Association between cadmium and calcium uptake and distribution during the moult cycle of female shore crabs, Carcinus maenas: an in vivo study

Net influxes into the haemolymph and tissue distribution of 45Ca and 109Cd were studied in vivo in female Carcinus maenas at different moult stages. Net influxes of 45Ca and 109Cd from water were higher in postmoult (A and B) C. maenas than in C3- and C4-intermoult crabs and the net influx of calcium was higher in C3-intermoult crabs than in C4-intermoult crabs. The net influxes of 45Ca and 109Cd increased in postmoult C. maenas with decreasing external calcium concentrations at constant salinity. At all external calcium concentrations a significant correlation existed between 45Ca and 109Cd accumulated in the haemolymph of individual animals. In vivo exposure of postmoult C. maenas to external lanthanum decreased the 45Ca and 109Cd uptake rates to 30 and 10%, respectively, of the control values. About 30% of injected 109Cd were found in the midgut gland, 10-20% in the gills and only a few (1-2) percent was lost to the seawater 24 h after injection. No major variations in tissue distribution of 109Cd were observed between moult stages in these tissues. Premoult crabs retained more cadmium in the haemolymph 24 h after injection than other moult stages, and postmoult crabs retained more in muscle. Between 20 and 40% of the injected 45Ca were excreted to the water, while only a few percent of the injected 45Ca were found in the soft tissues 24 h after injection. Large moult stage variations, however, were observed in the tissue distribution of internalised 45Ca. This study demonstrates that cadmium and calcium uptakes are elevated in postmoult C. maenas. The results indicate that cadmium and calcium in this stage are taken up via Ca2+-channels located in the apical membrane of gill epithelium cells. When internalised, however, cadmium and calcium are metabolised in fundamentally different ways, determined by the chemical properties and biological significance of the two metals.     

Cadmium in the shore crab Carcinus maenas: seasonal variation in cadmium content and uptake and elimination of cadmium after administration via food

The uptake and assimilation efficiency of cadmium administered via the food in the shore crab Carcinus maenas were investigated together with elimination kinetics and seasonal variations in cadmium content. The majority of shore crabs assimilated between 41 and 86% of the cadmium administered in their food. More than 90% of the cadmium taken up from food was retained in midgut gland. Elimination of cadmium after uptake from one meal of radioactively labelled soft parts of blue mussels could be described by a three-compartment model (percent 109Cd-retained = 64 x e(-0.001107 x t) + 25 x e(-0.0385 x t)+11 x e(-0.888 x t)). The biological half-life for cadmium in the most slowly exchanging compartment (containing 64% of the body burden) was 626 days. Groups of male and female shore crabs were collected from an uncontaminated site in the period May till October and the concentrations of cadmium in midgut gland and gills were determined. Male crabs had higher cadmium concentrations in the midgut gland in June and August (mean 2.7 microg Cd g(-1) dry weight) than they had in May, September and October (mean 1.7 microg Cd g(-1) dry weight). Females generally had slightly lower cadmium concentrations in the midgut gland than the males, except for a relatively high concentration in May. The cadmium concentrations in gills generally ranged between 0.3 and 0.5 microg Cd g(-1) dry weight) except for male values in October (mean 1 microg Cd g(-1) dry weight). Some of the seasonal changes in cadmium content of the crabs might plausibly be explained by changes in cadmium uptake from water, i.e. changes during the moult cycle and changes in cadmium uptake rates from water brought about by changes in ambient factors such as salinity and temperature. However, uptake of cadmium from water and transfer to the midgut gland take place at a rate that is two orders of magnitude too low to account for the increase in the cadmium concentrations in midgut gland in male crabs between May and June. The distribution of cadmium among tissues in crabs collected at uncontaminated sites also corresponds better with results obtained after administration of cadmium via the food than via water, and the exposure of the crabs to cadmium via the food is large enough to explain the increase in concentration between May and June.     

