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Hydraulic fracturing flowback and produced water (HF-FPW) has raised significant concerns owing to its potential impact on aquatic organisms and human health. Understanding the chemical composition of HF-FPW is crucial for developing appropriate management and remediation strategies. Herein, we performed nontarget screening on hydrophobic organic chemicals in the particulate phase of FPW (P-FPW) using gas chromatography-Orbitrap mass spectrometry coupled with cheminformatic analysis. In total, 5807 features were discovered, with 209 annotated with positive confidence levels, which were further classified based on their chemical taxonomy and functional use information. We found that benzenoids dominated the chemical class, followed by hydrocarbons. The annotated chemicals were classified into fragrances, catalysts, antimicrobials, antioxidants, and other classes. Chemical overlap across countries (China vs. Canada) and wells was observed, with most chemicals reaching peak intensity within 24 or 48 h after the initial flowback and gradually decreasing. Approximately two-thirds of the identified or annotated chemicals have not been previously reported or included in existing HF-related databases, indicating expanded chemical coverage by the current screening workflow. A Toxicological Priority Index (ToxPi) scheme, which integrates chemical properties, ecological toxicities, and in vivo exposure potentials, was adopted to prioritize chemicals for further evaluation. Seven chemicals were proposed as prioritized compounds, of which the ester derivative of perfluorobutanoic acid, octacosyl heptafluorobutyrate (confidence level 2), exhibited the highest ToxPi score. Notably, most prioritized substances have limited toxicological data and are beyond the routine monitoring of the HF industry, highlighting significant gaps in our understanding of HF-related chemical content and environmental risk associated with water management., 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 Ltd. All rights reserved.)

The mechanism(s) of sodium, chloride and pH regulation in teleost fishes has been the subject of intense interest for researchers over the past 100 years. The primary organ responsible for ionoregulatory homeostasis is the gill, and more specifically, gill ionocytes. Building on the theoretical and experimental research of the past, recent advances in molecular and cellular techniques in the past two decades have allowed for substantial advances in our understanding of mechanisms involved. With an increased diversity of teleost species and environmental conditions being investigated, it has become apparent that there are multiple strategies and mechanisms employed to achieve ion and acid-base homeostasis. This review will cover the historical developments in our understanding of the teleost fish gill, highlight some of the recent advances and conflicting information in our understanding of ionocyte function, and serve to identify areas that require further investigation to improve our understanding of complex cellular and molecular machineries involved in iono- and acid-base regulation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The consolidation of memory is thought to ultimately depend on the synthesis of new proteins, since translational inhibitors such as anisomycin and cycloheximide adversely affect the permanence of long-term memory. However, when applied directly in brain, these agents also profoundly suppress neural activity to an extent that is directly correlated to the degree of protein synthesis inhibition caused. Given that neural activity itself is likely to help mediate consolidation, this finding is a serious criticism of the strict de novo protein hypothesis of memory. Here, we test the neurophysiological effects of another translational inhibitor, emetine. Unilateral intra-hippocampal infusion of emetine suppressed ongoing local field and multiunit activity at ipsilateral sites as compared to the contralateral hippocampus in a fashion that was positively correlated to the degree of protein synthesis inhibition as confirmed by autoradiography. This suppression of activity was also specific to the circumscribed brain region in which protein synthesis inhibition took place. These experiments provide further evidence that ongoing protein synthesis is necessary and fundamental for neural function and suggest that the disruption of memory observed in behavioral experiments using translational inhibitors may be due, in large part, to neural suppression., (© 2024 The Author(s). Hippocampus published by Wiley Periodicals LLC.)

The freshwater sponge, Ephydatia muelleri, lacks a nervous or endocrine system and yet it exhibits a coordinated whole-body action known as a "sneeze" that can be triggered by exposure to L-glutamate. It is not known how L-glutamate is obtained by E. muelleri in sufficient quantities (i.e., 70 µM) to mediate this response endogenously. The present study tested the hypothesis that L-glutamate can be directly acquired from the environment across the body surface of E. muelleri. We demonstrate carrier mediated uptake of two distinct saturable systems with maximal transport rates (J max ) of 64.27 ± 4.98 and 25.12 ± 1.87 pmols mg -1  min -1 , respectively. The latter system has a higher calculated substrate affinity (K m ) of 2.87 ± 0.38 µM compared to the former (8.75 ± 1.00 µM), indicative of distinct systems that can acquire L-glutamate at variable environmental concentrations. Further characterization revealed potential shared pathways of L-glutamate uptake with other negatively charged amino acids, namely D-glutamate and L-aspartate, as well as the neutral amino acid L-alanine. We demonstrate that L-glutamate uptake does not appear to rely on exogenous sodium or proton concentrations as removal of these ions from the bathing media did not significantly alter uptake. Likewise, L-glutamate uptake does not seem to rely on internal proton motive forces driven by VHA as application of 100 nM of the VHA inhibitor bafilomycin did not alter uptake rates within E. muelleri tissues. Whether the acquired amino acid is used to supplement feeding or is stored and accumulated to mediate the sneeze response remains to be determined., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Concentrations of selenium that exceed regulatory guidelines have been associated with coal mining activities and have been linked to detrimental effects on aquatic ecosystems and the organisms therein. Although the major route of selenium uptake in macroinvertebrates is via the diet, the uptake of waterborne selenite (HSeO 3 - ), the prominent form at circumneutral pH, can be an important contributor to selenium body burden and thus selenium toxicity. In the current study, radiolabelled selenite (Se 75 ) was used to characterize the mechanism of selenite uptake in the water flea, Daphnia magna . The concentration dependence (1-32 μM) of selenite uptake was determined in 1-hour uptake assays in artificial waters that independently varied in bicarbonate, chloride, sulphate, phosphate and selenate concentrations. At concentrations representative of those found in highly contaminated waters, selenite uptake was phosphate-dependent and inhibited by foscarnet, a phosphate transport inhibitor. At higher concentrations, selenite uptake was dependent on waterborne bicarbonate concentration and inhibited by the bicarbonate transporter inhibitor DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid). These findings suggest that concentrations of phosphate in coal mining-affected waters could alter selenite uptake in aquatic organisms and could ultimately affect the toxic impacts of selenium in such waters., Competing Interests: The authors have no conflicts of interest to declare., (© The Author(s) 2024. Published by Oxford University Press and the Society for Experimental Biology.)

