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Rising CO 2 emissions have heightened the necessity for increased understanding of Earth's carbon cycle to predict future climates. The involvement of marine planktonic species in the global carbon cycle has been extensively studied, but contributions by marine fish remain poorly characterized. Marine teleost fishes produce carbonate minerals ('ichthyocarbonates') within the lumen of their intestines which are excreted at significant rates on a global scale. However, we have limited understanding of the fate of excreted ichthyocarbonate. We analyzed ichthyocarbonate produced by three different marine teleosts for mol%MgCO 3 content, size, specific gravity, and dissolution rate to gain a better understanding of ichthyocarbonate fate. Based on the species examined here, we report that 75 % of ichthyocarbonates are ≤0.91 mm in diameter. Analyses indicate high Mg 2+ content across species (22.3 to 32.3 % mol%MgCO 3 ), consistent with previous findings. Furthermore, ichthyocarbonate specific gravity ranged from 1.23 to 1.33 g/cm 3 , and ichthyocarbonate dissolution rates varied among species as a function of aragonite saturation state. Ichthyocarbonate sinking rates and dissolution depth were estimated for the Atlantic, Pacific, and Indian ocean basins for the three species examined. In the North Atlantic, for example, ~33 % of examined ichthyocarbonates are expected to reach depths exceeding 200 m prior to complete dissolution. The remaining ~66 % of ichthyocarbonate is estimated to dissolve and contribute to shallow water alkalinity budgets. Considering fish biomass and ichthyocarbonate production rates, our results support that marine fishes are critical to the global carbon cycle, contributing to oceanic alkalinity budgets and thereby influencing the ability of the oceans to neutralize atmospheric CO 2 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Marine bony fish are important participants in Earth's carbon cycle through their contributions to the biological pump and the marine inorganic carbon cycle. However, uncertainties in the composition and magnitude of fish contributions preclude their integration into fully coupled carbon-climate models. Here, we consider recent upwards revisions to global fish biomass estimates (2.7-9.5×) and provide new stable carbon isotope measurements that show marine fish are prodigious producers of carbonate with unique composition. Assuming the median increase (4.17×) in fish biomass estimates is linearly reflected in fish carbonate (ichthyocarbonate) production rate, marine fish are estimated to produce between 1.43 and 3.99 Pg CaCO 3 yr -1 , but potentially as much as 9.03 Pg CaCO 3 yr -1 . Thus, marine fish carbonate production is equivalent to or potentially higher than contributions by coccolithophores or pelagic foraminifera. New stable carbon isotope analyses indicate that a significant proportion of ichthyocarbonate is derived from dietary carbon, rather than seawater dissolved inorganic carbon. Using a statistical mixing model to derive source contributions, we estimate ichthyocarbonate contains up to 81 % dietary carbon, with average compositions of 28-56 %, standing in contrast to contents <10 % in other biogenic carbonate minerals. Results also indicate ichthyocarbonate contains 5.5-40.4 % total organic carbon. When scaled to the median revised global production of ichthyocarbonate, an additional 0.08 to 1.61 Pg C yr -1 can potentially be added to estimates of fish contributions to the biological pump, significantly increasing marine fish contributions to total surface carbon export. Our integration of geochemical and physiological analyses identifies an overlooked link between carbonate production and the biological pump. Since ichthyocarbonate production is anticipated to increase with climate change scenarios, due to ocean warming and acidification, these results emphasize the importance of quantitative understanding of the multifaceted role of marine fish in the global carbon cycle., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Technologies associated with hydraulic fracturing continue to be prevalent in many regions worldwide. As a result, the production of flowback and produced water (FPW) - a wastewater generated once pressure is released from subterranean wellbores - continues to rise in regions experiencing fracturing activities, while waste management strategies attempt to mitigate compounding burdens of increased FPW production. The heightened production of FPW increases the potential for release to the environment. However, relatively few studies have directly investigated how ecosystems and organisms may be latently affected long after exposures occur. The current study examines rainbow trout exposed in ovo at select critical cardiac developmental time points to differing dilutions and lengths of time (acute versus chronic) to determine how FPW-mediated exposure in ovo may alter later cardiac function and development. After exposure, we allowed fish to grow for ∼ 8 months post-fertilization and measured fish swimming performance, aerobic scope, and cardiac structure of juvenile trout. Acute 48 h embryonic 5% FPW exposure at either 3 days post-fertilization (dpf) or 10 dpf significantly reduced later swimming performance and aerobic scope in juvenile trout. In ovo exposure to 2.5% FPW at 3 dpf yielded significant decreases in these metrics as well, while exposing trout to 2.5% FPW at 10 dpf did not induce as significant effects. Morphometric analyses of heart muscle tissue in all treatments decreased compact myocardium thickness. Chronic 1% FPW in ovo exposure for 28 days induced similar reductions in swimming performance, aerobic scope, and decreased compact myocardium thickness as acute exposures. Overall, our results demonstrate that FPW exposure during egg development ultimately results in persistently impaired heart morphology and resulting physiological (swimming) performance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

