Influence of cypermethrin toxicity on ionic regulation and gill Na(+)/K(+)-ATPase activity of a freshwater teleost fish Cyprinus carpio

The effects of acute and sublethal toxicity of cypermethrin, a synthetic pyrethroid insecticide on plasma electrolytes (Na(+), K(+) and Cl(-)) levels and gill Na(+)/K(+)-ATPase activity in a common carp Cyprinus carpio were examined. The 24h LC(50) value of cypermethrin for C. carpio was 1.86ppm. During acute exposure, cypermethrin caused adverse effects in the form of hyponatreima, hypokalemia and hypochloremia and inhibition of gill Na(+)/K(+)-ATPase activity. In sublethal exposure to cypermethrin (0.186ppm for 35 days), plasma Na(+) was decreased throughout the exposure period except 7th day whereas plasma K(+) level was increased up to 28th day, then declined. However, plasma Cl(-) level was decreased. Likewise, gill Na(+)/K(+)-ATPase activity was decreased as the exposure period extended. The present study indicates that cypermethrin was highly toxic to freshwater fish and ion levels in blood as measured by specific ion concentrations (Na(+), K(+) and Cl(-)) and changes in gill Na(+)/K(+)-ATPase activity may represent a sensitive and useful nonspecific biomarkers of chemical exposure and effects.     

Acute and chronic physiological effects of silver exposure in three marine teleosts

This study evaluated the physiological effects of waterborne silver (added as AgNO(3)) on seawater fish, using acute (48-72 h) high level exposures (250-650 microg/l Ag) on tidepool sculpins (Oligocottus maculosus), and chronic (up to 21 day) low level exposures (1.5-50 microg/l Ag) on tidepool sculpins, plainfin midshipmen (Porichthys notatus), and rainbow trout (Oncorhynchus mykiss). Sculpins were tested at different salinities. Acclimation to lower salinity (18 vs 30 ppt) led to altered physiology, with higher ammonia excretion (J(Amm)), lower oxygen consumption, and lower branchial and intestinal Na(+)/K(+)-ATPase activities, but no difference in drinking rate. Short-term exposure to high silver levels tended to stimulate M(O(2)), J(Amm), and drinking rate. However, long-term exposure to low levels of silver depressed both J(Amm) and M(O(2)), and also led to decreased drinking rates. Both inhibition and stimulation of Na(+)/K(+)-ATPase activity occurred, dependent upon length and concentration of exposure, salinity (18 vs 30 ppt), tissue (gill vs intestine), and fish species (sculpin vs midshipmen vs rainbow trout). While the effects were variable, due to differing balances between inhibitory and compensatory responses, chronic silver exposure significantly altered Na(+)/K(+)-ATPase activity levels in almost all tests. In total, these findings reinforce the view that intestinal osmoregulatory function (drinking, Na(+)/K(+)-ATPase activity) is an important site of toxic impact for waterborne silver, that gill Na(+)/K(+)-ATPase activity is also a site of impact, and that chronic exposures at silver concentrations (1.5, 14.5 microg/l Ag) close to current or proposed water quality guidelines (albeit much higher than normal environmental levels), exert a variety of sublethal effects on marine teleosts.     

Copper toxicity in the spiny dogfish (Squalus acanthias): urea loss contributes to the osmoregulatory disturbance

Previous research showed that the spiny dogfish, Squalus acanthias, is much more sensitive to silver exposure than typical marine teleosts. The aim of the present study was to investigate if spiny dogfish were equally sensitive to copper exposure and whether the toxic mechanisms were the same. We exposed cannulated and non-cannulated spiny dogfish to measured concentrations of Cu (nominally 0, 500, 1000 and 1500 microg L(-1) Cu) for 72-96 h. All Cu exposures induced acidosis and lactate accumulation of either a temporary (500 microg L(-1)) or more persistent nature (1000 and 1500 microg L(-1)). At the two highest Cu concentrations, gill Na(+)/K(+)-ATPase activities were reduced by 45% (1000 microg L(-1)) and 62% (1500 microg L(-1)), and plasma Na(+) and Cl(-) concentrations increased by approximately 50 mM each. At the same time urea excretion doubled and plasma urea dropped by approximately 100 mM. Together with plasma urea, plasma TMAO levels dropped proportionally, indicating that the general impermeability of the gills was compromised. Overall plasma osmolarity did not change. Cu accumulation was limited with significant increases in plasma Cu and elevated gill and kidney Cu burdens at 1000 and 1500 microg L(-1). We conclude that Cu, like Ag, exerts toxic effect on Na(+)/K(+)-ATPase activities in the shark similar to those of teleosts, but there is an additional toxic action on elasmobranch urea retention capacities. With a 96 h LC(50) in the 800-1000 microg L(-1) range, overall sensitivity of spiny dogfish for Cu is, in contrast with its sensitivity to Ag, only slightly lower than in typical marine teleosts.     

