Repository for Oil and Gas Energy Research (ROGER)

Abstract:
Diluted bitumen, also known as dilbit, is transported by rail and pipeline across Canada and the United States. Due to the fewer number of studies characterizing the toxicity of dilbit, a dilbit spill poses an unknown risk to freshwater aquatic ecosystems. In the following study, we compared the impact of early-life exposure to conventional and unconventional crude oils on the optomotor behavior, reproductive success, and transgenerational differences in gene expression in zebrafish and their progeny. For exposures, water accommodated fractions (WAFs) of crude oil were generated using a 1:1000 oil to water ratio for 3 different crudes; mixed sweet blend (MSB), medium sour composite (MSC) and dilbit. All three oils generated unique volatile organic compound (VOC) and polycyclic aromatic compound (PAC) profiles. Of the WAFs tested, only dilbit decreased the eye size of 2 dpf larvae, and only MSB exposed larvae had an altered behavioral response to a visual simulation of a predator. Early-life exposure to crude oil had no lasting impact on reproductive success of adult fish; however, each oil had unique impacts on the basal gene expression of the somatically exposed offspring. In this study, the biological effects differed between each of the oils tested, which implied chemical composition plays a critical role in determining the sublethal toxicity of conventional and unconventional crude oils in freshwater ecosystems.

Abstract:
Hydraulic fracturing (“fracking”) is a process in which subterranean natural gas-laden rock is fractured under pressure to enhance retrieval of gas. Sand (a “proppant”) is present in the fracking fluid pumped down the well bore to stabilize the fissures and facilitate gas flow. The manipulation of sand at the well site creates respirable dust (fracking sand dust, FSD) to which workers are exposed. Because workplace exposures to FSD have exceeded exposure limits set by OSHA, a physico-chemical characterization of FSD along with comprehensive investigations of the potential early adverse effects of FSDs on organ function and biomarkers has been conducted using a rat model and related in vivo and in vitro experiments involving the respiratory, cardiovascular, immune systems, kidney and brain. An undercurrent theme of the overall hazard identification study was, to what degree do the health effects of inhaled FSD resemble those previously observed after crystalline silica dust inhalation? In short-term studies, FSD was found to be less bioactive than MIN-U-SIL® 5 in the lungs. A second theme was, are the biological effects of FSD restricted to the lungs? Bioactivity of FSD was observed in all examined organ systems. Our findings indicate that, in many respects, the physical and chemical properties, and the short-term biological effects, of the FSDs share many similarities as a group but have little in common with crystalline silica dust.

Abstract:
Hydraulic fracturing flowback and produced water (FPW) is a highly complex and heterogenous wastewater by-product of hydraulic fracturing practices. To date, no research has examined how FPW exposure to freshwater biota may affect energetic homeostasis following subsequent induction of detoxification processes. Rainbow trout (Oncorhynchus mykiss) were acutely exposed for 48 h to either 2.5% or 7.5% FPW, and hepatic metabolism was assessed either immediately or following a 3-week recovery period. Induction of xenobiotic metabolism was observed with an 8.8-fold increase in ethoxyresorufin-O-deethylase (EROD) activity after 48 h exposure to 7.5% FPW, alongside a 10.3-fold increase in the mRNA abundance of cyp1a, both of which returned to basal level after three weeks. Glucose uptake capacity was elevated by 6.8- and 12.9-fold following 2.5% and 7.5% FPW exposure, respectively, while alanine uptake was variable. Activity measurements and mRNA abundance of key enzymes involved in hepatic metabolism indicated that aerobic metabolism was maintained with exposure, as was glycolysis. Gluconeogenesis, as measured by phosphoenolpyruvate carboxykinase (PEPCK) activity, decreased by ~30% 48 h following 2.5% FPW exposure and ~20% 3 weeks after 7.5% FPW exposure. The abundance of pepck mRNA activity followed similar, yet non-significant, trends. Finally, a delayed increase in amino acid catabolism was observed, as glutamate dehydrogenase (GDH) activity was increased 2-fold in 7.5% FPW exposed fish when compared to saline control fish at the 3-week time point. We provide evidence to suggest that although hepatic metabolism is altered following acute FPW exposure, metabolic homeostasis generally returns 3-weeks post-exposure.

Abstract:
Publicly available toxicological studies on wastewaters associated with unconventional oil and gas (UOG) activities in offshore regions are nonexistent. The current study investigated the impact of hydraulic fracturing-generated flowback water (HF-FW) on whole organism swimming performance/respiration and cardiomyocyte contractility dynamics in mahi-mahi (Coryphaena hippurus—hereafter referred to as “mahi”), an organism which inhabits marine ecosystems where offshore hydraulic fracturing activity is intensifying. Following exposure to 2.75% HF-FW for 24 h, mahi displayed significantly reduced critical swimming speeds (Ucrit) and aerobic scopes (reductions of ∼40 and 61%, respectively) compared to control fish. Additionally, cardiomyocyte exposures to the same HF-FW sample at 2% dilutions reduced a multitude of mahi sarcomere contraction properties at various stimulation frequencies compared to all other treatment groups, including an approximate 40% decrease in sarcomere contraction size and a nearly 50% reduction in sarcomere relaxation velocity compared to controls. An approximate 8-fold change in expression of the cardiac contractile regulatory gene cmlc2 was also seen in ventricles from 2.75% HF-FW-exposed mahi. These results collectively identify cardiac function as a target for HF-FW toxicity and provide some of the first published data on UOG toxicity in a marine species.

