Cellular mechanisms of ion and acid-base regulation in teleost gill ionocytes.

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.

Evidence for transporter-mediated uptake of environmental L-glutamate in a freshwater sponge, Ephydatia muelleri.

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 (Jmax) 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 (Km) 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.

Mechanistic characterization of waterborne selenite uptake in the water flea, Daphnia magna, indicates water chemistry affects toxicity in coal mine-impacted waters.

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 (HSeO3-), 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 (Se75) 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.

Effects of Acute and Subchronic Waterborne Thallium Exposure on Ionoregulatory Enzyme Activity and Oxidative Stress in Rainbow Trout (Oncorhynchus mykiss).

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.

Mechanistic examination of thallium and potassium interactions in Daphnia magna.

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.

Accumulation of Thallium in Rainbow Trout (Oncorhynchus mykiss) Following Acute and Subchronic Waterborne Exposure.

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.

The Acute and Chronic Effects of a Sediment-Bound Synthetic Musk, Galaxolide, on Hyalella azteca, Chironomus dilutus, and Lumbriculus variegatus.

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 Kow (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.

Sub-chronic exposure to waterborne extracellular microcystin-LR impairs calcium homeostasis in rainbow trout.

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.

Latent impacts on juvenile rainbow trout (Oncorhynchus mykiss) cardio-respiratory function and swimming performance following embryonic exposures to hydraulic fracturing flowback and produced water.

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.

The roles of plasma accessible and cytosolic carbonic anhydrases in bicarbonate (HCO3-) excretion in Pacific hagfish (Eptatretus stoutii).

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 CO2). 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 CO2, hagfish can mount a rapid elevation of plasma HCO3- concentration (hypercarbia). Once PCO2 is restored, hagfish quickly excrete their HCO3- load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO3- dehydration into CO2 at the hagfish gills. We aimed to characterize the role of branchial CA in CO2/HCO3- clearance from the plasma at the gills of E. stoutii, under control and high PCO2 (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO3- excretion by measuring in situ [14C]-HCO3- 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. [14C]-HCO3- 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 HCO3- flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO3- fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO3- 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 PCO2 conditions. In summary, our data suggests that there are additional pathways for HCO3- excretion at the gills of hagfish that are independent of plasma-accessible CA.

Factors Affecting the Binding of Diltiazem to Rainbow Trout Plasma: Implications for the Risk Assessment of Pharmaceuticals in Aquatic Systems.

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.

Freshwater Sediment Toxicity Evaluation from Meso-Scale Spill Tests of Diluted Bitumen and Conventional Crude.

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.

Embryonic cardio-respiratory impairments in rainbow trout (Oncorhynchus mykiss) following exposure to hydraulic fracturing flowback and produced water.

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 (MO2), 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 MO2 through development until 15 dpf, where a switch to significantly reduced MO2 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 MO2; 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 MO2, 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.

Nanotechnology in agriculture: Comparison of the toxicity between conventional and nano-based agrochemicals on non-target aquatic species.

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.

Effect of thallium on phototactic behaviour in Daphnia magna.

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.

Dining on the dead in the deep: Active NH4 + excretion via Na+ /H+ (NH4 + ) exchange in the highly ammonia tolerant Pacific hagfish, Eptatretus stoutii.

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 NH4 + 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 NH4 + against steep inwardly directed ENH4 + (ΔENH4 + ~ 35 mV) and pNH3 (ΔpNH3 ~ 2000 µtorr) gradients. Active NH4 + excretion and plasma ammonia hypo-regulation were contingent on the presence of environmental Na+ , indicating a Na+ /NH4 + exchange mechanism. Active NH4 + 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.

Hypoxemia as the mechanism of acute cationic polymer toxicity in rainbow trout and prevention of toxicity using an anionic neutralizing polymer.

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 (LC50 ∼ 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 LC50 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.

Complex impacts of hydraulic fracturing return fluids on soil microbial community respiration, structure and functional potentials.

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.

Absence of some cytochrome P450 (CYP) and hydroxysteroid dehydrogenase (HSD) enzymes in hagfishes.

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-ßHSD and 3ß-HSD) derivatives of interest throughout the more derived fishes. Using trait-based ACR, we also found that hagfishes possess genes for 3ß-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.