Interactions of Pb and Cd mixtures in the presence or absence of natural organic matter with the fish gill.

Metal gill binding and toxicity can be modeled using the concentration addition model, in which the toxic unit (TU) concept is used to determine if constituent metals are acting in a strictly additive, less than, or greater than additive fashion. To test this hypothesis, rainbow trout (Oncorhynchus mykiss) were exposed to a matrix of Pb plus Cd mixtures (nominal concentrations=0.75, 1.5, 2.25, 3.0 µmol L(-1)), in the presence or absence of mainly terrigenous (allochthonous; 10 mg CL(-1)) natural organic matter (NOM), and metal-gill binding, and toxicity was quantified. Based on its greater affinity for metal-gill binding sites, Cd-gill binding was expected to exceed Pb-gill binding during metal mixture exposure, but this only occurred at the lowest metal concentrations (0.75 µmol L(-1)); at higher concentrations Pb-gill binding was greater than Cd-gill binding. These unexpected observations were because Pb and Cd likely bind to different populations of high affinity, low capacity binding sites on the gill, which was borne out in subsequent attempts to mathematically model metal-gill interactions during metal-mixture exposure. The presence of an additional low affinity, high capacity population of Pb-gill binding sites also contributed to higher Pb-gill accumulation. Metal-gill interactions were complicated by NOM, which exacerbated toxicity during Cd-only exposure despite lowering Cd-gill accumulation. NOM also promoted Cd-gill binding in the presence of low-moderate concentrations of Pb (0.75 and 1.50 µmol L(-1)). We suggest that direct interactions of Cd-NOM complexes with the gill, and increases in Cd bioavailability due to Pb outcompeting Cd for NOM-metal binding sites due to its greater affinity for such ligands, accounted for greater Cd-gill binding and toxicity. We conclude that interactions of Pb and Cd with the gill cannot be predicted using the concentration addition model, and that NOM is not universally protective against metal-gill binding and toxicity when fish are exposed to metal mixtures.

Effects of waterborne silver in a marine teleost, the gulf toadfish (Opsanus beta): effects of feeding and chronic exposure on bioaccumulation and physiological responses.

Marine teleosts drink seawater, and the digestive tract is a key organ of osmoregulation. The gastro-intestinal tract therefore offers a second site for the potential uptake and toxicity of waterborne metals, but how these processes might interact with the digestive functions of the tract has not been investigated previously. We therefore compared the responses of adult gulf toadfish (Opsanus beta, collected from the wild) to a chronic 22d exposure to waterborne Ag (nominally 200 microg L(-1)=1.85 micromol L(-1)), in the presence or absence of daily satiation feeding (shrimp). Ag exposure did not affect voluntary feeding rate. Feeding reduced the net whole body accumulation of Ag by >50%, with reductions in liver concentrations (high) and white muscle concentrations (relatively low) playing the largest quantitative roles. Feeding also protected against Ag buildup in the esophagus-stomach and kidney, and increased biliary and urinary Ag concentrations. The gill was the only tissue to show the opposite response. Although terminal plasma Na(+), Cl(-), and Mg(2+) concentrations were unaffected, there were complex interactive effects on osmoregulatory functions of the gastro-intestinal tract, including drinking rate, gut fluid volumes, and intestinal base secretion rates. At the end of the exposure, the plasma clearance kinetics of an arterially injected tracer dose of (110 m)Ag were faster in toadfish that had been chronically exposed to waterborne Ag, and (110 m)Ag accumulation in their red blood cells was reduced. After 32 h, higher amounts of (110 m)Ag were found in bile and urine, but lower amounts in the intestine of the Ag-exposed toadfish; there were no other differences in tissue-specific distribution. The results suggest that feeding reduces waterborne Ag uptake through the digestive tract and alters physiological responses, while chronic exposure enhances regulatory functions. The time-dependent actions of the liver in Ag scavenging and detoxification are highlighted.

A matter of potential concern: natural organic matter alters the electrical properties of fish gills.

