affected by the spill had significantly

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higher frequencies of morphological deformities and cytogenetic abnormalities than those

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originating from unaffected sites4. In the ensuing decades, and further propelled by the 2010

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Deepwater Horizon incident in the Gulf of Mexico, the effects of crude oils on developing fish

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have been extensively characterized at the cellular, organismal, and population level. Exposure

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of embryos of Gulf killifish (Fundulus grandis) and zebrafish (Danio rerio) to oiled sediments

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resulted in reduced hatch and developmental anomalies

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significantly impact early life stage in fishes.

5-6

, suggesting oiled sediments can



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The syndrome of developmental defects and mortality initially documented in field-collected

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herring and salmon larvae after the Exxon Valdez spill was linked to polycyclic aromatic

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hydrocarbons (PAHs), an abundant fraction of most crude oils 7-9. Subsequent research using the

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zebrafish model showed that the etiology of the syndrome was a disruption of embryonic cardiac

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function and morphogenesis

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PAHs

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the Iranian heavy crude oil (IHCO) spilled from the Hebei Spirit, disrupt heart development 12,13

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in a diversity of fish species

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calcium ion channels essential for excitation-contraction (E-C) coupling in heart muscle cells 16.

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It was further shown that externally normal embryos of zebrafish, Pacific herring, and pink

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salmon surviving trace crude oil exposures have permanently malformed hearts and reduced

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cardiorespiratory performance well into the first year of life

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reductions in adult herring and pink salmon populations observed in both field and laboratory

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studies following Exxon Valdez 18-21.

11,12

10

. This cardiotoxicity was specifically attributed to three-ringed

, and it is now established that crude oils from different geological sources, including

14,15

, by a mechanism involving the blockade of potassium and

17

, potentially explaining the

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These findings from the Exxon Valdez and Deepwater Horizon oil spills further warrant the

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investigation on effects of the HSOS on fisheries and aquaculture resources, and indicate the

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necessity for careful studies of the developmental cardiotoxicity of IHCO in indigenous species.

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In a previous study, we characterized the effects of the IHCO spilled from the Hebei Spirit in the

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zebrafish embryo model, and compared its developmental toxicity syndrome to the well studied

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ANSCO. Despite some differences in the physical and chemical properties of these two

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geologically distinct crude oils, the cardiotoxicity syndrome in developing zebrafish embryos

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caused by both oils was remarkably similar

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spotted sea bass (Lateolabrax maculatus) occur in the Pacific Ocean and Yellow Sea, especially

13

. Olive flounder (Paralichthys olivaceus) and


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off Korea, Japan, and China. Both species are of commercial value, and olive flounder is the

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main species augmented by artificial seedling production and juvenile release

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breeding seasons are from late November to December, coinciding with the HSOS. They

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produce buoyant, pelagic eggs that develop in the upper water column, and rendering them

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susceptible to exposure during the sea surface oil spill incident. Furthermore, there is no

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systematic comparative study on the toxicity under the environmentally relevant exposure

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system between fish species and weathering status. In this study, we evaluated ranges and

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interspecies differences in toxicity on fish embryos exposed to fresh and evaporated IHCO under

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environmentally

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malformation and the level of CYP1A expression were also quantified for this purpose.

relevant

laboratory

conditions.

Bioconcentration

of

22,23

selected

. Their

PAHs,

110 111

■ Materials and Methods

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Fish embryos and exposure.

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Eggs of olive flounder (Paralichthys olivaceus) and spotted sea bass (Lateolabrax maculates)

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were artificially fertilized in a commercial fishery stations (IHWASANGROK and

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GEUNGYANG fishery station). At six hours after fertilization, eggs were transported to the

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laboratory in Korea Institute of Ocean Science and Technology, Geoje, Korea. Eggs were

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acclimated for 24 h flowing seawater at 16°C on a 16 h light/ 8 h dark photoperiod in the

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laboratory prior to the beginning of experiments. Floating eggs were collected and separated for

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the exposure experiments. Developing embryos (post-fertilization: optic vesicle stage) were

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incubated in a 40 L exposure tank [40 cm (width)×45 cm (length) ×24 cm (height)].

