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Effects on Biotransformation, Oxidative Stress and Endocrine Disruption in Rainbow Trout (Oncorhynchus mykiss) Exposed to Hydraulic Fracturing Flowback and Produced Water Yuhe He, Erik J. Folkerts, Yifeng Zhang, Jonathan W. Martin, Daniel S Alessi, and Greg G Goss Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04695 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016
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Effects on Biotransformation, Oxidative Stress and Endocrine Disruption in Rainbow
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Trout (Oncorhynchus mykiss) Exposed to Hydraulic Fracturing Flowback and Produced
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Water
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Yuhe He1,†, Erik J. Folkerts1,†, Yifeng Zhang2, Jonathan W. Martin2, Daniel S. Alessi3,
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Greg G. Goss1, *
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Department of Biological Sciences, 2Department of Laboratory Medicine and Pathology and
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Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, Alberta,
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Canada, T6G 2E9
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* Corresponding author.
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† These authors contributed equally.
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ABSTRACT
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The effects of hydraulic fracturing (HF) flowback and produced water (HF-FPW), a complex
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saline mixture of injected HF fluids and deep formation water that return to the surface, was
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examined in rainbow trout (Oncorhynchus mykiss). Exposure to HF-FPWs resulted in significant
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induction of ethoxyresorufin-O-deethylase (EROD) activity in both liver and gill tissues.
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Increased lipid peroxidation via oxidative stress was also detected by Thiobarbituric acid reactive
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substances (TBARS) assay. The mRNA expressions of a battery of genes related to
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biotransformation, oxidative stress, and endocrine disruption were also measured using
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quantitative real-time polymerase chain reaction (Q-RT-PCR). The increased expression of
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cyp1a (2.49±0.28-fold), udpgt (2.01±0.31-fold), sod (1.67±0.09-fold) and gpx (1.58±0.10-fold)
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in raw sample exposure group (7.5%) indicated elevated metabolic enzyme activity, likely
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through aryl-hydrocarbon receptor pathway, and generation of reactive oxygen species. In
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addition, the elevated vtg and era2 expression demonstrated endocrine disrupting potential
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exerted by HF-FPW in rainbow trout. The overall results suggested HF-FPW could cause
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significant adverse effects on fish, and the organic contents might play the major role in its
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toxicity. Future studies are needed to help fully determine the toxic mechanism(s) of HF-FPW on
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freshwater fish, and aid in establishing monitoring, treatment, and remediation protocols for HF-
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FPW.
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INTRODUCTION
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Horizontal drilling with high-volume hydraulic fracturing (HF) is a practice being used in
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Alberta/Canada for improving the extraction of oil and gas from tight reservoirs. Energy
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production from these resources in North America is expected to continue, with estimated
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increases of 45% and 25% above current production levels for the US and Canada, respectively,
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in the next 25 years1,2. The rapid expansion of HF practices, together with its large quantity of
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water usage and process affected water production, poses potential environmental hazards to the
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environment, including contamination of surface and shallow groundwater aquifers via
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discharges and spills3,4,5,6, as well as subsurface gas migration7,8,9,10,11.
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However, there remain significant knowledge deficits on the environmental impacts and
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risks of the flowback and produced water (HF-FPW) to aquatic ecosystems12. HF-FPW is a
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complex, tripartite mixture of injected HF fluid components, deep formation water, and
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secondary by-products of downhole reactions with the formation environment11,13,14,15. HF-FPW
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brine may contain numerous inorganic and organic constituents, including high levels of metals
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(e.g., barium, strontium, chromium, cadmium, lead), radionuclides (e.g., radium and uranium),
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and a complex profile of organic compounds, theorized to be additive components in HF fluid,
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natural organics related to in situ formation hydrocarbons (e.g. polycyclic aromatic hydrocarbons
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(PAHs)), and even secondary chemical products from the interaction between the fracturing
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environment in the well (elevated temperature and/or pressure) and the deep saline
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groundwater16,17,18. Correspondingly, the concentrations of metals, radionuclides, and PAHs
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detected in FPW are often well above the maximum contamination level for water quality
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guidelines5,6,19. Together, these chemicals return to the surface with released oil and gas, and the
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HF-FPW is separated for treatment/reuse, or disposed in deep, sub-surface injection wells20.