Natural organic matter (NOM) is an important constituent of aquatic environments; however, its influence on aquatic biota remains poorly studied. In the current study, NOM was isolated from nine different sites in southern Ontario, Canada, by the on-site treatment of water by reverse osmosis, followed by cation exchange. NOM from each site was reconstituted to 10 mg of C/L and pH 7.0 and exposed to either adult rainbow trout implanted with indwelling catheters or to in vitro primary cultures of the gill epithelium grown on semipermeable membranes. In both the in vivo and in vitro preparations, NOM was found to hyperpolarize transepithelial potential (TEP), with the magnitude of this change correlating extremely well to the absorptivity of the NOM at 340 nm, which is an index of its aromaticity. Gill hyperpolarization appeared to be independent of Ca2+ complexation by the NOM in all but two samples tested. We argue that NOM has direct actions on the ionic transport and/or permeability properties of fish gills. While NOM effects on the bioavailability of contaminants are well-known, NOM actions on such fundamental physiological properties of the gills have previously been overlooked. These may be of comparable or greater magnitude than commonly reported for other water-quality variables (e.g., hardness, pH, salinity) and therefore of critical importance in ecological understanding and risk assessment.

Photodegradation of natural organic matter from diverse freshwater sources.

Natural organic matter (NOM) is significant in determining fate, transport and toxicity of metals in aqueous systems but NOM is not a static component; NOM can undergo photochemical changes in chemical structure. These changes will modify NOM quality and in turn influence how metals are transported in the environment, as well as their toxicity to aquatic organisms. Natural organic matter was collected from five freshwater sources using a portable reverse osmosis unit, diluted to about 10 mg CL(-1), then exposed for 13 days to summer temperatures either in the dark or exposed to sunlight. Light exposed NOM had decreases in total organic carbon (TOC) of 8-35% compared to samples kept in a refrigerator (dark, 4 degrees C), and the NOM became optically lighter, as shown by specific absorbance coefficients (SAC) taken at 340 nm (55-76% decreases in SAC(340)). In contrast, dark exposed NOM showed much smaller decreases in TOC (< or = 3%) or SAC(340) (

Short-term silver accumulation in tissues of three marine invertebrates: shrimp Penaeus duorarum, sea hare Aplysia californica, and sea urchin Diadema antillarum.

The present study was carried out to examine possible differential silver distribution among several tissues of three marine invertebrate species: the shrimp Penaeus duorarum, the sea hare Aplysia californica, and the sea urchin Diadema antillarum. Animals were exposed to sub-lethal concentrations of silver (1 or 10 microg/L) in seawater for 48 h. In gill-breathing species (shrimp and sea hare), higher silver accumulation in gills were associated with higher hemolymph silver levels. Furthermore, sea urchin showed lower hemolymph silver concentrations than shrimp and sea hare. These findings suggest that gills are an important route for silver uptake in marine invertebrates. In both sea hare and shrimp, hepatopancreas silver accumulation was concentration-dependent and this organ accumulated the most silver after 48 h of exposure, suggesting a possible involvement of the hepatopancreas in both silver accumulation and detoxification in marine invertebrates. In shrimp and sea hare, substantial silver accumulation in nervous tissues was detected, suggesting the need for further studies on possible behavioral effects of silver in these invertebrate species. In sea urchin, egg mass accumulated more silver than other tissues analyzed, indicating the need for future studies on possible reproductive effects of silver in sea urchin. In all three species, the lowest silver concentrations were observed in muscle, suggesting a low potential of this tissue for trophic transfer of silver.

Effects of using synthetic sea salts when measuring and modeling copper toxicity in saltwater toxicity tests.

