Developmental Toxicity of the Organic Fraction from Hydraulic

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Developmental Toxicity of the Organic Fraction from Hydraulic Fracturing Flowback and Produced Waters to Early Life Stages of Zebrafish (Danio rerio) Yuhe He, Chenxing Sun, Yifeng Zhang, Erik J. Folkerts, Jonathan W. Martin, and Greg G Goss Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06557 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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Environmental Science & Technology

Developmental Toxicity of the Organic Fraction from Hydraulic Fracturing Flowback and Produced Waters to Early Life Stages of Zebrafish (Danio rerio) Yuhe He1*, Chenxing Sun2,Yifeng Zhang2, Erik J. Folkerts1, Jonathan W. Martin2, Greg G. Goss1* 1

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada

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Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton,

Alberta, T6G 2G3, Canada

*Corresponding authors: Yuhe He Z508, 11455 Saskatchewan Drive, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada Email: [email protected] Tel: 1-780-492-1276 ORCID: 0000-0001-9211-4539

Greg G. Goss Z512, 11455 Saskatchewan Drive, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada Email: [email protected] Tel: 1-780-492-2381 ORCID: 0000-0003-0786-8868

1 ACS Paragon Plus Environment


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ABSTRACT

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Hydraulic fracturing (HF) has emerged as a major recovery method of unconventional oil and gas

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reservoirs and concerns have been raised regarding the environmental impact of releases of

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Flowback and Produced Water (FPW) to aquatic ecosystems. To investigate potential effects of HF-

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FPW on fish embryo development, HF-FPW samples were collected from two different wells and

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the organic fractions were isolated from both aqueous and particle phases to eliminate the

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confounding effects of high salinity. Each organic extract was characterized by non-target analysis

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with HPLC-Orbitrap-MS, with targeted analysis for polycyclic aromatic hydrocarbons provided as

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markers of petroleum-affected water. The organic profiles differed between samples, including

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PAHs and alkyl PAHs, and major substances identified by non-target analysis included

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polyethylene glycols, alkyl ethoxylates, octylphenol ethoxylates and other high molecular weight

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(C49-79) ethylene oxide polymeric material. Zebrafish embryos were exposed to various

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concentrations of FPW organic extracts to investigate acute (7-day) and developmental toxicity in

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early life stages. The acute toxicity (LD50) of the extracted FPW fractions ranged from 2.8× to 26×

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the original organic content. Each extracted FPW fraction significantly increased spinal

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malformation, pericardial edema, and delayed hatch in exposed embryos and altered the expression

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of a suite of target genes related to biotransformation, oxidative stress and endocrine-mediation in

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developing zebrafish embryos. These results provide novel information on the variation of organic

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profiles and developmental toxicity among different sources and fractions of HF-FPWs.

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TOC Abstract ART

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Introduction

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The rapid development of hydraulic fracturing (HF) for recovering oil and gas from tight reservoirs

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has raised significant public concerns regarding the potential contamination of surface water1,2 and

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shallow groundwater aquifers3,4. One of the key issues in hydraulic fracturing is the risk of spills

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associated with the generation and transport of large volumes of HF Flowback and Produced Water

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(HF-FPW), a complex mixture of wastewater returns to the surface after fracturing activity5,6. The

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chemical composition of HF-FPW is known to be very complicated and variable due to the

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geological nature of the formation, as well as various HF fluids applied during fracturing activities5.

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HF-FPW contains large amounts of salts, particles, metals, radionuclides, and numerous organic

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compounds1,5. The organic compounds can include natural petrogenic compounds (eg. polycyclic

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aromatic hydrocarbons (PAHs)), additive components of the HF fluid, as well as transformation

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products from chemical interactions under the extreme environment in the fracturing well (high heat

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and high pressure)7,8,9,. The concentrations of contaminants in HF-FPW samples are often above

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environmental guidelines for surface water quality1,2. The primary risks for HF-FPW ground and

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surface water contamination occur during transport and disposal, including pipeline leaks, truck

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transportation, and injection well integrity issues10. Millions of gallons of HF activity related

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wastewater have been accidentally released into the environment in various spill scenarios in United

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States and Canada11,12,13. Several publications have documented that disposal and/or spills of HF

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fluids or wastewaters can negatively affect aquatic organisms and create a potential long-term

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environmental health effect in aquatic ecosystems14,15,16,17.

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Recently, our research has demonstrated significant toxicity of HF-FPW to the zebrafish

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(Danio rerio)9, rainbow trout (Oncorhynchus mykiss)18,19 and water flea (Daphnia magna)20, with

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the raw samples shown to possess greater toxicity than the filtered particle free samples. These

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results demonstrate that HF-FPW spills/surface water contamination may pose significant hazards


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to both general and reproductive health of aquatic animals. A recent study predicted the potential

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reproductive and developmental toxicity of HF wastewater based on a systematic evaluation of

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endocrine disrupting chemicals (EDCs) used in HF fluids21. In our first study based on a real HF-

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FPW sample, endocrine disruptive effects were indicated through activation of vitellogenin

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transcription in rainbow trout18. We later reported decreased fecundity and reproduction in daphnia

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exposed to diluted HF-FPW20. More recently, it has also been suggested that EDCs involved in HF

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activity can disrupt endocrine-related nuclear receptor pathways22,23 and have the potential to affect

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reproduction and development in aquatic animals24.

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One significant uncertainty with previous studies using raw or diluted wastewater from HF

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activities is that the toxicological responses from organics are likely confounded by stress due to

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high salinity (200-300 ppm, 6-8× seawater)9,16. In fact, exposure to highly diluted HF-FPW could

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still cause death in experimental animals due to the effects of the remaining salts in the dilution

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water9,18,19,20. Moreover, to understand dose-response of sub-lethal adverse outcomes, including

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developmental toxicity and endocrine disruptive effects, exposures with concentrated HF-FPW are

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not practical due to the high native salinity.

