Evaluation of Gene Bioindicators in the Liver and Caudal Fin of

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Ecotoxicology and Human Environmental Health

Evaluation of gene bioindicators in the liver and caudal fin of juvenile Pacific coho salmon in response to low sulfur marine diesel seawater-accommodated fraction exposure Jacob J Imbery, Craig Buday, Rachel C Miliano, Dayue Shang, Jessica Round, Honoria Kwok, Graham Van Aggelen, and Caren C. Helbing Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b05429 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Environmental Science & Technology

Evaluation of gene bioindicators in the liver and caudal fin of juvenile Pacific coho salmon in response to low sulfur marine diesel seawater-accommodated fraction exposure

Jacob J. Imbery†, Craig Buday‡, Rachel C. Miliano‡, Dayue Shang‡, Jessica M. Round†, Honoria Kwok‡, Graham Van Aggelen‡, and Caren C. Helbing*†

†Department

of Biochemistry and Microbiology, University of Victoria, Victoria, British

Columbia, V8P 5C2, Canada ‡Pacific

& Yukon Laboratory for Environmental Testing, Pacific Environmental Science

Centre, Environment and Climate Change Canada, North Vancouver, British Columbia, V7H 1B1, Canada

*Corresponding

Author: Caren C. Helbing

E-mail: [email protected] Phone: 1 (250) 721-6146 Fax: 1 (250) 721-8855

Running title: Marine diesel biological effects in seawater Key words: oil spill; non-lethal sample; polycyclic aromatic hydrocarbons; salmon; sublethal effect; low sulfur marine diesel; sublethal biological effects; transcript; gene expression

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Abstract

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Low Sulfur Marine Diesel (LSMD) is frequently involved in coastal spills and monitoring

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ecosystem damage and the effectiveness of cleanup methods remains a challenge. The present

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study investigates the concentration and composition of polycyclic aromatic hydrocarbons

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(PAHs) dispersed in LSMD seawater accommodated fractions (WAF) and assesses the effects of

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exposure on juvenile coho salmon (Onchorhynchus kisutch). Three WAFs were prepared with

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333, 1067, and 3333 mg/L LSMD. The sum of 50 common PAHs and alkylated PAHs (tPAH50)

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measured by gas chromatography/triple quadrupole mass spectrometry showed saturation at

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~90 mg/L for all WAFs. These WAFs were diluted 30% for 96 h fish exposures. qPCR was

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performed on liver and caudal fin from the same genotypically-sexed individuals to evaluate

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PAH exposure, general and oxidative stress, estrogenic activity, and defense against metals.

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Excluding metal response, our analyses reveal significant changes in gene expression following

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WAF exposure on juvenile salmon with differential sensitivity between males and females. The

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3-methylcholanthrene responsive cytochrome P450-1a (cyp1a) transcript exhibited the greatest

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increase in transcript abundance in the caudal fin (10-18-fold) and liver (6-10-fold). This

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demonstrates that cyp1a is a robust, sex-independent bioindicator of oil exposure in caudal fin,

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a tissue that is amenable to non-lethal sampling.


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Introduction

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Petroleum oil products are a complex mixture of chemical compounds of tens of

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thousands of hydrocarbons and other related hetero-compounds including polycyclic aromatic

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hydrocarbons (PAHs).1,2 The general toxic, mutagenic, and carcinogenic properties of PAHs have

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been well-documented3,4 and analytical methods for quantifying many of the high-priority PAHs

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commonly dispersed from oil spills developed.5 However, the complexity and diversity of

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petroleum products has made toxicological assessments challenging, particularly in determining

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sublethal deleterious effects on marine organisms.

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Of particular note are identifying and monitoring biological effects as a consequence of

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exposure to petroleum oil-derived substances that partition into the aqueous phase after the

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spilled oil has been removed through clean-up efforts. Previous work has demonstrated that

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water-accommodated fractions (WAFs) of various petroleum oil types induce endocrine

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disruption leading to reproductive consequences6–8; impair growth, development, cardiac

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function, hypoxia tolerance, and behavior in marine fish9–12; and disturb microbiota13 and coral

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populations.14 During an oil spill response, there is a need to quickly and accurately determine

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whether or not clean-up efforts have been effective in mitigating biological effects.

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PAHs induce transcription of downstream aryl hydrocarbon receptor (AhR)-responsive

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genes in animals.15 Cytochrome P450-1a (cyp1a) gene induction by AhR is a widely-used

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biomarker of PAH exposure in aquatic organisms.16–18 Due to cross-talk between AhR and

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estrogen receptor (ER) pathways,19 PAH exposure also has a well-characterized estrogenic

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effect on exposed fish that induces differential expression of mRNA required for oocyte

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maturation.20,21 Measuring the abundance of sensitive gene transcripts can therefore indicate 3 ACS Paragon Plus Environment


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biological responses to PAHs, endocrine disruption, as well as stress.22–27 Typically, liver tissue is

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used to assess toxicological effects on these pathways, yet current methods of sampling require

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animal sacrifice or surgical methods that are not amenable to field sampling scenarios.17

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Non-lethally obtained tissue samples are more desirable to detect and assess pollutant

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exposure. The caudal fin is a readily accessible tissue present on all fish that can be sampled

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with minimal distress to the animal and allows for their immediate release following sample

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collection. Furthermore, caudal fin sampling allows for repeated measurements from a single

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organism during long-term exposures or monitoring. We have previously demonstrated that

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the caudal fin transcriptome is capable of indicating exposure to metals and estrogens in

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salmonids.28,29 It is plausible that the caudal fin could also be used to assess biological

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responses to oil spills.

