Copper uptake in adult rainbow trout irradiated during early life stages and in non-irradiated bystander trout which swam with the irradiated fish.

PURPOSE: This investigation forms part of a wider study into the legacy effects of exposure of rainbow trout eggs 38 h after fertilization, eyed eggs, yolk sac larvae (YSL) or first feeders to a single 0.5 Gy X-ray dose, including the induction of a bystander effect, by the irradiated fish, to non-irradiated fish. Fish may be exposed to multiple environmental stressors, including waterborne metals, during their lifespan and, while there are data on how the legacy of early life stage irradiation and bystander effect induction is affected by waterborne aluminum and cadmium, there are no studies into the effects radiation or the radiation induced bystander effect on metal uptake. Therefore the aim of this investigation was to determine if the legacy of early life stage irradiation included an effect on copper uptake by adult fish and by non-irradiated bystander adult trout which swam with the irradiated fish. METHODS: The four early life stages mentioned above were exposed to a single 0.5 Gy X-ray dose and then maintained, for two years with no further irradiation. At two years old the irradiated fish were allowed to swim, for 2 h with non-irradiated bystander trout (also two years old). After this time copper uptake was determined using 64Cu. RESULTS: Copper uptake was increased in adult trout irradiated as eggs at 48 h after fertilization and as first feeders but eyed egg or YSL irradiation had no effect. Copper uptake was also increased in the bystander trout which swam with trout irradiated as eggs at 48 h after fertilization and as eyed eggs but there was no effect on non-irradiated adult trout which swam with trout irradiated as YSL or first feeders. CONCLUSIONS: When put in context with the proteomic changes observed in these fish we propose the increased copper uptake in adult trout irradiated as eggs at 48 h after fertilization could be part of an anti-tumorigenic response and the increase in copper uptake in adult trout irradiated as first feeders could be part of a potentially protective pro-apoptotic response. Similarly we propose the increase in copper uptake in non-irradiated adult trout, induced by trout irradiated as eggs at 48 h after fertilization or as eyed eggs, was part of the universally anti-tumorigenic nature of the X-ray induced bystander effect in fish. However this was exclusive to embryonic irradiation.

Interactive effects of waterborne metals in binary mixtures on short-term gill-metal binding and ion uptake in rainbow trout (Oncorhynchus mykiss).

Metal binding to fish gills forms the basis of the biotic ligand model (BLM) approach, which has emerged as a useful tool for conducting site-specific water quality assessments for metals. The current BLMs are designed to assess the toxicity of individual metals, and cannot account for the interactive effects of metal mixtures to aquatic organisms including fish. The present study was designed mainly to examine the interactive effects of waterborne metals (Cd, Zn, Cu, Ag, and Ni) in specific binary combinations on short-term (3h) gill-metal binding and essential ion (Ca(2+) and Na(+)) uptake (a physiological index of toxicity) in fish, using juvenile freshwater rainbow trout (Oncorhynchus mykiss) as the model species. We hypothesized that binary mixtures of metals that share a common mode of uptake and toxicity (e.g., Cd and Zn - Ca(2+) antagonists, Cu and Ag - Na(+) antagonists) would reduce the gill binding of each other via competitive interactions and induce less than additive effects on ion transport. In addition, the mixture of metals that have different modes of uptake and toxicity (e.g., Cd and Cu, or Cd and Ni) would not exhibit any interactive effects either on gill-metal binding or ion transport. We found that both Zn and Cu reduced gill-Cd binding and vice versa, however, Ni did not influence gill-Cd binding in fish. Surprisingly, Ag was found to stimulate gill-Cu binding especially at high exposure concentrations, whereas, Cu had no effect on gill-Ag binding. The inhibitory effect of Cd and Zn in mixture on branchial Ca(2+) uptake was significantly greater than that of Cd or Zn alone. Similarly, the inhibitory effect of Cu and Ag in mixture on branchial Na(+) uptake was significantly greater than that of Cu or Ag alone. The inhibitory effects of Cd and Zn mixture on Ca(2+) uptake as well as Cu and Ag mixture on Na(+) uptake were found to follow the principles of simple additivity. In contrast, no significant additive effect on either Ca(2+) or Na(+) uptake was recorded in fish exposed to the mixture of Cd and Cu. Overall, we found that although the effects of metal mixture interactions on gill-metal binding did not always match with our original assumptions, the effects of metal mixtures on toxicity in fish were generally consistent with our predictions. The findings of the present study have important implications for improving the BLM approach to assess metal mixture toxicity in fish.