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In the current study, FPW organic contents were extracted from both particle and aqueous

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phases to allow examination of the potential for organic-substance mediated toxicity in the absence

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of salinity-related effects. Extracts were characterized by non-target HPLC-Orbitrap-MS method,

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and also by a targeted PAH analysis. Zebrafish embryos25 were used to evaluate median lethal

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concentrations (LC50), as well as potential developmental and teratogenic effects over a wide range

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of organic extract concentrations. Short-term embryonic exposure to petrogenic contaminants have

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been reported to cause a suite of toxic effects in fish larvae such as edema, hemorrhaging, spinal

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malformation, and impaired fitness26,27. Ample evidence indicates exposures to PAHs and

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petrogenic mixtures can alter normal fish development in multiple mechanisms of toxic action, and



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developmental endpoints, such as pericardial edema and spinal malformation, have been

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increasingly used for assessment of developmental abnormalities and potential long-term effects of

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developmental toxicants28,29. Additionally, the mechanism(s) behind the observed adverse outcomes

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was investigated by measurement of transcriptional responses related to xenobiotic metabolism,

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oxidative stress, and endocrine mediation using quantitative real-time polymerase chain reaction

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(Q-RT-PCR). This study will contribute towards an understanding of the hazards that organic

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constituents present in HF-FPW, and may aid future environmental risk assessments.

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Materials and methods

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HF-FPW samples. The HF-FPW samples (about 20 L for each) were collected from 2 different

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stimulated wells by Encana Service Company Ltd. Both wells are located in the Devonian-aged

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Duvernay Formation (Fox Creek, Alberta, Canada). Samples were collected at 7 days post-

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stimulation at the first well (HF-FPW-1) and 10 days post-stimulation at the second well (HF-FPW-

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2) by Encana’s technicians with our lab representatives on site to confirm authenticity and quality

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control. Stimulation is a technique of oil and gas well fracturing treatment that increases the flow of

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oil and/or gas to the wellbore. Following stimulation, the pressure was released and the well

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continuously flowed back, producing a large amount of HF-FPW. Raw water samples were directly

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stored in high-density polyethylene buckets (Pro-Western Plastic Ltd., Alberta). After cooling

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down, the sampling buckets were sealed and shipped to our lab. Sampling buckets were stored at

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room temperature and never opened prior to extraction. Blank test using warm salted facility water

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(with the same temperature and level of major salts as raw samples) stored in the same type of

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bucket overnight confirmed no organic chemicals were leached from the bucket. The geological and

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chemical information for these samples has been previously reported9,20. Some key compositional

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information on both samples is summarized in Table 1. 6 ACS Paragon Plus Environment

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Organic extraction. The organics from each 5 L sample of HF-FPW were isolated separately from

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the suspended solids (collected on 0.4 um filters) and the corresponding aqueous filtrate using

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methods provided in SI. In this study, 1-W and 1-S refer to the organic extracts from aqueous phase

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and particle phase from HF-FPW-1, respectively. For each sample, these two extracts were

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reconstituted in 166 µL Dimethyl Sulfoxide (DMSO) before further dilution. For pooled extract,

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given the similar recovery rates of water and sediment extraction process (data not shown), the

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same amount of aqueous and particle phase extracts (1:1 ratio) were combined and used as an

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equivalent of the concentrated organic extract from the original raw sample. For example, 1-P refers

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to the original total organics in HF-FPW-1 reconstituted by combining 1-W and 1-S in a 1:1 ratio

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(v/v). Similarly, 2-W, 2-S and 2-P are the corresponding extracts from HF-FPW-2. All organic

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extracts were further diluted with dilution water before exposure. All samples and extracts were

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then stored in glassware in the dark at 4 ºC prior to use.

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Organic compounds analysis by high performance liquid chromatography and orbitrap mass

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spectrometry (HPLC-Orbitrap-MS). Identification of the major organic compounds in the aqueous

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and particle phases was achieved by HPLC-Orbitrap-MS. Detailed method information is provided

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in SI. Briefly, diluted organic extract was injected to analytical reversed phase chromatography at

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25 ºC. The Orbitrap MS was operated in positive mode and using electrospray ionization, and

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acquisition was in full scan mode (m/z 100 to 2000) at 2.3 Hz, with resolving power set to 120,000

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at m/z 400. Tandem mass spectrometry was also performed using data dependent mode with

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collision-induced dissociation under various energies and modes.

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PAH analysis. Separate sub samples were used to analyze polycyclic aromatic compounds. Sixteen

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parent PAHs and 16 alkyl PAHs were targeted using authentic commercial standards for

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confirmation and quantification. Among these, 13 parent PAHs and 4 alkyl PAHs were detected in

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HF-FPW samples. In the following sections, the sum concentrations of 13 parent PAHs are referred

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to as Σ13PAH, and the sum concentrations of 4 alkyl-PAHs are referred to as Σ4alkyl-PAH, while 7 ACS Paragon Plus Environment


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the sum of Σ13PAH and Σ4alkyl-PAH is referred to as “total PAHs”. Detailed information on analyte

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standards and the GC-MS instrumental method are presented elsewhere30. Details of extraction and

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analysis are provided in SI.

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Exposure Conditions. A 168 h semi-static exposure (50% daily change) was performed in 50 mL

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beaker filled with 30 mL control/treatment water, with 10 fertilized embryos (1 h post fertilization,

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hpf) per beaker and 4 beakers per control or treatment, at 26±1 °C with 16h/8h day light cycle. This

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setting included 40 embryos in total for each control/treatment, and resulted in the replication

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number equal to 4. Embryos/larvae were exposed to 0.1% DMSO in dilution water as a control

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(Ctl), and various concentrations of each extract or the pooled organic extract, ranging from 0.1 to

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100 times (×) compared to the original concentrations in raw samples. Exposure was applied from

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1 to 168 hpf with 50% daily water change. Details of zebrafish husbandry and dilution water

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constituents are provided in SI. Daily mortality was recorded until the end of exposure. Semi-

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quantitative scoring systems have been used in assessment of teratogenicity in fish embryo model

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exposure31,32. In this study, incidences and degree of spinal malformation were recorded, and the

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severity of spinal malformation of each exposure group was calculated based on cumulative

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curvature score (CCS). Briefly, the more malformation points (scaled from 0 to 4; with 4

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documented as the most severe case) are given to more severe deformed embryos according to the

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degree of spine curvature, and the total score is calculated by the sum of all malformation points in

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the same exposure group. Details of scoring and calculation are provided in SI. The incidences of

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pericardial edema were also recorded at 96 hpf. The number of hatched embryos was also recorded

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every day. At the end of exposure, all living larvae in the 3× exposure group were frozen and stored

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at -80 °C for analysis of transcript levels by Q-RT-PCR.

