Transcriptome Profiling in Larval Fathead Minnow Exposed to

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Ecotoxicology and Human Environmental Health

Transcriptome profiling in larval fathead minnow exposed to commercial naphthenic acids and extracts from fresh and aged oil sands process-affected water Jennifer Loughery, Julie R. Marentette, Richard A. Frank, L. Mark Hewitt, Joanne L. Parrott, and Christopher J Martyniuk Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b01493 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 24, 2019

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Environmental Science & Technology

Transcriptome profiling in larval fathead minnow exposed to commercial naphthenic acids and extracts from fresh and aged oil sands process-affected water Jennifer R. Loughery1, Julie R. Marentette2, Richard A. Frank2, Larry Mark Hewitt2, Joanne L. Parrott2, Christopher J. Martyniuk1,3* 1Department

of Biological Sciences, University of New Brunswick, Saint John, NB, Canada Science and Technology Directorate, Environment and Climate Change Canada, Burlington, ON, Canada 3current address: Center for Environmental and Human Toxicology and Department of Physiological Sciences, UF Genetics Institute, College of Veterinary Medicine, University of Florida, FL, USA *corresponding author 2Water

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ACS Paragon Plus Environment


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Abstract

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Surface mining and extraction of oil sands results in the generation and need for storage of

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large volumes of oil sands process-affected water (OSPW). More structurally complex than

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classical naphthenic acids (NAs), naphthenic acid fraction components (NAFCs) are key toxic

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constituents of OSPW and changes in the NAFC profile in OSPW over time have been linked to

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mitigation of OSPW toxicity. Molecular studies targeting individual genes have indicated that

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NAFC toxicity is likely mediated via oxidative stress, altered cell cycles, ontogenetic

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differentiation, endocrine disruption, and immunotoxicity. However, the individual-gene

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approach results in a limited picture of molecular responses. This study shows that NAFCs,

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from aged or fresh OSPW, have a unique effect on the larval fathead minnow transcriptome and

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provides initial data to construct adverse outcome pathways for skeletal deformities. All three

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types of processed NAs (fresh, aged, and commercial) affected the immunome of developing

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fish. These gene networks included immunity, inflammatory response, B-cell response, platelet

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adhesion, and T-helper lymphocyte activity. Larvae exposed to both NAFCs and commercial NA

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developed cardiovascular and bone deformities and transcriptomic networks reflected these

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developmental abnormalities. Gene networks found only in NAFC-exposed fish suggest NAFCs

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may alter fish cardiovascular health through altered calcium ion regulation. This study improves

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understanding regarding the molecular perturbations underlying developmental deformities

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following exposure to NAFCs.

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Key words: Pimephales promelas, molecular responses, oxidative stress, naphthenic acid

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fraction components, acid extractable organics, oil sands, Athabasca River, immunome


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1. Introduction

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Oil (bituminous) sands, such as the deposits found in the Athabasca region McMurray formation

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of Alberta, Canada, contain a wide variety of compounds, including carboxylic acids and acid-

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extractable organic compounds (AEOs), hereafter referred to as ‘naphthenic acid fraction

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components’ (NAFCs)1-4. Surface mining of oil sands via the Clark caustic hot water extraction

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process, coupled with the reuse of process water, leads to high concentrations of NAFCs in

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large volumes of stored fluid tailings which also contain other compounds such as salts, trace

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metals and polycyclic aromatic hydrocarbons5-8. Classically defined as CnH2n+ZO2, where Z

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indicates hydrogen atoms lost to ring formation, commercially available naphthenic acids (NAs)

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are refined from petroleum products9-10. The molecular structures of NAFCs, however, are more

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complex than classical NAs, with elaborate three-dimensional cage-like structures and the

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incorporation of heteroatoms11-13. Comparisons of prominent structural components between

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NAFCs and NAs revealed higher proportions of oxygen species such as O2S, O4 and NO3, and

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a higher proportion of ring structures within NAFCs14.