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In North America’s Pacific northwestern coastal waters, low sulfur marine diesel (LSMD)

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is historically the primary petroleum oil product involved in large (>30 tonne) spills.30,31 This

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region is one of the busiest marine routes in the world that shares critical habitats, migration

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routes, and diverse fisheries including salmonid species.32 Compared to crude oils, relatively

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little is known about the toxic effects of LSMD in the context of seawater.

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Herein, we generated LSMD WAF in seawater using multiple LSMD concentrations and

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then measured the extent of PAH solubilisation using our recently developed micro-extraction

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method.33,34 To properly replicate ocean spill conditions, WAFs were made with seawater as

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salinity has a significant effect on PAH solubility.35

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We also investigated the sublethal effects of LSMD WAF exposure on genetically-verified male and female juvenile coho salmon (Onchorhynchus kisutch) by targeted gene transcript 4 ACS Paragon Plus Environment

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abundance changes indicative of PAH exposure, oxidative stress, general stress, estrogenic

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activity, and metal exposure in the liver and caudal fin. Coho salmon were chosen as they are

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an ecologically- and economically-relevant fish subject to the effects of oil spills. To our

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knowledge, this is the first report evaluating the effects of LSMD on gene transcript levels and

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the utility of the caudal fin in identifying LSMD exposure effects.

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Material and Methods

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Source of seawater

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A fresh supply of seawater was used for the toxicity testing at the Pacific & Yukon

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Laboratory for Environmental Testing (PYLET) in North Vancouver, British Columbia. The

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seawater supply was continuously pumped into the laboratory from the Burrard Inlet, BC, at a

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depth of 33 m and sand-filtered. The seawater typically has a salinity of 26.2 g/L and a pH of

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7.7-7.9.

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WAF generation WAF preparation followed the method of Singer et al36 with minor modifications37 as

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indicated below. Each oil loading concentration to generate the WAF was prepared individually

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with three test concentrations and a control based on a logarithmic series of increasing loading

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concentrations. In a range finding experiment, we tested fish exposure concentrations of WAF

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prepared with up to 3200 mg/L LSMD which is higher that what would be expected during a

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marine oil spill. No mortality was observed when fish were exposed to WAF prepared with this

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high LSMD concentration during a 96 h exposure. Based upon this and logistical considerations, 5 ACS Paragon Plus Environment


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three exposure concentrations were selected below this LSMD concentration and the WAFs

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were prepared as follows.

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The WAF was prepared in a covered holding area outside the laboratory in four large

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Nalgene® mixing vats which had spigots on the bottom to draw off the WAF without disrupting

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the surface layer of oil. The mixing vats were placed on large magnetic stir plates with 10 cm

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spin bars at the bottom of each vat. On Day -1, the mixing vats were filled with 155 L of

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seawater. The mixing speed was set to 200 rpm, which did not create a vortex. Three LSMD-

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containing WAFs (low, medium, and high concentration) were prepared by adding 51.62,

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165.39, and 516.62 g of LSMD to 155 L seawater for nominal loading concentrations of 333,

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1067 and 3333 mg/L LSMD. A seawater only vat was also prepared. The lids were sealed with

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duct tape and there was approximately 5 cm of headspace in the mixing vat. The fuel oils in the

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mixing vats were mixed for 18 h and then the WAF was allowed to settle for 6 h.

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On Day 0, a screw-on washer connected to Tygon® tubing was attached to the bottom

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spigot of each mixing vat. The WAF was drawn into four 90 L cubic vats lined with clear plastic,

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starting with the control and proceeding from the lowest concentration to highest

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concentration. At this time, the initial T = 0 h 40 mL subsamples (100% WAF) were sampled for

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chemistry analysis. The transfer vats were transported into the environmental test chambers

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where they were manually stirred with a Teflon® stir rod for several seconds.

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Marine-acclimated coho salmon exposure tests Adapted Environment Climate Change Canada standard methods38 were used in this study with coho salmon smolt substituted for rainbow trout as the test species. The fish were 6 ACS Paragon Plus Environment

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fed daily with 100 mL of BioVita Fry 1.2 mm Nutra Plus pellets per tank of approximately 500

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fish prior to the exposure. The fish were not fed during the exposure. All procedures were

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performed under the University of Victoria Animal Use Protocol #2017-011 under the auspices

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of the Canadian Council for Animal Care. The certified disease-free salmon were obtained from

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the Department of Fisheries and Oceans Canada Chilliwack Hatchery on June 30, 2017. The

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smolts were acclimated to seawater at PYLET with a salinity of approximately 26±2 g/L and a

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temperature of 13±2 °C prior to testing. Fish mortalities during the acclimation and holding

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period did not exceed the maximum of 2% per week preceding the toxicity tests.

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Using a submersible pump, the WAFs were transferred into the corresponding test

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vessels for the marine exposure tests arranged according to SI Figure S1. The test vessels

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contained 42 L of seawater and 18 L WAF for a final volume of 60 L at 15±1 °C corresponding to

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final nominal exposure loading concentrations of 0 (control), 100 (Low), 320 (Medium), and

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1000 (High) mg/L LSMD. No mortalities were observed during the experiment. Eight replicate

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tanks were prepared per treatment with 5 smolts per tank. The tanks were covered with lids to

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minimize evaporation and the loss of volatile hydrocarbons from the WAFs. The test solutions

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were aerated at 6.5±1 mL/min.L to maintain adequate levels of dissolved oxygen saturation for

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the smolts. The following water quality parameters were measured: pH, dissolved oxygen,

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temperature and salinity (SI Table S1). Phenol reference toxicant tests ensured that the

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organism sensitivity was within acceptable quality control warning chart limits.

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Chemistry subsampling



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Chemistry subsamples were taken at T = 0, 24, 48, 72, and 96 h. Twenty-five mL was

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taken from each tank and samples were pooled from paired tanks (SI Figure S1) for a total of

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four aliquots of 50 mL collected per concentration per day. Samples were collected into 60 mL

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trace clean clear glass vials using 10 mL Eppendorf automatic pipettes. Following sample

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collection, 2 mL of dichloromethane (DCM) was added immediately and then vortexed for

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sample preservation. All the samples were held between 1-23 days at 4 °C in the dark until

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analysis.