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Risks of ground and/or surface fresh water contamination are principally associated with on-site
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fluid handling, transportation of HF-FPW to disposal wells, and well integrity issues5. Accidental
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release of HF-FPW in certain regions is well documented, with more than 2500 spills in Alberta
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from 2011 to 201421. It has been suggested that the presence of endocrine disrupting chemicals in
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HF wastewater may be linked to reproductive and developmental impairment in laboratory
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animals based on the systematic evaluation of chemicals used in HF fluids22. While the potential
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biological risk and impacts of chemicals used during the fracturing process have been predicted
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and documented in several reviews23,24, there is very limited information regarding the toxicity of
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any real HF-FPW samples and the potential toxicological impacts of HF-FPW spills on
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freshwater organisms.
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The lack of available hazard assessment for HF-FPW spills in Canada and the United
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States hinders environmental impact and risk assessment of hydraulic fracturing activities 25.
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Mandatory disclosure of the chemical constituents of fracturing fluids for example, through the
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chemical disclosure registry, FracFocus, has somewhat improved our understanding but the
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toxicity data of many chemicals is often missing23,25. The environmental fates of those chemicals
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are further complicated by potential down-hole reactions and generation of secondary products18.
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Therefore, there exists an obvious need to investigate the toxicity on aquatic organisms. In this
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study, juvenile rainbow trout (Oncorhynchus mykiss), commonly used as a biologically relevant
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freshwater model for regulatory science, were used to determine responses to potential spills and
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leaks of HF-FPW in the aquatic environment. Acute exposures (48 hours) were conducted
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followed by measurements of a variety of endpoints including hepatic and branchial
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ethoxyresorufin-O-deethylase (EROD) activity, thiobarbituric acid reactive substance (TBARS)
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formation in various tissues, and mRNA abundance of a battery of genes related to
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biotransformation, oxidative stress, and endocrine disruption by quantitative real-time
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polymerase chain reaction (Q-RT-PCR). This is one of the first studies to investigate the
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physiological responses to HF-FPW exposure in a whole organism. Our study will help address
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the ecotoxicological hazards associated with HF-FPW, and provide potential biomarkers for
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water quality monitoring in areas affected by hydraulic fracturing activities.
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MATERIALS AND METHODS
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HF-FPW collection. The HF-FPW sample analyzed in this study was collected at 7 days post-
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stimulation from a horizontal, hydraulically fractured well in the Devonian-aged Duvernay
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Formation (Fox Creek, Alberta, Canada) by Encana Services Company Ltd. In this study, HF-
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FPW-S (abbreviated as S in figures, the same below) refers to the original, raw sample
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containing sediment and/or suspended particles. A summary of key compositional information of
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this sample is presented in Table 1. Detailed geological and chemical information of this sample
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is reported in a companion study18. All tests were conducted within 60 days of sample
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acquisition and samples were stored at room temperature to best reflect on-storage conditions.
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Sediment-free (HF-FPW-SF, or SF) was prepared by vacuum filtration of the raw sample
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through a 0.22 µm membrane, which also greatly reduced the levels of total organic
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contaminants in the sample. An activated-charcoal treated (HF-FPW-AC, or AC) sample was
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also prepared by treating the raw sample with activated charcoal, followed by vacuum filtration
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through a 0.22 µm membrane. This resulted in most of the organic contaminants being removed.
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We acknowledge that vacuum filtration may also have caused a loss of volatile dissolved
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components in SF and AC subsamples but this was necessary to remove the sediment fraction.
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Once we aliquoted a raw sample for either treatment or direct exposure (HF-FPW, HF-FPW-SF
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and HF-FPW-AC), these aliquots were stored at 4 °C until the start of the exposure period.
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Details of the sample preparation are described in a companion study18.
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Chemicals. All the chemicals were purchased from Sigma-Aldrich (USA). Details are provided
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in SI.
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Fish. Rainbow trout embryos were obtained from the Raven Brood Trout Station (Caroline, AB,
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Canada) and grown to the appropriate size (24.9±10.1 g) as juveniles for experimentation.
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Juvenile fish were maintained indoors in flow-through 450L tanks supplied with aerated and
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dechlorinated facilitate water (hardness as CaCO3: 1.6 mmol/l, alkalinity: 120 mg/l, NaCl: 0.5
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mmol/l, pH 8.2, 10±1 °C). Fish were fed ground dry commercial trout pellets (Purina trout chow)
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once daily and kept on a 14h/10h day/night photoperiod. All animal use was approved by the
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University of Alberta Animal Care Committee under Protocol AUP00001334.
