Effects of water chemistry variables on gill binding and acute toxicity of cadmium in rainbow trout (Oncorhynchus mykiss): A biotic ligand model (BLM) approach

This study investigated the short-term (3 h) cadmium binding characteristics of the gills, as well as the influence of various water chemistry variables [calcium, magnesium, sodium, pH, alkalinity and dissolved organic carbon (DOC)] on short-term gill accumulation and acute toxicity of cadmium in juvenile freshwater rainbow trout. The cadmium binding pattern revealed two types of cadmium binding sites in the gill: (i) saturable high affinity sites operating at a low range of waterborne cadmium concentration, and (ii) non-saturable low affinity sites operating at a higher range of cadmium concentration. Among the water chemistry variables tested, only calcium and DOC significantly reduced both gill accumulation and toxicity of cadmium. Interestingly, alkalinity (15-90 mg L(-1) as CaCO(3)) did not influence the gill cadmium accumulation but a significant increase in toxicity was recorded at a higher alkalinity level (90 mg L(-1)). Affinity constants (log K) for binding of competing cations (Cd(2+) and Ca(2+)) to the biotic ligand and for binding of Cd(2+) to DOC were derived separately from the 3 h gill binding tests and the 96 h toxicity tests. In general, the values agreed well, indicating that both tests targeted the same population of high affinity binding sites, which are likely Ca(2+) uptake sites on the gills. These parameters were then incorporated into a geochemical speciation model (MINEQL+) to develop a biotic ligand model for predicting acute toxicity of cadmium in trout. The model predictions exhibited a good fit with the measured toxicity data except for high alkalinity and pH.     

Influence of dissolved organic matter on copper binding, and calcium on cadmium binding, by gills of rainbow trout

Complexation of Cu by 5 mg Cl⁻¹ dissolved organic matter (DOM) from a marsh kept Cu from binding to gills of small rainbow trout Oncorhynchus mykiss in 9-day exposures to 0.5 μM Cu in soft water. The protective effect of DOM occurs because the formation of Cu-DOM complexes reduces the amount of free Cu in the water, so the disruptive effects of Cu on ionoregulation, such as inhibited Na uptake, cannot develop. The Cu-DOM complexes themselves do not bind to the gills. Calcium (1100 μm) reduced the accumulation of Cd by trout gills in short, 2-h exposures through competition for gill binding sites but not over longer, 7-day exposures to 0–14 μM Cd. However, the protective effect of Ca against Cd toxicity persisted throughout the longer experiment, likely due to the decrease in the electrochemical gradient for diffusive loss of Ca from the fish to the water. Rainbow trout and fathead minnows Pimephales promelas accumulated Cu and Cd on their gills in a similar manner; thus, binding constants for metal-gill interactions determined for one species of fish can be generalized to other fish species. When literature binding constants determined for fathead minnows were applied to our studies with rainbow trout, computer modelling of Cu-gill and Cu-DOM interactions simulated our results well. In contrast Cd-gill and Ca-gill modelling predicted the initial competitive effect of Ca against Cd accumulation by trout gills, but did not predict the longer-term accumulation of Cd by trout gills.     

A lead-gill binding model to predict acute lead toxicity to rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss, approximately 2 g) were exposed to 0.6-1.0 microM Pb (125-200 microgl(-1)) for 3 h in ion-poor water. Complexing ligands (citrate, ethylenediamine, organic matter (OM)) or competing cations (Ca, Mg, Na) were added to the water. After exposure, trout gills were removed and analyzed for accumulated Pb. From these exposures, a conditional equilibrium binding constant (K) for Pb-gill binding was calculated (log K(Pb-gillPb)=6.0), plus conditional binding constants for cationic competition at the Pb binding sites and for Pb binding to OM in the water. These log K values were entered into the MINEQL+ aquatic chemistry equilibrium program, to calculate binding of Pb by trout gills. Two versions of the Pb-gill binding model were generated, one of which took into account OM quality as indicated by a simple measure of OM aromaticity, the specific absorption coefficient. The two model versions were tested against acute Pb toxicity (as the time to reach 50% fish mortality; LT50) during 1-week exposures of trout to 3.9 microM Pb in water collected from across southern Ontario. Both versions of the model generated highly significant correlations between the LT50 values and gill Pb concentrations calculated from measured exposure water chemistry, with the OM quality version correlating slightly better. Water pH also correlated well with the LT50 values, because the Pb exposures were in the pH range (7-8) where there is a nearly linear relationship between water pH and inorganic complexation of Pb. Advantages of the Pb-gill binding model include its completeness and the flexibility inherent in its conceptual framework, for example the inclusion of competition by Ca and H(+) for Pb binding sites on gills, and inclusion of complexation of Pb in the water column by natural OM and by carbonate.     