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For sample extraction, 2 mL of acetonitrile (ACN) was added to each sample vial.

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Samples were vortexed and then centrifuged to separate the aqueous and organic layers. The

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bottom organic layer was transferred into a 4 mL liquid chromatography vial using a glass

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Pasteur pipette and the sample was made up to 2 mL with DCM. The samples were vortexed

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and then transferred into 2 mL amber glass gas chromatography vials for instrument analysis

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using gas chromatography/triple quadrupole mass spectrometry.5

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PAH analytical chemistry Our ability to completely characterize oil spill contaminants dispersed into the aqueous

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phase is limited due to the complexity of the sample and the limited number of available

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standards. Furthermore, the cocktail of compounds dispersed in an oil spill can vary greatly

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depending on the type of oil involved. Consequently, the extent of aqueous oil contamination is

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often quantified using a limited subset of more readily-characterized hydrocarbons, such as

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BTEX, or a broader subset including LEPH/HEPHs, PAHs, and related alkylated compounds.

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These subsets are considered to be the most important indicators of oil toxicity.39 8 ACS Paragon Plus Environment

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Because PAHs are typically more persistent in the environment than their monocyclic

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counterparts, oil contamination and exposure are often described in terms of individual PAH

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concentrations or by a sum of those individual PAH concentrations (i.e., tPAH).40

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A total of 50 PAHs (tPAH50) (23 parents and 27 alkyl homologs) were targeted for

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analysis (SI Table S2). This list includes all PAH and related hetero-polycyclic compounds

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reported as part of the standard PAH analysis performed for the Deepwater Horizon Natural

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Resource Damage Assessment (SI Table S3).41

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We developed a micro-extraction method33,34 to deliver timely analysis of 320 samples

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that were collected from the four WAF concentrations. Traditional PAH analysis requires mixing

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of 1 L water matrix with 200 mL of toxic DCM as the extraction solvent. Use of such large

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sample volumes would have compromised the exposure study due to volume loss in the fish

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tanks and long turn-around times. This trace level tPAH50 method requires only 50 mL of

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sample matrix and 2 mL of DCM as extraction solvent. Our method has passed eight separate

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rounds of proficiency testing organized by the Canadian Association for Laboratory

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Accreditation Inc. (CALA) and has been accredited under ISO17025. For several alkylated PAHs,

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we reported concentrations for both individual PAHs and grouped alkyl homolog PAHs (i.e., 1-

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methylnaphthalene and C1-naphthalenes, which includes 1-methylnaphthalene). To avoid

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double counting in the present study, the individual alkylated PAHs were excluded in the

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tPAH50 summations.

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The handing of volatile petroleum hydrocarbons (BTEX), light extractable and heavy

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extractable petroleum hydrocarbons (LEPH/HEPHs) are discussed in the Supporting Information.

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Dissections At 96 h, fish were individually euthanized using 0.3% sodium bicarbonate buffered (pH

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7) tricaine methane sulfonate (Aqua Life, Syndel Laboratories, Nanaimo, BC, Canada). The

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weight and fork length of each fish was measured and recorded immediately prior to

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dissection. Whole liver and caudal fin were collected into separate 1.5 mL microfuge tubes

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containing 1 mL RNALater (Ambion, Foster City, CA, USA). The tissues were stored at 4 °C

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overnight, then kept at -20 °C prior to the delivery to the University of Victoria for RNA isolation

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and sex genotyping.

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Isolation of total RNA and preparation of cDNA Caudal fin and liver tissue samples were randomized to remove processing bias. RNA

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was isolated from 2 cm tail tips or 3 mm3 liver samples using 700 µL TRIzol reagent, then

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homogenized for 6 min at 24 Hz in a Retsch MM301 mixer mill (ThermoFisher Scientific Inc.,

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Ottawa, ON, Canada) in microtubes containing a 3 mm tungsten carbide bead (Mixer mill rack

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was rotated 180 degrees halfway through homogenization). In order to increase RNA yield, 20

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µg glycogen was added to caudal fin samples prior to alcohol precipitation. RNA concentration

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was determined using a Nanodrop ND-1000 (ThermoFisher Scientific). The extracted RNA was

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stored at -80 ᵒC, and 1 µg was converted into cDNA for qPCR using the Applied Biosystems Inc.

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(ThermoFisher) High-Capacity cDNA reverse transcription kit with RNase inhibitor. cDNA

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samples were diluted 20-fold with RNase- and DNase-free water prior to qPCR analysis.

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qPCR analyses 10 ACS Paragon Plus Environment

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Primer sets that were previously used on Chinook and sockeye salmon42,43 were

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rigorously validated for use on coho liver and caudal fin samples as outlined in Veldhoen et al

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with reference to MIQE guidelines (SI Table S4).44,45 The primer sets chosen amplify transcripts

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sensitive to (1) PAH exposure: aryl hydrocarbon receptor α (ahr) and 3-methylcholanthrene

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responsive cytochrome P450 CYP1A1 (cyp1a); (2) oxidative stress: superoxide dismutase (sod);

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(3) general stress: heat shock protein 70 (hsp70); (4) estrogenic activity: vitellogenin A (vtg),

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vitelline envelope protein gamma (vepg), and cytochrome p450 family 19 (cyp19); and (5) metal

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exposure: metallothionein A (mta). Glyceraldehyde 3- phosphate dehydrogenase (gapdh),

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ribosomal protein L8 (rpl8), and ribosomal protein S10 (rps10) were used in a geometric mean

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normalization. The mta primer set did not pass our validation criteria for use with caudal fin

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samples so it was only used to assay transcript abundance in the liver.