An in vitro investigation of gastrointestinal Na(+) uptake mechanisms in freshwater rainbow trout.

In vitro gut-sac preparations of all four sections (stomach, anterior, mid, and posterior intestine) of the gastrointestinal tract (GIT) of freshwater rainbow trout, together with radiotracer ((22)Na) techniques, were used to study unidirectional Na(+) uptake rates (UR, mucosal â†’ blood space) and net absorptive fluid transport rates (FTR) under isosmotic conditions (mucosal = serosal osmolality). On an area-specific basis, unidirectional Na(+) UR was highest in the mid-intestine, but when total gut area was taken into account, the three intestinal sections contributed equally, with very low rates in the stomach. The theoretical capacity for Na(+) uptake across the whole GIT is sufficient to supply all of the animal's nutritive requirements for Na(+). Transport occurs by low affinity systems with apparent K m values 2-3 orders of magnitude higher than those in the gills, in accord with comparably higher Na(+) concentrations in chyme versus fresh water. Fluid transport appeared to be Na(+)-dependent, such that treatments which altered unidirectional Na(+) UR generally altered FTR in a comparable fashion. Pharmacological trials (amiloride, EIPA, phenamil, bafilomycin, furosemide, hydrochlorothiazide) conducted at a mucosal Na(+) concentration of 50 mmol L(-1) indicated that GIT Na(+) uptake occurs by a variety of apical mechanisms (NHE, Na(+) channel/H(+) ATPase, NCC, NKCC) with relative contributions varying among sections. However, at a mucosal Na(+) concentration of 10 mmol L(-1), EIPA, phenamil, bafilomycin, and hydrochlorothiazide were no longer effective in inhibiting unidirectional Na(+) UR or FTR, suggesting the contribution of unidentified mechanisms under low Na(+) conditions. A preliminary model is presented.

The effects of copper and nickel on the embryonic life stages of the purple sea urchin (Strongylocentrotus purpuratus).

The aim of this research was to generate data on the mechanisms of toxicity of copper [Cu (4-12 µg/L)] and nickel [Ni (33-40 µg/L)] during continuous sublethal exposure in seawater (32 ppt, 15 °C) in a sensitive test organism (Strongylocentrotus purpuratus) at its most sensitive life stage (developing embryo). Whole-body ions [calcium (Ca), sodium (Na), potassium (K), and magnesium (Mg)], metal burdens, Ca uptake, and Ca ATPase activity were measured every 12 h during the first 72-84 h of development. Ionoregulatory disruption was clearly an important mechanism of toxicity for both metals and occurred with minimal metal bioaccumulation. Most noteworthy was a significant disruption of Ca homeostasis, which was evident from an inhibition of unidirectional Ca uptake rates, whole-body Ca accumulation, and Ca ATPase activity intermittently during 72-84 h of development. At various times, Cu- and Ni-exposed embryos also displayed lower levels of K and increased levels of Na suggesting inhibition of Na/K ATPase activity. Greater levels of Mg during initial stages of development in Cu-exposed embryos were also observed and were considered a possible compensatory mechanism for disruptions to Ca homeostasis because both of these ions are important constituents of the developing spicule. Notably, most of these effects occurred during the initial stages of development but were reversed by 72-84 h. We therefore propose that it is of value to study the toxic impacts of contaminants periodically during development before the traditional end point of 48-72 h.

Zinc bioaccumulation and ionoregulatory impacts in Fundulus heteroclitus exposed to sublethal waterborne zinc at different salinities.

