The contribution of the gut to the ingestion, production, absorption, and excretion of the extra ammonia and urea-N associated with feeding (“exogenous” fraction) has received limited prior attention. Analysis of commercial pellet food revealed appreciable concentrations of ammonia and urea-N. Long term satiation-feeding increased whole trout ammonia and urea-N excretion rates by 2.5-fold above fasting levels. Blood was sampled from the dorsal aorta, posterior, mid, and anterior sub-intestinal veins, as well as the hepatic portal vein in situ. Ammonia, urea-N, and fluid flux rates were measured in vitro using novel gut sac preparations filled with native chyme. The sacs maintained the extreme physico-chemical conditions of the lumen seen in vivo. Overall, these results confirmed our hypothesis that the stomach and anterior intestine+pyloric caecae regions play important roles in ammonia and urea-N production and/or absorption. There was a very high rate of urea-N production in the anterior intestine+pyloric caecae, whereas the posterior intestine dominated for ammonia synthesis. The stomach was the major site of ammonia absorption, and the anterior intestine+pyloric caecae region dominated for urea-N absorption. Model calculations indicated that >50% of the exogenous ammonia and urea-N excretion associated with satiation-feeding was produced in the anaerobic gut. This challenges standard metabolic theory used in fuel use calculations. The novel gut sac preparations gained fluid during incubation, especially in the anterior intestine+pyloric caecae, due to marked hyperosmolality in the chyme. Thus, satiation-feeding with commercial pellets is beneficial to the water balance of freshwater trout.

Microplastics (MPs) are constantly degrading while moving through aquatic systems as a result of mechanical abrasion, thermal fluctuations, UV light, and chemical exposure. As such, fish may experience pulse exposures to differentially degraded plastics. This study addresses how pulse exposures, over the course of minutes, to differentially degraded microplastics alters a key ionoregulatory property of the goldfish gill. We used transepithelial potential (TEP) across the gills, a diffusion potential resulting from the differential permeability of cations versus anions, as a sensitive indicator of potential ionoregulatory effects. Virgin (non-degraded) MPs along with mechanically, UV, and thermally degraded plastics immediately depolarized the gills (less negative TEP), whereas chemically degraded MPs resulted in no change to TEP. To further explore the physicochemical interaction between the surface of the gill and MPs, combinations of MPs and a single source of dissolved organic carbon (DOC) were tested and revealed that the presence of DOC negated the effects of MPs at the gill regardless of whether DOC or MPs were introduced first. This study suggests that while MPs have the ability to cause ionoregulatory effects at the gill, the effects of ambient water quality, specifically the presence of DOC, are of greater influence.

Behavioral endpoints are of increasing interest in toxicology because of their sensitivity, but require clear guidance for experimental design. This study describes the design of a hypoxia chamber for use with pond snails, Lymnaea stagnalis. Studies assessing the switch from water- to air-breathing in hypoxic conditions have previously utilized methods that neglect intricacies of animal behavior such as handling stress and acclimation. The chamber provides a linear decline in dissolved oxygen, against which surfacing behavior for air-breathing can be precisely measured. The maximum biomass of snails suitable for use in the hypoxia chamber, such that the nitrogen-driven deoxygenation curve is not altered by the snails' own metabolism, was established to be greater than 10 adult snails. The capacity of most analysis softwares is below accurately tracking 10 individuals at once, indicating this is likely not a limitation. The size of snails determined the amount of time each episode of aerial respiration was, with smaller snails spending more time air-breathing. A proof-of-principle experiment using acute copper exposure (0 - 60 mu g/L) yielded a concentration-response curve, with greater copper concentrations inhibiting air-breathing. The chamber described in the present study provides an improved framework for assessing hypoxic response and is presented in a manner allowing for further modification to meet unique research needs.