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All qPCR reactions were run in quadruplicate and reactions were performed in a 15 μL

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volume consisting of 10 mM Tris-HCl (pH 8.3 at 20 °C), 50 mM KCl, 3 mM MgCl2, 0.01% Tween

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20, 0.8% glycerol, 40,000-fold dilution of SYBR Green I (Life Technologies), 69.4 nM ROX (Life

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Technologies), 5 pmol of each primer, 200 μM dNTPs (Bioline USA Inc, Taunton, MA, USA), 2 μL

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of 20-fold diluted cDNA, and one unit of Immolase Hot Start Taq DNA polymerase (Bioline).

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Amplification reactions were subject to the following thermocycle conditions: an initial 9 min

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activation step at 95 °C, followed by 40 cycles of 15 sec denaturation at 95 °C, 30 sec annealing

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at 60 °C, and 30 sec polymerization at 72 °C. Specificity of target amplification was measured by

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the inclusion of reactions lacking cDNA (no DNA template control) and by subjecting completed

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runs to thermodenaturation analysis to confirm correct targeting. An additional inter-plate

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control comprising a universal cDNA sample present in all qPCR runs was included for each 11 ACS Paragon Plus Environment


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gene target. Replicate data for each cDNA sample were averaged and test gene targets

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normalized to the geometric mean of gapdh, rps10, and rpl8 during application of the

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comparative Ct method (ΔΔCt).46 Suitability of these normalizer transcripts was determined

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using Cronbach’s α (http://languagetesting.info/statistics/spreadsheets/Alpha.xls), Norm Finder

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(moma.dk/normfinder-software), and Best Keeper analysis (www.gene-

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quantification.com/bestkeeper.html). Primer set efficiencies are presented in SI Table S5.

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Isolation of genomic DNA and sex genotyping Genomic DNA (gDNA) was isolated from RNAlater-preserved caudal fin tissue and

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analysed as previously described43 with the following modifications: gDNA concentration and

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integrity was determined by Nanodrop quantification and agarose gels, respectively (data not

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shown). To determine the genotypic sex of the juvenile coho salmon, we used two different

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primer sets: U-sdY and OtY2-WSU that have been developed to target the male-specific Y

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chromosome of salmonids (SI Table S3).47–49 qPCR genotyping was performed on gDNA samples

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in duplicate and followed the same reaction conditions as described above. A fragment of the

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gapdh gene was used to normalize the presence or absence of the Y chromosome markers for

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all gDNA samples (SI Table S3). Assay performance was verified using multiple gDNA samples of

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visually confirmed male and female coho salmon obtained from Hartley Bay, Kitimat River, and

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Salmon River, BC (n=4 fish of each sex per site, a generous gift from Dr. Ben Koop, University of

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Victoria).

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Statistical analyses 12 ACS Paragon Plus Environment

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For all statistical analyses, padj ≤ 0.05 was used to denote significance. Genetic sex was

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used to separate the fish into male and female groups. Distribution of male and female coho

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salmon across the four treatment conditions was evaluated using the Fisher Exact test

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(http://www.quantitativeskills.com/sisa/statistics/fiveby2.htm). Statistical analyses of the

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mRNA abundance data were performed using R version 3.4.1 (R Foundation for Statistical

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Computing, Vienna, Austria). Relative fold changes were examined for normal distribution

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(Shapiro-Wilk) and unequal variances (Levene’s) and the data were non-parametric.

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Significance was determined using the Kruskal-Wallis test and pairwise comparisons using the

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Mann-Whitney U test.

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Results and Discussion

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WAF tPAH50 concentration and chemical composition

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All measured tPAH50 concentrations were ~90 µg/L in the 100% WAF regardless of the

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initial LSMD loading concentration (SI Table S6), which suggest that saturation of these

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components can be easily reached within the context of a spill scenario. These concentrations

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are lower than tPAH50 concentrations of WAFs derived from heavier oils made under the same

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concentrations and conditions.5

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Each WAF was diluted to 30% for the fish exposures which translated to ~30 µg/L

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tPAH50 (Figure 1 and details of the PAH data in the exposure water preparations can be found

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in SI Table S6). The most dramatic reduction in hydrocarbons occurred in all three

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concentrations between 0 and 24 h, with a 60 to 70% loss. PAH concentrations continued to

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drop during the exposure, resulting in a final tPAH50 concentration of ~2 µg/L by 96 h (Figure 13 ACS Paragon Plus Environment


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1). Monthly water quality sampling and chemical analysis of the seawater confirmed very low

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concentrations of volatile hydrocarbon compounds and PAHs prior to WAF generation (SI Table

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S7).

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The primary PAH class observed in WAF was the water-soluble, two-ringed

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naphthalenes while the three-ringed and larger compounds were minor constituents (Figure 2

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and SI Table S6). This is in contrast to heavier, high-sulfur oils in which greater quantities of

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three- to five-ringed compounds partition into WAF due to the surfactant effects of sulfur on

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hydrophobic PAHs.5,50 Over time, the lighter hydrocarbons (i.e., naphthalenes) showed the

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greatest loss. This loss occurred as the result of several processes, including dissolution,

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evaporation, and biodegradation.51 In the field of oil spill forensics, the term “weathering” has

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been used to describe such a loss of the lighter hydrocarbons. The extent of weathering is often

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represented by the total mass loss in comparison to the starting point. Based on the tPAH50

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results, the WAF experienced a faster weathering rate compared to heavier oils5 due to the

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relatively high amount of volatile compounds found in the sample. BTEX and LEPH/HEPH

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measurements were ineffective due to the high volatility of these substances (see

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Supplementary Information).

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Fish mortalities and morphometrics There were no fish mortalities in any of the exposure conditions during the experiment.