The mechanisms of acute (96-hour) and subchronic (28-day) toxicity of the waterborne trace metal thallium (Tl) to rainbow trout (Oncorhynchus mykiss) were investigated. Specifically, effects on branchial and renal ionoregulatory enzymes (sodium/potassium adenosine triphosphatase [ATPase; NKA] and proton ATPase) and hepatic oxidative stress endpoints (protein carbonylation, glutathione content, and activities of catalase and glutathione peroxidase) were examined. Fish (19-55 g) were acutely exposed to 0 (control), 0.9 (regulatory limit), 2004 (half the acute median lethal concentration), or 4200 (acute median lethal concentration) µg Tl L -1 or subchronically exposed to 0, 0.9, or 141 (an elevated environmental concentration) µg Tl L -1 . The only effect following acute exposure was a stimulation of renal H + -ATPase activity at the highest Tl exposure concentration. Similarly, the only significant effect of subchronic Tl exposure was an inhibition of branchial NKA activity at 141 µg Tl L -1 , an effect that may reflect the interaction of Tl with potassium ion handling. Despite significant literature evidence for effects of Tl on oxidative stress, there were no effects of Tl on any such endpoint in rainbow trout, regardless of exposure duration or exposure concentration. Elevated basal levels of antioxidant defenses may explain this finding. These data suggest that ionoregulatory perturbance is a more likely mechanism of Tl toxicity than oxidative stress in rainbow trout but is an endpoint of relevance only at elevated environmental Tl concentrations. Environ Toxicol Chem 2024;43:87-96. © 2023 SETAC., (© 2023 SETAC.)

The trace element thallium (Tl) exerts its toxic effects, at least in part, through its mimicry of potassium (K + ) and subsequent impairment of K + homeostasis. However, the specific nature of this effect remains poorly understood, especially in aquatic biota that are threatened by elevated concentrations of Tl associated with mining and refining effluents. In this study experiments were conducted to mechanistically examine the relationship between Tl and K + in terms of uptake and toxicity in the regulatory model species Daphnia magna. In one set of experiments the effects of K + , the K + analog rubidium (Rb + ), and generalized K + channel blocker cesium (Cs + ) on Tl-induced acute toxicity were examined. The presence of increasing concentrations of K + and Rb + in exposure water reduced waterborne Tl toxicity, indicating that the actions of Tl were mediated at least in part through interactions with K + . However, in the presence of elevated water Cs + , the toxicity of Tl paradoxically increased. Pharmaceuticals with putative blocking actions on K + channels failed to alter whole-body K + of control organisms, but in the case of clozapine and chlorpropamide, whole-body K + status was significantly elevated relative to exposures with Tl alone, which tended to reduce this metric. These data identify inwardly rectifying and voltage gated K + channels as potential loci of Tl toxicity. Experiments using rubidium (Rb + ) as a tracer of K + , showed that waterborne Tl affects the uptake of K + , but the magnitude of inhibition by Tl was not sufficient to explain the effect on whole-body K + . While these data indicate interactions between Tl and K occur at K + transporters in D magna, they also indicate that environmental levels of K + are likely to ameliorate toxicity in most natural waters., 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 © 2023 Elsevier Inc. All rights reserved.)

The accumulation and tissue distribution of toxicants in aquatic biota can be determinative of their toxic impact to both exposed organisms and their potential human consumers. In the present study, accumulation of the trace metal thallium (Tl) in gill, muscle, plasma, and otoliths of rainbow trout (Oncorhynchus mykiss) following acute (96-h) and subchronic (28-day) waterborne exposures was investigated. Owing to known interactions between Tl and potassium ions (K + ), plasma and muscle K + concentrations were also determined. Branchial Tl accumulated in a dose-dependent manner in both acute and subchronic exposures, while plasma Tl was rapidly mobilized to tissues and accumulated only at exposure concentrations of 141 µg L -1 or higher. For muscle tissue, Tl concentrations at 28 days were markedly lower than those at 96 h at comparable exposure concentrations (0.9 µg L -1 ), indicating the presence of mechanisms that act to reduce Tl accumulation over time. However, after acute exposure, muscle Tl reached concentrations that, if consumed, would exceed acceptable daily intake values for this element, indicating some risk to human health from the consumption of fish from waters heavily contaminated with Tl. Otoliths showed Tl concentrations that reflected exposure concentration and length, confirming their capacity to provide insight into fish exposure history. No changes in tissue K + concentrations were observed, suggesting that accumulation of Tl in rainbow trout plasma and muscle does not occur at the expense of K + homeostasis. In addition to highlighting the capacity of rainbow trout to accumulate Tl to levels that exceed recommended dietary doses to human consumers, the present study provides the first data of tissue-specific Tl accumulation in an important regulatory species. Environ Toxicol Chem 2023;42:1553-1563. © 2023 SETAC., (© 2023 SETAC.)