Abstract:
Hydraulic fracturing (“fracking”) is a process used to enhance retrieval of gas from subterranean natural gas-laden rock by fracturing it under pressure. Sand used to stabilize fissures and facilitate gas flow creates a potential occupational hazard from respirable fracking sand dust (FSD). As studies of the immunotoxicity of FSD are lacking, the effects of whole-body inhalation (6 h/d for 4 d) of a FSD, i.e., FSD 8, was investigated at 1, 7, and 27 d post-exposure in rats. Exposure to 10 mg/m3 FSD 8 resulted in decreased lung-associated lymph node (LLN) cellularity, total B-cells, CD4+ T-cells, CD8+ T-cells and total natural killer (NK) cells at 7-d post exposure. The frequency of CD4+ T-cells decreased while the frequency of B-cells increased (7 and 27 d) in the LLN. In contrast, increases in LLN cellularity and increases in total CD4+ and CD8+ T-cells were observed in rats following 30 mg/m3 FSD 8 at 1 d post-exposure. Increases in the frequency and number of CD4+ T-cells and NK cells were observed in bronchial alveolar lavage fluid at 7-d post-exposure (10 mg/m3) along with an increase in total CD4+ T-cells, CD11b + cells, and NK cells at 1-day post-exposure (30 mg/m3). Increases in the numbers of B-cells and CD8+ T-cells were observed in the spleen at 1-day post 30 mg/m3 FSD 8 exposure. In addition, NK cell activity was suppressed at 1 d (30 mg/m3) and 27 d post-exposure (10 mg/m3). No change in the IgM response to sheep red blood cells was observed. The findings indicate that FSD 8 caused alterations in cellularity, phenotypic subsets, and impairment of immune function.

Abstract:
Hydraulic fracturing (HF) technology is increasingly utilized for oil and gas extraction operations. The widespread use of HF has led to concerns of negative impacts on both the environment and human health. Indeed, the potential endocrine disrupting impacts of HF chemicals is one such knowledge gap. Herein, we used structure-based molecular docking to assess the binding affinities of 60 HF chemicals to the human androgen receptor (AR). Five HF chemicals had relatively high predicted AR binding affinity, suggesting the potential for endocrine disruption. We next assessed androgenic and antiandrogenic activities of these chemicals in vitro. Of the five candidate AR ligands, only Genapol®X–100 significantly modified AR transactivation. To better understand the structural effect of Genapol®X–100 on the potency of AR inhibition, we compared the antiandrogenic activity of Genapol®X–100 with that of its structurally similar chemical, Genapol®X–080. Interestingly, both Genapol®X–100 and Genapol®X–080 elicited an antagonistic effect at AR with 20% relative inhibitory concentrations of 0.43 and 0.89 μM, respectively. Furthermore, we investigated the mechanism of AR inhibition of these two chemicals in vitro, and found that both Genapol®X–100 and Genapol®X–080 inhibited AR through a noncompetitive mechanism. The effect of these two chemicals on the expression of AR responsive genes, e.g. PSA, KLK2, and AR, was also investigated. Genapol®X–100 and Genapol®X–080 altered the expression of these genes. Our findings heighten awareness of endocrine disruption by HF chemicals and provide evidence that noncompetitive antiandrogenic Genapol®X–100 could cause adverse endocrine health effects.

Abstract:
Unconventional oil and natural gas extraction (UOG) combines directional drilling and hydraulic fracturing and produces billions of liters of wastewater per year. Herein, we review experimental studies that evaluated the potential endocrine-mediated health impacts of exposure to a mixture of 23 UOG chemicals commonly found in wastewater. The purpose of this manuscript is to synthesize and summarize a body of work using the same UOG-mix but with different model systems and physiological endpoints in multiple experiments. The studies reviewed were conducted in laboratory animals (mice or tadpoles) and human tissue culture cells. A key feature of the in vivo studies was the use of four environmentally relevant doses spanning three orders of magnitude ranging from concentrations found in surface and ground water in UOG dense areas to concentrations found in UOG wastewater. This UOG-mix exhibited potent antagonist activity for the estrogen, androgen, glucocorticoid, progesterone, and thyroid receptors in human tissue culture cells. Subsequently, pregnant mice were administered the UOG-mix in drinking water and offspring were examined in adulthood or to tadpoles. Developmental exposure profoundly impacted pituitary hormone concentrations, reduced sperm counts, altered folliculogenesis, and increased mammary gland ductal density and preneoplastic lesions in mice. It also altered energy expenditure, exploratory and risk-taking behavior, the immune system in three immune models in mice, and affected basal and antiviral immunity in frogs. These findings highlight the diverse systems affected by developmental EDC exposure and the need to examine human and animal health in UOG regions.

Abstract:
Although aquatic vertebrates and humans are increasingly exposed to water pollutants associated with unconventional oil and gas extraction (UOG), the long-term effects of these pollutants on immunity remains unclear. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable and sensitive model for evaluating the effects of waterborne pollutants. X. laevis tadpoles were exposed to a mixture of equimass amount of UOG chemicals with endocrine disrupting activity (0.1 and 1.0 μg/L) for 3 weeks, and then long-term effects on immune function at steady state and following viral (FV3) infection was assessed after metamorphosis. Notably, developmental exposure to the mixture of UOG chemicals at the tadpole stage affected metamorphic development and fitness by significantly decreasing body mass after metamorphosis completion. Furthermore, developmental exposure to UOGs resulted in perturbation of immune homeostasis in adult frogs, as indicated by significantly decreased number of splenic innate leukocytes, B and T lymphocytes; and a weakened antiviral immune response leading to increased viral load during infection by the ranavirus FV3. These findings suggest that mixture of UOG-associated waterborne endocrine disruptors at low but environmentally–relevant levels have the potential to induce long-lasting alterations of immune function and antiviral immunity in aquatic vertebrates and ultimately human populations.

Abstract:
The effluent produced during hydraulic fracturing (i.e. flowback and produced water; FPW), is a complex hyper-saline solution that is known to negatively impact the survival and the fitness of the water flea Daphnia magna, but to date effects on behavior are unstudied. In the current study, the effects of FPW on phototactic behavior of D. magna were examined. Exposure of naïve animals to FPW resulted in a dose-dependent increase in the speed of appearance of daphnids in the illuminated zone of the test apparatus (i.e. a faster positive phototaxis response). A similar dose-dependent response was observed in a test solution where the salt content of FPW was recreated in the absence of other components, suggesting that the effect was largely driven by salinity. The effect of FPW was significant when the raw FPW sample was diluted to 20% of its initial strength, while the effect of salt-matched solution was significant at a 10% dilution. A distinct effect was observed following FPW pre-exposure. After a 24 h pre-exposure to 1.5% FPW, Daphnia displayed a significantly inhibited positive phototaxis response when examined in control water, relative to control animals that were not pre-exposed to FPW. This effect was not observed in salinity pre-exposed animals, however these daphnids displayed a significantly reduced phototactic response when tested in saline waters, indicating a loss of the positive phototaxis seen in naïve organisms. These data indicate that FPW can induce perturbations in the behavior of aquatic invertebrates, an effect that may influence processes such as feeding and predation rates.