Waterborne vs. dietary copper uptake in rainbow trout and the effects of previous waterborne copper exposure

Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne Cu (22 microg/l) in moderately hard water for up to 28 days. Relative to control fish kept at background Cu levels (2 microg/l), Cu-preexposed fish displayed decreased uptake rates of waterborne Cu via the gills but not of dietary Cu via the gut during 48-h exposures to (64)Cu-radiolabeled water and diet, respectively. At normal dietary and waterborne Cu levels, the uptake rates of dietary Cu into the whole body without the gut were 0.40-0.90 ng. g(-1). h(-1), >10-fold higher than uptake rates of waterborne Cu into the whole body without the gills, which were 0.02-0.07 ng. g(-1). h(-1). Previously Cu-exposed fish showed decreased new Cu accumulation in the gills, liver, and carcass during waterborne (64)Cu exposures and in the liver during dietary (64)Cu exposures. A 3-h gill Cu-binding assay showed downregulation of the putative high-affinity, low-capacity Cu transporters and upregulation of the low-affinity, high-capacity Cu transporters at the gills in Cu-preexposed fish. Exchangeable Cu pools in all the tissues were higher during dietary than during waterborne (64)Cu exposures, and previous Cu exposure reduced waterborne exchangeable Cu pools in gill, liver, and carcass. Overall, these results suggest a quantitatively greater role for the dietary than for the waterborne route of Cu uptake, a key role for the gill in Cu homeostasis, and important roles for the liver and gut in the normal metabolism of Cu in fish.     

Hormonal and ion regulatory response in three freshwater fish species following waterborne copper exposure

We evaluated effects of sublethal copper exposure in 3 different freshwater fish: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). In a first experiment we exposed these fishes to an equally toxic Cu dose, a Cu level 10 times lower than their 96 h LC₅₀ value: 20, 65, and 150 µg/L Cu. In a second series we exposed them to the same Cu concentration (50 µg/L). Na⁺/K⁺-ATPase activity in gill tissue was disturbed differently in rainbow trout then in common and gibel carp. Rainbow trout showed a thorough disruption of plasma ion levels at the beginning of both exposures, whereas common carp and gibel carp displayed effects only after 3 days. Rainbow trout and common carp thyroid hormones experienced adverse effects in the beginning of the exposure. The involvement of prolactin in handling metal stress was reflected in changes of mRNA prolactin receptor concentrations in gill tissue, with an up regulation of this mRNA in rainbow trout and a down regulation in gibel carp, which was more pronounced in the latter. Overall, rainbow trout appeared more sensitive in the beginning of the exposure, however, when it overcame this first challenge, it handled copper exposure in a better manner then common and gibel carp as they showed more long term impacts of Cu exposure.     

THE TOXICITY OF ZINC SULPHATE TO RAINBOW TROUT

The toxicity of zinc sulphate to rainbow trout ( Salmo gairdnerii Richardson) has been investigated in waters of different chemical and physical properties.
Zinc sulphate was less toxic to rainbow trout in hard water than in soft water; when the log concentration of zinc was plotted against log median period of survival of the fish the dose response curve was linear in a very soft water, and curvilinear in a hard water, approaching an apparent threshold concentration. Solutions of zinc sulphate containing calcium chloride were less toxic than those containing an equivalent concentration of calcium as bicarbonate.
An increase in temperature decreased the survival time of rainbow trout in solutions of zinc sulphate in a hard water, but the threshold concentration was not appreciably affected by changes in temperature.
A reduction in the dissolved oxygen concentration of the water increased the toxicity of zinc sulphate, but the effect was reduced when the fish were previously acclimatized to the lower oxygen concentration of the test.
The cause of death of fish in solutions of zinc sulphate was not by the precipitation of mucus on the gills but probably by damage to the gill epithelium.     