Cadmium influx and efflux across perfused gills of the shore crab, Carcinus maenas

Cadmium influx across perfused gills of the shore crab Carcinus maenas exposed to 9.0 μM cadmium in the external medium ranged from 0.6 to 2.0 nmol Cd(2+) g(-1) gill wet wt. per h. Cadmium efflux across perfused gills exposed to 9.0 μM cadmium in the internal medium ranged from 0.3 to 0.9 nmol Cd(2+) g(-1) gill wet wt. per h. There was no significant difference between cadmium influx and efflux across the gills. Cadmium influx across the gills was not detectable after exposure to lanthanum in the external medium, whereas cadmium efflux was unaffected by external as well as internal lanthanum. Cadmium influx was not affected when the internal medium was changed to Na-free medium, whereas efflux showed a fourfold increase when the gills were perfused with Na-free medium. Low pH in the external medium did not exert any significant effect on cadmium influx and efflux.     

Temporal variations of manganese in the haemolymph and tissues of the Norway lobster, Nephrops norvegicus (L.)

The Norway lobster, Nephrops norvegicus, lives on sediments rich in manganese (Mn) and any dissolved Mn(2+) can readily be taken up by the animal. To investigate temporal fluctuations of bioavailable Mn, a N. norvegicus fishing ground on the Swedish west coast was repeatedly sampled every 2 months from September 1992 to November 1994. The lobsters collected contained on average 91.7 μg Mn g(-1) dry wt. (S.E. 4.2, n=156). The oxygen saturation of the bottom water proved to be negatively correlated with both the temperature of the water and the Mn (concentration and total content) of the animal's haemolymph. The temporal fluctuations in animal Mn load were however, small compared to spatial differences found in an earlier study. There was an increase in the Mn concentration of the lobster exoskeleton (from 56 to 340 μg Mn g(-1) dry wt. exoskeleton) and gills (from 34 to 160 μg Mn g(-1) dry wt. gill) in postmoult animals compared to premoult. The Mn concentrations of the lobsters' hepatopancreas and muscle tissue remained relatively constant throughout the moult cycle and appear to be more conservative in their Mn concentration and less affected by exposure to Mn.     

Dynamics of (Cd,Zn)-metallothioneins in gills, liver and kidney of common carp Cyprinus carpio during cadmium exposure

Cadmium concentrations, (Cd,Zn)-metallothionein (MT) concentrations, MT synthesis and the relative amounts of cadmium bound to (Cd,Zn)-MTs were determined in gills, liver and kidney of common carp Cyprinus carpio exposed to 0, 0.5 microM (0.06 mg.l(-1)), 2.5 microM (0.28 mg.l(-1)) and 7 microM (0.79 mg.l(-1)) Cd for up to 29 days. Cadmium accumulation was in the order kidney > liver > gills. Control levels of hepatic (Cd,Zn)-MT were four times higher compared to those of gills and kidney. No increases in (Cd,Zn)-MT concentrations were observed in liver during the exposure period. In comparison with control carp, (Cd,Zn)-MT concentrations increased up to 4.5 times in kidney and two times in gills. In both these organs, (Cd,Zn)-MT concentrations were linearly related with cadmium tissue levels and with the de novo synthesis of MTs. Hepatic cadmium was almost completely bound to (Cd,Zn)-MT, while percentages of non-MT-bound cadmium were at least 40% in gills and 25% in kidney. This corresponded with a total saturation of (Cd,Zn)-MT by cadmium in kidney and a saturation of approximately 50 and 60% in gills and liver, respectively. The final order of non-MT-bound cadmium was kidney > gills > liver. Our results indicate that cadmium exposure causes toxic effects, which cannot be correlated with the accumulated levels of the metal in tissues. Although cadmium clearly leads to the de novo synthesis of MT and higher (Cd,Zn)-MT concentrations, the role of this protein in the detoxification process is clearly organ-specific and its synthesis does not keep track with cadmium accumulation.     