Salmonids spawn in freshwater streams including those in urban areas that are impacted by human activities. In the Vancouver region of British Columbia, Canada, the extensive use of road salt (primarily NaCl) is associated with frequent 24-h "pulses" of salt in streams, some of which may exceed the provincial acute guideline for maximum chloride concentrations (600 mg L-1 Cl-) by up to 11-fold. For some salmonids, road salting coincides with critical developmental stages, as many species spawn between October and January. We explored the concentration-dependent effects of a 24-h salt pulse (600-9600 mg L-1 Cl-) on salmonid development using rainbow trout (Oncorhynchus mykiss). Salt pulses were imposed at one of three developmental time points: <1 h post-fertilization, the eyed-stage or 7 days post-hatch. Significant mortality occurred only in the <1 h post-fertilization treatment, at 2400, 4800 and 9600 mg L-1 Cl-, all environmentally relevant salt concentrations. Significant differences in whole-embryo ion concentrations at the end of the salt exposure and at the eyed-stage (17 days post-salt exposure) indicated lasting ionoregulatory effects on embryos. Co-exposure to CaCO3 during the salt pulse, at a level that increased dissolved Ca2+ by 2-to 3-fold in the ion poor Vancouver water, greatly reduced mortality and altered whole-embryo ion levels. These findings support the need for site-specific water quality guidelines, as toxicity varies with water's ionic composition. This research also highlights the need for improved road salting practices to reduce salt contamination and its potential adverse effects on developing salmonids.

Optical characterization of dissolved organic carbon (DOC) freshly collected from the circumneutral "white water" of the Rio Solimoes revealed that it had lower aromaticity, lower molecular weight, and a greater autochthonous content than DOC from the acidic "black water" of the Rio Negro. The tambaqui (Colossoma macropomum), a characid member of the Serrasalmidae, is a model neotropical fish that migrates annually between the two rivers. We analysed ionoregulatory responses of the tambaqui over 24 h in ion-poor water at pH 7.0 and pH 4.0 in the absence and presence of 10 mg L-1 Rio Solimoes DOC ("SOL DOC"). Measured parameters included transepithelial potential (TEP) across the gills, net flux rates, and plasma concentrations of Na+, Cl-, ammonia, and urea, and branchial Na+, K+ -ATPase, H+, ATPase, and carbonic anhydrase activities. Results were compared to our earlier study using similar protocols to examine the ionoregulatory effects of 10 mg L-1 DOC ("SGC DOC") collected from black water at S & atilde;o Gabriel da Cachoeira (SGC) in the upper Rio Negro. At pH 7.0, SOL DOC had no effect on the negative TEP across the gills. Exposure to pH 4.0 caused a marked depolarization of the TEP to positive values that was not ameliorated by the presence of SOL DOC. This contrasts with SGC DOC that drove TEP more negative at pH 7.0 and fully protected against the depolarization at pH 4.0. However, SOL DOC promoted positive balance of Na+ and Cl- at pH 7.0 and helped ameliorate the negative balance of these ions seen at pH 4.0. This again contrasts with SGC DOC that exacerbated ion losses at pH 4.0. The protective effects of SOL DOC on ion balance maybe related to increased v-type H+ ATPase activity in the gills, and unrelated to TEP. The very different responses to the two DOCs are discussed with respect to their optical properties, the time that they were in storage prior to testing (SOL <1 month, SGC 2 years), and the life history of the tambaqui in the two rivers.