Synthetic sea salts are often used to adjust the salinity of effluent, ambient, and laboratory water samples to perform toxicity tests with marine and estuarine species. The U.S. Environmental Protection Agency (U.S. EPA) provides guidance on salinity adjustment in its saltwater test guidelines. The U.S. EPA suggests using commercial sea salt brands, such as Forty Fathoms (now named Crystal Sea Marinemix, Bioassay Grade), HW Marinemix, or equivalent salts to adjust sample salinity. Toxicity testing laboratories in Canada and the United States were surveyed to determine synthetic sea salt brand preference. The laboratories (n = 27) reported using four brands: Crystal Sea Marinemix (56%), HW Marinemix (22%), Instant Ocean (11%), and Tropic Marin (11%). Saline solutions (30 g/L) of seven synthetic sea salts were analyzed for dissolved copper and dissolved organic carbon (DOC) content. Brands included those listed above plus modified general-purpose salt (modified GP2), Kent Marine, and Red Sea Salt. The synthetic sea salts added from < 0.1 to 1.2 microg Cu/L to the solution. Solutions of Crystal Sea Marinemix had significantly elevated concentrations of DOC (range = 5.4-6.4 mg C/L, analysis of variance, Tukey, alpha = 0.05, p < 0.001) while other brands generally contained < 1.0 mg C/L. The elevated DOC in Crystal Sea Marinemix was expected to reduce copper toxicity. However, the measured dissolved copper effective concentration 50% (EC50) for Crystal Sea Marinemix was 9.7 microg Cu/L, similar to other tested sea salts. Analysis indicates that the organic matter in Crystal Sea Marinemix differs considerably from that of natural organic matter. On the basis of consistently adding little DOC and little dissolved copper, GP2 and Kent Marine are the best salts to use.

Tissue-specific modulation of glucocorticoid receptor expression in response to salinity acclimation in rainbow trout.

While studies clearly point to a role for cortisol signaling in seawater adaptation, very little is known about salinity impact on glucocorticoid receptor (GR) expression in fish. To this end, we investigated the temporal GR expression in the gill and liver of rainbow trout (Oncorhynchus mykiss) to salinity exposure. Trout were subjected to gradual salinity increases (11 ppt for 1 d, 17 ppt for 2 d and 23 ppt for 2 d) over a five day period. Gill Na(+), K(+)-ATPase alpha-subunit mRNA showed a transient elevation with salinity exposure, while gill cystic fibrosis transmembrane conductance regulator mRNA was not significantly affected by salinity. Liver PEPCK transcript levels showed a transient increase at day 1, but not at day 3 or day 5 of salinity exposure, while the activity of this enzyme was significantly depressed at all time points. Liver glycogen content was also significantly reduced by salinity exposure compared to the freshwater group. Gill GR transcript levels were 3-fold greater upon salinity exposure and this level was maintained over the 5 day period, while gill GR protein content remained unchanged except for a significant drop at day 1 of salinity exposure. Liver GR transcript levels showed no significant change with salinity exposure, while GR protein content was transiently elevated at day 3, but not at day 1 or day 5 of salinity exposure. The tissue-specific GR transcript response in the gill leads us to hypothesize a role for osmosensory signal transduction pathway in the regulation of GR expression in fish. Collectively, salinity exposure modulates GR expression and glucocorticoid signaling in rainbow trout.

Influence of salinity and organic matter on silver accumulation in Gulf toadfish (Opsanus beta).

To help extend the freshwater based biotic ligand model for silver (Ag) into brackish and saltwater conditions, 50g Gulf toadfish (Opsanus beta) were acclimated to 2.5%, 5%, 10%, 20%, 40%, 80%, or 100% salt water and exposed for 6d to 1.0microM AgNO(3), with or without 10mg C/L organic matter. Suwannee River natural organic matter collected by reverse osmosis was used. Silver accumulation in toadfish gills and plasma decreased as salinity increased, indicating low bioavailability of AgCl complexes. Complexation of Ag by organic matter, normally important in freshwater conditions, was less important as salinity increased. Although relatively little intestinal Ag uptake was observed, both liver and bile accumulated Ag from water imbibed past the isosmotic salinity point ( approximately 1/3 salt water). Toadfish also produced intestinal carbonate pellets, minerals which did not influence Ag accumulation. Our results further stress the importance of Ag speciation, physiological mechanisms, and intestinal Ag uptake when modelling Ag uptake and toxicity beyond freshwater conditions.

Mechanism of acute silver toxicity in marine invertebrates.