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Quantitative Real-Time PCR assay. Q-RT-PCR assay was performed following a previous study18.

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Twenty-eight genes representing biotransformation, oxidative stress, and endocrine mediation were

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selected for screening. Fold changes of gene expression were quantified using ∆∆Ct method by 8 ACS Paragon Plus Environment

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normalizing to elongation factor 1a (elf1a). There was no difference in the expression of elf1a1

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among all the exposure groups (Figure S1). Gene name, abbreviation, sequences of primers,

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efficiency, and GeneBank reference number are listed in Table S1.

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Statistical Analysis. Statistical analyses were conducted by use of SPSS19.0 (SPSS, Chicago, IL).

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All data are expressed as mean ± standard error mean. Normality of each data set was assessed by

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use of Kolmogorov–Smirnov one-sample test, and homogeneity of variance was determined by use

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of Levene’s test. Log transformation was performed if necessary to meet the assumptions. The

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statistical difference of LC50 values of mortality data for zebrafish embryo (168 hpf) among various

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organic extracts exposures were analyzed using Litchfield-Wilcoxon method. Statistical differences

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among various organic extracts exposures in hatch delay and relative fold changes of gene

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expression were evaluated by one-way ANOVA followed by Tukey test. Statistical differences in

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CCS and percentage of pericardial edema among various organic extracts exposures and

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concentrations were analyzed by two-way ANOVA. Differences were considered significant at p

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1000) compounds ranging from C49H80O18 - C79H140O33 (i.e. detected as [M+H]+, [M+NH4]+, or

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[M+2NH4]2+), also containing repeating EO units. This group of ions could only be identified to

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Level 4 confidence based on the assigned molecular formula, and a lack of more detailed structural

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information.

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In both samples (Figure 1 at ~18.3 min) a series of C10-alkyl ethoxylates was also demonstrated by

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MS/MS, with EO units between 4 and 16 (Figure S3). These were assigned to Level 3 confidence

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with tentatively proposed structures, but were not confirmed by reference standards. Finally,

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another group of compounds eluting at 19.1 min in both samples were identified as octylphenol

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ethoxylates (OPEs), with EO units between 4 and 14 observed (Figure S4). OPEs with 7 and 8 EO

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units were identified to Level 1 based on matching retention times and mass spectra compared to

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authentic standards, while other OPEs were assigned Level 2 confidence because of the regular

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retention time intervals and the same MS fragmentation pattern, but without authentic standard

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matching.

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For both particle phase extracts, compared to blank control, no major organic compounds were

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observed, possibly because these organic compounds are non-polar and not effectively ionized

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under electrospray conditions (Figure S5). More detailed analysis of minor components in these

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samples is the subject of work in progress. 10 ACS Paragon Plus Environment

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PAH analysis

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PAH analysis was used as a marker of petrogenic impacts in the HF-FPW samples, but PAHs likely

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represented only a small fraction of total organics in each sample. Concentrations of PAHs in each

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organic extracts of the 2 samples are shown in Table 2, and detailed profiles are displayed in Table

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S2. The Σ13 PAH and Σ4 alkyl-PAH concentrations in 2-W and 2-S were much higher than in 1-W

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and 1-S, respectively (Table 2). For Σ13PAH, the distribution between phases (aqueous/particle) was

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similar between HF-FPW-1 (59%/41%) and HF-FPW-2 (68%/32%). However, for Σ4 alkyl-PAH,

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the distribution were much lower in HF-FPW-1 (8.5%/91.5%) than in HF-FPW-2 (49%/51%).

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Fluorene and phenanthrene were dominant parent PAHs, and their derivatives (1-methylfluorene

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and methyl/dimethyl phenanthrene) were dominant alkyl PAHs in 2-W and 2-S (Table S2, Figure

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S6). These results indicate that parent PAHs tend to be present primarily in the aqueous phase

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extract, while alkylated PAHs partition differently than the parent PAHs in the same sample. Of

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note, total PAHs in HF-FPW-2 (18,100 ng/L) were approximately 10 times higher than in HF-

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FPW-1 (1,770 ng/L), demonstrating that the composition of HF-FPW can be variable and likely

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depends on the well location and time of flowback collection.

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Mortality and LC50

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The organic extracts of HF-FPW displayed concentration-dependent acute toxicity to zebrafish

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embryos. Figure 2A illustrates the acute toxicity curves of each of the extracts applied in this study

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(Figure 2A). The lowest LC50 value occurred in the 2-P treatment group (2.8×) followed by 2-W

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(4.8×) < 2-S (7.2×) < 1-P (12×) ≤ 1-W (15×) < 1-S (26×) treatment group (Figure 2A, Table S3).

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The analysis of significant differences is marked in Figure 2A and detailed results were provided SI

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(Table S3).

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Spinal malformation



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Exposure to the organic extract of HF-FPW resulted in a significant increase of spinal malformation

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in zebrafish embryos. In order to better characterize this adverse outcome, both incidence rate and

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degree of spinal malformation were considered and used as factors to calculate the CCS as

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malformation index. A rubric for defining CCS (scale 1 to 4) is demonstrated in Figure 2E. Details

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of how to perform this analysis are shown in SI. For each organic extract, there was a clear dose-

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response relationship between the CCS and the exposure concentrations (Figure 2B). Detailed

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results of degree and incidence of spinal malformation are provided in Table S4. The results also

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demonstrated that extracts from HF-FPW-2 had the strongest ability to induce spinal malformation

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compared to any extract from HF-FPW-1. For example, in 10× group, exposure to 2-P resulted in

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CCS of 22.8 ± 1.8, followed by 2-W (21 ± 1.1), 2-S (17 ± 1.4), 1-P (13 ± 1.0), 1-W (9.5 ± 1.2), and

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1-S (7.0 ± 1.7) (Figure 2B, Table S4). The analysis of significant differences is marked in Figure 2B

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and detailed results are provided in SI (Table S5).