Abstract:
Hydraulic fracturing (fracking) chemicals are used to maximize the extraction of hard-to-reach underground energy resources. Large amounts of fracking fluid could escape to the surrounding environments, including underground and surface water resources, during the chemical mixing stage of the hydraulic fracturing water cycle due to equipment failure or human error. However, the impact of pollution resulting from operational discharges is difficult to assess in aquatic ecosystems. In this study, pathological investigations, chromosomal aberrations, DNA damage, and biochemical and hematological parameters were used to evaluate the effects of such chemicals on Nile tilapia. Chromosomal aberrations are considered very sensitive genetic markers of exposure to genotoxic chemicals and are used as indicators of DNA damage. The appearance of different types of chromosomal aberrations (gaps and breaks) due to chemical exposure was significantly reduced by treatment with spirulina. Various deleterious findings in Nile tilapia, in the current study, could attributed to the presence of fracking chemicals in the aquatic environment. However, the presence of spirulina in the diet reduced the hazards of such chemicals. In addition, cytogenetic studies in the current work revealed the importance of spirulina in ameliorating the genotoxic effects of a mixture of some chemicals used in fracking.

Abstract:
Background Thousands of chemicals exist in hydraulic-fracturing (HF) fluids and wastewater from unconventional oil gas development. The carcinogenicity of these chemicals in HF fluids and wastewater has never been systematically evaluated. Objectives In this study, we assessed the carcinogenicity of 1173 HF-related chemicals in the HF chemical data from the US Environmental Protection Agency (EPA). Methods We linked the HF chemical data with the agent classification data from the international Agency for Research on Cancer (IARC) at the World Health Organization (WHO) (N = 998 chemicals) to evaluate human carcinogenic risk of the chemicals and with the Carcinogenic Potency Database (CPDB) from Toxnet (N = 1534 chemicals) to evaluate potential carcinogenicity of the chemicals. Results The Chemical Abstract Service registry numbers for chemicals were used for data linkage. Among 1173 chemicals, 1039 were identified only in HF fluids, 97 only in wastewater, and 37 in both. Compared with IARC, we found information of 104 chemicals and 48 of them may have potentially carcinogenic risk to human, among which 14 are definitely carcinogenic, 7 probably carcinogenic and 27 possibly carcinogenic. Using the CPDB data, it suggests that 66 chemicals are potentially carcinogenic based on rats and mouse models.

Abstract:
Wastewater recovered from hydraulic fracturing is referred to as flowback and produced water (FPW), and is often saline, contains numerous organic and inorganic constituents, and may pose threats to groundwater resources. Hundreds of spills of FPW have been reported to the Alberta Energy Regulator each year. Recently, samples of FPW derived from hydraulic fracturing of the Duvernay Formation, AB, were found to contain a previously unidentified class of aryl phosphates, including diphenyl phosphate (DPP), triphenyl phosphate (TPP), and others. Aryl phosphates are also used in a variety of other industries and their constituents can be found in flame retardants, plasticizers, lubricants, hydraulic fluids, and oxidizers. Many of these aryl phosphates break down into DPP. Therefore, it is important to determine the environmental fate and potential impact of DPP if spilled in the near-surface, as DPP is an emerging contaminant in soil and groundwater systems. This study was aimed at determining 1) the sorption behavior of DPP onto various surficial sediments collected within the Fox Creek, AB region, and 2) the toxicity of DPP toward aquatic ecosystems. We report that the sorption of DPP onto both clay-rich soils and sandy sediment was low compared to that of other aryl phosphates, with an average log KOC value of 2.30 ± 0.42 (1σ). Therefore, the transport of DPP in groundwater would be rapid due to its low degree of sorption on surficial materials. We also determined the acute 96 h-LC50 of DPP on zebrafish embryos to be 50.0 ± 7.1 mg/L. Su et al. (2014) studied the toxic effects of DPP and TPP on chicken embryonic hepatocytes and found that DPP had less cytotoxic effects than TPP but altered more gene transcripts. From the results our study, we infer that DPP may pose an environmental risk to aquatic ecosystems if released into the environment.

Abstract:
Previous studies conducted in our laboratory have found altered adult health outcomes in animals with prenatal exposure to environmentally relevant levels of unconventional oil and gas (UOG) chemicals with endocrine-disrupting activity. This study aimed to examine potential metabolic health outcomes following a preconception, prenatal and postnatal exposure to a mixture of 23 UOG chemicals. Prior to mating and from gestation day 1 to postnatal day 21, C57BL/6J mice were developmentally exposed to a laboratory-created mixture of 23 UOG chemicals in maternal drinking water. Body composition, spontaneous activity, energy expenditure, and glucose tolerance were evaluated in 7-month-old female offspring. Neither body weight nor body composition differed in 7-month female mice. However, females exposed to 1.5 and 150 µg/kg/day UOG mix had lower total and resting energy expenditure within the dark cycle. In the light cycle, the 1500 µg//kg/day group had lower total energy expenditure and the 1.5 µg/kg/day group had lower resting energy expenditure. Females exposed to the 150 µg/kg/day group had lower spontaneous activity in the dark cycle, and females exposed to the 1500 µg/kg/day group had lower activity in the light cycle. This study reports for the first time that developmental exposure to a mixture of 23 UOG chemicals alters energy expenditure and spontaneous activity in adult female mice.