Galaxolide is one of the most frequently used synthetic polycyclic musks on the market and is commonly detected in aquatic waterways. Previous studies have mainly evaluated the toxicity of this emerging contaminant using water-only exposures; however, its high Log K ow (5.9) suggests that this compound is likely to partition to sediments. Three benthic invertebrates, Chironomus dilutus, Hyalella azteca, and Lumbriculus variegatus, were exposed to sediment-bound Galaxolide using both acute (10 d; survival) and chronic (28 d; survival and growth) bioassays. The acute and chronic LC50s for Galaxolide ranged from 238 to 736 mg/kg sediment (2400-7430 µg/g organic carbon [OC]) for all three species, which were above concentrations commonly detected in the environment (< 2.5 mg/kg). Growth effects (i.e., weight and/or length) were noted in two of the three organisms (with C. dilutus being the exception); however, these effects were also noted at concentrations above those that are environmentally relevant. Molecular level evaluations were conducted with surviving L. variegatus and C. dilutus collected from treatments near the LC50 value. Markers of oxidative stress (glutathione-s-transferase) and endocrine disruption (estrogen-related receptor) in C. dilutus were significantly decreased in the treatment group compared to controls by 0.7-fold and 1.9-fold, respectively. Although acute and chronic effects were largely absent at environmentally relevant concentrations, changes in endocrine response suggest that more sensitive endocrine-based endpoints, such as emergence (for C. dilutus) and molting (for H. azteca), are needed to ensure that the risk of this emerging contaminant is low at environmentally relevant concentrations., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fish mortality is associated with harmful algal blooms, although whether toxicity is related directly to the presence of cyanotoxins or the prevailing water chemistry remains unclear. Similarly, while planktivorous fish may be exposed to toxin through the diet, the hazard posed by waterborne extracellular toxin to carnivorous fish is less well understood. In this study rainbow trout (Oncorhynchus mykiss) were exposed for up to 28 d to waterborne microcystin-LR at nominal concentrations of 1.5 and 50 µg L -1 (measured values 2 and 49 µg L -1 , respectively). The former represents the Canadian drinking water guideline, and the latter an elevated environmental level. This study hypothesised that waterborne toxin exposure would specifically impact gill function, and given the importance of this tissue in freshwater fish ion regulation, effects on plasma ions and branchial ion transporter activity would be observed. Microcystin-LR exposure resulted in a significant and persistent hypocalcaemia at the higher exposure concentration, but plasma sodium and branchial activities of the sodium/potassium ATPase, proton ATPase and calcium ATPase enzymes remained unaffected. An in vitro assessment failed to show any effect of microcystin-LR on branchial calcium ATPase activity even at exposure concentrations as high as 1000 µg L -1 . A transient increase in hepatic alkaline phosphatase activity was also observed at 49 µg L -1 , but there were no effects of toxin exposure on branchial or hepatic lactate dehydrogenase activity. These results suggest that microcystin-LR exposure does not have a general effect on ion regulation, but instead produces a novel and specific impact on calcium metabolism in rainbow trout, although the mechanism underlying this effect remains unknown., 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 © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

The accumulation of organic toxicants in fish plasma, and how they partition between the bound and unbound fraction once absorbed, are important metrics in models that seek to predict the risk of such contaminants in aquatic settings. Rapid equilibrium dialysis of diltiazem, an ionizable weak base and important human pharmaceutical contaminant of freshwaters, was conducted with rainbow trout (Oncorhynchus mykiss) plasma. The effect of fed state, fish sex, fish strain/size, and dialysis buffer pH on the binding of radiolabeled diltiazem (9 ng ml -1 ) was assessed. In fed fish, 24.6%-29.5% of diltiazem was free, unbound to plasma proteins. Although starvation of fish resulted in a decrease in plasma protein, the bound fraction of diltiazem remained relatively constant. Consequently, the protein-bound concentration of diltiazem increased with length of starvation. In general, rainbow trout strain was a significant factor affecting plasma binding, although the two strains tested also differed markedly in size. Dialysis buffer pH significantly influenced plasma binding, with a higher unbound diltiazem fraction at pH 6.8 than pH 8.0. These data indicate that empirical measures of plasma binding in fish are important for accurate risk assessment and that the physiological status of a fish is likely to impact its sensitivity to toxicants such as diltiazem. Environ Toxicol Chem 2022;41:3125-3133. © 2022 SETAC., (© 2022 SETAC.)

Thallium (Tl) is a trace metal enriched in wastewaters associated with mining and smelting of base metals. The toxicity of Tl to aquatic biota is poorly understood, particularly with respect to its sublethal effects. In this study, phototactic behavioural responses of naïve (i.e. no previous exposure to Tl) Daphnia magna, a key regulatory freshwater crustacean species, were examined in waters containing Tl. Fed and fasted neonate daphnids (< 24 h old) and fed adults (10-15 days old) showed no significant response at any tested water Tl concentration. However, in fasted adults, an increase in the positive phototactic response (measured as a greater number of daphnids closer to the light source after a 5-min exposure) was seen at Tl concentrations of 917 and 2099 µg L -1 , values representative of extreme environmental Tl concentrations. The presence of Tl also decreased the swimming speed of adult Daphnia towards a light source. In the presence of cimetidine, a histamine receptor blocker, the increase in positive phototaxis induced by Tl disappeared, suggesting that Tl acts to perturb the phototaxis response through sensory inhibition. Conversely, although there was a trend towards enhanced activity, Tl had no significant effect on acetylcholinesterase, a marker of locomotor capacity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pacific hagfish (Eptatretus stoutii) are marine scavengers and feed on decaying animal carrion by burrowing their bodies inside rotten carcasses where they are exposed to several threatening environmental stressors, including hypercapnia (high partial pressures of CO 2 ). Hagfish possess a remarkable capacity to tolerate hypercapnia, and their ability to recover from acid-base disturbances is well known. To deal with the metabolic acidosis resulting from exposure to high CO 2 , hagfish can mount a rapid elevation of plasma HCO 3 - concentration (hypercarbia). Once PCO 2 is restored, hagfish quickly excrete their HCO 3 - load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO 3 - dehydration into CO 2 at the hagfish gills. We aimed to characterize the role of branchial CA in CO 2 /HCO 3 - clearance from the plasma at the gills of E. stoutii, under control and high PCO 2 (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO 3 - excretion by measuring in situ [ 14 C]-HCO 3 - fluxes. To accomplish this, we employed a novel surgical technique of individual gill pouch arterial perfusion combined with perifusion of the gill afferent to efferent water ducts. [ 14 C]-HCO 3 - efflux was measured at the gills of fish exposed to control, hypercapnic (48 h) and recovery from hypercapnia conditions (6 h), in the presence of two well-known pharmacological inhibitors of CA, the membrane impermeant C18 (targets membrane bound, plasma accessible CA) and membrane-permeant acetazolamide, which targets all forms of CA, including extracellular and intracellular cytosolic CAs. C18 did not affect HCO 3 - flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO 3 - fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO 3 - flux by 38%. The same pattern was observed for fish in recovery, where in all three experimental conditions, there was no significant inhibition of plasma-accessible CA. We also observed no change in CA enzyme activity (measured in vitro) in any of the experimental PCO 2 conditions. In summary, our data suggests that there are additional pathways for HCO 3 - excretion at the gills of hagfish that are independent of plasma-accessible CA., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oil and gas development and transportation in many areas of the world, such as the oil sands region of Alberta, Canada, are heavily monitored to minimize the environmental impacts of development and the risk of oil spills. However, oil spills to aquatic environments still occur. Although the science of oil spills has received considerable attention of late, uncertainty still remains in evaluating the fate and transport of oil spills as well as the effects of those spills on aquatic biota. Experiments using meso-scale spill tanks were used to examine the physical and chemical behavior of two types of oil, conventional crude (CC) and diluted bitumen (DB), under similar environmentally relevant scenarios (i.e., volume of spill, temperature, duration, wave action, and presence of river sediment). In addition, biological impact assessments via sediment toxicity testing collected from the oil spill tests were conducted. Sediments were evaluated for acute toxicity using three standard sediment test species: Hyalella azteca, Lumbriculus variegatus, and Chironomus dilutus. Sediments collected from the CC simulated spill showed a higher level of acute toxicity than sediments collected from spills with DB. Higher toxicity in the CC-contaminated sediment was supported by higher concentrations of low molecular weight polycyclic aromatic hydrocarbons (PAHs) when compared with the DB-contaminated sediment, while the remaining PAH profile was similar between the contaminated sediments. The use of a meso-scale spill tank in combination with sediment bioassays allowed for the evaluation of oil spills under controlled and environmentally relevant conditions (e.g., nearshore high sediment loading river), and in turn provides assessors with additional information to identify the appropriate mitigation and remediation efforts needed in the event of future spills. Environ Toxicol Chem 2022;41:2797-2807. © 2022 SETAC., (© 2022 SETAC.)