Tissue-specific accumulation of cadmium in subcellular compartments of eastern oysters Crassostrea virginica Gmelin (Bivalvia: Ostreidae)

Cadmium distribution was studied in different subcellular fractions of gill and hepatopancreas tissues of eastern oysters Crassostrea virginica. Oysters were exposed for up to 21 days to low sublethal Cd concentrations (25 microg L(-1)). Gill and hepatopancreas tissues were sampled and divided into organelle fractions and cytosol by differential centrifugation. Organelle content of different fractions was verified by activities of marker enzymes, citrate synthase and acid phosphatase for mitochondria and lysosomes, respectively. In both tissue types, there was a significant accumulation of cadmium in cytosol reaching 230-350 ng mg(-1) protein. Among organelles, mitochondria were the main target for Cd bioaccumulation in gills (250-300 ng mg(-1) protein), whereas in hepatopancreas tissues, the highest cadmium accumulation occurred in lysosomes (90-94 ng mg(-1) protein). Although 75-83% of total cadmium burden was associated with the cytosol reflecting high volume fraction of this compartment, Cd concentrations in organelle fractions reached levels that could cause dysfunction of mitochondria and lysosomes. Organ- and organelle-specific patterns of cadmium bioaccumulation support our previous in vivo studies, which showed adverse effects of cadmium exposures on mitochondrial oxidation in gills and on the lysosomal system of hepatopancreas. This may have important implications for the development of biomarkers of effect for heavy metals and for understanding the mechanisms of toxic effects of metals.     

Effects of long term sublethal Cd exposure in rainbow trout during soft water exposure: implications for biotic ligand modelling

The objectives of the study were to determine the physiological and toxicological effects of chronic cadmium exposure on juvenile rainbow trout in soft water. Particular attention focused on acclimation, on comparison to an earlier hard water study, and on whether a gill surface binding model, originally developed in dilute soft water, could be applied in this water quality to fish chronically exposed to Cd. Juvenile rainbow trout, on 3% of body weight daily ration, were exposed to 0 (control), 0.07, and 0.11 microg l(-1) Cd [as Cd(NO(3))(2).4H(2)O] in synthetic soft water (hardness=20 mg l(-1) as CaCO(3), alkalinity=15 mg l(-1) as CaCO(3), pH 7.2) for 30 days. Mortality was minimal for all treatments (up to 14% for 0.11 microg l(-1) Cd). No significant effects of chronic Cd exposure were seen in growth rate, swimming performance (stamina), routine O(2) consumption, or whole body/plasma ion levels. In contrast to the hard water study, no acclimation occurred in either exposure group in soft water, with no significant increases in 96-h LC(50) values. Cadmium accumulated in a time-dependent fashion to twice the control levels in the gills and only marginally in the liver by 30 days. No significant Cd accumulation occurred in the gall bladder or whole body. Cadmium uptake/turnover tests were run using radioactive 109Cd for acute (3 h) exposures. Saturation of the gills occurred for control fish but not for Cd-exposed fish when exposed to up to 36 microg l(-1) Cd for 3 h. Cd-exposed trout accumulated less 'new' Cd in their gills compared to controls and they internalized less 109Cd than control fish. This effect of lowered Cd uptake by the gills of acclimated trout was earlier seen for the fish acclimated to 10 microg l(-1) Cd in hard water. The affinity of the gill for Cd was greater in hard water (logK(Cd-gill)=7.6) than in soft water (logK(Cd-gill)=7.3) but the number of binding sites (B(max)=0.20 microg g(-1) gill) was similar in both media. In addition, there was a shift in affinity of the gill for Cd (i.e. lowered logK(Cd-gill)) and increased B(max) with chronic Cd exposure in both soft water and hard water. We conclude that the present gill modelling approach (i.e. acute gill surface binding model or Biotic Ligand Model) does work for soft and hard water exposures but there are complications when applying the model to fish chronically exposed to cadmium.     