Abstract:
Hydraulic fracturing flowback and produced water (FPW) samples were analyzed for toxicity and microbiome characterization over 220 days for a horizontally drilled well in the Denver-Julesberg (DJ) Basin in Colorado. Cytotoxicity, mutagenicity, and estrogenicity of FPW were measured via the BioLuminescence Inhibition Assay (BLIA), Ames II mutagenicity assay (AMES), and Yeast Estrogen Screen (YES). Raw FPW stimulated bacteria in BLIA, but were cytotoxic to yeast in YES. Filtered FPW stimulated cell growth in both BLIA and YES. Concentrating 25× by solid phase extraction (SPE) revealed significant toxicity throughout well production by BLIA, toxicity during the first 55 days of flowback by YES, and mutagenicity by AMES. The selective pressures of fracturing conditions (including toxicity) affected bacterial and archaeal communities, which were characterized by 16S rRNA gene V4V5 region sequencing. Conditions selected for thermophilic, anaerobic, halophilic bacteria and methanogenic archaea from the groundwater used for fracturing fluid, and from the native shale community. Trends in toxicity echoed the microbial community, which indicated distinct stages of early flowback water, a transition stage, and produced water. Biota in another sampled DJ Basin horizontal well resembled similarly aged samples from this well. However, microbial signatures were unique compared to samples from DJ Basin vertical wells, and wells from other basins. These data can inform treatability, reuse, and management decisions specific to the DJ Basin to minimize adverse environmental health and well production outcomes.

Abstract:
Potential effects of horizontal drilling combined with high-volume hydraulic fracturing (HF) have drawn significant public concern, especially on the handling, treatment, and disposal of HF flowback and produced water (HF-FPW). Previous studies indicated HF-FPW could significantly disrupt biotransformation and expressions of genes related to the endocrine system. This study focused on effects of organic extracts of HF-FPW on receptor binding activity using several transactivation assays. Six HF-FPW samples were collected from 2 wells (Well A and Well B, 3 time points at each well). These were separated by filtration into aqueous (W) and particulate (S) phases, and organics were extracted from all 12 subsamples. Of all the tested fractions, sample B1-S had the greatest Σ13PAH (11,000 ng/L) and B3-S has the greatest Σ4alkyl-PAHs (16,000 ng/L). Nuclear receptor binding activity of all the extracts on aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and androgen receptor (AR) were screened using H4IIE-luc, MVLN-luc, and MDA-kb2 cells, respectively. FPWs from various HF wells exhibited distinct nuclear receptor binding effects. The strongest AhR agonist activity was detected in B3-S, with 450 ± 20 μg BaP/L equivalency at 5 × exposure. The greatest ER agonist activity was detected in A1-W, with 5.3 ± 0.9 nM E2 equivalency at 10× exposures. There is a decreasing trend in ER agonist activity from A1 to A3 in both aqueous and particulate fractions from Well A, while there is an increasing trend in ER agonist activity from B1 to B3 in aqueous fractions from Well B. This study provides novel information on the sources of endocrine disruptive potentials in various HF-FPW considering both temporal and spatial variability. Results suggest that reclamation or remediation and risk assessment of HF-FPW spills likely requires multiple strategies including understanding the properties of each spill with respect to fractured geological formation and physiochemical properties of the injected fluid.

Abstract:
The process of extracting hydrocarbon resources by hydraulic fracturing is an increasingly utilised technique worldwide, resulting in an effluent called flowback and produced water (FPW). This effluent is a complex mixture of salts, metals and organic compounds, and has been shown to be highly toxic to aquatic biota, an effect attributed mainly to its salt and organic components. However, in the current study we show that the water flea, Daphnia magna, is physically impaired by, and rendered immobile at the surface of, test waters containing FPW. This effect occurs at concentrations significantly lower than the reported median lethal concentration for the same test FPW, and suggests that physical immobility is a more sensitive ecological indicator of adverse environmental effects associated with FPW exposure. We showed that this effect could be mediated by the dual action of waterborne surfactants, which decrease surface tension, and floating hydrocarbons, which adhere to daphnids that break through the water surface and prevent resubmergence. While mortality does not occur in physically impaired daphnids within the prescribed 48h, animals are unable to return to the water column, and thus cannot feed. Stranding at the water surface will also impair the capacity of the animals to shed the carapace, thus impeding reproduction. These results suggest that assessment of acute toxicity of FPW may need to be determined differently from traditional effluent toxicity assessments.

Abstract:
Unconventional oil and gas operations (UOG), which combine hydraulic fracturing (fracking) and directional drilling, involve the use of hundreds of chemicals including many with endocrine disrupting properties. Two previous studies examined mice exposed during early development to a 23-chemical mixture of UOG compounds (UOG-MIX) commonly used or produced in the process. Both male and female offspring exposed prenatally to one or more doses of UOG-MIX displayed alterations to endocrine organ function and serum hormone concentrations. We hypothesized that prenatal UOG-MIX exposures would similarly disrupt development of the mouse mammary gland. Female C57Bl/6 mice were exposed to approximately 3, 30, 300 or 3000 μg/kg/day UOG-MIX from gestational day 11 to birth. Although no effects were observed on the mammary glands of these females prior to puberty, in early adulthood, females exposed to 300 or 3000 μg/kg/day UOG-MIX developed more dense mammary epithelial ducts; females exposed to 3 μg/kg/day UOG-MIX had an altered ratio of apoptosis to proliferation in the mammary epithelium. Furthermore, adult females from all UOG-MIX-treated groups developed intraductal hyperplasia that resembled terminal end buds, i.e., highly proliferative structures typically seen at puberty. These results suggest that the mammary gland is sensitive to mixtures of chemicals used in unconventional oil and gas production, at exposure levels that are environmentally relevant. The impact of these findings on the long-term health of the mammary gland, including its lactational capacity and its risk of cancer, should be evaluated in future studies.