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Oil sands process-affected water (OSPW) is stored in settling basins to enable clarification for

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reuse in the extraction process, with the industry working under a zero-discharge practice. In the

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long term, remediation goals for the region include storage of OSPW in end pit lakes and

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hydrological reconnection back into the environment15. Given the large volumes of OSPW

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generated in the region (by 2013, 976 million m3 of OSPW had been generated, with a footprint

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of 220 km2 16), and the presence of NAFCs in groundwater near and far from oil sands mining

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operations17, an enhanced understanding of OSPW toxicity is an important step toward

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improving OSPW remediation8. However, OSPW toxicity is challenging to characterize due to

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temporal changes in chemical compositions (i.e., since final release into settling basins). Lower

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molecular weight (LMW) and relatively less complex NAFC mixtures are at their greatest

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concentrations in fresh OSPW, because they are readily lost to microbial degradation over



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time2,19,20. Commercial NA mixtures, in contrast to NAFCs, are largely composed of simple O2-

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dominated acids13,20-22. Importantly, these LMW compounds can be more acutely toxic than

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larger species23-24, so the loss of these smaller NAs or NAFCs often reduces the toxicity of these

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complex mixtures19,24-27 (but see references 14 and 29).

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NAFCs have long been recognized as a major contributor of the toxicity of OSPW to a wide

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variety of taxa10,28,30, but few studies have characterized molecular responses. Additionally,

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multiple modes of toxicity have been implicated for both OSPW and extracted NAFCs31.

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Oxidative stress and xenobiotic metabolic pathways, including several cytochrome P450 (CYP)

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enzymes, have emerged as common elements. For example, in exposures to fresh OSPW,

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early-life stages of fathead minnow (Pimephales promelas) increased expression of oxidative

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stress genes (glutathione-s-transferase, superoxide dismutase, caspase 932). However, these

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studies employed a targeted gene approach and may have captured a limited picture of the

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molecular responses due to NA and NAFC exposure.

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In this study, we determined the transcriptional responses in newly-hatched larval fathead

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minnow exposed as embryos to NAFCs extracted from both fresh and aged OSPW, and a

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commercial NA mixture. Prior work had established that commercial NAs were more acutely

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toxic than NAFCs to early-life stages of fathead minnow, but NAFCs from different sources and

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ages of OSPW showed similar levels of toxicity14. Transcriptomic responses were measured to

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probe whole organism responses to different NAFCs and a commercial NA. Transcriptomic

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studies provide mechanistic insight into chemicals with unknown modes of action33. Additionally,

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transcriptomic data can be grouped by related processes to gather a process-oriented view of

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the altered transcripts, referred to herein as gene networks. We also compared differentially

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expressed gene networks to morphometric characteristics of the larvae (growth, and

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deformities), data that can contribute to adverse outcome pathways for NA exposure in this

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environmentally relevant species.


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2. Materials and methods

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2.1 Preparation of Exposure Solutions

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Detailed methods are as described in Marentette et al. (2015) for exposure experiments. In

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brief, OSPW (~2000 L) was collected in 2011 from an industry settling basin in active use

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(extract termed 2011 Industry A Fresh, hereafter Fresh) and a test pond which had not received

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OSPW since its establishment in 1993 from the same industrial company34 (extract termed 2011

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Industry A Aged, or hereafter Aged). NAFCs were precipitated from OSPW bulk samples with

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concentrated sulfuric acid and then purified via diethylaminoethyl (DEAE) cellulose, weak anion

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exchanger, and liquid–liquid extraction clean-up using dichloromethane in a base solution of

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0.05 M NaOH35. For comparison, a commercial NA mixture was prepared in the laboratory at

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400 mg/L, also in a 0.05 M NaOH solution (Merichem Company: Acid no. 181 mg KOH/gm,