Exposure of Fundulus heteroclitus to an environmentally relevant Zn concentration (500 µg L⁻¹) at different salinities (0, 3.5, 10.5, and 35 ppt) revealed the following effects: (i) plasma [Zn] doubled after exposure at 0 ppt, a response which was eliminated at 35 ppt. Tissue [Zn] also increased in gill, liver, intestine, and carcass at 0 ppt. (ii) Both branchial and intestinal Ca2⁺ ATPase activities decreased in response to Zn at 0 ppt and were elevated at 35 ppt. Plasma [Ca] decreased by 50% at 0 ppt and by 30% at 3.5 ppt and increased by 20% at 35 ppt. Gill [Ca] decreased by 35% at 0 ppt and increased by about 30% at all higher salinities. (iii) Branchial Na⁺,K⁺ ATPase activity decreased by 50% at 0 ppt, increased by 30% and 90% at 10.5 and 35 ppt respectively. Intestinal Na⁺,K⁺ ATPase activity was reduced by 30% at 0 ppt. (iv) Plasma [Na] decreased by 30% at 0 ppt in Zn-exposed. Zn exposure also disturbed the homeostasis of tissue cations (Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺) in a tissue-specific and salinity-dependent manner. (v) Drinking rate was not altered by Zn exposure. In toxicity tests, acute Zn lethality (96-h LC50) increased in a close to linear fashion from 9.8 mg L⁻¹ at 0 ppt to 75.0 mg L⁻¹ at 35 ppt. We conclude that sublethal Zn exposure causes pathological changes in both Ca⁺⁺ and Na⁺ homeostases, and that increasing salinity exerts protective effects against both sublethal and lethal Zn toxicities.

Sublethal mechanisms of Pb and Zn toxicity to the purple sea urchin (Strongylocentrotus purpuratus) during early development.

In order to understand sublethal mechanisms of lead (Pb) and zinc (Zn) toxicity, developing sea urchins were exposed continuously from 3h post-fertilization (eggs) to 96 h (pluteus larvae) to 55 (±2.4) µgPb/L or 117 (±11)µgZn/L, representing ~ 70% of the EC50 for normal 72 h development. Growth, unidirectional Ca uptake rates, whole body ion concentrations (Na, K, Ca, Mg), Ca(2+) ATPase activity, and metal bioaccumulation were monitored every 12h over this period. Pb exhibited marked bioaccumulation whereas Zn was well-regulated, and both metals had little effect on growth, measured as larval dry weight, or on Na, K, or Mg concentrations. Unidirectional Ca uptake rates (measured by (45)Ca incorporation) were severely inhibited by both metals, resulting in lower levels of whole body Ca accumulation. The greatest disruption occurred at gastrulation. Ca(2+) ATPase activity was also significantly inhibited by Zn but not by Pb. Interestingly, embryos exposed to Pb showed some capacity for recovery, as Ca(2+)ATPase activities increased, Ca uptake rates returned to normal intermittently, and whole body Ca levels were restored to control values by 72-96 h of development. This did not occur with Zn exposure. Both Pb and Zn rendered their toxic effects through disruption of Ca homeostasis, though likely through different proximate mechanisms. We recommend studying the toxicity of these contaminants periodically throughout development as an effective way to detect sublethal effects, which may not be displayed at the traditional toxicity test endpoint of 72 h.

Ionic status, calcium uptake, and Ca2+-ATPase activity during early development in the purple sea urchin (Strongylocentrotus purpuratus).

Ionic status during early development was investigated in the purple sea urchin. Whole body cation concentrations (Ca(2+), Na(+), K(+), Mg(2+)), unidirectional Ca(2+) uptake rates measured with (45)Ca(2+), Ca(2+)-ATPase activity, and growth were examined at 12h intervals over the first 96h of development. Whole body Ca(2+) concentration was low initially but increased steadily by >15-fold through to the pluteus stage. Whole body Mg(2+), K(+) and Na(+) levels exhibited diverse patterns, but all increased at 72-96h. Ca(2+) uptake rates were low during initial cell cleavages at 12h but increased greatly at blastulation (24h) and then again at gastrulation (48h), declining thereafter in the pluteus stage, but increasing slightly at 96h. Ca(2+)-ATPase activity was initially low but increased at blastulation through gastrulation (24-48h) but declined thereafter in the pluteus stage. Embryonic weights did not change over most of development, but were significantly higher at 96h. Overall, the gastrulation stage displayed the most pronounced changes, as Ca(2+) uptake and accumulation and Ca(2+)-ATPase levels were the highest at this stage, likely involved in mineralization of the spicule. Biomarkers of Ca(2+) metabolism may be good endpoints for potential future toxicity studies.