Abstract:
Hydraulic fracturing (HF) has emerged as a major recovery method of unconventional oil and gas reservoirs and concerns have been raised regarding the environmental impact of releases of Flowback and Produced Water (FPW) to aquatic ecosystems. To investigate potential effects of HF-FPW on fish embryo development, HF-FPW samples were collected from two different wells and the organic fractions were isolated from both aqueous and particle phases to eliminate the confounding effects of high salinity. Each organic extract was characterized by non-target analysis with HPLC-Orbitrap-MS, with targeted analysis for polycyclic aromatic hydrocarbons provided as markers of petroleum-affected water. The organic profiles differed between samples, including PAHs and alkyl PAHs, and major substances identified by non-target analysis included polyethylene glycols, alkyl ethoxylates, octylphenol ethoxylates and other high molecular weight (C49-79) ethylene oxide polymeric material. Zebrafish embryos were exposed to various concentrations of FPW organic extracts to investigate acute (7-day) and developmental toxicity in early life stages. The acute toxicity (LD50) of the extracted FPW fractions ranged from 2.8× to 26× the original organic content. Each extracted FPW fraction significantly increased spinal malformation, pericardial edema, and delayed hatch in exposed embryos and altered the expression of a suite of target genes related to biotransformation, oxidative stress and endocrine-mediation in developing zebrafish embryos. These results provide novel information on the variation of organic profiles and developmental toxicity among different sources and fractions of HF-FPWs.

Abstract:
The widespread use of hydraulic fracturing (HF) in oil and gas extraction operations has led to concern over environmental risks posed by chemicals used in HF fluids. Here we employed a suite of stable luciferase reporter gene assays to investigate the potential for selected HF chemicals or geogenics to activate or antagonise nuclear receptor signalling. We screened three biocides (bronopol [BP], glutaraldehyde [GA], and tetrakis (hydroxymethyl)phosphonium sulfate [THPS]), a surfactant (2-butoxyethanol), a friction reducer (polyacrylamide), and a coal seam geogenic (o-cresol) for their potential to act as agonists or antagonists of the estrogen receptor, androgen receptor, progesterone receptor (PR), glucocorticoid receptor or peroxisome proliferator-activated receptor gamma (PPARγ). None of the chemicals induced luciferase activity in any of assays used in the study. In antagonistic mode, BP, GA and THPS caused reductions in luciferase activity in the reporter assays at higher concentrations (50–100 μM), while at low concentrations (2–10 μM) GA and THPS enhanced luciferase activity in some assays relative to controls. None of the other tested chemicals exhibited antagonism in the selected assays. In most cases, altered receptor signalling only occurred at concentrations exhibiting cytotoxicity. However, PPARγ activity, and to a lesser extent PR activity, were inhibited by THPS at sub-cytotoxic concentrations. The majority of binary combinations tested exhibited significantly less-than-additive cytotoxicity, and none of the combinations exhibited synergistic cytotoxicity. In summary, the results of the present study indicate that the selected chemicals are not likely to function as direct agonists of the nuclear receptors tested, and only one chemical, THPS was an apparent partial antagonist of two nuclear receptors.

Abstract:
Abstract. Chemicals associated with unconventional oil and gas (UOG) operations have been shown to contaminate surface and ground water with a variety of endoc

Abstract:
Unconventional oil and natural gas (UOG) operations have contributed to a surge in domestic oil and natural gas production in the United States, combining horizontal drilling with hydraulic fracturing to unlock previously inaccessible fossil fuel deposits. >1000 organic chemicals are used in the production process, and wastewater is produced following injection and for the life of the producing well. This wastewater is typically disposed of via injecting into disposal wells for long-term storage, treatment and discharge from wastewater treatment plants, and/or storage in open evaporation pits; however, wastewater spill rates are reported at 2–20% of active well sites across regions, increasing concerns about the environmental impacts of these wastewaters. This study assessed adipogenic activity (both triglyceride accumulation and pre-adipocyte proliferation) for a mixture of 23 commonly used UOG chemicals and a small subset of UOG wastewater-impacted surface water extracts from Colorado and West Virginia, using 3T3-L1 cells and a peroxisome proliferator activated receptor gamma (PPARγ) reporter assay. We report potent and efficacious adipogenic activity induced by both a laboratory-created UOG chemical mixture and UOG-impacted water samples at concentrations below environmental levels. We further report activation of PPARγ at similar concentrations for some samples, suggesting a causative molecular pathway for the observed effects, but not for other adipogenic samples, implicating PPARγ-dependent and independent effects from UOG associated chemicals. Taken together, these results suggest that UOG wastewater has the potential to impact metabolic health at environmentally relevant concentrations.

Abstract:

Abstract:
Chemicals used in unconventional oil and gas (UOG) operations have the potential to cause adverse biological effects, but this has not been thoroughly evaluated. A notable knowledge gap is their impact on development and function of the immune system. Herein, we report an investigation of whether developmental exposure to a mixture of chemicals associated with UOG operations affects the development and function of the immune system. We used a previously characterized mixture of 23 chemicals associated with UOG, and which was demonstrated to affect reproductive and developmental endpoints in mice. C57Bl/6 mice were maintained throughout pregnancy and during lactation on water containing two concentrations of this 23-chemical mixture, and the immune system of male and female adult offspring was assessed. We comprehensively examined the cellularity of primary and secondary immune organs, and used three different disease models to probe potential immune effects: house dust mite-induced allergic airway disease, influenza A virus infection, and experimental autoimmune encephalomyelitis (EAE). In all three disease models, developmental exposure altered frequencies of certain T cell sub-populations in female, but not male, offspring. Additionally, in the EAE model disease onset occurred earlier and was more severe in females. Our findings indicate that developmental exposure to this mixture had persistent immunological effects that differed by sex, and exacerbated responses in an experimental model of autoimmune encephalitis. These observations suggest that developmental exposure to complex mixtures of water contaminants, such as those derived from UOG operations, could contribute to immune dysregulation and disease later in life.