Increased crop production is necessary to keep up with rising food demand. However, conventional agricultural practices and agrochemicals are unable to sustain further increases without serious risk of adverse environmental consequences. The implementation of nanotechnology in agriculture practices has been increasing in recent years and has shown tremendous potential to boost crop production. The rapid growth in development and use of nano-agrochemicals in agriculture will inevitably result in more chemicals reaching water bodies. Some unique properties of nanoformulations may also alter the toxicity of the AI on aquatic organisms when compared to their conventional counterparts. Results from studies on conventional formulations may not properly represent the toxicity of new nanoformulations in the aquatic environment. As a result, current guidelines derived from conventional formulations may not be suitable to regulate those newly developed nanoformulations. Current knowledge on the toxicity of nano-agrochemicals on aquatic organisms is limited, especially in an ecologically relevant setting. This review complies and analyzes 18 primary studies based on 7 criteria to provide a comprehensive analysis of the available toxicity information of nano-agrochemicals and their conventional counterparts on aquatic organisms. Our analysis demonstrates that the overall toxicity of nano-agrochemicals on non-target aquatic species is significantly lower as compared to conventional counterparts. However, further dividing formulations into three categories (organic, bulk and ionic) shows that some nanoformulations can be more toxic when compared to bulk materials but less toxic as compared to ionic formulations while organic nanopesticides do not show a general trend in overall toxicity. Moreover, our analysis reveals the limitations of current studies and provides recommendations for future toxicity studies to ensure the effective and sustainable application of nano-agrochemicals, which will be beneficial to both the agrochemical industry and regulatory agencies alike., 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.)

Aim: Pacific hagfish are exceptionally tolerant to high environmental ammonia (HEA). Here, we elucidated a cellular mechanism that enables hagfish to actively excrete ammonia against steep ammonia gradients expected to be found inside a decomposing whale carcass., Methods: Hagfish were exposed to varying concentrations of HEA in the presence or absence of environmental Na + , while plasma ammonia levels were tracked. 14 C-methylammonium was used as a proxy for NH 4 + to measure efflux in whole animals and in isolated gill pouches; the latter allowed us to assess the effects of amiloride specifically on Na + /H + exchangers (NHEs) in gill cells. Western blotting and immunohistochemistry were utilized to evaluate the abundance and sub-cellular localization of Rhesus glycoprotein (Rh) channels in the response to HEA., Results: Hagfish actively excreted NH 4 + against steep inwardly directed E NH4 + (ΔE NH4 +  ~ 35 mV) and pNH 3 (ΔpNH 3  ~ 2000 μtorr) gradients. Active NH 4 + excretion and plasma ammonia hypo-regulation were contingent on the presence of environmental Na + , indicating a Na + /NH 4 + exchange mechanism. Active NH 4 + excretion across isolated gill pouches was amiloride-sensitive. Exposure to HEA resulted in decreased abundance of Rh channels in the apical membrane of gill ionocytes., Conclusions: During HEA exposure, hagfish can actively excrete ammonia against a steep concentration gradient using apical NHEs energized by Na + -K + -ATPase in gill ionocytes. Additionally, apical Rh channels are removed from the apical membrane, presumably to reduce ammonia loading from the environment. We suggest that this mechanism allows hagfish to maintain tolerable ammonia levels while feeding inside decomposing carrion, allowing them to exploit nutrient-rich food-falls., (© 2022 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The consequences of soils exposed to hydraulic fracturing (HF) return fluid, often collectively termed flowback and produced water (FPW), are poorly understood, even though soils are a common receptor of FPW spills. Here, we investigate the impacts on soil microbiota exposed to FPW collected from the Montney Formation of western Canada. We measured soil respiration, microbial community structure and functional potentials under FPW exposure across a range of concentrations, exposure time and soil types (luvisol and chernozem). We find that soil type governs microbial community response upon FPW exposure. Within each soil, FPW exposure led to reduced biotic soil respiration, and shifted microbial community structure and functional potentials. We detect substantially higher species richness and more unique functional genes in FPW-exposed soils than in FPW-unexposed soils, with metagenome-assembled genomes (e.g. Marinobacter persicus) from luvisol soil exposed to concentrated FPW being most similar to genomes from HF/FPW sites. Our data demonstrate the complex impacts of microbial communities following FPW exposure and highlight the site-specific effects in evaluation of spills and agricultural reuse of FPW on the normal soil functions., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Corticosteroids are synthesized from cholesterol by steroidogenic enzyme catalysts belonging to two main families: the cytochrome p450s (CYPs) and hydroxysteroid dehydrogenases (HSDs). The action of these steroidogenic enzymes allows the genesis of the terminal active corticosteroids 11-deoxycortisol (S), 1ɑ-hydroxycorticosterone (1α-OH-B), or cortisol in different fish species. However, for Cyclostomes like hagfishes, the terminal corticosteroid is still undefined. In this study, we examined the presence or absence of CYPs and HSDs as traits in fishes to gain insight about the primary corticosteroid synthesis pathways of the hagfishes. We used published cytochrome c oxidase I (COXI) amino acid sequences to construct a phylogeny of fishes and then mapped the CYPs and HSDs as morphological traits onto the tree to predict the ancestral character states through ancestral character reconstruction (ACR). There is a clear phylogenetic signal for CYP (i.e., CYP11a1, 17, 21, and 11b) and HSD (i.e., 11-βHSD and 3β-HSD) derivatives of interest throughout the more derived fishes. Using trait-based ACR, we also found that hagfishes possess genes for 3β-HSD, CYP11a1, CYP17, and CYP21. Importantly, the presence of CYP21 implies that hagfish can synthesize 11-deoxycorticosterone (11-DOC) and S. Previous research demonstrated that despite hagfish having CYP21, neither 11-DOC nor S could be detected in hagfish. This discrepancy between the presence of steroidogenic enzymes and products brings into question the expression and/or function of CYP21 in hagfishes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Industrial operations such as surface mining, road building, and aggregate washing result in high concentrations of suspended particles (Total Suspended Solids; TSS) in surface waters which must be treated prior to discharge into fish-bearing waters. A common industrial practice is to add flocculants to improve the efficacy and speed of TSS sedimentation. A significant environmental issue even small amounts of uncomplexed cationic polymer coagulant/flocculant remaining in treated water is highly toxic to fish at very low concentrations (LC 50 ∼ 0.3 mg L -1 ). Fingerling trout (Oncorhynchus mykiss) were exposed to (1) a cationic flocculant (Water Lynx 800 (WL800), (2) a Clearflow neutralizing polymer (CN369), and (3) a combination of WL800 and CN369 at various ratios with measured LC 50 as an index of toxicity. Acute toxicity was entirely reversed by addition of the neutralizing polymer at WL800:CN369 ratios >1:1.5 mg/L. Furthermore, we demonstrate that the proximal mechanism of acute cationic polymer toxicity is hypoxemia due to accumulation of polymer on the gill epithelia rather than gill damage. Exposure of 0.5 mg/L WL800 reduced oxygen consumption by >50% reduction by 12 h and this was accompanied by significantly increased blood, brain, and liver [lactate] and [glucose]. The development of an inexpensive amelioration technique preventing cationic polymer toxicity is a significant advancement in surface and industrial water treatment to prevent cationic polymer mediated fish kills., (Copyright © 2022 Elsevier B.V. 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.