During hydraulic fracturing, wastewaters - termed flowback and produced water (FPW) - are created as a by-product during hydrocarbon extraction. Given the large volumes of FPW that a single well can produce, and the history of FPW release to surface water bodies, it is imperative to understand the hazards that hydraulic fracturing and FPW pose to aquatic biota. Using rainbow trout embryos as model organisms, we investigated impacts to cardio-respiratory system development and function following acute (48 h) and sub-chronic (28-day) FPW exposure by examining occurrences of developmental deformities, rates of embryonic respiration (MO 2 ), and changes in expression of critical cardiac-specific genes. FPW-exposed embryos had significantly increased rates of pericardial edema, yolk-sac edema, and tail/trunk curvatures at hatch. Furthermore, when exposed at three days post-fertilization (dpf), acute 5% FPW exposures significantly increased embryonic MO 2 through development until 15 dpf, where a switch to significantly reduced MO 2 rates was subsequently recorded. A similar trend was observed during sub-chronic 1% FPW exposures. Interestingly, at certain specific developmental timepoints, previous salinity exposure seemed to affect embryonic MO 2 ; a result not previously observed. Following acute FPW exposures, embryonic genes for cardiac development and function were significantly altered, although at termination of sub-chronic exposures, significant changes to these same genes were not found. Together, our evidence of induced developmental deformities, modified embryonic MO 2 , and altered cardiac transcript expression suggest that cardio-respiratory tissues are toxicologically targeted following FPW exposure in developing rainbow trout. These results may be helpful to regulatory bodies when developing hazard identification and risk management protocols concerning hydraulic fracturing activities., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early vertebrates owing to their basal position in vertebrate phylogeny. Glucose is a ubiquitous cellular energy source that is transported into cells via two classes of carrier proteins: sodium-glucose-linked transporters (Sglt; Slc5a) and glucose transporters (Glut; Slc2a). Although previous pharmacological evidence has suggested the presence of both sodium-dependent and -independent transport mechanisms in the hagfish, the molecular identities were heretofore unconfirmed. We have identified and phylogenetically characterized both a Slc5a1-like and Slc2a-like gene in the Pacific hagfish ( Eptatretus stoutii ), the latter sharing common ancestry with other glucose-transporting isoforms of the Slc2a family. To assess the potential postprandial regulation of these glucose transporters, we examined the abundance and localization of these transporters with qPCR and immunohistochemistry alongside functional studies using radiolabeled d-[ 14 C]glucose. The effects of glucose or insulin injection on glucose transport rate and transporter expression were also examined to determine their potential role(s) in the regulation of intestinal glucose carrier proteins. Feeding prompted an increase in glucose uptake across the hindgut at both 0.5 mM (∼84%) and 1 mM (∼183%) concentrations. Concomitant increases were observed in hindgut Slc5a1 protein expression. These effects were not observed following either of glucose or insulin injection, indicating these postprandial factors are not the driving force for transporter regulation over this timeframe. We conclude that Pacific hagfish utilize evolutionarily conserved mechanisms of glucose uptake and so represent a useful model to understand early-vertebrate evolution of glucose uptake and regulation.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hydraulic fracturing has become widely used in recent years to access vast global unconventional sources of oil and gas. This process involves the injection of proprietary mixtures of water and chemicals to fracture shale formations and extract the hydrocarbons trapped within. These injection fluids, along with minerals, hydrocarbons, and saline waters present within the formations being drilled into, return to the surface as flowback and produced water (FPW). FPW is a highly complex mixture, containing metals, salts and clay, as well as many organic chemicals, including polycyclic aromatic hydrocarbons such as phenanthrene. The present study sought to determine the effects of temperature on the accumulation of phenanthrene in rainbow trout (Oncorhynchus mykiss). This model organism resides in rivers overlapping the Montney and Duvernay formations, both highly developed formations for hydraulic fracturing. Rainbow trout acclimated to temperatures of 4, 13 and 17 °C were exposed to either 5% or 20% FPW, as well as saline mixtures representing the exact ionic content of FPW to determine the accumulation of radiolabelled 14 C phenanthrene within the gill, gut, liver and gallbladder. FPW exposure reduced the overall accumulation of phenanthrene in a manner most often similar to high salinity exposure, indicating that the high ionic strength of FPW is the primary factor affecting accumulation. Accumulation was different at the temperature extremes (4 and 17 °C), although no consistent relationship was observed between temperature and accumulation across the observed tissues. These results indicate that several physiological responses occur as a result of FPW exposure and water temperature change which dictate phenanthrene uptake, particularly in the gills. Temperature (and seasonality) alone cannot be used to model the potential accumulation of polycyclic aromatic hydrocarbons after FPW spills., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Unconventional methods of oil and natural gas extraction have been a growing part of North America's energy sector for the past 20-30 years. Technologies such as horizontal hydraulic fracturing have facilitated the exploitation of geologic reserves that were previously resistant to standard drilling approaches. However, the environmental risks associated with hydraulic fracturing are relatively understudied. One such hazard is the wastewater by-product of hydraulic fracturing processes: flowback and produced water (FPW). During FPW production, transport, and storage, there are many potential pathways for environmental exposure. In the current review, toxicological hazards associated with FPW surface water contamination events and potential effects on freshwater biota are assessed. This review contains an extensive survey of chemicals commonly associated with FPW samples from shale formations across North America and median 50% lethal concentration values (LC 50 ) of corresponding chemicals for many freshwater organisms. We identify the characteristics of FPW which may have the greatest potential to be drivers of toxicity to freshwater organisms. Notably, components associated with salinity, the organic fraction, and metal species are reviewed. Additionally, we examine the current state of FPW production in North America and identify the most significant obstacles impeding proper risk assessment development when environmental contamination events of this wastewater occur. Findings within this study will serve to catalyze further work on areas currently lacking in FPW research, including expanded whole effluent testing, repeated and chronic FPW exposure studies, and toxicity identification evaluations., (© 2020. Springer Nature Switzerland AG.)