Abstract:
Hydraulic fracturing wastewaters discharged to surface water have led to elevated bromide and iodide levels, as well as enhanced formation of brominated trihalomethanes, haloacetic acids, haloacetonitriles, and iodo-trihalomethanes at downstream drinking water treatment plants, in chlorinated effluent from wastewater treatment plants, and in controlled laboratory studies. This enhanced formation of brominated and iodinated disinfection byproducts (DBPs) raises concerns regarding human health, because they are much more toxic than chlorinated DBPs. This study represents the first nontarget, comprehensive analysis of iodinated DBPs formed in chloraminated produced waters associated with hydraulic fracturing of shale and conventional gas formations. Fifty-six iodo-phenolics were identified, comprising three homologous series of mono-, di-, and tri-iodinated phenols, along with two new classes of DBPs: iodomethylphenols and iododimethylphenols. Four iodo-phenolics (2-iodophenol, 4-iodophenol, 2,4,6-triiodophenol, and 4-iodo-2-methylphenol) were investigated for mammalian cell cytotoxicity. All were cytotoxic, especially 2,4,6-triiodophenol, which was more cytotoxic than all trihalomethanes and most haloacetic acids. In addition, geogenic organic compounds present in the oil and gas produced waters, including methylphenol and dimethylphenol, were found to be potential precursors to these iodo-DBPs.

Abstract:
The human toxicity impact (HTI) of electricity produced from shale gas is lower than the HTI of electricity produced from coal, with 90% confidence using a Monte Carlo Analysis. Two different impact assessment methods estimate the HTI of shale gas electricity to be 1–2 orders of magnitude less than the HTI of coal electricity (0.016–0.024 DALY/GWh versus 0.69–1.7 DALY/GWh). Further, an implausible shale gas scenario where all fracturing fluid and untreated produced water is discharged directly to surface water throughout the lifetime of a well also has a lower HTI than coal electricity. Particulate matter dominates the HTI for both systems, representing a much larger contribution to the overall toxicity burden than VOCs or any aquatic emission. Aquatic emissions can become larger contributors to the HTI when waste products are inadequately disposed or there are significant infrastructure or equipment failures. Large uncertainty and lack of exposure data prevent a full risk assessment; however, the results of this analysis provide a comparison of relative toxicity, which can be used to identify target areas for improvement and assess potential trade-offs with other environmental impacts.

Abstract:
Hydraulic fracturing to extract oil and natural gas reserves is an increasing practice in many international energy sectors. Hydraulic fracturing flowback and produced water (FPW) is a hyper saline wastewater returned to the surface from a fractured well containing chemical species present in the initial fracturing fluid, geogenic contaminants, and potentially newly synthesized chemicals formed in the fracturing well environment. However, information on FPW toxicological mechanisms of action remain largely unknown. Both cardiotoxic and respirometric responses were explored in zebrafish (Danio rerio) embryos after either an acute sediment-free (FPW-SF) or raw/sediment containing (FPW-S) fraction exposure of 24 and 48 h at 2.5% and 5% dilutions. A 48 h exposure to either FPW fraction in 24–72 h post fertilization zebrafish embryos significantly increased occurrences of pericardial edema, yolk-sac edema, and tail/spine curvature. In contrast, larval heart rates significantly decreased after FPW fraction exposures. FPW-S, but not FPW-SF, at 2.5% doses significantly reduced embryonic respiration/metabolic rates (MO2), while for 5% FPW, both fractions reduced MO2. Expression of select cardiac genes were also significantly altered in each FPW exposure group, implicating a cardiovascular system compromise as the potential cause for reduced embryonic MO2. Collectively, these results support our hypothesis that organics are major contributors to cardiac and respiratory responses to FPW exposure in zebrafish embryos. Our study is the first to investigate cardiac and respiratory sub-lethal effects of FPW exposure, demonstrating that FPW effects extend beyond initial osmotic stressors and verifies the use of respirometry as a potential marker for FPW exposure.

Abstract:
Hydraulic fracturing (HF) has emerged as a major method of unconventional oil and gas recovery. The toxicity of hydraulic fracturing flowback and produced water (HF-FPW) has not been previously reported and is complicated by the combined complexity of organic and inorganic constituents in HF fluids and deep formation water. In this study, we characterized the solids, salts, and organic signatures in an HF-FPW sample from the Duvernay Formation, Alberta, Canada. Untargeted HPLC-Orbitrap revealed numerous unknown dissolved polar organics. Among the most prominent peaks, a substituted tri-phenyl phosphate was identified which is likely an oxidation product of a common polymer antioxidant. Acute toxicity of zebrafish embryo was attributable to high salinity and organic contaminants in HF-FPW with LC50 values ranging from 0.6% to 3.9%, depending on the HF-FPW fractions and embryo developmental stages. Induction of ethoxyresorufin-O-deethylase (EROD) activity was detected, due in part to polycyclic aromatic hydrocarbons (PAHs), and suspended solids might have a synergistic effect on EROD induction. This study demonstrates that toxicological profiling of real HF-FPW sample presents great challenges for assessing the potential risks and impacts posed by HF-FPW spills.

Abstract:
Hydraulic fracturing is an industrial process allowing for the extraction of gas or oil. To fracture the rocks, a proprietary mix of chemicals is injected under high pressure, which later returns to the surface as flowback and produced water (FPW). FPW is a complex chemical mixture consisting of trace metals, organic compounds, and often, high levels of salts. FPW toxicity to the model freshwater crustacean, Daphnia magna, was characterized utilizing acute (48 h median lethal concentrations; LC50) and chronic (21 d) exposures. A decrease in reproduction was observed, with a mean value of 18.5 neonates produced per replicate over a 21-d chronic exposure to 0.04% FPW, significantly decreased from the average of 64 neonates produced in controls. The time to first brood was delayed in the highest FPW (0.04%) treatment. Neonates exhibited an LC50 of 0.19% of full-strength FPW, making them more sensitive than adults, which displayed an LC50 value of 0.75%. Quantitative PCR highlighted significant changes in expression of genes encoding xenobiotic metabolism (cyp4) and moulting (cut). This study is the first to characterize chronic FPW toxicity and will help development of environmental monitoring and risk assessment of FPW spills.