Toxicity of lead and zinc to developing mussel and sea urchin embryos: critical tissue residues and effects of dissolved organic matter and salinity.

Lead (Pb) EC50 values in the very sensitive early development phases (48-72h post-fertilization) of the mussels Mytilus galloprovincialis and Mytilus trossolus and sea urchin Strongylocentrotus purpuratus in 100% sea water were: M. trossolus - 45 (95% C.I.=22-72) µgL(-1); M. galloprovincialis - 63 (36-94) µgL(-1); S. purpuratus - 74 (50-101) µgL(-1). Salinity thresholds for normal development varied: M. trossolus>21ppt; M. galloprovincialis>28ppt; S. purpuratus≥30ppt. Addition of two spectroscopically distinct dissolved organic matters (DOM) from fresh water (Nordic Reservoir) and sea water (Inshore) moderately decreased the toxicity of Pb to both mussels, but not in a concentration-dependent fashion, with only an approximate doubling of EC50 over the range of 1.4-11.2mgCL(-1). Independent Pb binding capacity determinations for DOC explained the lack of a relationship between DOM concentration and toxicity. Salinity had no effect on Pb toxicity down to 21ppt in M. trossolus, and low salinity (21ppt) did not enhance the protective effect of DOC. Both DOMs increased the toxicity of Pb in developing sea urchin embryos, in contrast to mussels. Relative to Pb, the organisms were 6-9 fold less sensitive to Zn on a molar basis in 100% seawater with the following Zn EC50s: M. trossolus - 135 (103-170) µgL(-1); M. galloprovincialis - 172 (126-227) µgL(-1), S. purpuratus - 151 (129-177) µgL(-1). Nordic Reservoir and Inshore DOM (2-12mgCL(-1)) had no significant effect on Zn toxicity to mussels, in accord with voltammetry data showing an absence of any strong ligand binding for Zn by DOMs. As with Pb, DOMs increased Zn toxicity to urchin larvae. Critical Tissue Residues (CTR) based on whole body concentrations of Pb and Zn were determined for M. galloprovincialis at 48h and S. purpuratus at 72h. The median lethal CTR values (LA50s), useful parameters for development of saltwater Biotic Ligand Models (BLMs), were approximately 4-fold higher on a molar basis for Zn than for Pb. The latter were not altered by DOM exposure, despite increased EC50 values, in accord with the tenets of the BLM.

Assimilation of water and dietary ions by the gastrointestinal tract during digestion in seawater-acclimated rainbow trout.

Recent studies focusing on the consequences of feeding for ion and water balance in freshwater fish have revealed the need for similar comparative studies in seawater fish. A detailed time course sampling of gastrointestinal (GI) tract contents following the ingestion of a single meal of a commercial diet revealed the assimilation of both water and dietary ions (Na(+), Cl(-), K(+), Ca(2+), Mg(2+)) along the GI tract of seawater-acclimated rainbow trout (Oncorhynchus mykiss) which had been fasted for 1 week. Consumption of the meal did not change the drinking rate. There was a large secretion of fluid into the anterior intestine and caecae (presumably bile and/or pancreatic secretions). As a result, net assimilation (63%) of the ingested water along the GI tract was lower than generally reported for fasted trout. Mg(2+) was neither secreted into nor absorbed from the GI tract on a net basis. Only K(+) (93% assimilated) and Ca(2+) (43% assimilated) were absorbed in amounts in excess of those provided by ingested seawater, suggesting that dietary sources of K(+) and Ca(2+) may be important to seawater teleosts. The oesophagus-stomach served as a major site of absorption for Na(+), Cl(-), K(+), Ca(2+), and Mg(2+), and the anterior intestine and caecae as a major site of net secretion for all of these ions, except Cl(-). Despite large absorptive fluxes of these ions, the ionic composition of the plasma was maintained during the digestion of the meal. The results of the present study were compared with previous work on freshwater-acclimated rainbow trout, highlighting some important differences, but also several similarities on the assimilation of water and ions along the gastrointestinal tract during digestion. This study highlights the complicated array of ion and water transport that occurs in the intestine during digestion while revealing the importance of dietary K(+) and Ca(2+) to seawater-acclimated rainbow trout. Additionally, this study reveals that digestion in seawater-acclimated rainbow trout appears to compromise intestinal water absorption.