Effects of acute and subchronic exposures to waterborne selenite on the physiological stress response and oxidative stress indicators in juvenile rainbow trout

Selenium (Se) is an essential element that may bioaccumulate to toxic levels. In fish, the major toxicity symptom is larval teratogenic deformities, but little is known about the effect of Se on other systems such as the physiological stress response and oxidative stress. To test the hypothesis that Se is a chemical stressor that causes toxicity through oxidative stress, juvenile rainbow trout were exposed to waterborne sodium selenite, and physiological stress response and stress-related parameters (plasma cortisol, glucose, T3 and T4, gill Na+/K+-ATPase, the ability of the head kidney to secrete cortisol, and condition factor) and hepatic oxidative stress indicators (reduced glutathione, glutathione peroxidase, and lipid peroxidation) were measured after 96 h (acute exposure to 0-2.67 mg/L Se) and 30 days (sub-chronic exposure to 0-0.16 mg/L). Acute exposure to waterborne sodium selenite significantly increased plasma cortisol levels (control=0.01+/-0.0 ng/mL, and 2.52 mg/L Se=73.5+/-22 ng/mL) and plasma glucose levels (control=0.75+/-0.1 mg/mL, and 3.60 mg/L Se=1.64+/-0.2 mg/mL), but gill Na+/K+-ATPase activities, plasma T3 and T4 levels, and condition factor were unchanged. The 96 h acute selenite exposure decreased hepatic reduced glutathione levels (control=18.4+/-1.5 micromol/mg protein, and 3.60 mg/L Se=12.4+/-1.1 micromol/mg protein). Lipid peroxidation levels (0.03-0.08 U/mg protein) and glutathione peroxidase (3.7-6.0 mU/mg protein) activities significantly varied with treatment. The 30 days sub-chronic exposure increased plasma cortisol, T3, and T4, but there was no effect on plasma glucose levels, gill Na+/K+-ATPase activity, the ability to secrete cortisol, and condition factor. The 30 days sub-chronic exposure to selenite did not alter antioxidant activities or lipid peroxidation levels. These experiments show, for the first time, that exposure to waterborne selenite up to 0.1mg/L, activates the physiological stress response in fish but does not impair cortisol secretion after 30 days. The decrease in reduced glutathione in juvenile rainbow trout subjected to the acute sodium selenite exposure suggests that oxidative stress may play an important role in the effects of Se in fish.     

Effects of copper on cortisol receptor and metallothionein expression in gills of Oncorhynchus mykiss

Effects of waterborne Cu (2.4 microM) on the expression of glucocorticoid receptor (GR) and metallothionein (MT) in the branchial epithelium of freshwater rainbow trout (Oncorhynchus mykiss) was studied by immunocytochemistry. After 5 days of Cu exposure, the number of GR-immunoreactive (GR-ir) cells in the gill epithelium had decreased, whereas the number of MT-ir cells had increased. Localization of GR in chloride cells was achieved by double staining for Na(+)/K(+)-ATPase; other cell types were identified on the basis of their topology. GRs were present in the chloride cells in both the filaments and lamellae, in respiratory cells in the lamellae, in pavement cells, basal layer cells and undifferentiated cells in the filaments. Co-localization of Na(+)/K(+)-ATPase and MT revealed chat MT was expressed in chloride cells, both in filaments and lamellae. Occasionally, MT immunoreactivity was found in pavement cells and in undifferentiated cells. By double staining for Na(+)/K(+)-ATPase and GR, for Na(+)/K(+)-ATPase and MT and for GR and MT, we can conclude that after 5 days of Cu stress there are chloride cells that express GR and MT, GR or MT alone or neither of the two proteins. This apparent functional heterogeneity of branchial chloride cells may reflect a limited window when chloride cell subpopulations show an adaptive response to Cu.     

Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout. 1: Iono-regulatory disturbance and metabolic costs

The relationships among growth, feeding behaviour, ion regulation, swimming performance and oxygen consumption in rainbow trout (Oncorhynchus mykiss) were compared during chronic exposure (up to 100 days) to sublethal levels of waterborne Cd (3 μg.l(-1)), Cu (75 μg.l(-1)) or Zn (250 μg.l(-1)) in moderately hard water (hardness of 140 mg.l(-1), pH 8). A pattern of disturbance, recovery and stabilization was evident for all three metal exposures, although the degree of disturbance, specific response and time course of events varied. Growth was unaffected by any of the metals under a regime of satiation feeding but appetite was increased and decreased in Cu- and Cd-exposed trout respectively. Critical swimming speed was significantly lowered in fish chronically exposed to Cu, an effect associated with elevated O(2) consumption rate at higher swimming speeds. Branchial Na(+)/K(+) ATPase activity was elevated in Cu-exposed fish but not in Cd-exposed trout. Disruption of carcass Na(+) and Ca(2+) balance was evident within 2 days of exposure to either Cd, Cu or Zn, with subsequent recovery to control levels. The loss of Ca(2+) in trout exposed to waterborne Cd persisted longest, and recovery took approximately a month. The physiological response of trout to chronic Cu exposure involves mechanisms that result in an associated metabolic cost. In comparison, Cd is neither a loading nor a limiting stress and acclimation to chronic Cd-exposure does not appear to involve a long term metabolic cost.     

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.     

Ionoregulatory disruption as the acute toxic mechanism for lead in the rainbow trout (Oncorhynchus mykiss)

The mechanism for acute toxicity of lead (Pb) in rainbow trout (Oncorhynchus mykiss) was investigated at Pb concentrations close to the 96 h LC50 of 1.0 mg dissolved Pb l(-1) (0.8-1.4, 95% C.I.) determined in dechlorinated Hamilton city tap water (from Lake Ontario, hardness=140 mg l(-1) CaCO(3)). Tissue Pb accumulation associated with death was highest in the gill, followed by kidney and liver. Significant ionoregulatory impacts were observed in adult rainbow trout (200-300 g) fitted with indwelling dorsal aortic catheters and exposed to 1.1+/-0.04 mg dissolved Pb l(-1). Decreased plasma [Ca(2+)], [Na(+)] and [Cl(-)] occurred after 48 h of exposure through to 120 h, with increases in plasma [Mg(2+)], ammonia, and cortisol. No marked changes in PaO(2), PaCO(2), pH, glucose, or hematological parameters were evident. Branchial Na(+)/K(+) ATPase activity in juvenile trout exposed to concentrations close to the 96 h LC50 was inhibited by approximately 40% after 48 h of Pb exposure. Calcium ion flux measurements using 45Ca as a radiotracer showed 65% inhibition of Ca(2+) influx after 0, 12, 24 or 48 h exposure to the 96 h LC50 concentration of Pb. There was also significant inhibition (40-50%) of both Na(+) and Cl(-) uptake, measured with 22Na and 36Cl simultaneously. We conclude that the mechanism of acute toxicity for Pb in rainbow trout occurs by ionoregulatory disruption rather than respiratory or acid/base distress at Pb concentrations close to the 96 h LC50 in moderately hard water.     

Effects of copper and cadmium on ion transport and gill metal binding in the Amazonian teleost tambaqui (Colossoma macropomum) in extremely soft water