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unsaponifiables 5.6%, water 0.13%). Final concentrations (mean ± SE) for the NAFC stock

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solutions were measured as 1998 mg/L (Fresh OSPW) and 242 mg/L (Aged OSPW), via liquid

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chromatography/quadrupole mass spectrometry with time of flight detection (LC/QToF) and

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using Merichem naphthenic acid reference material (Commercial NA) for calibration. As

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described in Marentette et al. (2015), nominal concentrations have been used throughout the

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following analyses because in related later exposures, measured exposure concentrations

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deviated only 16.5 ± 2.3% from nominal (mean ± SE).

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Daily exposure solutions were prepared from the NAFC and commercial NA stocks using 0.05

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M NaOH and carbon-filtered, dechlorinated and UV-sterilized municipal water, adjusted to a pH

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of 8.3 ± 0.1 with 1.0 M HCl and incubated to reach 25 ± 1°C overnight before use (nominal

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concentrations of 4.2 and 8.3 mg/L Fresh NAFC, 5.0 and 10.1 mg/L Aged NAFC and 1.25 and

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2.50 mg/L Commercial NA). Laboratory municipal water and pH-adjusted 0.5 M NaOH solutions



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(i.e., a salt control) were used as control exposures for the tests. Each salt control was tailored

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to match the concentration of NaOH used for the corresponding test (therefore NaOH and HCl

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concentrations are internally consistent within each toxicity test, but vary across tests of different

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NAFCs/NAs). Water quality parameters of municipal water and exposure solutions, including

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salt controls are reported in Marentette et al. (2015).

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2.2 Fish husbandry and experimental design

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Toxicity tests were conducted as described previously in Marentette et al. 2015. In brief, newly-

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fertilized fathead minnow embryos (20 per group) were washed with test solution and pipetted

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into exposure vessels (24-well transparent polystyrene tissue culture plates, Falcon, Becton,

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Dickenson and Co., New Jersey, USA). Eggs and larvae were incubated (one per well, 1 mL

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solution per well) at 25 ± 1 °C with a 16 h light, 8 h dark light cycle. Tests were run in triplicate;

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each replicate contained one laboratory control plate, one salt control plate, and five to six

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dilutions of NA or NAFC. Exposure solutions were renewed daily and tests were terminated

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within one day of hatch (approximately 4-5 days in freshwater at 25°C36) to encompass the

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eleutheroembryonic (chorion-free, yolk-dependent) period. Animal handling and experimental

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procedures were approved by the Animal Care Committee in the National Water Research

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Institute, Canada Centre for Inland Waters (AUP # 1310).

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This study used a subset (n = 136) of fathead minnow larvae from the toxicity tests of

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Marentette et al. (2015). Larvae were selected from newly-hatched individuals randomly

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selected from test plates, focusing on salt controls (i.e., 0 mg/L NA or NAFC) and two exposure

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groups as close to the test LC50 as possible; higher concentrations could not be sampled as

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mortality rates ensured not enough larvae were available. In most instances (21 of 27 exposure

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groups), five larvae were selected per plate (i.e., 15 larvae total over three replicates). Only four

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were available from each of three plates and six to seven larvae were collected from each of the

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remainder.


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2.3 Larval Assessment

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Newly hatched larvae were assessed as described in Marentette et al. 2015. Briefly, each larva

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was inspected for deformities within one day of hatch. Inspection was not blind to the treatment

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group. Mild deformities included: slight enlargement of the pericardial sac, single (pinpoint)

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hemorrhages, mild spinal curvature and malformation of the medial/caudal finfold. Severe

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deformities were designated as: pronounced edemas of the pericardium and/or yolk-sac, and

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spinal curvature associated with mobility problems. Such severe deformities often co-occurred

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with hemorrhages including hemostasis, abnormal (tube-shaped) hearts, and craniofacial

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abnormalities, such as microphthalmia, edema around the ocular sockets, and/or abnormally

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small or large jaws. After assessment, larvae were euthanized in a solution of tricaine methane-

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sulfonate (TMS) and flash frozen in liquid nitrogen before storage at –80 °C.