Mechanistic characterization of gastric copper transport in rainbow trout.

An in vitro gut-sac technique and (64)Cu as a radiotracer were used to characterize gastric copper (Cu) transport. Cu transport was stimulated by low luminal pH (4.0 vs. 7.4), to a greater extent than explained by the increased availability of the free Cu(2+) ion. At pH = 4.0, uptake kinetics were indicative of a low affinity (K (m) = 525 µmol L(-1)), saturable carrier-mediated component superimposed on a large linear (diffusive and/or convective) component, with about 50% occurring by each pathway at Cu = 50 µmol L(-1). Osmotic gradient experiments showed that solvent drag via fluid transport may play a role in Cu uptake via the stomach, in contrast to the intestine. Also unlike the intestine, neither the Na(+) gradient, high Ag, nor phenamil had any influence on gastric Cu transport, and a tenfold excess of Fe and Zn failed to inhibit Cu uptake. These findings indicate that neither Na(+)-dependent pathways nor DMT1 are likely candidates for carrier-mediated Cu transport in the stomach. We have cloned a partial cDNA sequence for the copper transporter Ctr1, and show its mRNA expression in all segments of the trout gastrointestinal tract, including the stomach. Based on the fact that this transporter is functional at low pH conventionally found in the stomach lumen, we suggest Ctr1 is a pathway for gastric Cu transport in trout. Extreme hypoxia inhibited Cu uptake. High P(CO2) levels (7.5 torr) increased Cu uptake and acetazolamide (100 µmol L(-1)) significantly inhibited Cu uptake, indicating carbonic anhydrase activity was involved in gastric Cu transport. Transport of Cu was insensitive to bafilomycin (10 µmol L(-1)) suggesting a V-ATPase did not play a direct role in the process. Expression (mRNA) of H (+) , K (+)-ATPase, carbonic anhydrase 2, and the α-3 isoform of Na (+)-K (+)-ATPase were observed in the stomach. We suggest these enzymes facilitate Cu transport in the stomach indirectly as part of a physiological mechanism exporting H(+) to the cell exterior. However, pre-treatment with the H (+) , K (+)-ATPase proton pump blocker omeprazole did not affect gastric Cu transport, suggesting that other mechanisms must also be involved.

Characterization of dietary Ni uptake in the rainbow trout, Oncorhynchus mykiss.

We characterized dietary Ni uptake in the gastrointestinal tract of rainbow trout using both in vivo and in vitro techniques. Adult trout were fed a meal (3% of body mass) of uncontaminated commercial trout chow, labeled with an inert marker (ballotini beads). In vivo dietary Ni concentrations in the supernatant (fluid phase) of the gut contents averaged from 2 micromoll(-1) to 24 micromoll(-1), and net overall absorption efficiency of dietary Ni was approximately 50% from the single meal, similar to that for the essential metal Cu, adding to the growing evidence of Ni essentiality. The stomach and mid-intestine emerged as important sites of Ni uptake in vivo, accounting for 78.5% and 18.9% of net absorption respectively, while the anterior intestine was a site of net secretion. Most of the stomach uptake occurred in the first 4h. In vitro gut sac studies using radiolabeled Ni (at 30 micromoll(-1)) demonstrated that unidirectional uptake occurred in all segments, with area-weighted rates being highest in the anterior intestine. Differences between in vivo and in vitro results likely reflect the favourable uptake conditions in the stomach, and biliary secretion of Ni in the anterior intestine in vivo. The concentration-dependent kinetics of unidirectional Ni uptake in vitro were biphasic in nature, with a saturable Michaelis-Menten relationship observed at 1-30 micromoll(-1) Ni (K(m) - 11 micromoll(-1), J(max) - 53 pmolcm(-2)h(-1) in the stomach and K(m) - 42 micromoll(-1), J(max) - 215 pmolcm(-2)h(-1) in the mid-intestine), suggesting mediation by a channel or carrier process. A linear uptake relationship was seen at higher concentrations, indicative of simple diffusion. Ni uptake (at 30 micromoll(-1)) into the blood compartment was significantly reduced in the stomach by high Mg (50 mmoll(-1)), and in the mid-intestine by both Mg (50 mmoll(-1)) and Ca (50 mmoll(-1)). In both regions, kinetic analysis demonstrated reductions in J(max) with unchanged K(m), suggesting non-competitive interactions. Therefore the Mg and Ca content of the food will be an important consideration affecting the availability of Ni.