Urban freshwater streams across northern latitudes are undergoing increasing salinization due, in part, to road salt inputs during winter months. Road salt contamination has been monitored across Canada for over 40 years; however, the scale of contamination in the Pacific Northwest, which experiences relatively mild and rainy winters, is not well understood. A network of almost 40 water quality loggers in the Lower Mainland of Vancouver, B.C., Canada (VLM) was leveraged to better understand the scale of road salt inputs to local streams and identify factors that influence the magnitude and occurrence of these contamination events. Specific conductance data from these loggers indicate that road salt is entering creeks, resulting in brief salt pulses that typically last 1 day or less. Road salt pulses occur as frequently as three times per week in winter months and can attain maximum chloride concentrations above British Columbia’s acute guideline for chloride (600 mg/L Cl−) by as much as 11-fold in streams. The amount of road salt entering creeks is influenced by the extent of impervious surface in the surrounding catchment basin, with more urbanized creeks receiving higher inputs. Interestingly, cumulative salt inputs do not correlate with winter severity and remain consistent even during mild winters. Acute pulses of road salt occur in VLM streams between November and March, coinciding with the spawning and incubation period of locally important Pacific salmon species such as coho and chum salmon. This timing poses a direct risk to developing salmonids, and the benthic invertebrates which sustain them later in development.

The tambaqui (Colossoma macropomum, G. Cuvier 1818) thrives both in the ion-poor waters of the Amazon and in commercial aquaculture. In both, environmental conditions can be harsh due to low ion levels, occasional high salt challenges (in aquaculture), low pH, extreme PO2 levels (hypoxia and hyperoxia), high PCO2 levels (hypercapnia), high ammonia levels (in aquaculture), and high and low temperatures. Ion transport across the gill is affected by active transport processes, passive diffusive permeability, ion concentrations (the chemical gradient), and transepithelial potential (TEP, the electrical gradient). The latter is a very important indicator of ionoregulatory status but is rarely measured. Using normoxic, normocapnic, ion-poor, low-dissolved organic carbon (DOC) well water (27°C, pH 7.0) as the acclimation and reference condition, we first confirmed that the strongly negative TEP (-22.3 mV inside relative to the external water) is a simple diffusion potential. We then evaluated the effects on TEP of more complex waters from the Rio Negro (strong hyperpolarization) and Rio Solimões (no significant change). Additionally, we have quantified significant effects of acute, realistic changes in environmental conditions-low pH (depolarization), hypercapnia (depolarization), hypoxia (depolarization), hyperoxia (hyperpolarization), elevated NaCl concentrations (depolarization), and elevated NH4Cl concentrations (depolarization). The TEP responses help explain many of the changes in net Na+ flux rates reported in the literature. We have also shown marked effects of temperature on TEP and unidirectional Na+ flux rates (hyperpolarization and decreased fluxes at 21°C, depolarization and increased fluxes at 33°C) with no changes in net Na+ flux rates. Calculations based on the Nernst equation demonstrate the importance of the TEP changes in maintaining net Na+ balance.

This study aimed to investigate how exposure to elevated water temperature and metal concentration jointly affect the physiology of Amazonian fish. Aboard a research vessel in the Amazon, we evaluated the effects of water temperature (river T°C at 31.5°C and a + 4°C increase to 35.5°C) and of 3-h copper (Cu) exposure (up to 600 μg/L) in juvenile Tambaqui (Colossoma macropomum) exposed in freshly collected Rio Negro ('black water') and Rio Solimões ('white water') waters. In Cu-free water, the +4°C raise accelerated physiological Na+ influx and efflux rates, but only in Rio Negro water. Temperature had no effects on the other physiological fluxes (Cl-, K+ and ammonia fluxes). Cu exposure led to net losses of Na+ (via increased efflux), Cl- and K+ and decrease in acute upper thermal tolerance (CTmax). These Cu effects were more prominent in Rio Negro water, where Cu bioavailability was the greatest. The +4°C change had no effect on gill Cu accumulation and, overall, there was limited evidence that warming worsened Cu-induced ionoregulatory disturbances. However, in Rio Negro, as Cu and heat both separately promoted Na+ net losses, fish Na+ balance was the most compromised in the presence of the two stressors. Altogether, the impaired thermotolerance and ionoregulation under combined Cu and heat exposures suggest a cumulative physiological interaction between two stressors that are increasing threats to the Amazon basin.