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 ( U crit ) 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.

The role of the marine elasmobranch gastrointestinal tract in nitrogen-recycling and osmotic homeostasis has become increasingly apparent, with the gut microbial community likely playing a significant role converting urea, an important osmolyte in elasmobranchs, into ammonia. The Pacific spiny dogfish can experience and tolerate reduced environmental salinities, yet how this environmental challenge may affect the microbiome, and consequently nitrogen transport across the gut, is as of yet unknown. In the present study, excised gut sac preparations were made from dogfish acclimated to the following: full-strength seawater (C), low salinity for 7 days (LS), and after acute transfer of LS-acclimated fish to full-strength SW for 6 h (AT). Significantly reduced microbial derived urease activity was observed in the mucosal saline of gut sac preparations from the LS (by 81%) and AT (by 89%) treatments relative to the C treatment. Microbial derived cellulase activity from mucosal saline samples tended to follow similar patterns. To further ensure an effective decrease in the spiral valve microbial population, an antibiotic cocktail was applied to the mucosal saline used for in vitro measurements of ion, water, and nitrogen flux in these gut sac preparations. This caused a further 57-61% decrease in the mucosal saline urease activity of the C and LS treatments. Overall, we observed relatively little flux across the stomach for all measured parameters aside from water movement, which switched from a net efflux in control fish to a net influx in acutely transferred fish, indicative of drinking. While no significant differences were observed in terms of nitrogen flux (urea or ammonia), we tended to see the accumulation of ammonia in the spiral valve lumen and a switch from efflux to influx of urea in control versus acutely transferred fish. The increased ammonia production likely occurs as a result of heightened metabolism in a challenging environment, while the retention and acquisition of urea is suggestive of nitrogen scavenging under nitrogen-limiting conditions.

Produced water is the largest waste stream associated with oil and gas operations. This complex fluid contains petroleum hydrocarbons, heavy metals, salts, naturally occurring radioactive materials and any remaining chemical additives. In the United States, west of the 98th meridian, the federal National Pollutant Discharge Elimination System (NPDES) exemption allows release of produced water for agricultural beneficial reuse. The goal of this study was to quantify mutagenicity of a produced water NPDES release and discharge stream. We used four mutation assays in budding yeast cells that provide rate estimates for copy number variation (CNV) duplications and deletions, as well as forward and reversion point mutations. Higher mutation rates were observed at the discharge and decreased with distance downstream, which correlated with the concentrations of known carcinogens detected in the stream (e.g., benzene, radium), described in a companion study. Mutation rate increases were most prominent for CNV duplications and were higher than mutations observed in mixtures of known toxic compounds. Additionally, the samples were evaluated for acute toxicity in Daphnia magna and developmental toxicity in zebrafish. Acute toxicity was minimal, and no developmental toxicity was observed. This study illustrates that chemical analysis alone (McLaughlin et al., 2020) is insufficient for characterizing the risk of produced water NPDES releases and that a thorough evaluation of chronic toxicity is necessary to fully assess produced water for beneficial reuse., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the present study, we compared the toxicity and associated chemical characterizations of flowback and produced water (FPW) collected from a single horizontal hydraulically fractured well at different time points during FPW production. Since few studies on whole mixture toxicity related to FPW exist, our aims were to determine both overall toxicity of the FPW mixture in a suite of organisms (Daphnia magna, Lumbriculus variegatus, Danio rerio, and Oncorhynchus mykiss) and also determine if toxicity changes depending on variation in FPW chemical properties as a function of time sampled (1.33, 72, and 228 h FPW samples collected immediately post-well production onset were analyzed in current study). FPW chemical composition was determined via quadra-pole inductively coupled plasma - mass spectrometry/mass spectrometry (ICP-MS/MS), full-scan high performance liquid chromatography/Orbitrap mass spectrometry (HPLC/Orbitrap-MS), and gas chromatography-mass spectrometry (GC-MS). We observed that FPW sampled later in the production process contained higher ion and total dissolved solids concentrations, whereas the highest concentrations of dissolved organic compounds were observed in the earliest FPW sample analyzed. Toxicity associated with FPW exposure was deemed to be species-specific to a certain extent, but general trends revealed the earliest FPW sampled contained highest toxic potential. Accordingly, we theorize that although the saline conditions of FPW are the foremost toxicological drivers to freshwater organisms, dissolved organics associated with FPW significantly contribute to the overall toxicity of exposed organisms., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