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The weight and fork length were similar between treatment groups and ranged between 6.7-

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7.4 g and 8.4-8.6 cm on average, respectively (SI Table S8).

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Sex genotyping The proportion of males and females was approximately 50:50 within each treatment

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group with no significant difference in overall distribution of sex between treatment groups (SI

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Table S9). The OtY2-WSU and the U-sdY qPCR results correctly identified known male and

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female controls (Figure 3). The OtY2-WSU primer set was able to sex-genotype with a greater

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resolution between male and females (minimum 3 Ct difference in transcript abundance) than

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the U-sdY primer set (minimum 2 Ct difference in transcript abundance). Thus, for coho salmon,

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the OtY2-WSU primer set provided greater certainty in differentiating between males and

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females than the U-sdY primer set (SI Figure S3).

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Sex genotyping is important in interpreting phenotype and can substantially impact

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transcriptomic results.43 This information is often overlooked in the evaluation of pollutant

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exposure effects, particularly in juvenile fish as they lack obvious physical sex characteristics.

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Despite the lack of phenotypic difference between juvenile males and females, sex-based

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differential response to WAF exposure is possible. The results from the sex genotyping were

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used to group juvenile fish as male or female for subsequent targeted transcript analyses.

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Transcript abundance of biological indicators in liver and caudal fin

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Assuming that PAH toxicity is proportional to PAH concentration52 and given the

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apparent equivalency in tPAH concentrations of the three WAF concentrations, we expected to

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see equivalent molecular responses between the fish exposed to the three LSMD WAF

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concentrations. Using routine qPCR methods,28 we examined established gene transcripts that

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are indicators of PAH exposure, general and oxidative stress, estrogenic activity, and metal 15 ACS Paragon Plus Environment


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exposure.42,43 We analysed paired liver and caudal fin samples to assess the efficacy of the

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caudal fin response to the response of a conventional tissue used of monitoring purposes.

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To assess the responsiveness of the liver and caudal fin tissues to PAH exposure, two

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well-documented PAH-responsive genes, ahr and cyp1a, were amplified (Figure 4). Ahr is a

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constitutively expressed gene that encodes the AhR protein which binds planar aromatic

308

hydrocarbons.16 Upon PAH binding, the ligand-receptor complex translocates into the nucleus

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and dimerizes with the AhR nuclear translocator (ARNT) inducing changes in the transcription of

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target genes such as cyp1a.53 The cyp1a gene encodes the cytochrome P450 CYP1A protein

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involved in PAH metabolism.17

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The levels of ahr and cyp1a transcripts were similar in both the liver and caudal fin of

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male and female seawater control fish (Figure 4). Significant ahr response to WAF exposure was

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observed in both the liver and caudal fin although the observed patterns differed between

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sexes and tissues (Figure 4). The liver was the most responsive tissue (~2-fold increase) with

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similar significant increases observed in males at all WAF concentrations (Figure 4). A similar

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magnitude significant increase was also observed in female liver in the medium WAF

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concentration while the low and high WAF concentration results were approaching significance

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(p=0.1 and 0.08, respectively; Figure 4). Transcript abundance of ahr was significantly increased

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by ~2-fold in the male caudal fin at all WAF concentrations whereas female caudal fin ahr

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transcript levels were not significant at any WAF concentration although the medium

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concentration approached significance (p=0.09; Figure 4). Our results are consistent with a

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modest induction of ahr transcript abundance reported in the liver of juvenile rainbow trout54


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and Japanese medaka embryos exposed to diluted bitumen WAF55 but neither study separated

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sexes.

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We found a significant increase between 6- to 10-fold in cyp1a transcript abundance in

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the liver of male and female juveniles with no significant difference between WAF

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concentrations (Figure 4). The cyp1a abundance levels were more strongly induced (11- to 18-

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fold) in the caudal fin compared to the liver by LSMD WAF exposure regardless of sex (Figure 4).

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The cyp1a transcript responses observed are consistent with previous findings of diesel,

331

diluted bitumen, and crude oil WAF exposure in rainbow trout liver, Atlantic cod larvae and

332

liver, and Japanese medaka embryos.18,54–56 Madison et al observed a strong relationship

333

between developmental malformations and cyp1a mRNA level in newly hatched Japanese

334

medaka.55 While reasonable concordance between hepatic cyp1a mRNA and CYP1A protein

335

induction as a consequence of marine diesel WAF exposure has been observed in rainbow trout

336

and Atlantic cod18,54, these studies both noted the difficulties in relating abundances to 7-

337

ethoxy-resorufin O-deethylation (EROD) activity; an assay that is commonly used to assess

338

CYP1A enzyme activity. These authors pointed out the difficulties in relating EROD activity to

339

transcript and protein levels due to the effects of antagonists present in the complex oil

340

mixtures on EROD activity.18,54 Regardless of the relationship, activation of the AhR-CYP1A

341

pathway is indicative of a biological response to diesel exposure requiring energetic resources

342

allocated to this detoxification pathway perhaps to deal with the reduced capacity to

343

metabolize oil components. The fact that the caudal fin shows a stronger cyp1a transcript

344

induction than the liver indicates that this tissue may be more sensitive to WAF exposure than

345

the liver for testing, without the need for animal sacrifice. These results are very promising for 17 ACS Paragon Plus Environment


346

use of the caudal fin as a quick, non-lethal sampling method that would be a great advantage

347

for biological assessment of fish in the environment.