In freshwater crustaceans and in both freshwater and marine fish, the key mechanism of acute silver toxicity involves ionoregulatory impairment. An inhibition of the Na+ ,K+-ATPase located at the basolateral membrane of the gill epithelium seems to be the key site for silver toxicity. However, studies to determine if the same mechanism of toxicity is occurring in marine invertebrates, which also are ionoregulators, had not been done. Thus, the present study was carried out to determine acute silver effects on hemolymph osmo- and ionoregulation in three marine invertebrates: the shrimp Penaeus duorarum, the sea hare Aplysia californica, and the sea urchin Diadema antillarum. Animals were exposed to silver (1 or 10 microg/L), as silver nitrate, in seawater for 48 h. Results show that acute silver exposure did not affect hemolymph osmolality or ion concentration (Na+, Cl-, K+, Ca2+ and Mg2+) in the three species studied. However, silver induced significant changes in the water content in shrimp gill and sea hare gill and hepatopancreas. Silver also caused significant changes in Na+ ,K+-ATPase activity and in both total and intracellular ion (Cl-, Na+, K+, Mg2+, and Ca2+) concentrations in different tissues of the three species studied. Overall, these results show that the key mechanism of acute silver toxicity in marine invertebrates is not associated with an osmotic or ionoregulatory impairment at the hemolymph level, as observed in freshwater fish and crustaceans and in seawater fish. However, they indicate that acute waterborne silver induces significant changes in Na+ ,K(+)-ATPase activity and probably affects other mechanisms involved in water and ion transport at the cell membrane level, inducing impairments in water and ion regulation at the cellular level in different tissues of marine invertebrates. These results indicate the need to consider other "toxic sites" than gills in any future extension of the biotic ligand model (BLM) for seawater.

Influence of water chemistry and natural organic matter on active and passive uptake of inorganic mercury by gills of rainbow trout (Oncorhynchus mykiss).

To distinguish physiologically regulated uptake from passive uptake of inorganic Hg in fish, rainbow trout (Oncorhynchus mykiss) were exposed to inorganic Hg (0.5, 1, or 2 microM total Hg) in ion-poor water with various treatments. Addition of ions to the water (mM concentrations of Ca, K, Cl) did not consistently alter Hg accumulation by trout gills, although there was a trend to higher Hg accumulation at higher ion concentrations. The apical Ca channel blockers Verapamil and lanthanum also did not consistently affect Hg accumulation by trout gills. Pre-treatment of trout with the Na channel blocker Phenamil decreased Hg uptake by about half. These results suggest a combination of physiologically regulated and passive uptake of Hg by trout gills. Strong complexing agents of Hg (EDTA, NTA, ethylenediamine, cysteine) decreased Hg-binding by trout gills in a dose-dependent manner. From these data, a conditional equilibrium binding constant for Hg to the gills was estimated as logK(Hg-gill) = 18.0, representing very strong binding of Hg to the gills. This value is a first step in creating a biotic ligand model (BLM) for inorganic Hg and fish. Natural organic matter (2-10 mg C/L) also decreased Hg-binding by trout gills, although mM concentrations of Na, K, and Cl interfered with this effect. At low concentrations of these ions, natural organic matter samples isolated from various sources bound Hg to similar degrees, as judged by Hg accumulation by trout gills. A conditional binding constant to natural organic matter (NOM) was estimated as logK(Hg-NOM) = 18.0 with about 0.5 micromol binding sites per mg C, representing strong binding of Hg to NOM.

Heterogeneity of natural organic matter amelioration of silver toxicity to Daphnia magna: effect of source and equilibration time.

Despite the heterogeneity of natural organic matter (NOM) in the aquatic environment, current models that predict metal toxicity to aquatic biota treat these important metal-complexing agents in a homogeneous manner. In this investigation, the ability of 11 commercial and naturally isolated NOM samples to ameliorate silver toxicity to the freshwater crustacean Daphnia magna was examined. The commercially available Aldrich humic acid (AHA) increased the 48-h median lethal concentration for daphnid neonates from nominally NOM-free levels of 0.29 to 3.80 microg/L (at 6.9 mg C/L) in a concentration-dependent manner. Three of the tested samples exhibited similar protective effects, but the additional seven NOM samples displayed significantly stronger ameliorative actions. In fact, four samples of both commercial and naturally isolated origin demonstrated greater than fourfold increases in protection compared to that of AHA. Additional investigations showed that increased silver-AHA equilibration time resulted in decreased toxicity. Increased equilibration time also decreased whole-body silver accumulation at NOM levels less than 1 mg C/L. The present results suggest that heterogeneity of NOM and silver-NOM equilibration time will have to be accounted for in future models of silver toxicity to D. magna and that laboratory toxicity testing using NOM and metals should account for the effects of metal-NOM equilibration time.