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2.4 Microarray analysis

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Microarray analysis was performed using an 8x15 K fathead minnow microarray (GEO #

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GPL9248) manufactured by Agilent Technologies (Santa Clara, CA, USA). Larval samples

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were pooled by exposure group and replicate (sample sizes within each replicate ranged

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between 3 and 7 larvae as noted above), resulting in N = 3 pooled samples for each of the

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following groups: salt controls (fresh NAFC, aged NAFC and commercial NA 0 mg/L), low dose

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(fresh NAFC 4.2 mg/L, aged NAFC 5.0 mg/L and commercial NA 1.25 mg/L) and high dose

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(fresh NAFC 8.3 mg/L, aged NAFC 10.1 mg/L and commercial NA 2.5 mg/L). Extraction of RNA

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from embryo samples was performed using 1 mL TRIzol® Reagent (Life Technologies,

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Carlsbad, CA, USA) as per manufacturer’s protocol. Immediately after extraction, RNA pellets

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were dissolved in 10 µL of RNAse-DNAse free water and purified through the RNeasy Mini Kit

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column, as per manufacturer’s protocol (Qiagen, Valencia, CA, USA). Purified RNA samples

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were assessed for quality using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA,

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USA). All samples used in this study had a RIN >9.4. The mean RIN value was (9.9 ± 0.14). The



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concentration of RNA was determined using the NanoDrop-2000 (Thermo Scientific, USA);

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260/280 and 260/230 ratios were examined to confirm sample purity.

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Following verification of high quality RNA, the RNeasy Mini Kit was used to purify RNA prior to

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labeling as per manufacture’s protocol (Qiagen, Mississauga, ON, CAN). RNA concentrations

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were determined using the NanoDrop-2000 spectrophotometer (Thermo Scientific). Microarray

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hybridizations were performed according to the One-Color Microarray-Based Gene Expression

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Analysis Low Input Quick Amp Labeling kit (Agilent V6.5, May 2010) and 125 ng total RNA per

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sample was used for labelling and hybridization as per our previous protocol37. Microarrays

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were scanned at 5 μm with the Agilent G2505 B Microarray Scanner, and Agilent Feature

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Extraction Software (v. 9.5) was used to extract raw signal intensities from microarray images.

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Microarray data were evaluated by manual inspection of the quality control parameters. All

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arrays were deemed high quality. Raw microarray data have been deposited into the NCBI

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Gene Expression Omnibus (GEO) database (Geo Accession Series GSE85994).

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Raw intensity data were imported into JMP® Genomics v 7.0 (SAS Institute Inc., Cary, NC,

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USA). Intensity data were normalized using quantile normalization. Control spots were filtered

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out prior to identifying differentially expressed genes (DEGs) and the limit of detection was set

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to an intensity of 3.5 based on the Agilent spike in controls. Therefore, any probe falling below

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this value was assigned a normalized intensity of 3.5. DEGs were identified using a one-way

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analysis of variance (ANOVA) followed by a false discovery rate (FDR) set at 5.0%, with α =

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0.05. Cluster analysis was conducted in JMP Genomics v 7.0 and involved clustering of the

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standardized least square means for differentially expressed transcripts.

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Pathway Studio 9.0 (Elsevier) and ResNet 10.0 were utilized for sub-network enrichment

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analysis (SNEA)37-38. A total number of 11, 378 fathead minnow transcripts were mapped to the

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program using the official gene name (Name + Alias). SNEA was performed to identify cell

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process networks that were affected in the FHM embryos following exposure to commercial NAs


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and aged NAFCs39. Highest fold change, best p value was used for duplicated probes. The

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enrichment P-value for a gene network was set at P < 0.05.