Page 18 of 45

348

In male caudal fin, a similar strength cyp1a transcript response was observed in all WAF

349

concentrations (Figure 4). However, a distinctive induction pattern was observed in the female

350

caudal fin (Figure 4). While the cyp1a gene transcripts were also significantly elevated in all

351

three WAF concentrations, a significantly greater response was observed in the medium and

352

high WAF concentrations relative to the low concentration (Figure 4). This result is intriguing

353

because of the apparent PAH saturation in all of the LSMD WAFs (Figure 1 and SI Table S6). This

354

observation suggests that while the overall tPAH50 values may be the same between the LSMD

355

WAF concentrations tested, it is possible that the application amount influenced partitioning of

356

individual PAHs or other components (e.g. acid extractable organics) in such a way to lead to

357

differential biological effects. For example, anthracene levels are consistently 4-6 times higher

358

in the low concentration WAF compared to the high concentration WAF throughout the

359

experiment (SI Table S6). Further analyses of WAF composition and the relationship to

360

biological effects are warranted.

361

To evaluate WAF exposure-induced stress, we measured the transcript levels of genes

362

involved in general and oxidative stress pathways: hsp70 and sod (Figure 5). The hsp70 gene

363

encodes a chaperone protein involved in stabilizing partially folded proteins and protein

364

remodeling and has been linked to general stress pathways.57 The sod gene encodes superoxide

365

dismutase which catalyses the conversion of superoxide to less toxic hydrogen peroxide.58

366

General and oxidative stress in fish is strongly associated with hsp70 and sod biomarker up-

367

regulation and can cause reduced disease resistance and growth, and increased mortality.59 18 ACS Paragon Plus Environment

Page 19 of 45

368


No significant difference between male and female fish in seawater alone was observed

369

for hsp70 or sod transcripts in either liver or caudal fin (Figure 5). WAF exposure induced a

370

significant increase in both hsp70 (1.4-fold) and sod (2-fold) response in the liver of males at all

371

concentrations (Figure 5). Male caudal fin showed no significant response in hsp70 transcript

372

levels and a 1.4-fold increase in sod transcript abundance at the high WAF concentration

373

(Figure 5). The medium WAF concentration for male caudal fin sod transcripts was approaching

374

significance (p=0.1; Figure 5).

375

Female livers had a similar magnitude increase in hsp70 transcript abundance relative to

376

the controls in the medium and high WAF concentrations, but these values did not quite reach

377

significance (p=0.07 and p=0.1, respectively; Figure 5). A significant 1.7-fold increase in sod

378

transcript levels was observed in female livers at only the medium WAF concentration with a

379

trend towards a similar increase at the high WAF concentration (p=0.08; Figure 5). WAF

380

exposure did not induce a significant response in either of these gene transcripts in the female

381

caudal fin although the low WAF concentration approached a significant 0.8-fold decrease for

382

hsp70 transcript levels compared to the control (p=0.09; Figure 5).

383

Previous work on Atlantic cod larvae and juveniles and Japanese medaka embryos

384

evaluated exposures to WAF prepared from heavier oils and found evidence of general and

385

oxidative stress using a variety of bioindicators including increased abundance of hsp70 and sod

386

transcripts.18,56,60 Holth et al also examined the effects of ship-diesel WAF on mixed-sex juvenile

387

Atlantic cod and found evidence for increased glutathione S-transferase antioxidant enzyme

388

activity and lipid peroxidation in the gill; both indicators of oxidative stress.18



389

The present study indicates a modest stress response induced by LSMD WAF with males

390

being more sensitive than females. While previous fish studies did not distinguish between

391

sexes, increased sensitivity of oxidative stress pathways to PAH exposure has previously been

392

reported in male Nile crocodiles compared to females.61 As the genotypic sex is not often

393

reported in juvenile fish studies, our results indicate that sex should be taken into account

394

when evaluating stress responses to PAHs.

395

Page 20 of 45

These stress bioindicator results also indicate possible tissue-specific differences as the

396

liver had a greater response than the caudal fin. The caudal fin is in direct contact with the

397

WAF, so it is possible that a stress response did occur in this tissue, but at a much earlier time

398

point than that selected in the present study. Another possibility is that the caudal fin may not

399

be as sensitive to stress induced by LSMD WAF exposure. Based on previous studies mentioned

400

above, we expect that exposure to WAFs prepared from other oil types with different

401

compositions (such as heavy oils) may induce a stronger stress response in this tissue. Further

402

analyses of earlier time points and comparison of LSMD to other oil WAF exposures are needed

403

to address these possibilities.

404

The effect of WAF exposure on estrogenic activity was evaluated by measuring female-

405

biased gene transcripts encoding proteins involved in egg (vtg: vitellogenin A, vepg: vitelline

406

envelope protein gamma) and estrogen (cyp19: cytochrome p450 family 19) production (Figure

407

6). Adult female salmon express substantially higher levels of these three gene transcripts in

408

the liver than adult males.43 All three transcripts were detected and validated in both liver and

409

caudal fin in both sexes (Figure 6). Of these transcripts, only cyp19 abundance in the caudal fin

410

showed a female bias with a 1.5-fold significantly higher abundance in female caudal fin 20 ACS Paragon Plus Environment

Page 21 of 45


411

compared to males in the seawater controls (Figure 6). The liver did not show this female bias

412

in these immature juvenile fish (Figure 6).

413

A 2-fold increase in vepg transcript abundance in the liver tissue of males was induced

414

at the low and medium WAF concentrations (Figure 6). The abundance of vepg transcripts in

415

female liver or caudal fin of either sex were not affected by WAF exposure (Figure 6). However,

416

at high WAF concentrations, the vepg transcript levels trended downwards to 0.6-fold in the

417

caudal fin (p=0.08) and upwards to 1.4-fold in the liver (p=0.1) compared to controls in female

418

fish (Figure 6).

419

There was no significant effect of LSMD WAF exposure on vtg transcripts in any

420

condition, sex, or tissue although the female caudal fin showed a tendency toward a 0.6-fold

421

reduction in vtg transcript levels at the high WAF concentration (p=0.08; Figure 6).