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Exposure design. Exposure was conducted in 8L glass tanks filled with 4L aerated
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control/treatment water. Water temperature was maintained at 10±1 °C by partial immersion of
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tanks in a water bath with constant facility water flow. 50% control/treatment water changes
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were made in each tank every 24h. Fish were fasted 3 days prior to and during experimentation.
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Fish were exposed to control/treatment waters in triplicate tanks, with each tank containing 2
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individuals. The concentrations of low dose (2.5%) and high dose (7.5%) were selected based on
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the results of preliminary range finding test of HF-FPW-S using finger-length juvenile rainbow
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trout (Figure S1). Fish were exposed to facility water as a control (Ctl), HF-FPW-AC (2.5% and
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7.5% dilutions in facility water, the same below), HF-FPW-SF (2.5% and 7.5%) and HF-FPW-S
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(2.5% and 7.5%), as well as benzo[a]pyrene (BaP) (0.33and 1 µM) as a positive control for 24
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and 48 h. At the end of exposure, fish were euthanized by cephalic blow and decapitated. Gill
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filament and liver samples were collected and immediately assayed for EROD activity.
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Subsamples (0.25 g) of gill, liver and kidney in all 48 h exposure groups were placed into 1.5 mL
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Eppendorf tubes containing 500 µL phosphate buffer (100 mmol l-1 KH2PO4, 5mmol l-1 EDTA,
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pH = 7.5), frozen in liquid nitrogen and stored at -80 °C for TBARS assay. Subsamples of liver
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in all 48 h exposure groups were frozen and stored at -80 °C for Q-RT-PCR assay. The exposure
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water from each treatment was sampled and stored in dark at 4°C prior to PAHs analysis.
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PAH analysis. Subsamples of exposure water at 24 h and 48 h (500 mL), including control,
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AC (2.5% and 7.5%), SF (2.5% and 7.5%), and S (2.5% and 7.5%), were collected for polycyclic
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aromatic compound analysis by liquid-liquid extraction method as described previously26.
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Detailed methodology is provided in the SI and in Table S1.
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Hepatic and Branchial EROD assays. The hepatic EROD activity was determined via a
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modified method from Hudson et al. (1991)27. The gill-filament-based EROD assay was
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performed followed the method described by Jönsson et al. (2002)28. Details are provided in SI.
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TBARS assay. TBARS assay using fish tissues was performed following a previous study with
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minor modification29. Briefly, the supernatant of the homogenized samples were treated with
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thiobarbituric acid and TBARS formation was quantified by fluorescence measurement at 531
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and 572 nm (excitation and emission respectively). In addition, the intrinsic oxidative potentials
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of HF-FPW fractions alone were also determined via a modified protocol from Biaglow et al.
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(1997)30. HF-FPW samples were incubated with a final concentration of 4 mM 2-deoxy-d-ribose
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(2-DR) for 1 hour at 25°C under ambient room light, followed by fluorescence measurement of
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TBARS formation. Details are provided in SI.
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Quantitative Real-Time PCR assay. Total RNA was extracted from liver samples and
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cDNA was prepared for Quantitative real-time PCR (Q-RT-PCR) measurement using SYBR
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Green master mix system (Applied Biosystems, CA). Details of RNA extraction, cDNA
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synthesis, and Q-RT-PCR reactions were provided in SI. Eleven genes representing
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biotransformation, oxidative stress, and endocrine disruption in rainbow trout were selected for
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screening. Changes in abundances of transcripts of target genes were quantified by normalizing
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to elongation factor 1a (elf1a). There was no difference in the expression of elf1a among all the
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exposure groups (Figure S2). Gene name, abbreviation, sequences of primers, efficiency, and
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GeneBank reference number are listed in Table S2.
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Statistical Analysis. Juvenile rainbow trout were exposed to the control/treatment solutions in
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triplicate tanks, with each tank containing 2 fish. No differences in responses to the same
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treatment between fish in the same tanks were observed. Therefore, each individual fish is
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considered an experimental unit. Statistical analyses were conducted by use of SPSS16.0 (SPSS,
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Chicago, IL). All data are expressed as mean ± standard error mean. Log transformation was
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performed if necessary to meet the assumptions. Statistical differences were evaluated by one-
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way ANOVA followed by posthoc Tukey test. Differences were considered significant at ρ