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Pericardial edema

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Exposure to organic extracts of HF-FPW also caused significant increases in the incidence of

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pericardial edema in zebrafish embryo. Similar to the results for spinal malformation, there was an

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obvious dose-dependent relationship between the incidence of pericardial edema and the exposure

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concentrations (Figure 2C, Table S6). A representative sample of pericardial edema is demonstrated

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in Figure 2F. The pooled extracts tend to have greater ability to induce pericardial edema compared

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to the parallel concentrations of each of the aqueous or particle phase extracts. For example, 2-P

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was significantly stronger in inducing pericardial edema than 2-W, and 2-S. Exposure to 2-P

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caused 55% ± 6.5% of embryos displaying pericardial edema in 10× exposure group, followed by

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2-W (40% ± 4.1%) and 2-S (33% ± 4.8%). In addition, similar to CCS, extracts from HF-FPW-2

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caused more cardiac edema compared to those from HF-FPW-1. For example, 2-P with 10×

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exposure concentration caused more incidences (55% ± 6.5%) compared to that of 1-P (28% ±


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4.8%). The analysis of significant differences is marked in Figure 2C and detailed results are

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provided SI (Table S6 and S7).

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Hatch delay

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Zebrafish embryos exposed to the organic extracts of HF-FPW also resulted in a delay in hatch.

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Exposure to 3× of 1-W and 1-S resulted in average 72 h hatch rates of 33% ± 2.5% and 65% ±

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2.9%, respectively (Figure 2D, Table S8), which were significantly lower compared to the average

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hatching rate at 72 hpf of 80% ± 4.1%. Exposure to 1× of 2-W and 2-P resulted in average hatch

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rates of 38% ± 4.8% and 53% ± 4.8%, respectively, while exposure to 3× of 2-W, 2-S, and 2-P

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resulted in average hatch rates of 25% ± 2.9%, 58% ± 4.8% and 50% ± 4.1%, respectively, each

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were significantly lower compared to the average hatching rate at 72 hpf (Figure 2D, Table S8). In

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addition, for the 3× exposure groups using extracts from HF-FPW-1 sample, the average hatching

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rate in 1-W group was significantly lower than those of 1-S and 1-P groups. The same pattern was

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also observed in extracts from HF-FPW-2 sample (Figure 2D) suggesting the organic compounds

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extracted from aqueous phase might also have adverse effects on embryo hatch.

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Quantitative Real-time PCR assay

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Biotransformation genes. Exposure to organic extracts of HF-FPW significantly affected the

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abundance of transcripts of genes related to biotransformation including nuclei receptors (aryl

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hydrocarbon receptor, ahr; and pregnane-x-receptor, pxr), phase I (cytochrome p450s, cyp’s) and

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Phase II enzymes (uridine 5’-diphospho-glucuronosyltransferase, udpgt) (Table 3, Figure S7). The

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abundance of transcripts of ahr were significantly elevated in embryos exposed to 1-S, 1-P, 2-W, 2-

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S, and 2-P extracts, with the highest induction fold (3.1 ± 0.3-fold) in 2-P group. The abundance of

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transcripts of pxr were also significantly elevated in embryos exposed to 1-S, 1-P, 2-W, 2-S, and 2-

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P extracts, with the highest induction fold of 2.3 ± 0.3-fold in 2-P group. The expression of several

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cytochrome p450 genes, including cyp1a, cyp1b, cyp1c1, cyp1c2, cyp2aa12 and cyp3a65 were also 13 ACS Paragon Plus Environment


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significantly upregulated in exposed embryos (Table 3). Exposure to 2-P resulted in 127 ± 20-fold

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induction in cyp1a, which was the highest induction fold observed in all genes (Table 3). Exposure

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to organic extracts of HF-FPW also caused significant induction of udpgt1a1 (Table 3). Exposure

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to 2-P resulted in significantly higher expression of ahr, pxr, and cyp3a65, compared to 1-P,

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suggesting HF-FPW-2 had stronger effect on activation of biotransformation genes than HF-FPW-

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1.

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Oxidative stress genes. Abundances of transcripts of several genes involved in responses to

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oxidative stress were also significantly affected by exposure to organic extracts of HF-FPW,

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including (superoxidase dismutase 1, sod1; glutathione peroxidase 1b, gpx1b; glutathione

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synthetase, gss; glutathione reductase, gsr; and glutathiones-transferase p1, gstp1) (Table 3, Figure

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S8). Generally, the pooled extracts caused higher fold induction compared to the aqueous or particle

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phase extract.HF-FPW-2 had significantly stronger effect compared to HF-FPW-1 sample, as

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clearly demonstrated by extract 2-P versus 1-P on sod1 and gss expression (Table 3).

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Endocrine-mediated genes. Exposure to organic extracts of HF-FPW also resulted in significantly

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elevated expressions of several endocrine-mediated genes, including estrogen receptor 2a, esr2a;

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estrogen receptor 2b, esr2b; androgen receptor, ar; aromatase a1a, cyp19a1a; aromatase a1b,

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cyp19a1b, and vitellogenin 1, vtg1 (Table 3, Figure S9). The expression of vtg1 were elevated in all

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the exposure groups with the highest fold induction (9.6 ± 0.6-fold) in the 2-P group, which is

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significantly higher than 1-P. However, for other endocrine-mediated genes (ar, cyp19a1a, and

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cyp19a1b), the fold change in transcript levels were significantly higher in 1-P compared to 2-P

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group. (Table 3).

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The abundance of transcripts for cyp2aa1, cyp2aa2, udpgt5g1, cat, sod2, gpx1a, gstm, and esr1

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were not significantly different amongst any of the control/treatment groups (Figure S10). The


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detailed results of fold change of transcript abundance for each gene in all exposure groups are

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listed in Table S9.

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Discussion

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The two HF-FPW samples tested in this study were obtained from the same geological region

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(Duvernay Formation, Fox Creek, Alberta, Canada). Both samples have extremely high salinity (~

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250,000-280,000 ppm), but HF-FPW-2 had higher pH, higher total organic carbon compared to HF-

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FPW-1, and the organic chemical compositions were shown to be quite different by targeted PAH

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analysis and non-target mass spectrometry. High resolution MS has previously been shown useful

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for identification of organic substances in HF-FPW34,35,36, and here the application of HPLC-

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Orbitrap-MS was useful for identifying major components of the aqueous phase. The much higher

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mass spectral response of the total ion chromatogram for the organic extract of HF-FPW-2,

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compared to HF-FPW-1, is consistent with the higher total organic carbon in HF-FPW-2. PEGs

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were detected in both samples, which has been previously reported by our group9, and was also

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found in HF water samples collected from other shale gas sites36.