Metal toxicity in fish is expected to be most severe in soft waters because of the low availability of cations (particularly Ca(2+)) to out-compete the metal forms for binding sites on the gills. Natural waters in the Amazon basin are typically soft due to regional geochemistry, but few studies have focused on metal toxicity in fish native to the basin. We assessed the ionoregulatory effects of waterborne copper (Cu) and cadmium (Cd) on tambaqui (Colossoma macropomum) in extremely soft water (10 micromoll(-1) Ca(2+)). Tambaqui had a very high tolerance to Cu (50-400 microgl(-1)), as indicated by a complete lack of inhibition of Na(+) uptake and an ability to gradually recover over 6h from elevated diffusive Na(+) losses caused by Cu. The insensitivity of active Na(+) influx to Cu further supports the notion that Amazonian fish may have a unique Na(+) transport system. Addition of 5-10 mgCl(-1) of dissolved organic matter (DOM) did not prevent initial (0-3h) negative Na(+) balance in tambaqui exposed to Cu. Exposure to 40 mgCl(-1) DOM prevented Na(+) losses in tambaqui even at 400 microgl(-1) Cu, probably because most Cu was complexed to DOM. Tambaqui exposed to waterborne Cd (10-80 microgl(-1)) experienced an average of 42% inhibition in whole body Ca(2+) uptake relative to controls within 3h of exposure to the metal. Inhibition of Ca(2+) uptake increased over time and, at 24h, Ca(2+) uptake was suppressed by 51% and 91% in fish exposed to 10 and 80 microgl(-1) Cd, respectively. Previous acclimation of fish to either elevated [Ca(2+)] or elevated [DOM] proved to be very effective in protecting against acute short-term metal accumulation at the gills of tambaqui in soft water (in the absence of the protective agent during metal exposure), suggesting a conditioning effect on gill metal binding physiology.     

Physiological and behavioural responses of Gammarus pulex (Crustacea: Amphipoda) exposed to cadmium

The aim of this study was to investigate the effects of cadmium on physiological and behavioural responses in Gammarus pulex. In a first experiment, cadmium LC50s for different times were evaluated in 264 h experiment under continuous mode of exposure (LC50(96 h)=82.1 microgL(-1), LC50(120 h)=37.1 microgL(-1), LC50(168 h)=21.6 microgL(-1), LC50(264 h)=10.5 microgL(-1)). In a second experiment, the physiological and behavioural responses of the amphipod exposed to cadmium (0, 7.5 and 15 microgL(-1)) were investigated under laboratory conditions. The mortality and the whole body cadmium concentration of organisms exposed to cadmium were significantly higher than in controls. Concerning physiological responses, cadmium exposure exerted a significant decrease on osmolality and haemolymph Ca(2+) concentration, but not on haemolymph Na(+) and Cl(-) concentrations, whereas the Na(+)/K(+)-ATPase activity was significantly increased. Behavioural responses, such as feeding rate, locomotor and ventilatory activities, were significantly reduced in Cd exposed organisms. Mechanism of cadmium action and consequent energetic reallocation in favour of maintenance functions (i.e., osmoregulation) are discussed. The results of this study indicate that osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystem and to assess the health of organisms.     

The influence of ration size on copper homeostasis during sublethal dietary copper exposure in juvenile rainbow trout,Oncorhynchus mykiss

The influence of ration size on homeostasis and sublethal toxicity of copper (Cu) was assessed in rainbow trout (Oncorhynchus mykiss) during dietary Cu exposure in synthetic soft water. A constant dietary dose of 0.24 micromol Cu per g fish per day as CuSO(4).5H(2)O was delivered via diets containing 15.75, 7.87, and 5.24 micromol Cu g(-1) fed at 1.5, 3.0, and 4.5% wet body weight daily ration, respectively. Juvenile rainbow trout showed clear effects of ration but not Cu on growth suggesting that growth is hardly a sensitive endpoint for detection of sublethal dietary Cu exposure. All Cu-exposed fish accumulated the same total metal load when expressed on a per fish basis. This suggests that differences in tissue and whole-body Cu concentrations among the treatments reflected the differences in the fish size rather than total Cu accumulation, and demonstrate that absorption and accumulation of Cu from the gut during dietary exposure are independent of the food quantity in which the Cu is delivered. Fish fed a high ration exhibited greater mass-specific unidirectional uptake of waterborne Cu than fish fed a low ration indicating an increased need for Cu for growth processes in rapidly growing fish. Stimulated excretion of Cu was indicated by greater Cu accumulation in the bile of the Cu-exposed fish. Branchial Na(+), K(+)-ATPase was not affected by dietary Cu exposure or ration but gut Na(+), K(+)-ATPase activities showed stimulatory effects of increasing ration but not of Cu exposure. The 96-h LC50 for waterborne Cu (range 0.17-0.21 micromol l(-1) (10.52-13.20 microg l(-1)) was the same in all treatment groups indicating that ration size was unimportant and that dietary Cu did not induce an increase in tolerance to waterborne Cu. Taken together these results suggest that the nutritional status, fish size, and growth rates should be considered when comparing whole-body and tissue Cu concentration data for biomonitoring and risk assessment. Moreover, expressing the exposure as total metal dose rather than metal concentration in the diet is more appropriate.     