422

While there were no significant changes in cyp19 transcript levels in male or female

423

livers or male caudal fin (Figure 6), a significant decrease in cyp19 transcript abundance up to

424

2.5-fold was observed in the female caudal fin (Figure 6). The low WAF concentration just

425

missed the significance cut-off at p=0.07 due to slightly higher variation in the data compared

426

to the other WAF concentrations.

427

Previous work on individual PAHs indicates that these substances can act as estrogen

428

receptor agonists possibly due to cross-talk between the AhR- and ER-mediated

429

pathways.19,20,62 The significant and nearly-significant differences compared to controls

430

observed in these estrogen-responsive gene transcripts (i.e. increase in vepg mRNA in male

431

livers, decrease of cyp19 mRNA in female caudal fin, and near significant decrease of vtg mRNA

432

in female liver) is suggestive that there was a perturbation in the estrogen signaling pathway. 21 ACS Paragon Plus Environment


Page 22 of 45

433

Previous work using subtractive suppressive hybridization on the liver of female guppies,

434

Poecilia vivipara, after a 24 h exposure to 10% (v/v) diesel WAF63 identified 27 differentially

435

expressed gene fragments that included increased cyp1a and decreased vtg consistent with our

436

findings.

437

As the majority of information regarding xenoestrogen effects on these gene transcripts

438

in differentiated tissues has come from adult male livers, it is becoming increasingly evident

439

that juvenile fish are responsive to estrogenic substances, perhaps more so than adult

440

males.28,64 We previously examined the response of juvenile rainbow trout liver and caudal fin

441

to a concentration range of 17α-ethinyl estradiol and established that, while the liver was more

442

sensitive, both the liver and caudal fin responded to this potent estrogen with increased vtg

443

and vepg transcript abundance while cyp19 transcripts were not affected.28 It is currently

444

unclear how these rainbow trout responses relate to the juvenile coho salmon in the present

445

study as the rainbow trout work did not distinguish between genetic sexes and the juveniles

446

were different ages. Further elucidation of the juvenile response to other estrogens and PAHs,

447

again keeping genotypic sex in mind, through analysis of additional time points and a broader

448

range of response gene transcripts is warranted.

449

The extent of heavy metal interaction resulting from WAF exposure was evaluated

450

through measurement of metallothionein A (mta) transcript levels (SI Figure S4). Our mta

451

primer set only passed our validation for use with the liver tissue and not for use with the

452

caudal fin (SI Table S5). In the liver, there was no significant change in mta transcript

453

abundance following exposure to WAF. Although not significant, there was a slight trend (up to

454

1.3-fold) toward increased mta transcript abundance at low and high WAF concentrations 22 ACS Paragon Plus Environment

Page 23 of 45


455

(p=0.08; SI Figure S4). These results suggest that the trace elements of heavy metals in LSMD

456

WAF did not affect the exposed fish to an appreciable degree.

457

In summary, LSMD WAF exposure clearly showed effects on PAH detoxification, stress,

458

and estrogenic pathways and the liver showed a broader range of significant transcript

459

responses compared to the caudal fin. Male salmon were particularly sensitive, accentuating

460

the importance of taking genotypic sex into account in biological effects assessments in juvenile

461

fish. Our data showed that when cyp1a is up-regulated in the liver of juvenile coho salmon, that

462

this up-regulation is apparent and even enhanced in the caudal fin of the same individuals.

463

However, the other limited targeted gene transcripts used in the present study did not show

464

this relationship and rather accentuated the differences in responsiveness between tissue

465

types. Nevertheless, the qPCR-based tools developed and validated herein for both liver and

466

caudal fin are poised for broader comparative WAF effects analyses with different oils. With the

467

limited targeted gene transcript approach presented herein, it is likely that additional affected

468

pathways were not identified with the targeted approach taken so far. For example, there is

469

some suggestion that diesel exposure can affect immune system components and metabolic

470

functioning in guppies and coral reef fish.63,65 Further in-depth RNA-seq analyses are in progress

471

to identify additional transcripts and pathways affected in the liver and caudal fin upon LSMD

472

WAF exposure.

473

Given the results of this present study and from previous experiments, the salmonid

474

caudal fin is a useful tissue for assessing response to a variety of contaminant exposures, such

475

as: estradiol, cadmium, heavy metals field contamination, and now LSMD WAF exposure.28,29 As

476

we learn more about how the caudal fin responds to different scenarios, the utility of this easily 23 ACS Paragon Plus Environment


Page 24 of 45

477

accessible tissue for non-lethal biomonitoring assays will further increase with the anticipated

478

discovery of more bioindicators to monitor pollutant exposure in the environment. This will be

479

a great boon, not only for oil spill cleanup initiatives, but for tracking aquatic pollutant effects

480

on fish while preserving their populations.

481 482 483

Supporting Information PAH analytical chemistry methods for volatile petroleum hydrocarbons (BTEX) and light

484

and heavy extractable petroleum hydrocarbons (LEPH/HEPHs), water chemistry before and

485

after WAF exposure, a list of the 50 PAHs and alkylated PAHs included in the tPAH50 analysis, a

486

comparison of the PAH analyte list to US EPA and NOAA lists, a qPCR primers list, qPCR primer

487

efficiency scores, detailed PAH data in the exposure water and WAF preparations, water

488

chemistry of the marine seawater supply, salmon weight and fork length, and sex genotyping

489

results, the tank setup and analytical composite water samples taken during the fish exposures,

490

the distribution of the PAHs grouped by number of aromatic rings over time, OtY2 and sdY

491

amplification curves, and mta gene transcript abundance in liver tissue.

492 493 494

Acknowledgements This work was supported by a grant from the National Contaminants Advisory Group,

495

Department of Fisheries and Oceans Canada. The authors thank Dr. B. Koop of University of

496

Victoria for generously providing DNA from confirmed male and female coho salmon used in

497

sex genotyping primer validation as well as L. Purdey for technical assistance.