Flowback and produced water (FPW) is an end-product of the hydraulic fracturing method of oil and gas extraction that is highly enriched in alkaline earth metals such as strontium (Sr). While Sr concentrations in FPW can exceed toxic thresholds for fish, the accompanying high concentrations of calcium (Ca) in FPW may ameliorate any toxicity. In this study, Sr bioaccumulation and molecular, biochemical, and physiological changes in ionoregulatory endpoints were investigated in rainbow trout (Oncorhynchus mykiss). Exposures were conducted over a 96-h period at Sr concentrations ranging from 1.7 to 1948 µM, with effects at the highest Sr exposure concentration also separately examined in waters of varying Ca concentration (10 to 958 µM). Plasma and gill Sr burdens increased as a function of increasing waterborne Sr, and accumulation increased further as water Ca concentrations were lowered. Despite this, there was no consistent, dose-dependent effect of Sr on plasma or gill Ca concentrations, although impacts on plasma and branchial sodium (Na) concentrations were observed. Waterborne Sr significantly inhibited branchial Ca 2+ -ATPase activity, albeit only at the highest tested Sr concentration (1948 µM). In exposure treatments where Sr was highly elevated and water Ca was reduced, the hepatic gene expression of Ca signaling receptors β-2 adrenergic receptor (Adrb2) and inositol-1,4,5-triphosphate receptor-2 (Itpr2) were inhibited, highlighting novel potential pathways of Sr toxicity in rainbow trout. Overall, these data indicate that water Ca has a strong effect on Sr bioavailability, but over an acute exposure period there is limited evidence for an effect of Sr on Ca homeostasis. Although Sr is elevated in effluents associated with the oil and gas industry, the co-occurrence of high Ca concentrations might protect freshwater fish against acute effects related to Sr exposure., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Aim: To determine whether Na + uptake in adult zebrafish (Danio rerio) exposed to acidic water adheres to traditional models reliant on Na + /H + Exchangers (NHEs), Na + channels and Na + /Cl - Cotransporters (NCCs) or if it occurs through a novel mechanism., Methods: Zebrafish were exposed to control (pH 8.0) or acidic (pH 4.0) water for 0-12 hours during which 22 Na + uptake ( J Na in ), ammonia excretion, net acidic equivalent flux and net K + flux ( J H net ) were measured. The involvement of NHEs, Na + channels, NCCs, K + -channels and K + -dependent Na + /Ca 2+ exchangers (NCKXs) was evaluated by exposure to Cl - -free or elevated [K + ] water, or to pharmacological inhibitors. The presence of NCKXs in gill was examined using RT-PCR., Results: J Na in was strongly attenuated by acid exposure, but gradually recovered to control rates. The systematic elimination of each of the traditional models led us to consider K + as a counter substrate for Na + uptake during acid exposure. Indeed, elevated environmental [K + ] inhibited J Na in during acid exposure in a concentration-dependent manner, with near-complete inhibition at 10 mM. Moreover, J H net loss increased approximately fourfold at 8-10 hours of acid exposure which correlated with recovered J Na in in 1:1 fashion, and both J Na in and J H net were sensitive to tetraethylammonium (TEA) during acid exposure. Zebrafish gills expressed mRNA coding for six NCKX isoforms., Conclusions: During acid exposure, zebrafish engage a novel Na + uptake mechanism that utilizes the outwardly directed K + gradient as a counter-substrate for Na + and is sensitive to TEA. NKCXs are promising candidates to mediate this K + -dependent Na + uptake, opening new research avenues about Na + uptake in zebrafish and other acid-tolerant aquatic species., (© 2022 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Development of elastin-like polypeptide (ELP) biomaterials is widespread, but information critical for clinical deployment is limited, with biocompatibility studies focused on a narrow cross-section of ELP sequences. Macrophages can impair biomaterial systems by degrading or isolating the biomaterial and by activating additional immune functions. Their phagocytic response will reveal early immune biocompatibility of ELP nanoparticles (NPs). This study examines that response, induced by the adsorbed protein corona, as a function of ELP guest amino acid, chain length and NP diameter. The breadth of proteins adsorbed to ELP NPs varied, with valine-containing ELP NPs adsorbing fewer types of proteins than leucine-containing constructs. Particle diameter was also a factor, with smaller leucine-containing ELP NPs adsorbing the broadest range of proteins. Macrophage viability was unaffected by the ELP NPs, and their phagocytic capabilities were unimpeded except when incubated with a 500 nm valine-containing 40-mer. This NP significantly decreased the phagocytic capacity of macrophages relative to the control and to a corresponding 500 nm leucine-containing 40-mer. NP size and the proportion of opsonin to dysopsonin proteins likely influenced this outcome. These results suggest that certain combinations of ELP sequence and particle size can result in an adsorbed protein corona, which may hinder macrophage function., (© 2021 Wiley Periodicals LLC.)