Physiological effects of copper in the euryhaline copepod Acartia tonsa: waterborne versus waterborne plus dietborne exposure

The physiological effects of waterborne and waterborne plus dietborne copper exposure were determined in the euryhaline copepod Acartia tonsa at different salinities (5, 15 and 30ppt). Copepods were exposed (48h) to a reported 48-h LC50 for copper (CuCl(2)), which had been previously determined under the same experimental conditions. Whole body copper accumulation, ion concentrations (Na(+), Cl(-), Mg(2+)), and Na(+), K(+)-ATPase activity were the endpoints measured in all experimental groups. Feeding rate was also measured in fed experimental groups. In copper-exposed copepods, whole body copper accumulation was dependent on salinity, decreasing as salinity increased. However, it was similar in copepods exposed to waterborne and waterborne plus dietborne copper, irrespective the salinity tested. Waterborne copper exposure induced a disturbance of the whole body Na(+) concentration in all salinities tested. This effect was characterized by an increased whole body Na(+) concentration in seawater (salinity 30ppt) and a decreased whole body Na(+) concentration at lower salinities (5 and 15ppt). The ionoregulatory imbalance in low salinity (5ppt) was associated with an inhibition of the whole body Na(+), K(+)-ATPase activity, as observed in freshwater fish and crustaceans. When copepods were exposed to waterborne plus dietborne copper, the physiological effects described were only observed at a low salinity (5ppt) and were associated with a marked inhibition of the feeding rate. Taken altogether, the data suggest that the physiological effects induced by waterborne copper exposure in A. tonsa acclimated to higher salinities (15 and 30ppt) are due to a combined effect of food restriction and copper exposure. Differential physiological responses to waterborne and waterborne plus dietborne copper cannot be ascribed to differences in whole body copper burden.     

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.     

Effects of Cu on plasma cortisol and cortisol secretion by adrenocortical cells of rainbow trout (Oncorhynchus mykiss)

Fish are exposed to multiple stressors, often acting concurrently, in their environment. To evaluate the potential of Cu to act as a chemical stressor, rainbow trout (Oncorhynchus mykiss) were exposed to Cu (30 or 80 microg/l) for 30 days in the laboratory and they were subjected to a physical stressor (1 min air exposure) before sampling. Physiological stress indicators in the whole fish as well as cortisol secretion by adrenocortical cells in vitro were measured. Fish exposed to Cu had a lower condition factor, hepatosomatic index, plasma glucose, hepatic glycogen and gill Na(+)/K(+)-ATPase activity compared to controls. Exposure to Cu did not have an effect on basal plasma cortisol (fish sampled without air exposure stress) however, the air exposure-induced increase in plasma cortisol was lower in fish exposed to Cu. Cortisol secretion stimulated by ACTH in vitro was greater in adrenocortical cells isolated from fish exposed to Cu in vivo but in vitro exposure to Cu consistently impaired cortisol secretion. Our results indicate that Cu at high concentrations disrupts cortisol secretion through a direct toxic effect on adrenocortical cells while low concentrations resulting from a 30-day exposure to environmentally relevant Cu concentrations enhances cortisol secretion in response to ACTH in vitro.     