Abstract:
Unconventional oil and gas operations using hydraulic fracturing can contaminate surface and groundwater with endocrine-disrupting chemicals. We have previously shown that 23 of 24 commonly used hydraulic fracturing chemicals can activate or inhibit the estrogen, androgen, glucocorticoid, progesterone, and/or thyroid receptors in a human endometrial cancer cell reporter gene assay and that mixtures can behave synergistically, additively, or antagonistically on these receptors. In the current study, pregnant female C57Bl/6 dams were exposed to a mixture of 23 commonly used unconventional oil and gas chemicals at approximately 3, 30, 300, and 3000 μ g/kg·d, flutamide at 50 mg/kg·d, or a 0.2% ethanol control vehicle via their drinking water from gestational day 11 through birth. This prenatal exposure to oil and gas operation chemicals suppressed pituitary hormone concentrations across experimental groups (prolactin, LH, FSH, and others), increased body weights, altered uterine and ovary weights, increased heart weights and collagen deposition, disrupted folliculogenesis, and other adverse health effects. This work suggests potential adverse developmental and reproductive health outcomes in humans and animals exposed to these oil and gas operation chemicals, with adverse outcomes observed even in the lowest dose group tested, equivalent to concentrations reported in drinking water sources. These endpoints suggest potential impacts on fertility, as previously observed in the male siblings, which require careful assessment in future studies.

Abstract:
Currently, > 95% of end disposal of hydraulic fracturing wastewater from unconventional oil and gas operations in the US occurs via injection wells. Key data gaps exist in understanding the potential impact of underground injection on surface water quality and environmental health. The goal of this study was to assess endocrine disrupting activity in surface water at a West Virginia injection well disposal site. Water samples were collected from a background site in the area and upstream, on, and downstream of the disposal facility. Samples were solid-phase extracted, and extracts assessed for agonist and antagonist hormonal activities for five hormone receptors in mammalian and yeast reporter gene assays. Compared to reference water extracts upstream and distal to the disposal well, samples collected adjacent and downstream exhibited considerably higher antagonist activity for the estrogen, androgen, progesterone, glucocorticoid and thyroid hormone receptors. In contrast, low levels of agonist activity were measured in upstream/distal sites, and were inhibited or absent at downstream sites with significant antagonism. Concurrent analyses by partner laboratories (published separately) describe the analytical and geochemical profiling of the water; elevated conductivity as well as high sodium, chloride, strontium, and barium concentrations indicate impacts due to handling of unconventional oil and gas wastewater. Notably, antagonist activities in downstream samples were at equivalent authentic standard concentrations known to disrupt reproduction and/or development in aquatic animals. Given the widespread use of injection wells for end-disposal of hydraulic fracturing wastewater, these data raise concerns for human and animal health nearby.

Abstract:
2,2-Dibromo-3-nitrilopropionamide (DBNPA) is a major biocide in hydraulic fracturing fluids. Most biocides in fracturing fluids are considered to have low acute toxicity to mammals, but little information is available in the literature regarding the toxic actions of DBNPA on mammalian cells. This information is important to suggest the DBNPA toxicity on wild mammals. In this study, the effects of DBNPA on rat thymocytes were studied using flow cytometric techniques in order to further characterize the cytotoxicity of DBNPA for its safe use. DBNPA at 3–7.5 μM produced a steep concentration-dependent increase in cell lethality. At 5 μM, DBNPA significantly depolarized the membranes with a disturbance of the asymmetrical distribution of membrane phospholipids. The lethal effect of DBNPA was completely abolished under cold conditions, and was augmented in the presence of ethanol. It is suggested that the lethal action of DBNPA is linked to changes in membrane fluidity. Because the concentration-dependent change of DBNPA-induced lethal action was very steep under in vitro conditions, the adverse actions of DBNPA on wild mammals are concerning, even though such reports have not yet surfaced.

Abstract:
The United States Environmental Protection Agency (EPA) identified 1,173 chemicals associated with hydraulic fracturing fluids, flowback, or produced water, of which 1,026 (87%) lack chronic oral toxicity values for human health assessments. To facilitate the ranking and prioritization of chemicals that lack toxicity values, it may be useful to employ toxicity estimates from quantitative structure-activity relationship (QSAR) models. Here we describe an approach for applying the results of a QSAR model from the TOPKAT program suite, which provides estimates of the rat chronic oral lowest-observed-adverse-effect level (LOAEL). Of the 1,173 chemicals, TOPKAT was able to generate LOAEL estimates for 515 (44%). To address the uncertainty associated with these estimates, we assigned qualitative confidence scores (high, medium, or low) to each TOPKAT LOAEL estimate, and found 481 to be high-confidence. For 48 chemicals that had both a high-confidence TOPKAT LOAEL estimate and a chronic oral reference dose from EPA’s Integrated Risk Information System (IRIS) database, Spearman rank correlation identified 68% agreement between the two values (permutation p-value = 1x10-11). These results provide support for the use of TOPKAT LOAEL estimates in identifying and prioritizing potentially hazardous chemicals. High-confidence TOPKAT LOAELs were available for 389 of 1,026 hydraulic fracturing-related chemicals that lack chronic oral RfVs and OSFs from EPA-identified sources, including a subset of chemicals that are frequently used in hydraulic fracturing fluids.

Abstract:
Concerns have been raised about potential public health effects that may arise if hydraulic fracturing-related chemicals were to impact drinking water resources. This study presents an overview of the chronic oral toxicity values?specifically, chronic oral reference values (RfVs) for noncancer effects, and oral slope factors (OSFs) for cancer?that are available for a list of 1,173 chemicals that the United States (US) Environmental Protection Agency (EPA) identified as being associated with hydraulic fracturing, including 1,076 chemicals used in hydraulic fracturing fluids, and 134 chemicals detected in flowback or produced waters from hydraulically fractured wells. EPA compiled RfVs and OSFs using six governmental and intergovernmental data sources. 90 (8%) of the 1,076 chemicals reported in hydraulic fracturing fluids and 83 (62%) of the 134 chemicals reported in flowback/produced water had a chronic oral RfV or OSF available from one or more of the six sources. Furthermore, of the 36 chemicals reported in hydraulic fracturing fluids in at least 10% of wells nationwide (identified from EPA?s analysis of the FracFocus Chemical Disclosure Registry 1.0), 8 chemicals (22%) have an available chronic oral RfV. The lack of chronic oral RfVs and OSFs for the majority of these chemicals highlights the significant knowledge gap that exists to assess the potential human health hazards associated with hydraulic fracturing.