Hagfishes may encounter low dissolved oxygen in their natural habitats, a consequence of association with hypoxic sediments and their feeding behaviour. In teleost fish, hypoxia exposure decreases ion uptake, speculated to be a mechanism for energy conservation. Although hagfishes osmoconform, they do regulate extracellular fluid concentrations of divalent cations such as calcium. The current study hypothesised that exposure of hagfish to hypoxia (0.4 kPA, 24 h) would reduce calcium uptake (determined via in vitro isolated skin and gut epithelial transport assays) and calcium accumulation (determined by in vivo whole animal exposures, using radiolabelled calcium ( 45 Ca) to assess newly acquired calcium). A decrease in in vitro epidermal uptake was observed at sub-environmental calcium levels (10 μM), but not at environmental calcium levels (10 mM). No changes in gut calcium uptake were determined. Conversely, hypoxia led to a more rapid in vivo accumulation of calcium in tissues (skin, muscle, liver, heart, plasma, brain), mediated mostly by a significant increase in accumulation at the gill. These differences were only apparent after 1-h of exposure to the radiolabel (i.e., the last hour of the 24-h hypoxia exposure) and were not observed after 3 and 24 h periods of radiolabel exposure. This outcome was the opposite of the hypothesised effect. The reasons for a more rapid accumulation of calcium in hypoxic hagfish are unknown but may relate to roles for calcium in enhancing hypoxia tolerance in hagfishes or could be a consequence of changes in ventilatory frequency., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hydraulic fracturing flowback and produced water (HF-FPW), which contains polyaromatic hydrocarbons (PAHs) and numerous other potential contaminants, is a complex wastewater produced during the recovery of tight hydrocarbon resources. Previous studies on HF-FPW have demonstrated various toxicological responses of aquatic organisms as consequences of combined exposure to high salinity, dissolved organic compounds and particle/suspended solids-bound pollutants. Noteworthy is the lack of studies illustrating the potentially toxic effects of the FPW suspended solids (FPW-SS). In this study, we investigated the acute and sublethal toxicity of suspended solids filtered from six authentic FPW sample collected from two fracturing wells, using a sediment contact assay based on early-life stages of zebrafish (Danio rerio). PAHs profiles and acute toxicity tests provided initial information on the toxic potency of the six samples. Upon exposure to sediment mixture at two selected doses (1.6 and 3.1 mg/mL), results showed adverse effects in larval zebrafish, as revealed by increased Ethoxyresorufin-O-deethylase (EROD) activity. Transcriptional alterations were also observed in xenobiotic biotransformation (ahr, pxr, cyp1a, cyp1b1, cyp1c1, cyp1c2, cyp3a65, udpgt1a1, udpgt5g1), antioxidant response (sod1, sod2, gpx1a, gpx1b) and hormone receptor signaling (esr1, esr2a, cyp19a1a, vtg1) genes. The results demonstrated that even separated from the complex aqueous FPW mixture, FPW-SS can induce toxicological responses in aquatic organisms' early life stages. Since FPW-SS could sediment to the bottom of natural wetland acting as a continuous source of contaminants, the current findings imply the likelihood of long-term environmental risks of polluted sediments on aquatic ecosystems due to FPW spills., (Copyright © 2021. Published by Elsevier Ltd.)

We hypothesized that the skin acts as an extrabranchial route for ammonia excretion in adult rainbow trout (Oncorhynchus mykiss) following high environmental ammonia (HEA) exposure. Trunks of control or HEA-exposed trout were perfused with saline containing 0 or 1 mmol l -1 NH 4 + . Cutaneous ammonia excretion rates increased 2.5-fold following HEA exposure, however there was no difference in rates between trunks perfused with 0 or 1 mmol l -1 NH 4 + . The skin is therefore capable of excreting its own ammonia load, but it does not clear circulating ammonia from the plasma., (© 2021 Fisheries Society of the British Isles.)

Alterations in water chemistry can challenge resident fish species. More specifically, chemical changes that disrupt ion balance will negatively affect fish health and impact physiological and ecological performance. However, our understanding of which species and populations are at risk from ionoregulatory disturbances in response to changing freshwater environments is currently unclear. Therefore, we propose a novel framework for incorporating ionoregulatory physiology into conservation management of inland fishes. This framework introduces the concepts of fundamental chemical niche, which is the tolerable range of chemical conditions for a given species based on laboratory experiments, and realized chemical niche, which is the range of chemical conditions in which a species resides based on distribution surveys. By comparing these two niches, populations that may be at risk from ionoregulatory disturbances and thus require additional conservation considerations can be identified. We highlight the potential for commonly measured ionoregulatory traits to predict fundamental and realized chemical niches but caution that some traits may not serve as accurate predictors despite being important for understanding ionoregulatory mechanisms. As a sample application of our framework, the minimum pH distribution (realized niche) and survival limit pH (fundamental niche) of several North American fishes were determined by systematic review and were compared. We demonstrate that ionoregulatory capacity is significantly correlated with a realized niche for many species, highlighting the influence of ionoregulatory physiology on fish distribution patterns along chemical gradients. Our aim is that this framework will stimulate further research in this field and result in a broader integration of physiological data into conservation management decisions for inland waters., (© The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology.)