Salinity acclimation affects the somatotropic axis in rainbow trout.

In this study, we set out to examine the role of the somatotropic axis in the ion-regulation process in rainbow trout. Specifically, our objective was to examine whether plasma insulin-like growth factor-binding proteins (IGFBPs) are modulated by gradual salinity exposure. To this end, freshwater (FW)-adapted rainbow trout were subjected to gradual salinity increases, up to 66% seawater, over a period of 5 days. During this acclimation process, minimal elevations in plasma Ca2+ and Cl- were seen in the salinity-acclimated groups compared with FW controls. There were no changes in plasma Na+ levels, and only a minor transient change in plasma cortisol levels was seen with salinity exposure. The salinity challenged animals responded with elevations in plasma growth hormone (GH) and IGF-I levels and gill Na+-K+-ATPase activity. We identified IGFBPs of 21, 32, 42, and 50 kDa in size in the plasma of these animals, and they were consistently higher with salinity. Despite the overall increase in IGFBPs with salinity, transient changes in individual BPs over the 5-day period were noted in the FW and salinity-exposed fish. Specifically, the transient changes in plasma levels of the 21-, 42-, and 50-kDa IGFBPs were different between the FW and salinity groups, while the 32-kDa IGFBP showed a similar trend (increases with sampling time) in both groups. Considered together, the elevated plasma IGFBPs suggest a key role for these binding proteins in the regulation of IGF-I during salinity acclimation in salmonids.

An in vitro biotic ligand model (BLM) for silver binding to cultured gill epithelia of freshwater rainbow trout (Oncorhynchus mykiss).

"Reconstructed" gill epithelia on filter supports were grown in primary culture from dispersed gill cells of freshwater rainbow trout (Oncorhynchus mykiss). This preparation contains both pavement cells and chloride cells, and after 7-9 days in culture, permits exposure of the apical surface to true freshwater while maintaining blood-like culture media on the basolateral surface, and exhibits a stable transepithelial resistance (TER) and transepithelial potential (TEP) under these conditions. These epithelia were used to develop a possible in vitro version of the biotic ligand model (BLM) for silver; the in vivo BLM uses short-term gill binding of the metal to predict acute silver toxicity as a function of freshwater chemistry. Radio-labeled silver ((110m)Ag as AgNO(3)) was placed on the apical side (freshwater), and the appearance of (110m)Ag in the epithelia (binding) and in the basolateral media (flux) over 3 h were monitored. Silver binding (greater than the approximate range 0-100 mug l(-1)) and silver flux were concentration-dependent with a 50% saturation point (apparent K(d)) value of about 10 mug l(-1) or 10(-7) M, very close to the 96-h LC50 in vivo in the same water chemistry. There were no adverse effects of silver on TER, TEP, or Na(+), K(+)-ATPase activity, though the latter declined over longer exposures, as in vivo. Silver flux over 3 h was small (<20%) relative to binding, and was insensitive to water chemistry. However, silver binding was decreased by elevations in freshwater Na(+) and dissolved organic carbon (humic acid) concentrations, increased by elevations in freshwater Cl(-) and reductions in pH, and insensitive to elevations in Ca(2+). With the exception of the pH response, these effects were qualitatively and quantitatively similar to in vivo BLM responses. The results suggest that an in vitro BLM approach may provide a simple and cost-effective way for evaluating the protective effects of site-specific waters.

Bioavailability of silver and its relationship to ionoregulation and silver speciation across a range of salinities in the gulf toadfish (Opsanus beta).