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ethoxylates, with 4 to 16 EO units were also detected in both samples, and similar MS/MS spectra

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were also observed by Thurman et al36. Moreover, OPEs were detected in both samples. This group

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of chemicals, commercially referred to as Triton-X, are commonly used as surfactants in hydraulic

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fracturing fluids, which are readily microbial-degraded to octylphenol, a well-known EDC37.

In addition, C10 alkyl

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In this study, the parent and alkylated PAHs were analyzed as a marker of petroleum-related

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hydrocarbons in HF-FPW samples. It has been demonstrated that even short-term exposure to

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PAHs and their methylated derivatives can be highly toxic and teratogenic to fish embryo26,38, 39.

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For both samples, parent PAHs (16 USEPA PAHs) tend to partition into the aqueous phase, while

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alkylated PAHs tend to partition into particle phase. However, certain PAHs, including pyrene,

310

fluoranthene, fluorine, methylphenanthrene, differentially partition between the aqueous and 15 ACS Paragon Plus Environment


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particle phases extracts due to the different physiochemical properties of the solution as well as the

312

associated particles in these samples. This also indicates not only certain PAHs, but other organic

313

contaminants in these wastewaters could also partition differentially between different extracts.

314

However, the actual proportions of total PAHs with respect to the total organic constituent

315

concentration were extremely low. The total organic carbon in HF-FPW-1 and HF-FPW-2 were

316

estimated to be 211 and 737 mg/L, respectively9,20. Based on approximation, total PAHs only

317

account for 0.00084% and 0.0025% of total organic carbon in HF-FPW-1 and HF-FPW-2,

318

respectively, and likely serve as a marker of other petrogenic chemicals in the samples. HF-FPW-2

319

contains much higher organic pollutants, including PAHs and their derivatives, and we found in

320

general that extracts from HF-FPW-2 induced more adverse effects than those from HF-FPW-1.

321

The results of the current study suggest that exposure to the organic constituents in HF-FPW alone,

322

including but not limited to PAHs, are associated with major developmental adverse outcomes in

323

zebrafish embryogenesis.

324

These effects of the organic extracts of HF-FPW on zebrafish embryos are consistent with

325

the results noted in a previous study9. Several studies have reported lower survival and

326

reduced/delayed hatching success in a variety of fish embryos exposed to PAHs, oil sands process-

327

affected water, petroleum oil and extracts of crude oil38,40,41. The LC50 values obtained from this

328

study using organic extracts exposure are much higher than those in previous studies using

329

raw/diluted samples exposure9,14,17,20, indicating the acute toxicity of HF activity related water in

330

our previous study was mainly driven by salt content. In both HF-FPW samples, the pooled extracts

331

result in the highest toxicity and the greatest degree of adverse effects in exposed embryo.

332

Moreover, HF-FPW-2, which has more organic content (e.g. ~10× more total PAHs), caused greater

333

adverse effects compared to HF-FPW-1.

334

resulted in greater adverse effects than the co-incident particle phase extract. Nevertheless, the

For both samples tested, the aqueous phase extract


Page 17 of 36


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particle phase extract, despite being a very small fraction by volume, carried a substantial portion of

336

the acute toxicity. For example, extract 1-W resulted in LC50 value of 15×, which is significantly

337

lower than extract 1-S (26×) (Table S3). Similarly, extract 2-W resulted in LC50 value of 4.8×,

338

which is significantly lower than extract 2-S (7.2×) (Table S3). We hypothesize that these

339

differences between the water-associated and particle-associated toxicity may be due to the greater

340

hydrophilicity and bioavailability and/or more potent organic chemicals present in the aqueous

341

phase extract compared to the particle phase extract. In the case of a direct spill into a flowing

342

stream, we hypothesize that the accumulated FPW particles could result in a local environment (eg.

343

stream bed) with contaminant concentrations well above those in the wastewater. An analogous

344

exposure in the aquatic ecosystem is foreseeable. Future characterization of the detailed chemical

345

profile of HF-FPW samples with focus towards understanding classes of organics associated with

346

either the sediment or aqueous phase extracts will aid in assessing hazard and ultimately,

347

understanding the risk associated with handling different HF-FPWs.

348

In the current study, the spinal malformation and pericardial edema observed during

349

development of embryos exposed to either HF-FPW organic extracts were consistent with the

350

findings of other studies using petroleum-related extracts. For example, previous studies in fish

351

embryos exposed to petroleum contaminated waters reported delayed hatch, hemorrhaging,

352

pericardial edema, and malformation of the spine40,42,43,44. These deformities have been compared to

353

symptoms of “blue sac disease”, which is induced when PAHs and other dioxin-like compounds

354

activate the Aryl hydrocarbon Receptor (AhR)45. The mechanism of toxicity due to activation of

355

AhR signaling includes induction of CYP1A, as well as correlated oxidative stress resulting in

356

DNA/tissue damage in both embryos and visibly healthy post-hatch fry46. The observed spinal

357

malformation and pericardial edema in exposed embryos strongly suggest activation of AhR

358

signaling and associated oxidative stress as one of the main toxicity pathway activated by FPW

359

organic extracts. To further investigate the involvement of AhR and oxidative stress pathways as 17 ACS Paragon Plus Environment


Page 18 of 36

360

potential mechanism(s) of toxicity, a Q-RT-PCR array was designed to measure the expression

361

changes of a battery of genes in exposed embryos. Significantly elevated ahr was indeed observed

362

in zebrafish embryos exposed to each FPW organic extract, with the highest expression fold change

363

in 2-P. These results are also consistent with the PAH data suggesting the presence of AhR

364

agonist(s) (including but not limited to PAHs) in both HF-FPW samples. In addition, we have also

365

demonstrated significantly elevated expression of cyp1a, cyp1b1, cyp1c1, cyp1c2, further

366

supporting the activation of AhR as a significant pathway induced by FPW organic extracts.