Post-prandial metabolic alkalosis in the seawater-acclimated trout: the alkaline tide comes in.

The consequences of feeding and digestion on acid-base balance and regulation in a marine teleost (seawater-acclimated steelhead trout; Oncorhynchus mykiss) were investigated by tracking changes in blood pH and [HCO3-], as well as alterations in net acid or base excretion to the water following feeding. Additionally the role of the intestine in the regulation of acid-base balance during feeding was investigated with an in vitro gut sac technique. Feeding did not affect plasma glucose or urea concentrations, however, total plasma ammonia rose during feeding, peaking between 3 and 24 h following the ingestion of a meal, three-fold above resting control values (approximately 300 micromol ml(-1)). This increase in plasma ammonia was accompanied by an increase in net ammonia flux to the water (approximately twofold higher in fed fish versus unfed fish). The arterial blood also became alkaline with increases in pH and plasma [HCO3-] between 3 and 12 h following feeding, representing the first measurement of an alkaline tide in a marine teleost. There was no evidence of respiratory compensation for the measured metabolic alkalosis, as Pa CO2 remained unchanged throughout the post-feeding period. However, in contrast to an earlier study on freshwater-acclimated trout, fed fish did not exhibit a compensating increase in net base excretion, but rather took in additional base from the external seawater, amounting to approximately 8490 micromol kg(-1) over 48 h. In vitro experiments suggest that at least a portion of the alkaline tide was eliminated through increased HCO3- secretion coupled to Cl- absorption in the intestinal tract. This did not occur in the intestine of freshwater-acclimated trout. The marked effects of the external salinity (seawater versus freshwater) on different post-feeding patterns of acid-base balance are discussed.

In vitro examination of interactions between copper and zinc uptake via the gastrointestinal tract of the rainbow trout (Oncorhynchus mykiss).

An in vitro gut sac technique was used to investigate whether reciprocal inhibitory effects occurred between Cu and Zn uptake in the gastrointestinal tract of the rainbow trout and, if so, whether there was regional variation among the stomach, anterior intestine, mid intestine, and posterior intestine in the phenomena. Metal accumulation in surface mucus and in the mucosal epithelium and transport into the blood space were assayed using radiolabeled Cu or Zn at environmentally realistic concentrations of 50 micromol L(-1) in the luminal saline, with 10-fold higher levels of the other metal (nonradioactive) as a potential inhibitor. Zn transport rates were generally higher than Cu transport rates in all compartments except the stomach, where they were lower. High [Zn] reduced Cu transport into the blood space in the mid and posterior intestines by 67% and 33%, respectively, whereas high [Cu] reciprocally reduced Zn transport into the blood space in these same sections by 54% and 78%. No inhibitions occurred in either the anterior intestine or the stomach. In these segments, elevated concentrations of the other metal stimulated Cu and Zn transport into the blood space and/or the mucosal epithelium by 50-100%, possibly by displacement from intracellular binding sites, thereby raising local concentrations at other transport sites. None of the treatments affected metal accumulation in surface mucus. The results indicate that one or more shared high-affinity pathways (possibly DMT1) occur in the mid and posterior intestine, which transport both Cu and Zn. These pathways appear to be absent from the stomach and anterior intestine, where other transport mechanisms may occur.

Toxicity of dissolved Cu, Zn, Ni and Cd to developing embryos of the blue mussel (Mytilus trossolus) and the protective effect of dissolved organic carbon.