Increases in anthropogenic activities in the Amazon have led to pollution from trace metals, including copper. Dissolved organic carbon (DOC) is known to protect against metal toxicity and ionoregulatory disturbances in Amazonian fish, particularly at low pH. However, little is known about the effects of DOC and trace metals, such as copper, on the branchial water transport pathways. Water moves across the gills of fish through two distinct pathways: paracellularly through tight junctions and transcellularly by diffusion through aquaporins. In the present study, we evaluated the effects of copper (nominally 200 μg L −1 ) on diffusive water flux rate (transcellular water movement), paracellular permeability ([ 3 H]‐polyethylene glycol‐4000 clearance), ion balance (net sodium, potassium and chloride fluxes) and nitrogenous waste (ammonia and urea) excretion in the dwarf cichlid, Apistogramma agassizii. Exposures were conducted in control water (low ions, very low DOC), in filtered Rio Negro (RN) blackwater (low ions, high DOC) and in filtered Rio Solimões (RS) whitewater (higher ions, intermediate DOC) at pH 7 and pH 4. Copper increased ion losses in control water, especially at low pH; RN water protected against these effects, whereas RS water did not, reflecting greater complexation of free Cu 2+ ions by RN DOC. Our results are the first to show that copper tends to inhibit urea‐N excretion as well as ammonia excretion, and also decreases branchial water transport both transcellularly and through tight junctions. The protective effects of DOC against the disturbances caused by copper were dependent on the source of the DOC and the water pH.

Over the past 20 years, the Amazon has experienced extreme floods, droughts and warmer temperatures due to climate change. Water temperature in the Rio Negro, a major tributary of the Amazon River, reached it highest October value during the 2023 drought. In Lake Tefé, connected to the Rio Solimões (another major tributary of the Amazon river), water temperatures reached 39°C. Increasing temperatures, and associated decreases in oxygen, will plague the Amazon and other regions, altering and accelerating links in the carbon cycle, such as photooxidation of dissolved organic carbon (DOC). We determined the response of DOC photooxidation rate in the Rio Negro (black-water) and Rio Solimões (white-water) to increases in water temperature between 20°C and 40°C and oxygen concentration between 0.01 mg O2.l-1 and 8 mg O2.l-1. The temperature coefficient (Q10) averaged 1.165, indicative of the dominance of diffusive processes, presumably of reactive oxygen species involved in photooxidation. Direct kinetic release of CO2 was 15% to 21% of normoxic CO2 production, and did not respond to temperature. The activation energy (Ea) of photooxidation was 13.14 kJ.mol-1 in the Rio Solimões and14.09 kJ.mol-1 in the Rio Negro. The Eas were not significantly different, suggesting no differences in the cost of photooxidation between the two rivers. They align with UVB Eas. Photooxidative production of CO2 only became oxygen limited between 0.5 mgO2.l-1 – 0.8 mgO2.l-1 (1.2 kPa – 1.9 kPa, 23°C). Thus, near-surface levels of oxygen are unlikely to directly depress CO2 production of DOC as temperatures rise.

Freshwater salinization is increasing globally through seawater intrusion, road de-icing, and changes in anthropogenic land uses. Concurrently, freshwaters are browning with the rise in dissolved organic carbon (DOC) concentrations, while water pH is falling. Elevations in external major ion concentrations (Na+ or Ca2+) and low pH, independently disturb osmoregulatory homeostasis in freshwater organisms. Several studies have demonstrated that DOC often mitigates osmoregulatory stress responses to acidic pH. However, the interactive effects of these three water quality parameters together have been relatively understudied. Transepithelial potential (TEP), the electrical gradient across the gills between the animal and the external water, can be used as an index of osmoregulatory stress. We investigated whether DOC and exposure to elevated major ions interact with TEP responses at circumneutral and low environmental pH in the freshwater rainbow trout. Two natural DOCs, one allochthonous and the other autochthonous, were used. To aid interpretation, three model compounds of known chemical structure were also employed (tannic acid, sodium dodecyl sulfate, bovine serum albumin), based on the criteria that they structurally resemble or functionally behave like certain chemical moieties of humic or fulvic acids, major components of DOC. The Multi-Ion Toxicity Model predicts that a disturbance in absolute TEP is indicative of salt toxicity; however, recent studies have shown that ΔTEP (the change in TEP relative to the baseline) may be more predictive. Our data followed a pattern that could be described by the Michaelis–Menten equation. Therefore, considering Michaelis–Menten constants (Km and ΔTEPmax), absolute TEP and ΔTEP, we used a weight of evidence approach to predict how DOC and pH will influence Na+ or Ca2+ toxicity. We conclude that key chemical moieties of DOC will likely play pH-dependent roles in both Na+ and Ca2+ toxicity.