Abstract:
Hydraulic-fracturing fluids and wastewater from unconventional oil and natural gas development contain hundreds of substances with the potential to contaminate drinking water. Challenges to conducting well-designed human exposure and health studies include limited information about likely etiologic agents. We systematically evaluated 1021 chemicals identified in hydraulic-fracturing fluids (n=925), wastewater (n=132), or both (n=36) for potential reproductive and developmental toxicity to triage those with potential for human health impact. We searched the REPROTOX database using Chemical Abstract Service registry numbers for chemicals with available data and evaluated the evidence for adverse reproductive and developmental effects. Next, we determined which chemicals linked to reproductive or developmental toxicity had water quality standards or guidelines. Toxicity information was lacking for 781 (76%) chemicals. Of the remaining 240 substances, evidence suggested reproductive toxicity for 103 (43%), developmental toxicity for 95 (40%), and both for 41 (17%). Of these 157 chemicals, 67 had or were proposed for a federal water quality standard or guideline. Our systematic screening approach identified a list of 67 hydraulic fracturing-related candidate analytes based on known or suspected toxicity. Incorporation of data on potency, physicochemical properties, and environmental concentrations could further prioritize these substances for future drinking water exposure assessments or reproductive and developmental health studies.

Abstract:
Oil and natural gas operations have been shown to contaminate surface and ground water with endocrine-disrupting chemicals. In the current study, we fill several gaps in our understanding of the potential environmental impacts related to this process. We measured the endocrine-disrupting activities of 24 chemicals used and/or produced by oil and gas operations for five nuclear receptors using a reporter gene assay in human endometrial cancer cells. We also quantified the concentration of 16 of these chemicals in oil and gas wastewater samples. Finally, we assessed reproductive and developmental outcomes in male C57BL/6J mice after the prenatal exposure to a mixture of these chemicals. We found that 23 commonly used oil and natural gas operation chemicals can activate or inhibit the estrogen, androgen, glucocorticoid, progesterone, and/or thyroid receptors, and mixtures of these chemicals can behave synergistically, additively, or antagonistically in vitro. Prenatal exposure to a mixture of 23 oil and gas operation chemicals at 3, 30, and 300 μ g/kg · d caused decreased sperm counts and increased testes, body, heart, and thymus weights and increased serum T in male mice, suggesting multiple organ system impacts. Our results suggest possible adverse developmental and reproductive health outcomes in humans and animals exposed to potential environmentally relevant levels of oil and gas operation chemicals.

Abstract:
The rapid development of high-volume horizontal hydraulic fracturing for mining natural gas from shale has posed potential impacts on human health and biodiversity. The produced flow back waters after hydraulic stimulation are known to carry high levels of saline and total dissolved solids. To understand the toxicity and potential carcinogenic effects of these wastewaters, flow back waters from five Marcellus hydraulic fracturing oil and gas wells were analyzed. The physicochemical nature of these samples was analyzed by inductively coupled plasma mass spectrometry and scanning electron microscopy/energy dispersive X-ray spectroscopy. A cytotoxicity study using colony formation as the endpoint was carried out to define the LC50 values of test samples using human bronchial epithelial cells (BEAS-2B). The BEAS-2B cell transformation assay was employed to assess the carcinogenic potential of the samples. Barium and strontium were among the most abundant metals in these samples and the same metals were found to be elevated in BEAS-2B cells after long-term treatment. BEAS-2B cells treated for 6 weeks with flow back waters produced colony formation in soft agar that was concentration dependent. In addition, flow back water-transformed BEAS-2B cells show better migration capability when compared to control cells. This study provides information needed to assess the potential health impact of post-hydraulic fracturing flow back waters from Marcellus Shale natural gas mining.

Abstract:

Abstract:
The rapid rise in natural gas extraction using hydraulic fracturing increases the potential for contamination of surface and ground water from chemicals used throughout the process. Hundreds of products containing more than 750 chemicals and components are potentially used throughout the extraction process, including more than 100 known or suspected endocrine-disrupting chemicals. We hypothesized that a selected subset of chemicals used in natural gas drilling operations and also surface and ground water samples collected in a drilling-dense region of Garfield County, Colorado, would exhibit estrogen and androgen receptor activities. Water samples were collected, solid-phase extracted, and measured for estrogen and androgen receptor activities using reporter gene assays in human cell lines. Of the 39 unique water samples, 89%, 41%, 12%, and 46% exhibited estrogenic, antiestrogenic, androgenic, and antiandrogenic activities, respectively. Testing of a subset of natural gas drilling chemicals revealed novel antiestrogenic, novel antiandrogenic, and limited estrogenic activities. The Colorado River, the drainage basin for this region, exhibited moderate levels of estrogenic, antiestrogenic, and antiandrogenic activities, suggesting that higher localized activity at sites with known natural gas–related spills surrounding the river might be contributing to the multiple receptor activities observed in this water source. The majority of water samples collected from sites in a drilling-dense region of Colorado exhibited more estrogenic, antiestrogenic, or antiandrogenic activities than reference sites with limited nearby drilling operations. Our data suggest that natural gas drilling operations may result in elevated endocrine-disrupting chemical activity in surface and ground water., AffiliationsDepartment of Obstetrics, Gynecology and Women's Health and Division of Biological Sciences (C.D.K., A.M.H., S.C.N.), University of Missouri, Columbia, Missouri 65211; US Geological Survey (D.E.T.), Columbia Environmental Research Center, Columbia, Missouri 65201; and Departments of Statistics and Health Management and Informatics (J.W.D.), University of Missouri, Columbia, Missouri 65211