Sodium-dependent copper uptake across epithelia: a review of rationale with experimental evidence from gill and intestine

The paper reviews the evidence for apparent sodium-dependent copper (Cu) uptake across epithelia such as frog skin, fish gills and vertebrate intestine. Potential interactions between Na(+) and Cu during transfer through epithelial cells is rationalized into the major steps of solute transfer: (i) adsorption on to the apical/mucosal membrane, (ii) import in to the cell (iii) intracellular trafficking, and (iv) export from the cell to the blood. Interactions between Na(+) and Cu transport are most likely during steps (i) and (ii). These ions have similar mobilities (lambda) in solution (lambda, Na(+), 50.1; Cu(2+), 53.6 cm(2) Int. ohms(-1) equiv(-1)); consequently, Cu(2+) may compete equally with Na(+) for diffusion to membrane surfaces. We present new data on the Na(+) binding characteristics of the gill surface (gill microenvironment) of rainbow trout. The binding characteristics of Na(+) and Cu(2+) to the external surface of trout gills are similar with saturation of ligands at nanomolar concentrations of solutes. At the mucosal/apical membrane of several epithelia (fish gills, frog skin, vertebrate intestine), there is evidence for both a Cu-specific channel (CTR1 homologues) and Cu leak through epithelial Na(+) channels (ENaC). Cu(2+) slows the amiloride-sensitive short circuit current (I(sc)) in frog skin, suggesting Cu(2+) binding to the amiloride-binding site of ENaC. We present examples of data from the isolated perfused catfish intestine showing that Cu uptake across the whole intestine was reduced by 50% in the presence of 2 mM luminal amiloride, with 75% of the overall inhibition attributed to an amiloride-sensitive region in the middle intestine. Removal of luminal Na(+) produced more variable results, but also reduced Cu uptake in catfish intestine. These data together support Cu(2+) modulation of ENaC, but not competitive entry of Cu(2+) through ENaC. However, in situations where external Na(+) is only a few millimoles (fish gills, frogs in freshwater), Cu(2+) leak through ENaC is possible. CTR1 is a likely route of Cu(2+) entry when external Na(+) is higher (e.g. intestinal epithelia). Interactions between Na(+) and Cu ions during intracellular trafficking or export from the cell are unlikely. However, effects of intracellular chloride on the Cu-ATPase or ENaC indicate that Na(+) might indirectly alter Cu flux. Conversely, Cu ions inhibit basolateral Na(+)K(+)-ATPase and may increase [Na(+)](i).     

The role of dissolved organic carbon in moderating the bioavailability and toxicity of Cu to rainbow trout during chronic waterborne exposure

We examined the influence of dissolved organic carbon (DOC) on the bioavailability of waterborne Cu to rainbow trout (Oncorhynchus mykiss) during chronic sublethal exposure. Juvenile rainbow trout were exposed to Cu (as CuSO(4)) and DOC as humic acid (HA, as sodium salt) for one month in synthetic soft water to give treatments with varying combinations of free ionic and HA complexed Cu. The total Cu concentration was 7 microg/l for all treatments (except controls) and HA was added at levels of 0, 2.5 and 7.5 mg/l which corresponded to DOC levels of 1.2, 2.2 and 4.0 mg/l. Fish grew well in all treatments and no mortalities occurred. Cu was highly bioavailable in the treatment with no added HA; gill and liver Cu accumulation occurred as well as a disruption of Na(+) regulation. In Cu treatments with additions of both 2.5 and 7.5 mg/l HA, there was no significant tissue accumulation of Cu. The addition of HA alleviated and delayed the disruption of iono-regulatory mechanisms. A recovery of plasma Na(+) losses was observed and this was associated with an increase in gill Na(+)/K(+) ATPase activity by the end of the exposure. Following the month of chronic exposure the uptake and turnover rates of Cu at the gills and into various tissue compartments were measured through radioisotopic techniques ((64)Cu). While chronic Cu exposure did not result in acclimation (i.e. increased LC50), the uptake rate and extent of Cu uptake into the gills and liver was increased. This study demonstrates that growth and tissue accumulation of Cu are poor predictors of the chronic effects of Cu, and illustrates that HA moderates chronic Cu bioavailability. The lack of a link between Cu bioaccumulation and Cu impact and the role of organic matter in reducing the bioavailability of Cu are important considerations in the context of ecological risk assessment.     