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 (C 49-79 ) 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 (LD 50 ) 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.

Hydraulic fracturing flowback and produced water (FPW) is a wastewater produced during fracturing activities in an operating well which is hyper saline and chemically heterogeneous in nature, containing both anthropogenic and petrogenic chemicals. Determination of FPW associated toxicity to embryonic fish is limited, while investigation into how embryonic exposures may affect later life stages is not yet studied. Zebrafish embryos (24hrs post fertilization) were acutely exposed to 2.5% and 5% FPW fractions for either 24 or 48hrs and returned to freshwater. After either 24 or 48h exposures, embryos were examined for expression of 3 hypoxia related genes. Erythropoietin (epoa) but not hypoxia inducible factor (hif1aa) nor hemoglobin -ß chain (hbbe1.1) was up-regulated after either 24 or 48h FPW exposure. Surviving embryos were placed in freshwater and grown to a juvenile stage (60days post fertilization). Previously exposed zebrafish were analyzed for both swim performance (U crit and U max ) and aerobic capacity. Fish exposed to both sediment containing (FPW-S) or sediment free (FPW-SF) FPW displayed significantly reduced aerobic scope and U crit /U max values compared to control conditions. Our results collectively suggest that organics present in our FPW sample may be responsible for sub-lethal fitness and metabolic responses. We provide evidence supporting the theory that the cardio-respiratory system is impacted by FPW exposure. This is the first known research associating embryonic FPW exposures to sub-lethal performance related responses in later life fish stages., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 (MO 2 ), while for 5% FPW, both fractions reduced MO 2 . 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 MO 2 . 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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; LC 50 ) and chronic (21 day) exposures. A decrease in reproduction was observed, with a mean value of 18.5 neonates produced per replicate over a 21 day chronic exposure to 0.04% FPW, which was a significant decrease from the average of 64 neonates produced in the controls. The time to first brood was delayed in the highest FPW (0.04%) treatment. Neonates exhibited an LC 50 of 0.19% of full-strength FPW, making them more sensitive than adults, which displayed an LC 50 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 with the development of environmental monitoring and risk assessment of FPW spills.

The effects of hydraulic fracturing (HF) flowback and produced water (HF-FPW), a complex saline mixture of injected HF fluids and deep formation water that return to the surface, was examined in rainbow trout (Oncorhynchus mykiss). Exposure to HF-FPWs resulted in significant induction of ethoxyresorufin-O-deethylase (EROD) activity in both liver and gill tissues. Increased lipid peroxidation via oxidative stress was also detected by thiobarbituric acid reactive substances (TBARS) assay. The mRNA expressions of a battery of genes related to biotransformation, oxidative stress, and endocrine disruption were also measured using quantitative real-time polymerase chain reaction (Q-RT-PCR). The increased expression of cyp1a (2.49 ± 0.28-fold), udpgt (2.01 ± 0.31-fold), sod (1.67 ± 0.09-fold), and gpx (1.58 ± 0.10-fold) in raw sample exposure group (7.5%) indicated elevated metabolic enzyme activity, likely through the aryl hydrocarbon receptor pathway, and generation of reactive oxygen species. In addition, the elevated vtg and era2 expression demonstrated endocrine disrupting potential exerted by HF-FPW in rainbow trout. The overall results suggested HF-FPW could cause significant adverse effects on fish, and the organic contents might play the major role in its toxicity. Future studies are needed to help fully determine the toxic mechanism(s) of HF-FPW on freshwater fish, and aid in establishing monitoring, treatment, and remediation protocols for HF-FPW.