The mechanisms underlying ionoregulation in fishes have been studied for nearly a century, and reductionist methods have been applied at all levels of biological organization in this field of research. The complex nature of ionoregulatory systems in fishes makes them ideally suited to reductionist methods and our collective understanding has been dramatically shaped by their use. This review provides an overview of the broad suite of techniques used to elucidate ionoregulatory mechanisms in fishes, from the whole-animal level down to the gene, discussing some of the advantages and disadvantages of these methods. We provide a roadmap for understanding and appreciating the work that has formed the current models of organismal, endocrine, cellular, molecular, and genetic regulation of ion balance in fishes and highlight the contribution that reductionist techniques have made to some of the fundamental leaps forward in the field throughout its history., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hydraulic fracturing creates large volumes of flowback and produced water (FPW). The waste is a complex mixture of organic and inorganic constituents. Although the acute toxicity of FPW to freshwater organisms has been studied, few have attempted to discern the interaction between organic and inorganic constituents within this matrix and its role in toxicity. In the present study, bioaccumulation assays (7-d uptake and 7-d elimination period) with FPW (1% dilution) were conducted with the freshwater oligochaete, Lumbriculus variegatus, to evaluate the toxicokinetics of inorganic elements. To evaluate the interacting role of organics, bioaccumulation of elements in unmodified FPW was compared to activated carbon treated FPW (AC-modified). Differences in uptake and elimination rates as well as elimination steady state concentrations between unmodified and AC-modified treatments indicated that the organics play an important role in the uptake and depuration of inorganic elements in FPW. These differences in toxicokinetics between treatments aligned with observed growth rates in the worms which were higher in the AC-modified treatment. Whether growth differences resulted from increased accumulation and changes in toxicokinetic rates of inorganics or caused by direct toxicity from the organic fraction of FPW itself is still unknown and requires further research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

There is considerable debate about the sustainability of the hydraulic fracturing (HF) water cycle in North America. Recently, this debate has expanded to China, where HF activities continue to grow. Here, we provide a critical review of the HF water cycle in China, including water withdrawal practices and flowback and produced water (FPW) management and their environmental impacts, with a comprehensive comparison to the U.S. and Canada (North America). Water stress in arid regions, as well as water management challenges, FPW contamination of aquatic and soil systems, and induced seismicity are all impacts of the HF water cycle in China, the U.S., and Canada. In light of experience gained in North America, standardized practices for analyzing and reporting FPW chemistry and microbiology in China are needed to inform its efficient and safe treatment, discharge and reuse, and identification of potential contaminants. Additionally, conducting ecotoxicological studies is an essential next step to fully reveal the impacts of accidental FPW releases into aquatic and soil ecosystems in China. From a policy perspective, the development of China's unconventional resources lags behind North America's in terms of overall regulation, especially with regard to water withdrawal, FPW management, and routine monitoring. Our study suggests that common environmental risks exist within the world's two largest HF regions, and practices used in North America may help prevent or mitigate adverse effects in China.

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.)

Soluble adenylyl cyclase (sAC) is a HC O 3   - -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid-base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by HC O 3   - at physiologically relevant levels for fish internal fluids (EC 50 ∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid-base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The Advanced Oxidation System (AOS) is a novel electrochemical advanced oxidation process that effectively removes bacterial and organic contaminants from wastewater. However, potential formation of secondary oxidative species may pose additional hazards to aquatic organisms living in the receiving water affected by the post-treatment effluent. The effect of exposure to AOS treated water, especially the potential long-term effects on aquatic organisms, requires further investigation to demonstrate both efficacy and safety of this process. To examine the potential adverse effects of AOS treated water, three aquatic species, including daphnia, zebrafish, and rainbow trout, were exposed to treated and untreated municipal wastewater effluent (MWE) spiked with one of two model organic contaminants, benzo[a]pyrene (BaP) and 17β-estradiol (E2). The results indicated AOS treatment significantly reduced the adverse effects caused by exposure to MWE and model organic contaminants to baseline levels in daphnia (reduced fecundity), zebrafish embryo (elevated EROD activity), and rainbow trout (elevated plasma vitellogenin). The Ames test was also conducted to confirm the removal efficacy of carcinogenicity of BaP spiked in MWE. Overall, this study demonstrated that AOS treatment is a promising and environmentally friendly technology for wastewater treatment, remediation, and management., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

There is limited information regarding the toxicity of the trace element thallium (Tl) to aquatic biota, most of which assesses acute toxicity and bioaccumulation. The relative lack of chronic Tl toxicity data compromises the establishment of water quality criteria for this trace metal. In the presented work, chronic toxicity endpoints (final body weight (a proxy measure of growth), survival, and reproduction) and Tl body burden were measured in the freshwater crustacean Daphnia magna during a 21-day exposure to dissolved Tl. Thallium caused complete mortality in daphnids between exposure concentrations of 424 and 702 μg L -1 . In contrast with previously published work examining acute Tl toxicity, exposure to Tl for 21 days was not associated with changes in whole-body potassium concentration. This was despite a 710-fold increase in Tl body burden in animals exposed to 424 μg L -1 relative to the control. Median effect concentrations (EC 50 's) for growth and reproduction (total neonates produced), were 1.6 (95% confidence interval: 1.0-3.1) and 11.1 (95% confidence interval: 5.5-21.8) μg Tl L -1 , respectively. A no observable effect concentration (NOEC) of 0.9 μg Tl L -1 for growth, and a NOEC range of 0.9-83 μg Tl L -1 for a variety of reproductive metrics, was measured. A lowest observable effect concentration (LOEC) of 8.8 μg Tl L -1 was determined for the effects of Tl on growth and most of the reproductive endpoints examined. These data indicate that under controlled laboratory conditions D. magna is significantly less sensitive to Tl than the species on which the current Canadian Council of Ministers of the Environment regulatory guideline value of 0.8 μg L -1 is based., 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. Published by Elsevier Ltd.)

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.

Nanoparticles (NPs) that are exposed to blood are coated with an assortment of proteins that establish their biological identity by forming the interface between the NP and the cells and tissues of the body. The biological relevance of this protein corona is often overlooked during toxicological assessments of NPs. However, accurate interpretation of biological outcomes following exposure to NPs, including activation of coagulation, opsonization of pathogens, and cellular phagocytosis, must take this adsorbed proteome into account. In this study, we examined protein coronas on the surface of five poly(acrylic acid) (PAA) metal-oxide NPs (TiO 2 , CeO 2 , Fe 2 O 3 , ZnO, and PAA-capsules) following exposure to human plasma for key markers of various host response pathways, including humoral immunity and coagulation. We also evaluated the impacts of pre-exposing serum proteins to PAA-NPs on the opsonization and phagocytosis of bacteria by two immune cell lines. Results demonstrated that each PAA-NP type adsorbed a unique profile of blood proteins and that protein-coated PAA-NPs significantly inhibited human plasma coagulation with PAA-zinc oxide NPs and their associated proteome fully abrogating clotting. Protein-coated PAA-NPs also resulted in a 50% increase in phagocytic activity of RBL-2H3 cells and a 12.5% increase in phagocytic activity in the RAW 264.7 cell line. We also identified numerous structural, coagulation, and immune-activating proteins in the adsorbed protein corona, which resulted in altered biological function. Overall, our findings demonstrate that the formation of protein coronas on the surface of NPs plays an important role in directing the biological outcomes of opsonization, cell phagocytosis, and blood coagulation.