Comparative proteomics of copper exposure and toxicity in rainbow trout,common carp and gibel carp

Species specific differences in transporters, chaperones, metal binding proteins and other targets are important in metal toxicity. Therefore, we have studied the effects of copper exposure on the proteome of gill tissue from Oncorhynchus mykiss, Cyprinus carpio and Carassius auratus gibelio, which have different sensitivities toward copper. Fish were exposed to the Flemish water quality standard for surface waters, being 50 μg/L, for 3 days. Sampled gill tissue was subjected to a 2D-Dige and an iTRAQ analysis. While gibel carp showed more positive responses such as increased apolipoprotein A-I, transferrin and heat shock protein 70, common carp's gill tissue on the other hand displayed a changed actin cytoskeleton, and indications of a changed metabolism. These last two traits were evident in rainbow trout as well, together with decreased expressions of transferrin and albumin. urthermore, the Weighted Gene Co-Expression Network Analysis of rainbow trout data revealed a network of 98 proteins related to Cu accumulation in gill, of which the occurrence of proteins related to oxidative stress, such as superoxide dismutase and cytochrome c were promising. Additionally, the outcome of the different proteomics techniques demonstrates the usefulness of iTRAQ analysis compared to 2D-Dige and the need for fully annotated genomes.     

Radiotracer Studies on Waterborne Copper Uptake,Distribution, and Toxicity in Rainbow Trout and Yellow Perch: A Comparative Analysis

Rainbow trout (Oncorhynchus mykiss) are often used to estimate important biotic ligand model (BLM) parameters, such as metal-binding affinity (log K) and capacity (B_max). However, rainbow trout do not typically occupy metal-contaminated environments, whereas yellow perch (Perca flavescens) are ubiquitous throughout most of North America. This study demonstrates that dynamic processes that regulate Cu uptake at the gill differ between rainbow trout and yellow perch. Rainbow trout were more sensitive to acute aqueous Cu than yellow perch, and toxicity was exacerbated in soft water relative to similar exposures in hard water. Whole body Na loss rate could account for acute Cu toxicity in both species, as opposed to new Cu uptake rate that was not as predictive. Time course experiments using radiolabelled Cu (⁶⁴Cu) revealed that branchial Cu uptake was rather variable within the first 12 h of exposure, and appeared to be a function of Cu concentration, water hardness, and fish species. After 12 h, new branchial Cu concentrations stabilized in both species, suggesting that metal exposures used to estimate BLM parameters should be increased in duration from 3 h to 12+ h. In rainbow trout, 71% of the new Cu bound to the gill was exchangeable (i.e., able to either enter the fish or be released back to the water), as opposed to only 48% in yellow perch. This suggests that at equal exposure concentrations, proportionally more branchial Cu can be taken up by rainbow trout than yellow perch, which can then go on to confer toxicity. These qualitative differences in branchial Cu handling between the two species emphasize the need to develop BLM parameters for each species of interest, rather than the current practice of extrapolating BLM results derived from rainbow trout (or other laboratory-reared species) to other species. Data reported here indicate that a one-size-fits-all approach to predictive modeling, mostly based on rainbow trout studies, may not suffice for making predictions about metal toxicity to yellow perch—that is, a species that inhabits metal-contaminated lakes around northern Canadian industrial operations.     

Influence of temperature and membrane lipid composition on the osmotic water permeability of teleost gills.