Silver is taken up as a Na(+) analog (Ag(+)) by freshwater organisms, but little is known about its bioavailability in relation to salinity. Adult Opsanus beta were acclimated to 2.5, 5, 10, 20, 40, 60, 80, and 100% seawater (Cl(-)=545 mM) and exposed for 24 h to 2.18 microg L(-1) silver as (110m)Ag-labelled AgNO(3), a concentration close to the U.S. EPA marine criterion and less than 0.1% of the acute 96-h LC50 in seawater. Plasma osmolality, Na(+), and Cl(-) remained approximately constant from 100% down to 20-40% seawater, thereafter declining to 89% (osmolality) and 82% (Na(+), Cl(-)) of seawater values at the lowest salinity (2.5% seawater), while plasma Mg(2+) was invariant. Ionic measurements in intestinal fluids and urine supported the view that above the isosmotic point (about 32% seawater), toadfish drink the medium, absorb Na(+), Cl(-), and water across the gastrointestinal tract, actively excrete Na(+) and Cl(-) across the gills, and secrete Mg(2+) into the urine. Below this point, toadfish appear to stop drinking, actively take up Na(+) and Cl(-) at the gills, and retain ions at the kidney. Silver accumulation varied greatly with salinity, by nine-fold (whole body), 26-fold (gill tissue), and 18-fold (liver), with the maxima occurring in 2.5% seawater, the minima in 40% seawater (close to the isosmotic point), and slightly greater values at higher salinities. Highest silver concentrations occurred in liver, second highest in gills, intermediate concentrations in kidney, spleen, and gastrointestinal tissues, and lowest in swim bladder and white muscle, though patterns changed with salinity. There were substantial biliary but minimal urinary levels of silver. The salinity-dependent pattern of silver accumulation best correlated with the abundance of the neutral complex AgCl(0), though the presence of small amounts of Ag(+) at the lowest salinities may also have been important. In contrast, silver accumulation in the esophagus-stomach was greatest in 100% seawater and least at the isosmotic salinity (five-fold variation), a pattern probably explained by drinking and silver uptake into the blood through the gills. Models of silver bioavailability across salinity must consider the presence of silver-binding ligands on both gills and gastrointestinal tract, changing silver speciation, and the changing ionoregulatory physiology of the organism.

Physiological action of dissolved organic matter in rainbow trout in the presence and absence of copper: sodium uptake kinetics and unidirectional flux rates in hard and softwater.

We investigated the physiological effects of dissolved organic matter (DOM) on sodium (Na+) transport in juvenile Oncorhynchus mykiss (approximately 2.5 g) in the presence and absence of simultaneous acute exposure to copper (Cu2+; 0, 70, and 300 microg l(-1)). Trout were acclimated in either hardwater (approximately 1000 microM Ca2+) or softwater (approximately 100 microM Ca2+), and DOM was tested at approximately 8 mg C l(-1) using a natural (NOM) and a commercial (AHA) source. Ion transport was evaluated based on kinetics estimates (maximum Na+ uptake rates, Jmax; substrate affinity, Km) and unidirectional flux measurements (Jin, Jout, Jnet). Jmax was higher and unidirectional flux rates were greater in softwater-acclimated trout. Fish exposed to DOM alone in hardwater exhibited an increased Na+ transport capacity indicated by both the kinetics (67% higher Jmax for AHA) and Jin measurements (153% higher for AHA and 125% higher for NOM). In softwater, the effects of DOM alone on kinetic parameters and unidirectional flux rates were negligible. Cu2+ affected Na+ uptake by a mixed-type inhibition (both non-competitive and competitive). In hardwater, only Km was increased (i.e., affinity decreased), whereas in softwater, Km was increased and Jmax was decreased, with more marked effects at the higher Cu2+ level. In hardwater, the stimulatory effect of AHA on Jmax persisted even in the presence of 300 microg l(-1) Cu2+, whereas both AHA and NOM prevented the increase in Km caused by Cu2+; these effects were reflected in Jin measurements. In softwater, AHA helped to protect against the increased Km caused by high Cu2+, but there was no protection against the inhibition of Jmax. Unidirectional flux measurements indicated that in softwater, Cu2+ inhibited Jin at 70 microg l(-1), whereas at 300 microg l(-1) Cu2+, Jout was also stimulated. Fish were more affected by Cu2+ in softwater, as indicated by the inability to control diffusive losses of Na+ and a reduced ability to take up Na+, but in the presence of DOM, losses were better controlled at the end of 6 h exposure. We conclude that DOM has direct effects on the gills, as well as protecting fish against acute Cu2+ toxicity. This occurs because DOM complexes Cu2+, and because it acts on the transport and permeability properties of the gills. These effects differ depending on both water hardness and the nature of the DOM source.