367

Organic compounds in HF-FPW may also act as agonists of the Pregnane-X-Receptor

368

(PXR). PXR is a pleiotropic nuclear receptor activated by endogenous and exogenous chemicals

369

that regulates various enzymes involved in biotransformation of xenobiotics47. Primary targets of

370

PXR activation include several phase I enzymes (eg. CYP2s and CYP3s), as well as phase II

371

enzymes (eg. UDPGTs) involved in xenobiotic biotransformation and secretion48. In addition, PXR

372

also interacts with factors binding to the antioxidant response element and this up-regulates

373

expression of phase II conjugating enzymes such as Glutathione S-Transferase (GST)49. It has been

374

reported that Triclosan, a biocide and well-known PXR agonist50, was used in HF fluid to prevent

375

bioclogging51, but we cannot directly identify Triclosan in any of our samples. In the current study,

376

the transcripts abundance of pxr, together with cyp3a65 and udpgt1a1 were significantly greater in

377

embryos exposed to FPW extracts compared to control. As discussed below, the abundance of

378

transcripts of gstp1 was also greater in exposed embryos. Albeit indirect activation of PXR is also

379

possible, these findings are compelling evidence of the presence of PXR agonist(s) in HF-FPW

380

samples.

381

The malformations in embryos exposed to organic extracts of HF-FPW are also consistent

382

with those caused by oxidative stress, including spinal malformation and pericardial edema40.

383

Oxidative stress results when antioxidant defense mechanisms become saturated and concentrations


Page 19 of 36


384

of reactive oxygen species (ROS) exceed the levels produced during normal functioning of cells,

385

overwhelming the capacity of cells to reduce ROS and ultimately resulting in damage to tissues and

386

cells52. Several phase II enzymes, including superoxide dismutase (SOD), Glutathione Peroxidase

387

(GPX), Glutathione Synthetase (GSS), Glutathione Reductase, and Glutathione-S-transferase (GST)

388

facilitate detoxification of drugs and play key functions in clearance of ROS. ROS generation in

389

microsomes due to biotransformation has been correlated with total P450 content and CYP3A

390

activity53. Overall, our observation of increased transcript levels for 5 different ROS related genes

391

strongly suggest the oxidative stress is one of the major toxicity mechanisms following HF-FPW

392

exposure.

393

Exposure to petroleum-contaminated waters have been previously demonstrated to be

394

associated with upregulation of endocrine related genes54,55, suggesting that EDC related effects are

395

a possible consequence from HF-FPW exposure. In vitro estrogen and androgen receptors binding

396

activities have been reported in chemicals used in HF fluid as well as in ground and surface water in

397

drilling region and disposal site22,23. It has also been reported that exposure to HF chemicals mixture

398

can cause adverse reproductive and developmental health outcomes in mice56,57. In the current

399

study, the expression of seven endocrine-related genes was examined and the results suggest the

400

organic extracts of HF-FPW samples have significant endocrine disruptive properties for at least six

401

different genes.

402

Exposure to organic extracts of HF-FPWs resulted in significantly elevated expression of vtg1,

403

consistent with the previous study in rainbow trout18. Moreover, extract 2-P resulted in the highest

404

fold induction in vtg1 expression, suggesting HF-FPW-2 contains more estrogenic compounds than

405

HF-FPW-1. There are 3 estrogen receptor (ER) isoforms identified in zebrafish: esr1, esr2a, and

406

esr2b. It has been reported that all three isoforms can be altered in transcriptional level and their

407

upregulation usually means estrogenic effects58. In the current study, upregulation was observed in

408

esr2a and esr2b, together with vtg1, suggesting the presence of estrogenic compounds in HF-FPWs.

Vitellogenin is a sensitive indicator of environmental estrogenic effects58.



Page 20 of 36

409

The altered expression of androgen receptor (ar) has also been used to indicate EDC effects59. In

410

our study, the expression of ar was also up-regulated in exposed embryos, suggesting the presence

411

of androgenic chemicals in HF-FPW, which is consistent with the presence of androgenic chemicals

412

applied in HF fluid as demonstrated by Kassotis et al.56. To support our findings that estrogenic and

413

androgenic compounds may be present in HF-FPW, we also examined the expression of 2 gonadal

414

aromatase isoforms, cyp19a1a and cyp19a1b and found them to be upregulated in exposed

415

embryos. Aromatase is an enzyme that plays an important role in the endocrine system by

416

converting androgen to estrogen. The upregulation of the aromatase gene(s) has been linked to

417

various environmental estrogenic compounds, including flame retardants, pesticides, and petroleum

418

related produced water60,61. In our study, the highest fold increase in expression of ar, cyp19a1a and

419

cyp19a1b was found in the 1-P exposure group. Considering there are ~10 times higher total PAHs

420

content and more other organics in HF-FPW-2, it was a bit surprising that the highest expressions of

421

ar, cyp19a1a and cyp19a1b were found in extract 1-P exposure group. This finding implies that

422

using total PAHs or total organic carbon cannot be used markers of hazards due to the significant

423

endocrine disrupting potentials of this wastewater. A 2011 study documented approximately 120

424

known or suspected EDCs out of 353 chemicals used in oil and gas operation chemicals with

425

Chemical Abstract Service (CAS) numbers62. More importantly, a lot of the chemicals remain

426

proprietary information63, greatly limiting the toxicity study and risk assessment for HF activity

427

related wastewater. It has been determined that certain chemicals used in oil and gas operation,

428

such as Ethylene glycol, Naphthalene, Bronopol, have significant EDC activity and could

429

potentially cause adverse health outcomes56. In the current study, OPEs were detected in both

430

samples, whose degradation product, octylphenol is a documented EDC. However, the identities of

431

other EDCs in HF-FPW organic extracts or in real HF activity related wastewater still require

432

further investigation. Given the complexity of HF-FPW in terms of different sampling timepoints,

433

differing fracturing fluids applied, underground temperature profiles governing downhole reaction


Page 21 of 36


434

chemistry and variations in geological location/ionic and petrogenic profile of the fracturing wells,

435

understanding the presence and nature of EDC-related effects of HF-FPW will be a significant

436

challenge.

437

The results of the current study demonstrate the compositional variation between two real

438

HF-FPW samples. OPEs, a typical group of EDCs, were detected in both samples. The results also

439

demonstrate that exposure to organic extracts of HF-FPW caused adverse effects in developing

440

zebrafish embryos. Compared to our previous studies using diluted samples9,18,19,20, the exposure

441

using organic extracts of HF-FPW showed much lower lethal toxicity, suggesting that the salt

442

content is the major lethal component in acute toxicity. Although some sub-lethal effects were

443

suggested in previous studies, the current study using concentrated organic extracts allows better

444

characterizations on the development and endocrine mediated effects in exposed embryo. In the

445

current study, greater incidences of mortality, spinal malformation, pericardial edema, and hatch

446

delay were caused by exposure to organic extracts of HF-FPW. Transcriptional responses of a

447

variety of genes related to biotransformation, oxidative stress and endocrine mediation were also

448

significantly changed in embryos exposed to organic extracts of HF-FPW. The results suggest that

449

oxidative stress resulting from metabolism of substrates by cytochrome P450 enzymes induced by

450

activation of both AhR and PXR is the primary mechanism of effects on embryo. In addition, the

451

various unknown organic compounds in HF-FPW may also pose various EDC effects in the early

452

life stage of zebrafish. Future study is necessary to investigate the EDC effects on adult fish in

453

fecundity and reproduction, and detailed chemical analysis is also required to characterize the

454

identity and origin of EDC(s) present in HF-FPW samples.