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2.5 Real time PCR

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Purified, high quality RNA, 500 ng, was used for cDNA synthesis using iScript (BioRad),

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following the manufacturer’s protocol. After addition of nuclease free water, sample volume was

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20 µL. No reverse transcriptase (NRT) and no template controls (NTC) were prepared in the

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same fashion, except that the NRT contained no reverse transcriptase, and the NTC contained

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no RNA. Nuclease free water replaced the volumes of reverse transcriptase and RNA,

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respectively, to conserve a final reaction volume of 20 µL. Once prepared, samples were placed

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in a T100™ Thermal Cycler (BioRad, USA). The cDNA was generated using the following steps:

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25 °C for 5 min, 42 °C for 30 min, 85 °C for 5 min, and a final cycle of 4 °C for 5 min.

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Primer sets for target genes were collected from literature (Table S1). Newly developed primer

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sets were designed by Primer-BLAST (NCBI). The genes investigated in this study included

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cytochrome b5 type a, cyb5a; member 1, cytochrome P450 family 2 subfamily J, polypeptide 30,

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cyp2j30; apoptosis-inducing factor, mitochondrion-associated, 2, aifm2; and dihydrodiol

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dehydrogenase, dhdhl. Proteins encoded by cyb5a have been shown to be related to lipid

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metabolism40, while aifm2 translation products are related to caspase-independent apoptosis41.

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The protein encoded by cyp2j30 is an epoxygenase enzyme associated with the metabolism of

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arachidonic acid to epoxyeicosatrienoic acids and expression has been shown to be elevated in

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cardiovascular and vascular tissue (orthologous to human cyp2j2)42. Lastly, dhdhl is associated

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with xenobiotic metabolism, formation of reactive oxygen species and cancer43. Selection

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criteria can be found in Supporting Information 1. Real-time PCR (qPCR) was performed using

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the CFX96™ Real-Time PCR Detection System (BioRad) with SSoFast™ EvaGreen®

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Supermix (BioRad, Hercules, CA, USA), 100 nM of each forward and reverse primer, and 5 µL

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of cDNA (diluted 20-fold prior to real-time analysis). The two-step thermal cycling parameters



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were as follows: initial 1-cycle Taq activation at 95 °C for 30 s, followed by 95 °C for 5 s, and

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primer annealing for 5 s (temperature specified in Table S1). After 40 cycles, a dissociation

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curve was generated, starting at 65.0 and ending at 95.0°C, with increments of 0.5 °C every 5 s.

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Three reference genes (b-actin, ef1a, and rps18) were assessed for normalization. Expression

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values were evaluated statistically using a Kruskal-Wallis test, to determine whether expression

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levels varied across experimental groups. This was based upon total RNA input for the cDNA

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synthesis. The mean expression levels of b-actin and ef1a were determined to be the most

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stable combination of reference genes to normalize target genes. The target stability function in

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the CFX96 software determined that the combined M-value for b-actin and ef1 was 0.84 (CV =

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0.29). Each primer set was tested for linearity and efficiency using a 4 or 5 point standard curve

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generated by a dilution series from a cDNA pool of FHM embryo. The qPCR analysis included 4

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NRT samples and 1 NTC sample. Negative controls indicated that RNA isolation and column

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purification sufficiently removed genomic DNA, so that it did not significantly impact gene

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expression analyses. Normalized gene expression was extracted using CFX Manager™

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software with the relative ΔΔCq method (baseline subtracted)44. All primers used in the qPCR

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analysis amplified one product, indicated by a single melt curve. Samples were analyzed using

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a Kruskal-Wallis test following by a Dunn’s post hoc test for multiple comparisons to the control

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group. Correlation was determined with a Spearman matrix using normalized gene expression

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data. All statistical analyses were performed in Prism (v. 6.0).