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 most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO 2 ) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO 2 excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin's capacity to bind with oxygen (O 2 )]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.

The response of microbial communities to releases of hydraulic fracturing flowback and produced water (PW) may influence ecosystem functions. However, knowledge of the effects of PW spills on freshwater microbiota is limited. Here, we conducted two separate experiments: 16S rRNA gene sequencing combined with random forests modelling was used to assess freshwater community changes in simulated PW spills by volume from 0.05% to 50%. In a separate experiment, live/dead cell viability in a freshwater community was tested during exposure to 10% PW by volume. Three distinct patterns of microbial community shifts were identified: (i) indigenous freshwater genera remained dominant in <2.5% PW, (ii) from 2.5% to 5% PW, potential PW organic degraders such as Pseudomonas, Rheinheimera and Brevundimonas became dominant, and (iii) no significant change in the relative abundance of taxa was observed in >5% PW. Microbial taxa including less abundant genera such as Cellvibrio were potential bioindicators for the degree of contamination with PW. Additionally, live cells were quickly damaged by adding 10% PW, but cell counts recovered in the following days. Our study shows that the responses of freshwater microbiota vary by spill size, and these responses show promise as effective fingerprints for PW spills in aquatic environments., (© FEMS 2020.)

Hydraulic fracturing of horizontal wells is a cost effective means for extracting oil and gas from low permeability formations. Hydraulic fracturing often produces considerable volumes of flowback and produced water (FPW). FPW associated with hydraulic fracturing has been shown to be a complex, often brackish mixture containing a variety of anthropogenic and geogenic compounds. In the present study, the risk of FPW releases to aquatic systems was studied using the model benthic invertebrate, Lumbriculus variegatus and field-collected FPW from a fractured well in Alberta. Acute, chronic, and pulse toxicity were evaluated to better understand the implications of accidental FPW releases to aquatic environments. Although L.variegatus is thought to have a high tolerance to many stressors, acute toxicity was significant at low concentrations (i.e. high dilutions) of FPW (48 h LC50: 4-5%). Chronic toxicity (28 d)of FPW in this species was even more pronounced with LC50s (survival/reproduction) and EC50s (total mass) at dilutions as low as 0.22% FPW. Investigations evaluating pulse toxicity (6 h and 48 h exposure) showed a significant amount of latent mortality occurring when compared to the acute results. Additionally, causality in acute and chronic bioassays differed as acute toxicity appeared to be primarily driven by salinity, which was not the case for chronic toxicity, as other stressors appear to be important as well. The findings of this study show the importance of evaluating multiple exposure regimes, the complexity of FPW, and also shows the potential aquatic risk posed by FPW releases., Competing Interests: Declaration of competing interest The authors of this study declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The use of nanotechnology to enhance pesticide formulations holds the promise of reduced pesticide use, reduced mobility in soils, and overall improvements in agricultural practices while simultaneously maintaining yields. However, the toxicity of nano-enabled pesticides, including azoxystrobin (Az), has not been well studied compared with their conventional form. This study investigates both lethal and sub-lethal endpoints in zebrafish embryos up to 120 h post-fertilization (hpf) under either laboratory light or simulated UV light. The median lethal concentration (LC50) value of nano-enabled Az (nAz) was significantly lower than the conventional form (Az). Interestingly, artificial UV light significantly increased toxicity (decreased LC50) of both Az and nAz. Malformations were not observed but the remaining yolk sac volume was significantly increased in both types of Az at both light conditions. This decreased yolk consumption is in agreement with reduced oxygen consumption and heart rate. Catalase enzyme activity was only reduced to UV light while superoxide dismutase activity was significantly reduced by co-exposure of UV light, and either type of Az at a nominal concentration of 100 μg L -1 . The co-exposure of Az at 100 μg L -1 and UV light significantly upregulated sod1 , sod2, and gpx1b expression and both types of Az significantly reduced gpx1a expression. Lipid peroxidation was significantly increased in nAz and Az at 100 μg L -1 under laboratory light, while UV light induced even higher level of lipid peroxidation. The results will provide important information on the toxicity of nAz under ecologically realistic conditions.

Although hagfishes osmoconform, concentrations of calcium in their extracellular fluids are maintained at levels lower than those of seawater. Ultimately, calcium homeostasis is a product of relative rates of influx and efflux into the animal, but little is known regarding these processes in hagfish. Using in vitro approaches, calcium influx across gut and skin epithelia of the Pacific hagfish, Eptatretus stoutii, was characterised. Analysis of concentration-dependent kinetics showed that accumulation into skin tissue was linear at mucosal calcium concentrations up to 10 mM. However, movement into the serosal compartment was saturable, with a maximal transport rate of 59 nmol cm -2  h -1 and an affinity (substrate concentration to give half maximal transport rate) of 1139 µM. Calcium accumulation into gut tissue and uptake into the gut serosal compartment was also saturable, with maximal transport rates of 5.1 and 2.8 nmol cm -2  h -1 , and affinities of 6417 and 3327 µM, respectively. A strong correlation between epidermal and intestinal calcium tissue uptake was noted in assays where tissues were taken from the same individual. Intestinal calcium influx was inhibited by nickel, but not significantly affected by other putative inorganic (lanthanum, zinc) or organic (diltiazem, verapamil) transport blockers. Inhibitors had no effect on skin calcium handling. These data indicate that both gut and skin epithelia mediate calcium influx in hagfishes, albeit via distinct mechanisms and with differences in responsiveness to potential modifying factors.

This study used 3 H- L -alanyl- L -alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration-dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (J max -like) of 70 ± 3 nmol cm -2 h -1 and an affinity constant (K m -like) of 1072 ± 81 μM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a K m distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium-proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical L -alanyl- L -alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later-diverging vertebrates such as teleosts and mammals., (© 2019 The Fisheries Society of the British Isles.)