Page 25 of 45


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729 730

Figure Legends

731

Figure 1. Similar tPAH50 concentrations were observed for all WAF concentrations: Low,

732

medium (Med), and High concentration WAF (equivalent to 100, 320, and 1000 mg/L LSMD,

733

respectively), over a test period of 96 h. PAHs were quantified from WAF subsamples taken

734

every 24 h from start to end of the exposure. Decreasing grey scale intensity indicate

735

progression of time from 0-96 h of exposure in 24 h increments. Bars represent the median

736

concentration observed from composite samples from 8 different tanks of the same WAF

737

concentration. Error bars represent the median absolute deviation.

738

Figure 2. LSMD WAF is mostly composed of diaromatic hydrocarbons. The concentrations of

739

PAH analytes grouped by number of rings quantified from the Medium (320 mg/L LSMD) WAF

740

concentration over time are shown. The minimum quantifiable limit of PAH concentration was

741

0.04 µg/L. The low and high WAF concentrations show similar PAH profiles as grouped by ring

742

size (SI Figure S2). Refer to SI Table S2 for compound identification and SI Table S6 for detailed

743

PAH data. Medians (bars) and median absolute deviation (whiskers) are depicted. 36 ACS Paragon Plus Environment

Page 37 of 45


744

Figure 3. Use of OtY2-WSU and U-sdY genotypic sex markers clearly separates fish into two

745

groups corresponding to males (M) and females (F). Each black circle represents an individual

746

fish and the ovals indicate individuals grouped into either M or F categories for gene expression

747

analysis. Red circles represent confirmed adult male and female coho salmon.

748

Figure 4. The abundance of ahr and cyp1a transcripts in both caudal fin and liver tissue is

749

significantly affected by WAF exposure but the response patterns differ by sex and tissue type.

750

The normalized relative fold change of PAH-response gene transcript abundance are shown

751

after exposure to different WAF concentrations as determined by qPCR analysis. The bevel

752

represents increasing WAF concentrations (equivalent to 100, 320, and 1000 mg/L LSMD,

753

respectively). Each open circle represents an individual animal, the median is represented by a

754

horizontal bar, and the median absolute deviation is defined by the whiskers. Relative fold

755

changes are expressed in a log2 scale. “a” denotes significant difference between control and

756

treatment (p < 0.05); “b” denotes significant difference from both control and the low WAF

757

concentration.

758

Figure 5 The abundance of hsp70 and sod transcripts is significantly affected by WAF exposure

759

in male liver with reduced responsiveness in the caudal fin. Female liver and caudal fin are

760

generally non-responsive. The normalized relative fold change are shown of general stress-

761

related gene transcript abundance after exposure to different WAF concentrations as

762

determined by qPCR analysis. See Figure 4 legend for additional details.

763

Figure 6. WAF exposure elicits a modest response in male liver and female caudal fin to some

764

estrogen-responsive transcripts. The normalized relative fold change are shown of estrogen

765

response gene transcripts, vepg, vtg, and cyp19, in caudal fin and liver tissue after exposure to 37 ACS Paragon Plus Environment


Page 38 of 45

766

different WAF concentrations as determined by qPCR analysis. See Figure 4 legend for

767

additional details. “c” denotes significant difference between male and female seawater control

768

animals.


Page 39 of 45


0h 24 h 48 h 72 h 96 h

tPAH50 concentration (µg/L)

30

20

10

0

Low

Med

WAF Concentration

ACS Paragon Plus Environment

High

NAP NAP1 NAP2 NAP3 NAP4 BIP DBF APY APE FLR FLR1 FLR2 FLR3 ANT PHEN PHEN1 PHEN2 PHEN3 PHEN4 DBT DBT1 DBT2 DBT3 DBT4 BDF FLT PYR FLT1 FLT2 FLT3 FLT4 NBT NBT1 NBT2 NBT3 NBT3 NBT4 BAA CT BAA1 BAA2 BAA3 BAA4 BBF BJK BAF BEP BAP DAH ICDP ICDP DAH BGHIP

Concentration (µg/L)


2 Ring 3 Ring tPAH compounds


Page 40 of 45

12

0h

8

4

0

12

24 h

8

4

0

12

48 h

8

4

0

12

72 h

8

4

0

12

96 h

8

4

0

4 Ring 5-6 Ring

Page 41 of 45


OtY-2 normalized to gapdh (Ct)

14

M

12 10 8 R² = 0.9947

6 4

R² = 0.971

F

2 0

0

2

4

6

8

10

12

sdY normalized to gapdh (Ct)


14


Page 42 of 45

Caudal fin

Liver

M

F

M

8.0

a a

a

F a

a

a

a

Relative Fold Change

2.0

ahr 0.50

0.125 128.0

a

a

a

a

b

b

a

a

a

a

a

a

32.0 8.0

cyp1a

2.0

0.50 0.125 0.03125

0

0

0

WAF Concentration


0

Page 43 of 45


Liver

Caudal fin M

F

M

F

8.0 a

a

a


2.0

hsp70 0.50

0.125

a

8.0

a

a

a

a

sod

2.0

0.50

0.125

0

0

0

WAF Concentration


0


Page 44 of 45

Liver

Caudal fin

M

32.0

F

M a

8.0

F a

2.0

vepg

0.50


0.125 0.03125 32.0 8.0

vtg

2.0 0.50 0.125 0.03125 16.0 a 4.0

a

c

cyp19

1.0 0.250 0.0625

0

0

0

WAF Concentration


0

Page 45 of 45


TOC graphic 84x47mm (300 x 300 DPI)


Evaluation of Gene Bioindicators in the Liver and Caudal Fin of

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