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Experimental groups were control (no oil-treated gravel column), fresh IHCO (FIHCO), and

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weathered (or evaporated) IHCO (EIHCO) treated gravel columns. EIHCO was prepared 5 ACS Paragon Plus Environment


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according to Jokuty et al. 24 by evaporating fresh oil to simulate the effect of volatilization, by

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which considerable fraction of oil is removed during the initial weathering process immediately

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after a spill 25. In short, 200 ml of fresh oil in 10 L flask was placed in a rotary evaporator with

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water bath filled with distilled water of 80±5℃. The flask was run at full speed (135 rpm) with

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an air flow of approximately 13 L/min through the flask. The flow rate was maintained by

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leaving the vacuum release stopcock open to atmospheric pressure while a small vacuum pump

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was running. The oil was evaporated for 48 hours to reach the almost constant total weight loss

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of approximately 30%. Gravels were coated with fresh or evaporated IHCO according to Jung et

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al., 13. Three gram of fresh or evaporated IHCO was loaded to 2 kg gravel by manually shaking

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them together in an uncoated stainless steel container. Coated gravels were air dried to a thin

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layer of soaked oil for 24 h and loaded into a generator column. Filtered water was continuously

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percolated (0.6 L h-1) through the column to produce oil contaminated seawater for toxicity

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studies. Water quality variables (i.e., pH, salinity and dissolved oxygen) were monitored daily.

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Incubation temperature was maintained at 16°C. Embryos were examined under a dissecting

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microscope, and abnormal or dead individuals were removed prior to the beginning of exposure.

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Embryos exposed for 48 hours to FIHCO and EIHCO gravel effluents. Toxicological endpoints

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evaluated were pericardial edema, spinal curvature, tail fin defect and developmental delay. In

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each experimental group, about 30,000 embryos were used in triplicates.

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Imaging of embryos

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Confocal microscopy

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Developmental defects of embryos from oiled gravel effluent on the pericardial edema, tail

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fin defect, spinal curvature and developmental delay were observed at 24 h and 48 h after the

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onset of exposure. Five hundred embryos from each triplicate were imaged and counted. At least


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ten embryos were randomly selected, mounted in 1.5% methylcellulose for viewing on the Zeiss

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Axioplan 2 compound microscope. Edema was quantified by measuring the pericardial area in

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left lateral images (collected on the stereoscope at 60x total magnification) using ImageJ

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(rsbweb.nih.gov/ij/) as described previously by Incardona et al.,

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increase in pericardial area by subtracting the average pericardial area measured in control

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embryos from all measures (controls and exposed), following conversion from pixels to µm2

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based on a stage micrometer image. Thirty embryos were fixed in 4% phosphate-buffered

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paraformaldehyde and processed for CYP1A and myosin heavy chain. Immunohistochemical

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staining was performed using rainbow trout CYP1A monoclonal antibody (Biosense Co. USA)

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and myosin heavy chain cardiac, muscle staining (Biosense Co. USA) for primary antibodies and

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goat anti-mouse IgG3 (g3) (Invitrogen Co., USA) and goat anti mouse IgG2b (g2b labelled Alexa

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488), (Invitrogen Co., USA) for secondary antibodies. Staining intensity and occurrence of

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CYP1A were evaluated by fluorescence microscopy in each embryo. Immuno-labelled embryos

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were mounted in 3% methylcellulose and imaged using a Zeiss LSM 780 Carl Zeiss Confocal

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system with Ar and HeNe lasers (Germany) (100 x total magnification) as described by Jung et

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al., 13.

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Quantitative comparison of AhR2 and CYP1A mRNAs among groups

26

. Edema was measured as an

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Total RNA was extracted from embryos (30 mg wt.) by Isogen (Wako, Japan). The purified

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total RNA was reverse transcribed into cDNA using a first-strand cDNA synthesis kit

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(Invitrogen, USA). Quantitative PCR was performed using a two-step procedure. The β-actin

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gene was used as a positive sham for real-time PCR. PCR was performed using an initial

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denaturization step for 5 min at 95°C, and then 40 cycles were run as follows: 30 s

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denaturization at 95°C, 30 s of annealing at 55°C, and 1 min of extension at 72°C. The ratio of 7 ACS Paragon Plus Environment


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optical density (OD) 260/280 was about 1.9 and the ratio of 260/230 was about 1.8 to 1.9. To

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quantify the mRNA expression level, the comparative CT methods (2-ΔΔCT method) was used in

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Roto-Gene Q (Qiagen, Germany) according to the manufacturer's instruction. All experiments

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were performed in triplicate. The specific primers and probes for flounder AhR2 (fAhR 2), sea

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bass AhR2 (sbAhR2), flounder CYP1A (fCYP1A) and sea bass CYP1A (sbCYP1A) sequences

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were designed using the ABI PRISM Primer Express software (Applied Biosystems, USA) and

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the details are shown in Table 1.