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3.0 Results and Discussion

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3.1 Survival and deformities

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Nominal concentrations used in this study are reported in Table S2. The commercial NA mixture

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(EC50 of 1.9 mg/L, CI95 1.7-2.2) was slightly more toxic to newly hatched fathead minnow than

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NAFCs from either fresh or aged OSPW (EC50s of 5.0 (CI95 4.2-6.0) and 12.4 mg/L, CI95 11.2-


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13.7, respectively)14. NAFCs from OSPW and commercial NAs also induced different types of

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deformities. Over the entire toxicity test, exposure to commercial NAs produced elevated rates

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of finfold deformities, while NAFC exposure resulted in elevated rates of cardiovascular (fresh

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and aged NAFCs) and spinal deformities (aged alone)14. A similar but non-significant pattern

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was reflected in the smaller subset of larvae used in the present analysis (no significant

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differences via chi-square comparison of deformity proportions in the larvae; commercial NA χ2

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= 2.5, p = 0.287; fresh NAFC χ2 = 2.35, p = 0.309; aged NAFC χ2 = 2.61, p = 0.270; Figure 1).

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Deformities tended to be lowest in number or severity in larvae from salt controls relative to

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exposed groups. While cardiovascular abnormalities (edemas and hemorrhages) were seen in

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subsampled larvae from all three tests, spinal curvature was only observed in two individuals

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exposed to NAFCs from aged OSPW, and finfold malformations were only observed in three

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larvae exposed to commercial NAs (Figure 1).

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NAFC exposure resulted in a higher percentage of larvae with cardiovascular and spinal

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abnormalities, consistent with other findings. Walleye embryos exposed to NAFC, from 1 day

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post-fertilization to hatch, developed spinal curvature, cardiovascular, craniofacial, and finfold

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abnormalities45. Fathead minnow and Japanese medaka (Oryzias latipes) exposed to AEO from

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OSPW showed that higher molecular weight fractions were more toxic than low molecular

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weight fractions. Some fractions delayed hatch time, shortened larval length at hatch, and

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exposed larvae showed yolk sac and pericardial edema in both species31. Yellow perch (Perca

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flavescens) reared in Mildred Lake settling basin water, fresh OSPW, developed spinal, eye and

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head deformities. Japanese medaka reared in the same water displayed pericardial edema,

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tube heart, and decreased yolk uptake46. In another study, early life stage zebrafish (Danio

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rerio) exposed to 2.50 mg/L NA extracted from Daqing oil exploration area, China, showed

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delayed hatch time and decreased survival. Some embryos developed yolk sac and pericardial

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edema and/ spine malformations47. Even in the absence of deformities, exposure to OSPW



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decreased expression of genes associated with cardiac development and function in embryonic

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zebrafish48.

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Conversely, commercial NA exposure induced finfold abnormalities. However, this has not been

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consistently reported in the literature. Peters et al. (2007) reported yellow perch exposed to

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sodium naphthenate developed spinal, eye and head deformities, while Japanese medaka

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developed pericardial edema, tube heart and decreased yolk uptake. While there were species

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differences, each species developed similar deformities as larvae exposed to Mildred Lake

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settling basin water. Wang et al. (2015) reported that embryo zebrafish exposed to commercial

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NA developed yolk sac edema and spinal malformations. However, the percentage of

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incidences was lower when compared to embryos exposed to commercial NA extracted from oil,

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possibly related to the compositional differences between these mixtures14. Taken together,

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these data suggest that both NAs and NAFCs can nevertheless induce significant

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developmental defects and delays in different species of fish.

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3.2 Transcriptome profiling of NAFC and NA treatments

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In larvae exposed to fresh NAFCs, there were 34 transcripts that were differentially expressed

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with exposure to 4.2 mg/L NAFCs, and 75 transcripts differentially expressed with exposure to

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8.3 mg/L NAFCs, compared to salt controls (p

Transcriptome Profiling in Larval Fathead Minnow Exposed to

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