455

Acknowledgements

456

The project was funded by Natural Sciences and Engineering Research Council of Canada

457

(NSERC) Collaborative Research and Development (CRD) grant CRDPJ 469308-14, with support



Page 22 of 36

458

from the Encana Corporation, to Danial S. Alessi, Jonathan W. Martin, and Greg G. Goss. As per

459

our research agreement, oversight by from Encana is limited to a maximum 60 days review period

460

prior to publication only . Encana provides samples and sample collection data but does not provide

461

input into either research directions or interpretation of results generated. We would like to thank

462

Science Animal Support Services for assistance in animal care.

463

Disclosure

464

The authors declare no competing financial interest.

465

Supporting Information

466

The Supporting Information, including methods, and supplementary tables and figures, is available

467

free of charge via the Internet at http://pubs.acs.org.

468

Reference

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44. Incardona, J.P.; Gardner, L.D.; Linbo, T.L.; Brown, T.L.; Esbaugh, A.J.; Mager, E.M.; Stieglitz,

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J.D.; French, B.L.; Labenia, J.S.; Laetz, C.A.; Tagal, M.; Sloan, C.A.; Elizur, A.; Benetti, D.D.;

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Grosell, M.; Block, B.A.; Scholz, N.L. Deepwater Horizon crude oil impacts the developing

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hearts of large predatory pelagic fish. Proc. Natl. Acad. Sci. U.S.A. 2014, 111(15), E1510-1518.

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45. Fernandez-Salguero, P.M.; Hilbert, D.M.; Rudikoff, S.; Ward, J.M.; Gonzalez, F.J. Aryl-

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hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-

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induced toxicity. Toxicol. Appl. Pharmacol. 1996, 140(1), 173-179.

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46. Cantrell, S.M.; Joy-Schlezinger, J.; Stegeman, J.J.; Tillitt, D.E.; Hannink, M. Correlation of

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2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptotic cell death in the embryonic vasculature

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with embryotoxicity. Toxicol. Appl. Pharmacol. 1998, 148(1), 24-34.

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47. Kliewer, S.A.; Goodwin, B.; Willson, T.M. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr. Rev. 2002, 23(5), 687-702.

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48. Kubota, A.; Goldstone, J.V.; Lemaire, B.; Takata, M.; Woodin, B.R.; Stegeman, J.J. Role of

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pregnane X receptor and aryl hydrocarbon receptor in transcriptional regulation of pxr, CYP2,

619

and CYP3 genes in developing zebrafish. Toxicol. Sci. 2015, 143(2), 398-407.

620

49. Higgins, L.G.; Hayes, J.D. Mechanisms of induction of cytosolic and microsomal glutathione

621

transferase (GST) genes by xenobiotics and pro-inflammatory agents. Drug Metab. Rev. 2011,

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43(2), 92-137.

623

50. Hernandez, J.P.; Mota, L.C.; Baldwin, W.S. Activation of CAR and PXR by Dietary,

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Environmental and Occupational Chemicals Alters Drug Metabolism, Intermediary Metabolism,

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and Cell Proliferation. Curr. Pharmacogenomics Person Med. 2009, 7(2), 81-105.


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51. Kahrilas, G.A.; Blotevogel, J.; Stewart, P.S.; Borch, T. Biocides in hydraulic fracturing fluids: a

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critical review of their usage, mobility, degradation, and toxicity. Environ. Sci. Technol. 2015,

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49(1), 16-32.

629 630 631 632

52. Lushchak, V.I. Contaminant-induced oxidative stress in fish: a mechanistic approach. Fish Physiol. Biochem. 2016, 42(2), 711-747. 53. Zangar, R.C.; Davydov, D.R.; Verma, S. Mechanisms that regulate production of reactive oxygen species by cytochrome P450. Toxicol. Appl. Pharmacol. 2004, 199(3), 316-331.

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54. He, Y.; Wiseman, S.B.; Wang, N.; Perez-Estrada, L.A.; El-Din, M.G.; Martin, J.W.; Giesy, J.P.

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Transcriptional responses of the brain-gonad-liver axis of fathead minnows exposed to untreated

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and ozone-treated oil sands process-affected water. Environ. Sci. Technol. 2012, 46(17), 9701-

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9708.

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55. Salaberria, I.; Brakstad, O.G.; Olsen, A.J.; Nordtug, T.; Hansen, B.H. Endocrine and AhR-

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CYP1A pathway responses to the water-soluble fraction of oil in zebrafish (Danio rerio

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Hamilton). J. Toxicol. Environ. Health A 2014, 77(9-11), 506-515.

640

56. Kassotis, C.D.; Klemp, K.C.; Vu, D.C.; Lin, C.H.; Meng, C.X.; Besch-Williford, C.L.; Pinatti,

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L.; Zoeller, R.T.; Drobnis, E.Z.; Balise, V.D.; Isiguzo, C.J.; Williams, M.A.; Tillitt, D.E.;

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Nagel, S.C. Endocrine-Disrupting Activity of Hydraulic Fracturing Chemicals and Adverse

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Health Outcomes After Prenatal Exposure in Male Mice. Endocrinology 2015, 156(12), 4458-

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4473.

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57. Kassotis, C.D.; Bromfield, J.J.; Klemp, K.C.; Meng, C.X.; Wolfe, A.; Zoeller, R.T.; Balise,

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V.D.; Isiguzo, C.J.; Tillitt, D.E.; Nagel, S.C. Adverse Reproductive and Developmental Health

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Outcomes Following Prenatal Exposure to a Hydraulic Fracturing Chemical Mixture in Female

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C57Bl/6 Mice. Endocrinology 2016, 157(9), 3469-3481.