Siluriform fishes collected from the Rio Negro and Rio Solimões proved to be highly resistant to aquatic hypoxia. In all four species analysed in this study, aquatic oxygen consumption significantly decreased from normoxic levels at water PO 2 values near 1 kPa. Air‐breathing activity was observed only in Sturisoma sp. (Rio Negro). In this species, under severe hypoxia, oxygen uptake from the air dominated, but total oxygen uptake was significantly lower than that under normoxic conditions. In Anadoras weddellii (Rio Solimões), aquatic surface respiration was detected. However, the other species ( Tympanopleura atronasus and three members of the family of Sternopygidae; Rio Solimões) showed no attempt to supplement aquatic oxygen uptake, even under severe hypoxia. In all species tested, neither ammonia nor urea‐N excretion was affected by the decreasing water PO 2 . At the lowest water PO 2 levels, the reduction in total oxygen uptake in the face of unchanged nitrogenous waste excretion resulted in extraordinary high nitrogen quotient (NQ) ratios. In normoxia, NQ ratios ranged from 0.16 to 0.34. Urea‐N excretion contributed between 19% and 28% to total nitrogen excretion and appeared to be unrelated to natural diet as indicated by the gut length‐to‐fork length ratio or to plasma urea‐N levels. Overall, our data underline the quantitative importance of urea‐N for nitrogen excretion in siluriform fishes.

Fish face a functional trade-off at the gills between minimizing ion movement and maximizing oxygen uptake – the osmorespiratory compromise, but the extent of this trade-off remains poorly understood in elasmobranchs. Using the Pacific dogfish shark, we assessed the impacts of progressive hypoxia in animals acclimated to 25, 30 and 36 ppt for 4 days at 12 °C. Plasma osmolality increased with water osmolality at 36 ppt (osmoconformation) and decreased at 25 ppt. Plasma urea decreased at 25 ppt, though to a lesser extent than plasma Cl−, while plasma urea increased to a greater extent than plasma Cl− at 36 ppt. In normoxia, oxygen consumption rate (MO2) was elevated by 60

The Amazonian loricariid fish Pterygoplichthys gibbiceps, from the Rio Negro, and Pterygoplichthys pardalis, from the Rio Solimões, are facultative air-breathers that can use the stomach as an air-breathing organ. Measurement of oxygen uptake under progressive aquatic hypoxia revealed a relatively high hypoxia resistance of both species. In both species, air-breathing was initiated at aquatic PO2 values below 3 kPa. In hypoxia, aerial oxygen uptake was dominant, but in P. gibbiceps total oxygen uptake was reduced to 55 ± 5% of the normoxic values, and in P. pardalis to only 43 ± 4% of the normoxic value. P. pardalis took a greater percentage of its total O2 consumption from air (92 ± 2%) than did P. gibbiceps (85 ± 3%). Air-breath volume increased with body mass in P. gibbiceps, whereas in P. pardalis air-breathing frequency increased with body mass. The minimal breath volume required to account for aerial oxygen uptake was calculated as 24.9 ± 2.1 mL*kg-1 for P. pardalis, and 17.3 ± 1.1 mL*kg-1 for P. gibbiceps. In both species, ammonia and urea-N excretion were not significantly modified under hypoxic conditions, and urea-N excretion contributed a relatively high percentage (23%) to total nitrogen excretion. Measurement of unidirectional and net Na+ flux rates during normoxia, hypoxia and subsequent normoxic recovery in P. gibbiceps revealed a significant decrease in Na+ influx rate under hypoxic conditions, followed by a significant increase during recovery compared to the control period, with no changes in net Na+ balance. The data suggest that a reduction in energy-consuming processes may contribute to the observed hypoxia resistance.