Marine water quality criteria for metals are largely driven by the extremely sensitive embryo-larval toxicity of Mytilus sp. Here we assess the toxicity of four dissolved metals (Cu, Zn, Ni, Cd) in the mussel Mytilus trossolus, at various salinity levels while also examining the modifying effects of dissolved organic carbon (DOC) on metal toxicity. In 48 h embryo development tests in natural seawater, measured EC50 values were 6.9-9.6 microg L(-1) (95% C.I.=5.5-10.8 microg L(-1)) for Cu, 99 microg L(-1) (86-101) for Zn, 150 microg L(-1) (73-156) for Ni, and 502 microg L(-1) (364-847) for Cd. A salinity threshold of >20 ppt (approximately 60% full strength seawater) was required for normal control development. Salinity in the 60-100% range did not alter Cu toxicity. Experimental addition of dissolved organic carbon (DOC) from three sources reduced Cu toxicity; for example the EC50 of embryos developing in seawater with 20 mg C L(-1) was 39 microg Cu L(-1) (35.2-47.2) a 4-fold increase in Cu EC50. The protective effects of DOC were influenced by their distinct physicochemical properties. Protection appears to be related to higher fulvic acid and lower humic acid content as operationally defined by fluorescence spectroscopy. The fact that DOC from freshwater sources provides protection against Cu toxicity in seawater suggests that extrapolation from freshwater toxicity testing may be possible for saltwater criteria development, including development of a saltwater Biotic Ligand Model for prediction of Cu toxicity.

Influence of acclimation and cross-acclimation of metals on acute Cd toxicity and Cd uptake and distribution in rainbow trout (Oncorhynchus mykiss).

The development of chronic metal toxicity models for fresh water fish is complicated by the physiological adjustments made by the animal during exposure which results in acclimation. This study examines the influence of a pre-exposure to a chronic sublethal waterborne metal on acclimation responses as well as the uptake and distribution of new metal into juvenile rainbow trout. In one series of tests, trout were exposed to either 20 or 60 microg/L Cu, or 150 microg/L Zn for a month in moderately hard water and then cross-acclimation responses to Cd were measured in 96 h LC(50) tests. Cu exposed trout showed a cross-acclimation response but Zn exposed trout did not. Using these results, a detailed examination of Cd uptake and tissue distribution in metal-acclimated trout was done. Trout were exposed to either 75 microg/L Cu or 3 microg/L Cd for 1 month to induce acclimation and subsequently, the uptake and distribution of new Cd was assessed in both Cd- and Cu-acclimated fish using (109)Cd. The pattern of accumulation of new metal was dramatically altered in acclimated fish. For example, in 3 h gill Cd binding experiments, Cd- and Cu-acclimated trout both had a higher capacity to accumulate new Cd but only Cu-acclimated fish showed a higher affinity for Cd compared to unexposed controls. Experiments measuring Cd uptake over 72 h at 3 microgCd/L showed that the Cd uptake rate was lower for Cd-acclimated fish compared to both Cu-acclimated fish and unexposed controls. The results demonstrate the phenomenon of cross-acclimation to Cd and that chronic sublethal exposure to one metal can alter the uptake and tissue distribution of another. Understanding how acclimation influences toxicity and bioaccumulation is important in the context of risk assessment. This study illustrates that knowledge of previous exposure conditions is essential, not only for the metal of concern, but also for other metals as well.

The alkaline tide goes out and the nitrogen stays in after feeding in the dogfish shark, Squalus acanthias.

In light of previous work showing a marked metabolic alkalosis ("alkaline tide") in the bloodstream after feeding in the dogfish shark (Squalus acanthias), we evaluated whether there was a corresponding net base excretion to the water at this time. In the 48 h after a natural voluntary meal (teleost tissue, averaging 5.5% of body weight), dogfish excreted 10,470 micromol kg(-1) more base (i.e. HCO3- equivalents) than the fasted control animals (which exhibited a negative base excretion of -2160 micromol kg(-1)). This large activation of branchial base excretion after feeding thereby prevented a potentially fatal alkalinization of the body fluids by the alkaline tide. The rate peaked at 330 micromol kg(-1) h(-1) at 12.5-24 h after the meal. Despite a prolonged 1.7-fold elevation in MO2 after feeding ("specific dynamic action"), urea-N excretion decreased by 39% in the same 48 h period relative to fasted controls. In contrast, ammonia-N excretion did not change appreciably. The N/O2 ratio declined from 0.51 in fasted animals to 0.19 in fed sharks, indicating a stimulation of N-anabolic processes at this time. These results, which differ greatly from those in teleost fish, are interpreted in terms of the fundamentally different ureotelic osmoregulatory strategy of elasmobranchs, and recent discoveries on base excretion and urea-retention mechanisms in elasmobranch gills.