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58. Söffker, M.; Tyler, C.R. Endocrine disrupting chemicals and sexual behaviors in fish - a critical review on effects and possible consequences. Crit. Rev. Toxicol. 2002, 42(8), 653-668.



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60. Cheshenko, K.; Brion, F.; Le Page, Y.; Hinfray, N.; Pakdel, F.; Kah, O.; Segner, H.; Eggen, R.I.

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Expression of zebra fish aromatase cyp19a and cyp19b genes in response to the ligands of

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estrogen receptor and aryl hydrocarbon receptor. Toxicol. Sci. 2007, 96(2), 255-67.

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61. Wang, J.; Cao, X.; Huang, Y.; Tang, X. Developmental toxicity and endocrine disruption of

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naphthenic acids on the early life stage of zebrafish (Danio rerio). J. Appl. Toxicol. 2015,

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35(12), 1493-1501.

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62. Colborn, T.; Kwiatkowski, C.; Schultz, K.; Bachran, M. Natural gas operations from a public health perspective. Hum Ecol Risk Assess. 2011, 17(5), 1039–1056. 63. Shonkoff, S.B.; Hays, J.; Finkel, M.L. Environmental public health dimensions of shale and tight gas development. Environ. Health Perspect. 2014, 122(8), 787–795.

663


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664

TABLE

665

Table 1. Summary of basic water chemistry for HF-FPW-1 and HF-FPW-2. (Data obtained from

666

companion studies 9,18). TDS: total dissolved solid, TN: total nitrogen, TOC: total organic carbon.

HF-FPW-1

HF-FPW-2

7 days

10 days

4.78

5.86

243 g/L

183 g/L

2.2 mg/L CaCO3

14.3 mg/L CaCO3

TN

498 mg/L

425 mg/L

TOC

211 mg/L

737 mg/L

Time pH TDS Alkalinity

667

668



Page 32 of 36

669

Table 2. Total concentrations of 13 EPA parent PAHs and 4 alkylated PAHs in organic fractions of

670

HF-FPW Samples.

HF-FPW-1

HF-FPW-2

1-W

1-S

1-P

2-W

2-S

2-P

810

560

1370

5000

2400

7400

59%

41%

100%

68%

32%

100%

33

360

390

5200

5500

10700

Pooled %b

8.5%

92%

100%

49%

51%

100%

Total PAHs

840

930

1770

10200

7900

18100

ng/L Σ13PAH a Pooled %

b

Σ4alkyl-PAHc

671

a

672

b

673

extracts.

674

c

675

Dimethylphenanthrene and 1-Methylpyrene.

Thirteen PAHs (out of 16 USEPA PAHs) were detected from HF-FPW-1 and HF-FPW-2. Data in pooled extracts (1-P and 2-P) are generated by the sum of aqueous and particle phases

Four alkyl PAHs were measured, i.e., 1-Methylfluorene, 1-Methylphenanthrene, 3,6-

676


Page 33 of 36


677

Table 3. Transcriptional responses of biotransformation, oxidative stress and endocrine mediation

678

related genes in zebrafish larvae (168 hpf) exposed to various organic extracts of HF-FPW. Data in

679

bold indicate significant difference from control. Asterisk indicate significant difference between 1-

680

P and 2-P (n = 4, p < 0.05). Gene

Ctl

1-W

1-S

1-P

2-W

2-S

2-P

ahr pxr cyp1a cyp1b1 cyp1c1 cyp1c2 cyp2aa12 cyp3a65 udpgt1a1 sod1 gpx1b gstp1 gss gsr cyp19a1a cyp19a1b ar esr2a esr2b vtg1

1.0

1.3

1.7

1.8

2.0

1.9

*3.1

1.0

1.3

1.5

1.6

1.9

1.8

*2.3

1.0

75.1

64.6

92.0

101.6

78.4

127.0

1.0

47.2

31.0

55.7

35.1

24.3

51.2

1.0

12.1

10.5

14.2

15.6

10.3

16.7

1.0

15.4

13.7

13.1

13.5

7.7

15.6

1.0

2.0

2.0

2.6

2.9

2.4

2.7

1.0

1.6

3.7

3.8

3.4

4.0

*4.2

1.0

2.1

2.6

3.1

2.8

2.8

3.5

1.0

0.8

1.3

1.3

1.6

1.8

*1.9

1.0

0.7

1.8

2.4

1.8

1.8

3.7

1.0

2.7

2.3

3.7

2.9

2.3

3.9

1.0

0.7

1.4

1.0

1.9

1.7

*2.3

1.0

1.4

2.4

1.8

1.7

1.8

2.6

1.0

1.9

6.6

*10.1

1.8

1.3

1.3

1.0

1.5

2.1

*3.2

1.3

0.9

1.0

1.0

1.4

3.8

*5.6

1.9

1.8

2.1

1.0

2.0

2.5

4.0

2.9

3.4

3.8

1.0

1.8

2.3

3.9

2.1

2.0

2.7

1.0

2.6

4.3

6.3

6.7

5.3

*9.6

681

682



683

Page 34 of 36

FIGURE

684

685 686

Figure 1. HPLC-Orbitrap-MS total positive ion chromatograms of organic extracts from aqueous

687

phases of (A) HF-FPW-1, and (B) HF-FPW-2.


Page 35 of 36


688 689

Figure 2. Observation of the effects of HF-FPW on zebrafish embryos exposed to various organic

690

fractions (water fraction, sediment fraction and pooled) extracted from two different HF-FPW

691

samples (HF-FPW-1 and HF-FPW-2). Exposures of all the concentrations and one control were

692

conducted at the same time. A) Mortality curves. B) Cumulative curvature scores for spinal

693

malformation. C) Incidences of pericardial edema. D) Hatch delay at 72 hpf. E) Cumulative

694

curvature score scale as applied to document degrees of spinal malformation occurring in zebrafish

695

embryo following HF-FPW exposure. F) Example of pericardial edema (PE) in zebrafish embryo

696

following HF-FPW exposure. . Different letters in A, B, and C indicate significance obtained from 35 ACS Paragon Plus Environment


Page 36 of 36

697

two-way ANOVA. Different letters in D indicate significant differences within group obtained from

698

one-way ANOVA, and asterisk (*) represents significant differences from control.


Developmental Toxicity of the Organic Fraction from Hydraulic

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