Influence of natural organic matter source on copper speciation as demonstrated by Cu binding to fish gills, by ion selective electrode, and by DGT gel sampler.

Rainbow trout (Oncorhynchus mykiss, 2 g) were exposed to 0-5 microM total copper in ion-poor water for 3 h in the presence or absence of 10 mg C/L of qualitatively different natural organic matter (NOM) derived from water spanning a large gradient in hydrologic residence time. Accumulation of Cu by trout gills was compared to Cu speciation determined by ion selective electrode (ISE) and by diffusive gradients in thin films (DGT) gel sampler technology. The presence of NOM decreased Cu uptake by trout gills as well as Cu concentrations determined by ISE and DGT. Furthermore, the source of NOM influenced Cu binding by trout gills with high-color, allochthonous NOM decreasing Cu accumulation by the gills more than low-color autochthonous NOM. The pattern of Cu binding to the NOM measured by Cu ISE and by Cu accumulation by DGT samplers was similar to the fish gill results. A simple Cu-gill binding model required an NOM Cu-binding factor (F) that depended on NOM quality to account for observed Cu accumulation by trout gills; values of Fvaried by a factor of 2. Thus, NOM metal-binding quality, as well as NOM quantity, are both important when assessing the bioavailability of metals such as Cu to aquatic organisms.

Using multiple metal-gill binding models and the toxic unit concept to help reconcile multiple-metal toxicity results.

Metal-gill binding models and biotic ligand models (BLMs) in general are designed to predict metal toxicity to aquatic organisms. These models calculate the amount of a metal-binding to a sensitive biological membrane, such as a fish gill, which equates with metal toxicity. Cation competition at the metal-binding site and anionic complexation in the water decrease metal-binding to the membrane, decreasing metal toxicity. These models have, to date, been developed for individual metals. To assess how these models handle multiple-metal interactions, metal-gill binding models for two to six metals were created and their behavior tested against the toxic unit (TU) concept assuming strict additivity. The multiple-metal models yield greater than strict additivity at low aqueous metal concentrations (Sigma < 1 TU), strict additivity at intermediate metal concentrations (Sigma = 1 TU), and less than strict additivity at high metal concentrations (Sigma > 1 TU), independent of the combination of metals. Deviations from strict additivity are due to the non-linear nature of the models, where greater than linear filling of binding sites occurs at low metal concentrations, and where strong competition for binding sites occurs at high metal concentrations, with a point of strict additivity between, where the metals sum to one toxic unit. Simulations with natural organic matter (NOM) show similar trends but are complex. Mathematical modeling of multiple-metal interactions may help in the interpretation of toxicity results from mixed-metal exposures to aquatic organisms.

Time course analysis of the mechanism by which silver inhibits active Na+ and Cl- uptake in gills of rainbow trout.

A time course analysis using (110m)Ag, (24)Na(+), and (36)Cl(-) examined gill silver accumulation and the mechanism by which waterborne silver (4.0 x 10(-8) M; 4.3 microg/l) inhibits Na(+) and Cl(-) uptake in gills of freshwater rainbow trout. Analyses of gill and body fluxes allowed calculation of apical uptake and basolateral export rates for silver, Na(+), and Cl(-). To avoid changes in silver bioavailability, flow-through conditions were used to limit the buildup of organic matter in the exposure water. For both Na(+) and Cl(-) uptake, apical entry, rather than basolateral export, was the rate-limiting step; Na(+) and Cl(-) uptake declined simultaneously and equally initially, with both uptakes reduced by approximately 500 nmol.g(-1).h(-1) over the 1st h of silver exposure. There was a further progressive decline in Na(+) uptake until 24 h. Carbonic anhydrase activity was inhibited by 1 h, whereas Na(+)-K(+)-ATPase activity was not significantly inhibited until 24 h of exposure. These results indicate that carbonic anhydrase inhibition can explain the early decline in Na(+) and Cl(-) uptake, whereas the later decline is probably related to Na(+)-K(+)-ATPase blockade. Contrary to previous reports, gill silver accumulation increased steadily to a plateau. Despite the rapid inhibition of apical Na(+) and Cl(-) uptake, apical silver uptake (and basolateral export) increased until 10 h, before decreasing thereafter. Thus silver did not inhibit its own apical uptake in the short term. These results suggest that reduced silver bioavailability is the mechanism behind the pattern of peak and decline in gill silver accumulation previously reported for static exposures to silver.