The depuration of newly accumulated metal from the crab Carcinus maenas (inter-moult stage) was studied, with a particular focus on the carapace, in light of recent findings that it is a major site for direct uptake and incorporation of Ca, Zn, and Ni from the external sea water. Crabs were exposed for 24 h to calcium ([Ca] = 389 mg L−1 or 9.7 mmol L−1), zinc ([Zn] = 82 μg L−1 or 1.25 μmol L−1), and nickel ([Ni] = 8.2 μg L−1 or 0.14 μmol L−1) with the addition of radio-labeled metal (45Ca, 65Zn, 63Ni) in sea water (12 °C, 32 ppt), then transferred to clean sea water for ≥48 h. After 24 h of metal exposure, the carapace accounted for ≥85 % of the total body burden of all three newly accumulated metals. For Ca, depurations from the carapace and whole crab were negligible, though levels in soft tissues (gills, hemolymph, and muscle) fell quickly. In contrast, newly accumulated Zn levels in carapace and gills declined by ~60 % over 48 h, while muscle, hepatopancreas, and hemolymph burdens increased, reflecting shifts from gills and/or carapace to internal tissues. Newly accumulated Ni concentrations in the carapace and gills declined by ~50 % over 48 h, reaching >75 % loss by 10 days. As for Zn, Ni levels increased in the hemolymph and initially in the hepatopancreas, indicative of internal redistribution. Acute temperature increase (12 °C to 22 °C) had negligible effects on depuration rates (Q10 values ~1.0). Depuration from the carapace was unchanged in recently euthanized crabs, or when the carapace was shielded with a membrane. We conclude that physicochemical processes are the rate-limiting steps for metal elimination from the carapace, and that metals newly incorporated in the carapace can leave only through the internal surface of the carapace; the external surface is not a depuration site.

Pacific spiny dogfish, Squalus suckleyi, move to shallow coastal waters during critical reproductive life stages and are thus at risk of encountering hypoxic events which occur more frequently in these areas. For effective conservation management, we need to fully understand the consequences of hypoxia on marine key species such as elasmobranchs. Because of their benthic life style, we hypothesized that S. suckleyi are hypoxia tolerant and able to efficiently regulate oxygen consumption, and that anaerobic metabolism is supported by a broad range of metabolites including ketones, fatty acids and amino acids. Therefore, we studied oxygen consumption rates, ventilation frequency and amplitude, blood gasses, acid-base regulation, and changes in plasma and tissue metabolites during progressive hypoxia. Our results show that critical oxygen levels (P crit) where oxyregulation is lost were indeed low (18.1% air saturation or 28.5 Torr at 13°C). However, many dogfish behaved as oxyconformers rather than oxyregulators. Arterial blood PO2 levels mostly decreased linearly with decreasing environmental PO2. Blood gases and acid-base status were dependent on open versus closed respirometry but in both set-ups ventilation frequency increased. Hypoxia below Pcrit resulted in an up-regulation of anaerobic glycolysis, as evidenced by increased lactate levels in all tissues except brain. Elasmobranchs typically rely on ketone bodies as oxidative substrates, and decreased concentrations of acetoacetate and β-hydroxybutyrate were observed in white muscle of hypoxic and/or recovering fish. Furthermore, reductions in isoleucine, glutamate, glutamine and other amino acids were observed. After 6 hours of normoxic recovery, changes persisted and only lactate returned to normal in most tissues. This emphasizes the importance of using suitable bioindicators adjusted to preferred metabolic pathways of the target species in conservation physiology. We conclude that Pacific spiny dogfish can tolerate severe transient hypoxic events, but recovery is slow and negative impacts can be expected when hypoxia persists.