Mechanisms of dietary Cu uptake in freshwater rainbow trout: evidence for Na-assisted Cu transport and a specific metal carrier in the intestine.

Copper (Cu) is both a vital nutrient and a potent toxicant. The objective of this study was to analyze the mechanistic nature of intestinal Cu transport in rainbow trout using radiolabeled Cu (64Cu) and an in vitro gut sac technique. Reduction of mucosal NaCl levels inhibited Cu transport while increase caused stimulation; Na(2)SO(4) had an identical effect, implicating Na(+) rather than the anion. These responses were unrelated to solvent drag, osmotic pressure or changes in transepithelial potential. The presence of elevated luminal Ag stimulated Cu and Na(+) uptake. Phenamil caused a partial inhibition of both Cu and Na(+) uptake while hypercapnia stimulated Na(+) and Cu transport. Cu uptake was sensitive to luminal pH and inhibited by a tenfold excess of Fe and Zn. These factors had no effect on Na(+ )uptake. On the basis of these results we propose a novel Na(+)-assisted mechanism of Cu uptake wherein the Na(+) gradient stimulates an increase in the H(+) concentration of the brushborder creating a suitable microenvironment for the effective transport of Cu via either DMT1 or Ctr1.

Physical characterization of high-affinity gastrointestinal Cu transport in vitro in freshwater rainbow trout Oncorhynchus mykiss.

This study investigated the transport of copper (Cu) in the gut of trout. Examination of the spatial distribution of Cu along the digestive tract and a physical characterization of the uptake process was carried out using an in vitro gut sac technique and (64)Cu as a tracer. Unidirectional Cu uptake was highest in the anterior intestine followed in decreasing order by the posterior intestine, mid intestine and the stomach. Cu uptake was resistant to hypoxia and appeared to be fueled equally well by Cu(II) or Cu (I) at Cu concentrations typically found in the fluid phase of the chyme in vivo in the trout intestine. Transport demonstrated saturation kinetics (e.g. K (m) = 31.6 microM, J (max) = 17 pmol cm(-2) h(-1), in mid intestine) at low Cu levels representative of those measured in the chyme in vivo, with a diffusive component at higher Cu concentrations. Q (10) analysis indicated Cu uptake is via diffusion across the apical membrane and biologically mediated across the basolateral membranes of enterocytes. The presence of L-histidine but not D-histidine stimulated both Cu and Na uptake suggesting a common pathway for the transport of Cu/Na with L-histidine.

Gastrointestinal assimilation of Cu during digestion of a single meal in the freshwater rainbow trout (Oncorhynchus mykiss).

Gastrointestinal processing and assimilation of Cu in vivo was investigated by sequential chyme analysis over a 72 h period following ingestion of a single satiation meal (3% body weight) of commercial trout food (Cu content=0.42 micromol g(-1)) by adult rainbow trout. Leaded glass ballotini beads incorporated into the food and detected by X-ray radiography were employed as an inert marker in order to quantify net Cu absorption or secretion in various parts of the tract. Cu concentrations in the supernatant (fluid phase) fell from about 0.06 micromol mL(-1) (63 microM) in the stomach at 2 h to about 0.003 micromol mL(-1) (3 microM) in the posterior intestine at 72 h. Cu concentrations in the solid phase were 10 to 30-fold higher than in the fluid phase, and increased about 4-fold from the stomach at 2 h to the posterior intestine at 72 h. By reference to the inert marker, overall net Cu absorption from the ingested food by 72 h was about 50%. The mid-intestine, and posterior intestine emerged as important sites of net Cu and water absorption and a potential role for the stomach in this process was also indicated. The anterior intestine was a site of large net Cu addition to the chyme, probably due to large net additions of Cu-containing fluids in the form of bile and other secretions in this segment. The results provide valuable information about sites of Cu absorption and realistic concentrations of Cu in chyme fluid for future in vitro mechanistic studies on Cu transport in the trout gastrointestinal tract.