The time course of silver accumulation in rainbow trout during static exposure to silver nitrate: physiological regulation or an artifact of the exposure conditions?

The pattern of gill silver accumulation in rainbow trout during waterborne silver exposure has been reported to be unusual, reaching a peak in the first few hours of silver exposure followed by a marked decline with continued exposure. The potential causes of the pattern were investigated. Rainbow trout (1-5g) were exposed in a static system to 110mAg labeled AgNO(3) at a total concentration of 1.92microg Agl(-1) for 24h in synthetic soft water. Periodically throughout the exposure, gill and body 110mAg accumulation, gill and body 24Na uptake (from which whole body Na(+) uptake was calculated), gill Na(+)K(+)-ATPase activity, plus water silver (total and dissolved), Cl(-) and total organic carbon (TOC) concentrations were measured. Gill silver levels rapidly increased, peaked at 3h of exposure and then decreased until a plateau was reached at 12h of exposure. Body (minus gills) silver levels increased steadily over the exposure period until 18h of exposure. Whole body Na(+) uptake decreased, was maximally inhibited by 3h of exposure but recovered by 12h despite continued silver exposure. Gill Na(+)K(+)-ATPase activity was not inhibited until 5h of exposure. The water dissolved silver concentration declined by approximately 70% over the 24h exposure period and the TOC content of the water increased over three-fold during the first 2h of exposure. There was a decrease in the calculated contribution of Ag(+) (from 20.9 to 2.5%) and an increase in the calculated contribution of Ag-TOC complexes (from 77 to 97.3%) to the total water silver concentration over the first 2h of exposure. Apical silver uptake into the gills decreased over the initial 2.5h of exposure while basolateral silver export out of the gills to the body remained constant throughout the exposure. The results of this study suggest that: (1) physiological regulation of silver movement may explain the pattern of gill silver accumulation observed in rainbow trout, although not by a mechanism coupled to Na(+)K(+)-ATPase inhibition as originally proposed; (2) alternatively or additionally, a decreased bioavailability of silver, due to the static exposure conditions, may explain the pattern of gill accumulation; (3) the early inhibition of whole body Na(+) uptake observed during silver exposure occurs via a mechanism other than Na(+)K(+)-ATPase inhibition; and (4) gill silver accumulation may be an appropriate endpoint for biotic ligand modeling.

Influence of natural organic matter source on acute copper, lead, and cadmium toxicity to rainbow trout (Oncorhynchus mykiss).

Natural organic matter (NOM) was concentrated from various sites across Canada using a portable reverse-osmosis unit to obtain a range of NOM types, from mainly allochthonous (terrestrially derived) to mainly autochthonous (aquatically derived) NOM. The addition of NOM to Cu exposures in ion-poor water always decreased Cu toxicity to rainbow trout (Oncorhynchus mykiss, approximately 1 g) over a 96-h period, and the degree of protection varied with respect to NOM source. A good correlation was found between the specific absorbance coefficient (SAC) and time to reach 50% mortality (LT50; p < 0.001), indicating that more optically dark, allochthonous-like NOM decreases Cu toxicity better than does optically light, more autochthonous-like NOM. A similar, good relationship between NOM source and Pb toxicity was seen (p < 0.001), once confounding effects of Ca binding to NOM were accounted for. No significant relationship between Cd toxicity and NOM optical quality was seen (p = 0.082), and in toxicity tests with Cd the presence of some of the NOM sources increased Cd toxicity compared to Cd-only controls. Specific absorbance coefficients were used as a proxy measurement of NOM aromaticity in our study, and fluorescence indices were run on some NOM samples to obtain percent aromaticity for each sample. A good correlation was found between SAC and percent aromaticity, indicating that the simple SAC measurement is a reasonable indication of NOM aromaticity and of metal binding by NOM.