The pirarucu is one of the very few obligate air-breathing fish, employing a gigantic, highly vascularized air-breathing organ (ABO). Traditionally, the ABO is thought to serve mainly for O2 uptake (ṀO2), with the gills providing the major route for excretion of CO2 (ṀCO2) and N-waste. However, under aquatic hypercapnia, a common occurrence in its natural environment, branchial ṀCO2 to the water may become impaired. Under these conditions, does the ABO become an important route of ṀCO2 excretion to the air? We have answered this question by measuring ṀCO2 and ṀO2 in both air and water phases, as well as the pattern of air-breathing, in pirarucu under aquatic normocapnia and hypercapnia (3

There is a consensus that electroneutral Na+/H+ exchangers (NHEs) are important in branchial Na+ uptake in freshwater fish. There is also widespread belief, based on mammalian data, that EIPA [5-(N-ethyl-N-isopropyl)-amiloride]], and HMA [5-(N,N-hexamethylene)-amiloride)] are more potent and specific in blocking Na+ uptake than amiloride. We evaluated this idea by testing the three drugs at 10-7 to 10-4 M, i.e. 0.1 to 100 μM in two model species, rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus), using 22Na+ to measure unidirectional Na+ influx and efflux rates. In both species, the potency order for inhibiting unidirectional Na+ influx was HMA > amiloride > EIPA (IC50 values in the 10-70 μM range), very different from in mammals. At 100 μM, all three drugs inhibited Na+ influx by >90% in both species, except for amiloride in goldfish (65%). However, at 60-100 μM, all three drugs also stimulated unidirectional Na+ efflux rates, indicating non-specific effects. In trout, HMA and EIPA caused significant increases (2.1- to 2.3-fold) in efflux rates, whereas in goldfish, significant efflux elevations were greater (3.1- to 7.2-fold) with all three drugs. We conclude that the inhibitory potency profile established in mammals does not apply to the NHEs in fish gills, that non-specific effects on Na+ efflux rates are a serious concern, and that EIPA and HMA offer no clear benefits in terms of potency or specificity. Considering its much lower cost, we recommend amiloride as the drug of choice for in vivo experiments on freshwater fishes.

Dissolved organic carbon (DOC) is a complex mixture of molecules that varies in composition based on origin as well as spatial and temporal factors. DOC is an important water quality parameter as it regulates many biological processes in freshwater systems, including the physiological function of the gills in fish. These effects are often beneficial, especially at low pH where DOCs mitigate ion loss and protect active ion uptake. DOCs of different compositions and quality have varied ionoregulatory effects. The molecular variability of DOCs can be characterized using optical and chemical indices, but how these indices relate to the physiological effects exerted by DOCs is not well understood. We tested the effects of five naturally sourced DOCs, at both pH 7 and pH 4, on transepithelial potential (TEP) (a diffusion potential between the blood plasma and the external water) in rainbow trout. The five chosen DOCs have been well characterized and span large differences in physicochemical characteristics. Each of the DOCs significantly influenced TEP, although in a unique manner or magnitude which was likely due to their physicochemical characteristics. These TEP responses were also a function of pH. With the goal of determining which physicochemical indices are predictive of changes in TEP, we evaluated correlations between various indices and TEP at pH 7 and pH 4. The indices included: specific absorbance coefficient at 340 nm, molecular weight index, fluorescence index, octanol–water partition coefficient, molecular charge, proton binding index,