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Water and embryo concentration of PAHs

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PAH analyses

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The analyses were performed on two sample types: effluent water and fish embryo. Effluent

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water samples (approximately 2 liter) were serially extracted three times with 50 mL of

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dichloromethane using a separatory funnel. Fish embryos were freezing dried and samples (0.2 –

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1.0 g dry weight) were extracted in a Soxhlet for 16 h with 200 mL of dichloromethane. Samples

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were spiked with surrogate standards (naphthalene-d8, acenaphthene-d10, dibenzothiophene-d8,

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phenanthrene-d10, chrysene-d12 and perylene-d12) before extraction. A 10 mL aliquot of the

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extract was used for lipid content determination with the gravimetric method. The remaining

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extracts were extensively cleaned-up using deactivated silica/alumina column and HPLC with

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size exclusion columns. The eluted samples were concentrated and taken up in n-hexane, and

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then terphenyl-d14 was added before the chemical analysis using a gas chromatography

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(Hewlett–Packard HP6890) coupled with a quadrupole mass spectrometry (Hewlett–Packard

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HP5972). Quantification of the individual PAH components in the samples was performed using

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selected ion monitoring described in Yim et al.,27. Sixteen priority PAHs by US EPA and selected

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alkyl-substituted PAHs were analyzed as follows: (C0~C4) naphthalene, acenaphthylene, 8 ACS Paragon Plus Environment

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acenaphthene, (C0~C3) fluorene, (C0~C4) phenanthrene, anthracene, fluoranthene, pyrene,

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benz[a]anthracene,

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benzo[a]pyrene,

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(C0~C3) dibenzothiophene.

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Quality control/quality assurance

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Surrogate standards were used for accounting the recovery of PAHs in each sample. Recoveries

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of target PAHs (Sixteen priority PAHs) from the analyzed samples were 47~82%. For quality

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assurance of the fish embryo sample analysis the certified reference materials (CRM) NIST 1947

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provided by National Institute of Standards and Technology (Gaithersburg, MD, USA) were

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analyzed. PAH concentrations in the CRM were within the certified ranges. The PAH

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concentrations in the blank samples were not detected or lower than the detection limits of the

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method, which were 0.13 to 3.36 ng/g for biota and 0.09 to 5.67 ng/L for seawater. Relative

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percent differences of duplicate samples ranged from 0.19 (2-methylnaphthalene) to 19% (1-

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methylphenanthrene) for biota and from 0.13 (C4-fluorenes) to 21% (benzo[b]fluoranthene) for

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seawater.

(C0~C3)

chrysene,

indeno[1,2,3-cd]pyrene,

benzo[b]fluoranthene, dibenz[a,h]anthracene,

benzo[k]fluoranthene,

benzo[ghi]perylene,

and

207 208

Statistical evaluation

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All the data are expressed as mean ± s.d. In exposures involving treatment groups (e.g. control

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gravel and IHCO gravel), means were compared by a t-test. Differences between the control and

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exposed-groups were analyzed using one-way ANOVA followed by Duncan's test. Differences

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showing p< 0.05 were considered as significant. SPSS ver. 17.0 (SPSS Inc., USA) software

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package was used for statistical analysis.

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■ Results

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PAH concentrations in oiled gravel effluents and embryos

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Initial concentrations of total PAHs (∑PAHs) in oiled gravel effluent (OGE) were 12,100 ng/L

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for FIHCO and 10,800 ng/L for EIHCO (Table 2), declining to 4340 and 6420 ng/L for FIHCO

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and EIHCO, respectively, after 48 h of column flow. This was 41- to 46- times higher ∑PAH

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concentration than in the effluent of control clean gravel, which remained below 300 ng/L. The

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observed concentrations of individual PAHs also agreed well with those predicted using Raoult’s

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law 28. Relative compositions of PAH compounds were also similar for both FIHCO and EIHCO

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effluents (Table S1), with a dominance of naphthalenes (C0- to C4-) followed by

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dibenzothiophene, fluorine and phenanthrene groups. However, the artificial weathering of

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EIHCO did result in lower levels of the more volatile naphthalenes, and lower alkyl-substituted

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compounds, with a slight increase in less volatile, higher molecular weight compounds, such as

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C2- to C4-phenanthrenes. After 48 h column flow concentrations of C0- to C2-naphthalenes

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were decreased in both FIHCO and EIHCO to a much higher degree than the more hydrophobic

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alkyl-phenanthrenes and alkyl-dibenzothiophenes (Table S1).