Cadmium affects the social behaviour of rainbow trout,Oncorhynchus mykiss

The present study investigated both the effects of cadmium on the social interactions of rainbow trout and the differential accumulation of waterborne cadmium among social ranks of fish. Fish exposed to waterborne cadmium concentrations of 2 microg l(-1) for 24 h, followed by a 1, 2 or 3 day depuration period in clean water, had a decreased ability to compete with non-exposed fish. However, the competitive ability of exposed fish given a 5 day depuration period was not significantly impaired. Cadmium accumulated in the olfactory apparatus of fish exposed to waterborne cadmium for 24 h and decreased significantly only after 5 days depuration in clean water. Among groups of ten fish held in stream tanks, where all fish were exposed to cadmium, there were significant effects on social behaviour and growth rate. Dominance hierarchies formed faster among fish exposed to cadmium than among control fish, and overall growth rates were higher in the cadmium treatment. In groups of ten fish, social status also affected tissue accumulation of cadmium during waterborne exposure, with dominant fish accumulating more cadmium at the gill. In conclusion, exposure to low levels of cadmium, affects the social behaviour of fish, in part due to accumulation in the olfactory apparatus, and dominant fish accumulate more gill cadmium than subordinates during chronic waterborne exposure.     

Food selection, growth and physiology in relation to dietary sodium chloride content in rainbow trout (Oncorhynchus mykiss) under chronic waterborne Cu exposure

Waterborne Cu is toxic to Na(+) and Cl(-) regulation in freshwater fish, and Cu is taken up, at least in part, via the Na(+)-transport pathway in the gills. Therefore, we hypothesized that freshwater fish may mitigate the toxic effects of waterborne Cu by selecting a NaCl-enriched diet over a normal diet. We tested this hypothesis in juvenile rainbow trout (Oncorhynchus mykiss) by offering them the choice between NaCl-enriched (1.9 mmol g(-1)Na(+)) and normal (0.2 mmol g(-1)Na(+)) diets under a chronic waterborne Cu exposure of 55 microg L(-1) for a period of 28 days. Contrary to expectation, trout exhibited a preference for NaCl-enriched diet under control conditions, while exposure to chronic waterborne Cu severely disrupted their normal feeding pattern with an accompanying loss of preference for the NaCl-enriched diet. Waterborne Cu exposure also severely affected appetite and growth. Both appetite and growth gradually recovered with time, but remained significantly impaired relative to Cu-unexposed fish until the end of the exposure. Waterborne Cu exposure also significantly increased Cu accumulations in target organs (gill, liver, and gut), plasma and whole body. However, Cu accumulation decreased substantially towards the end of the exposure in target organs and whole body as well as in plasma in Cu-exposed fish with dietary choice relative to Cu-exposed fish with normal diet. These adjustments were concurrent with the gradual recovery of appetite, which also led to increased ingestion of the NaCl-enriched diet. Interestingly, this elevated dietary uptake of NaCl produced significant stimulation of Na(+) efflux in Cu-exposed fish. Subsequently, it also led to significant elevation of Na(+) levels in target organs and whole body, and restored the decrease of plasma Na(+) and Cl(-) levels in Cu-exposed fish. The NaCl supplemented diet appeared to be beneficial in compensating Na(+) and Cl(-) losses from the body induced by waterborne Cu. Overall, these results demonstrate that a NaCl-enriched diet, although consumed in relatively reduced quantities due to the impairment of food selection and appetite, can help to protect freshwater fish against chronic waterborne Cu toxicity.     

Inhibition of eel enzymatic activities by cadmium

The aim of the present work was to study the in vitro effect of cadmium on enzymes, such as intestinal and branchial carbonic anhydrase (CA) and Na(+)-K(+)-ATPase which play a key role in salt- and osmoregulation and acid-base balance in the teleost fish, Anguilla anguilla. Carbonic anhydrase activities in gill and intestinal homogenates were significantly inhibited by CdCl(2), the gill CA being more sensitive to the heavy metal (IC(50) for the branchial CA=9.97+/-1.03x10(-6) M, IC(50) for the intestinal CA=3.64+/-1.03x10(-5) M, P<0.01). With regards to the intestinal CA activity, it has been shown in a previous study (Maffia et al., 1996) that two isoforms exist, a cytosolic and a brush-border membrane bound. These two isoforms show a different sensitivity to cadmium, with the membrane-bound enzyme less sensitive with respect to the cytosolic one, since it showed still an incomplete inhibition at the highest cadmium concentration tested. The inhibition of all the CA activity tested revealed a time-dependence since it required at least 10 min (1 h for the membrane-bound isoform) preincubation with the heavy metal to appear. Na(+)-K(+)-ATPase enzymatic activities, measured in intestinal and branchial homogenates, were inhibited by cadmium in a dose-dependent manner, with the branchial activity being more sensitive to the action of the heavy metal than the intestinal one (IC(50) for the branchial enzyme=1.38+/-0.09x10(-7) M, IC(50) for the intestinal enzyme=2.86+/-0.02x10(-7) M, P<0.01). The most of inhibition of the enzyme appeared without any preincubation with the heavy metal. Mg(2+)-ATPase activity was not significantly altered by the in vitro cadmium exposure either in the gills or in the intestine. These findings observed in vitro could be useful in the understanding of the toxic effects that cadmium elicits on aquatic organisms in vivo. In fact, the impairment of the activity of enzymes which carry out key physiological roles could cause alterations of the physiology of the whole organism.     