Osmotic water uptake was measured gravimetrically in isolated, ligated gill arches from trout (acclimated to and incubated at 5 degrees and 20 degrees C) and tilapia (21.5 degrees and 33 degrees C). For both species, incubation of arches at the higher temperature led to 1.5- to 3-fold greater measures of water weight gain. However, gills from warmer-acclimated trout and tilapia had 1- to >3-fold lower the initial rate and 1.5- to >2.5-fold lower the extent of water uptake seen in colder-acclimated conspecifics. Both the incubation temperature sensitivity and the acclimation effects are consistent with transmembrane water permeation. Calcium-free incubations (permitting paracellular water movement) also indicated that interfacial cell membranes contribute to gill permeability characteristics; without calcium, trout gill osmotic water uptake values increased 1.5- to 2-fold, and the temperature dependence of water uptake decreased (initial rate) or was eliminated (extent). The specific contribution of cholesterol to restricting barrier membrane water permeability was indicated by concentration-dependent increases in water uptake in the presence of either nystatin (a cholesterol-complexing, pore-forming agent) or methyl-beta-cyclodextrin (which selectively depletes membrane cholesterol). In addition, a cholesterol-specific cytochemical probe (filipin) intensely labeled the apical surface membranes of trout and tilapia gill epithelium. In summary, these studies implicate membrane cholesterol in determining water permeability in fish gills.     

The effect of pH and/or calcium-enriched freshwater on gill Ca2+-ATPase activity and osmotic water inflow in rainbow trout (Salmo gairdneri)

Rainbow trout (Salmo gairdneri) were exposed to pH 5.0-5.1, 6.6 and/or calcium-enriched freshwater for 14 days. Hematocrit, gill Ca2+-ATPase enzyme activities, gill osmotic water inflow, plasma calcium and osmolarity were measured. No significant changes in plasma calcium ion levels were found. The typical increase in hematocrit usually associated with exposure of fish to acidified water was not found in the present study and is discussed. Plasma osmolarity decreased in fish exposed to calcium-enriched freshwater (60 mg Ca2+ X 1(-1) ) in comparison to fish exposed to control freshwater conditions (2 mg Ca2+ X 1(1) ), irrespective of the pH level. Gill Ca2+-ATPase enzyme activities were measured for both low affinity (3 mM Ca2+) and high affinity (100 microM) activity. Exposure of rainbow trout to low pH (pH 5.0-5.1) did not affect the specific activity of Ca2+-ATPase enzyme. However, low affinity Ca2+-ATPase activity in fish exposed to calcium-enriched freshwater did show a significant reduction. The increase in gill osmotic water permeability in fish exposed to calcium-enriched freshwater is interpreted as a result of the increase in osmolarity of the ambient media.     

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.     

鲤鱼鳃部微环境的pH特征及其对吸附态铜的解吸影响

研究了不同pH条件下鲤鱼（Cyprinus carpio)鳃部微环境pH及CO2的变化情况以及鳃部微环境出水对水铝矿吸附态铜的淋洗效率,结果表明,鱼鳃微环境的平衡pH为8.0,出外环境pH低于或高于该值时,鱼鳃生理作用可以起到缓冲作用,造成鳃部微环境于外环境之间高达0.4个pH单位的差别,CO2含量也表现出相似的变化规律,在碱性条件下,鱼鳃微环境出水对吸附态铜的洗脱效应显著高于pH值相对较高的外环境水。     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na⁺/K⁺ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions     

Time course of osmotic adaptation and gill energetics of rainbow trout (Salmo gairdneri R.) following abrupt changes in external salinity

Summary The physiological and biochemical responses of rainbow trout (mean weight 250 g) to abrupt increases in salinity have been investigated. An initial crisis period lasting about 30 h was characterized by an increase in plasma and muscle ions, a rapid gill dehydration and a pronounced acidosis following a transient alkalosis. Mortality was low during this period. During the following days, gradual changes resulted in new steady state levels for most parameters examined.Analysis of adenylate pool (ATP, ATP/ADP ratio and energy charge) in the gills demonstrated an increased energy demand exhibiting two phases (4 h and 3 days), and a return to freshwater values. The gill respiration rate was constant during 3 days in sea water and decreased slightly later on. It was not influenced during the reverse transfer of seawater adapted fish into fresh water at the level of either isolated gills or perfused heads.