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Flounder embryos accumulated higher levels of PAHs than did sea bass, and this species also

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differed in the changes of embryo PAH concentrations over time (Table 3). At 24 hours of

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exposure, sea bass embryos accumulated ∑PAH of 62,700 ng/g lipid and 67,900 ng/g lipid from

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FIHCO and EIHCO OGEs, respectively, with embryo concentrations increasing to 87,400 ng/g

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lipid and 103,000 ng/g lipid, respectively at 48 hours. ∑PAH concentrations in control sea bass

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embryos were only 15,800 ng/g lipid. In contrast, embryo PAH levels peaked in flounder

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embryos at 24 hours, with ∑PAH 369,000 ng/g lipid and 883,000 ng/g lipid from FIHCO and

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EIHCO OGEs, respectively, dropping (but remaining higher than sea bass) at the 48-hr time 10 ACS Paragon Plus Environment

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point to 280,000 ng/g lipid and 397,000 ng/g lipid, respectively. ∑PAH levels were 83,200 ng/g

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lipid in flounder embryos exposed to control effluent. Embryos exposed to control effluents

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exhibited only 3 to 14 times lower ∑PAH than the embryos exposed to oiled gravel effluent,

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while ∑PAH in the oiled effluent water was 41 to 46 times higher than in the effluent from

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control clean gravel. This relatively small difference in PAH concentration between oiled and

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control embryos is mainly due to elevated levels in the control embryos. The high levels in

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control embryos are most likely due to maternal transfer of PAHs from adult fish to embryo. It

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also should be noted that PAH in flounder embryos after 24 h exposure to EIHCO is highly

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elevated when compared with those exposed to FIHCO, while the concentration difference is not

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evident in the exposure waters. Due to a dynamic condition in the exposure system, embryos

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which are floating on the water surface can be contaminated by oil slicks or droplets which are

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not completely removed during the initial wash-out of oil-coated gravels prior to the exposure. In

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spite of the discrepancy, all presented PAH concentration data exhibit consistent trend.

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The most dominant PAHs in the both species were dibenzothiophene homologues

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(4,450~351,000 ng/g lipid) followed by naphthalene group (3,600~274,000 ng/g lipid) (Table

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S2). Examination of individual embryo PAH concentrations (Table S2) revealed marked

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differences in the concentration ratios, calculated as the ratio of embryo PAH to water PAH, both

255

between sea bass and flounder and among individual PAHs within each species (Table S3). In sea

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bass, most of the embryo to water concentration ratios were below 20 and followed an expected

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trend of increasing ratios with increasing hydrophobicity (increasing molecular weight and

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degree of alkylation). In contrast, the concentration ratios for flounder were both markedly

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higher and did not follow a simple pattern associated with hydrophobicity, e.g., above 100 for

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phenanthrenes and as high as 254 for 2/3-methyldibenzothiophene, but more similar to sea bass 11 ACS Paragon Plus Environment


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for naphthalenes and fluorenes (Table S3).

262 263 264

Morphological abnormalities from crude oil exposure A suite of defects, including pericardial edema, reduced finfolds, dorsal body axis curvature

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and developmental delay were observed in embryos of both species exposed to FIHCO and

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EIHCO gravel effluent (Fig. 1). By some measures flounder embryos were more severely

267

affected. At 48 h after exposure, pericardial edema was observed at very high frequencies,

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99.5±0.7 and 97.5±3.5 % in flounder (FIHCO and EIHCO, respectively), and 98.2±2.4 and

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98.5±2.0 % in spotted sea bass (FIHCO and EIHCO, respectively) (Fig. 1a). Frequencies of

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reduced finfold growth was similarly high in both species, at 93.5±2.0 and 88.6±16.1%

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(FIHCO and EIHCO, respectively) in flounder, and 91.6±11.8 and 84.8±20.0 in embryonic sea

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bass (FIHCO and EIHCO, respectively) (Fig. 1b). There were no statistically significant

273

differences between embryonic flounder and sea bass in either exposure group. Developmental

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delay, indicated by reduced dorsal rotation of the head, immature pectoral fin buds, and pigment

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pattern, was statistically significant more frequent in embryonic flounder than spotted sea bass,

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occurring in 27.9±18.4% and 30.4 ±12.1 % of FIHCO and EIHCO exposure groups in

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flounder, respectively (Fig. 1c). Dorsal body axis curvature was also observed more frequently in

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embryonic flounder, occurring in 96±10.3% and 97±8.9 of FIHCO and EIHCO exposure

279

groups, respectively, compared to 66 ± 5.2% and 79 ± 8.1% in sea bass (Fig. 1d). There were

280

statistically significant differences between embryonic flounder and sea bass in either exposure

281

group (p

Differential Toxicokinetics Determines the Sensitivity of Two Marine

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