Sensitivity of the spiny dogfish (Squalus acanthias) to waterborne silver exposure

The physiological effects of waterborne silver exposure (added as AgNO(3)) on spiny dogfish, Squalus acanthias, were evaluated at 30, 200 and 685 microg silver per l in 30 per thousand seawater. These concentrations cover the toxic range observed for freshwater teleosts, where silver is extremely toxic, to seawater teleosts which tolerate higher silver concentrations. However, these levels are considerably higher than those that occur in the normal environment. At 685 microg l(-1), dogfish died within 24 h. Causes of death were respiratory as well as osmoregulatory failure. Arterial P(a)O(2) rapidly declined below 20 Torr, and blood acidosis (both respiratory and metabolic) occurred. Urea excretion increased dramatically and plasma urea dropped from 340 to 225 mM. There were pronounced increases in plasma Na(+), Cl(-), and Mg(2+), indicative of ionoregulatory failure due to increased diffusive permeability as well as inhibited NaCl excretion. At 200 microg l(-1), fish died between 24 and 72 h of silver exposure. The same physiological events occurred with a small time delay. At 30 microg l(-1), effects were much less severe, although slight mortality (12.5%) still occurred. Respiratory alkalosis occurred, together with moderate elevations in plasma Na(+) and Cl(-) levels. Silver accumulated to the highest concentrations on gills, with only low levels in the intestine, in accord with the virtual absence of drinking. Na(+)/K(+)-ATP-ase activities of gill and rectal gland tissue were impaired at the highest silver concentration. Normal gill function was impaired due to swelling and fusion of lamellae, lamellar aneurism and lifting of the lamellar epithelium. Our results clearly indicate that this elasmobranch is much more sensitive (about 10-fold) to silver than marine teleosts, with silver's toxic action exerted on the gill rather than on the intestine, in contrast to the latter.     

Metallothionein and cortisol receptor expression in gills of Atlantic salmon, Salmo salar, exposed to dietary cadmium

Commercial fish feeds may contain significant levels of cadmium (Cd). However, little is known about the effects of dietary cadmium on fish organs, especially gills, the key osmoregulatory organ. We therefore studied the effects of dietary cadmium on metallothionein (MT) and cortisol receptor (GR) immunoreactivity in the branchial epithelium of the Atlantic salmon (Salmo salar). Cadmium was daily administered via food at 0.2mg (control), 5mg (low dose) and 125 mg (high dose) Cd per kilogram dry pellet weight. Fish were sampled after four and eight weeks. After both four and eight weeks, plasma cadmium concentration had increased significantly only in fish fed the high cadmium dose. Plasma calcium, sodium, chloride and cortisol levels were not affected. In the controls, most MT was colocated with the chloride cell marker, Na(+)/K(+)-ATPase, but some MT was present in pavement and respiratory cells. GR expression was found in chloride, pavement, respiratory and undifferentiated cells in all fish groups, but cadmium accumulation and a marked stimulation of MT expression were seen only in the chloride cells in the gills of fish fed the high cadmium dose. Cadmium treatment did not alter GR expression. When the double staining technique for MT and GR was applied, a marked heterogeneity became apparent in the chloride, pavement and respiratory cells of both groups of cadmium-treated fish and in the control fish. Some fish showed double staining, others stained only for one of the antibodies, whereas other cells were negative for both. We conclude that cadmium entering the gut also enters the gills, where it accumulates in chloride cells and stimulates MT expression.