Morphological and biochemical changes in the gills of Tilapia (Oreochromis mossambicus) to ambient cadmium exposure

Tilapia, Oreochromis mossambicus, were reared in freshwater and exposed to 40, 80 and 160 ppb ambient cadmium for a period of 7 days to investigate the effects of short-term Cd exposure on the cellular function of gill chloride cells. Gills were sampled after 3 and 7 days exposure. The accumulated Cd concentration in gills was analyzed using a graphite furnace atomic absorption spectrophotometer while the morphological changes of pavement and chloride cells were examined using scanning and transmission electron microscopes. Gill Cd concentration was significantly increased in samples (10.03-44.36 ppb) at 3 and 7 days exposure. Accompanying this was an augmentation of microridge in pavement cells, and an increase in the density (1964-3603 /mm(2)) and apical membrane area (11.57-46.32 μm(2)) in chloride cells, indicating an adaptational modification of the cell morphology in assisting gaseous transfer and Ca(2+) uptake, respectively. However, biochemical analyses of the gill tissues enumerated a decrease in both the activities of alkaline and high-affinity Ca(2+)-ATPases. This indicated a reduction in the Ca(2+)-transport capacity per unit chloride cell, suggesting chloride cells being the primary target of Cd which subsequently lead to fish hypocalcemia.     

Modes of metal toxicity and impaired branchial ionoregulation in rainbow trout exposed to mixtures of Pb and Cd in soft water

Models such as the Biotic Ligand Model (BLM) predict how natural organic matter (NOM) and competing ions (e.g., Ca(2+), H(+) and Na(+)) affect metal bioavailability and toxicity in aquatic organisms. However, such models focus upon individual metals, not metal mixtures. This study determined whether Pb and Cd interact at the gill of rainbow trout (Oncorhynchus mykiss) when trout were exposed to environmentally relevant concentrations of these metals (Cd<100nmolL(-1); Pb<500nmolL(-1)) in soft (<100mumolCa(2+)L(-1)), moderately acidic (pH 6.0) water. The 96-h LC50 for Pb was 482nmolL(-1), indicating that Pb was one-order of magnitude more toxic in soft, acidic water than in harder, circumneutral pH waters. The LC50 for Cd alone was also low, 6.7nmolL(-1). Surprisingly, fish acclimated to soft water had multiple populations of Pb-gill and Cd-gill binding sites. A low capacity, high affinity population of Pb-gill binding sites had a B(max) of 18.2nmolg(-1) wet weight (ww) and apparent logK(Pb-gill)=7.05, but a second low affinity population could not be saturated up to free Pb concentrations approaching 4000nmolL(-1). Two populations of Cd-gill binding sites were characterized: a high affinity, low capacity population with an apparent logK(Cd-gill)=7.33 and B(max)=1.73nmolg(-1) ww, and a low affinity, high capacity population with an apparent logK(Cd-gill)=5.86, and B(max)=13.7nmolg(-1) ww. At low concentrations, Cd plus Pb accumulation was less than additive because Cd out-competed Pb for gill binding sites, which were likely apical Ca(2+)-channels. While disturbances to Ca(2+) influx were caused by Cd alone, Pb alone had no effect. However, Pb exacerbated Cd-induced disturbances to Ca(2+) influx demonstrating that, although Pb- plus Cd-gill binding was less than additive due to competition, the effects (ionic disturbances) were more than additive (synergistic). Pb was also likely binding to intracellular targets, such as branchial carbonic anhydrase, which led to inhibited Na(+) influx. This ionic disturbance was exacerbated by Cd. We conclude that exposure to environmentally relevant concentrations of Pb plus Cd results in less than additive metal-gill binding in soft, moderately acidic waters. However, ionic disturbances caused by Cd plus Pb are greater than additive, and this may ultimately increase the toxicity of Cd-Pb mixtures to fishes. Our findings suggest that it may be necessary to re-evaluate water quality criteria and assumptions of the BLM for fish exposed to mixtures of Pb and Cd in the acidic, soft waters found in the Canadian Shield, Scandinavia and other sensitive regions.     

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.     

Cadmium-handling strategies in two chronically exposed indigenous freshwater organisms--the yellow perch (Perca flavescens) and the floater mollusc (Pyganodon grandis)

Laboratory experiments on a variety of aquatic organisms suggest that metallothionein-like proteins (MT) play an important role in the regulation of essential metals, and in the sequestration and detoxification of non-essential metals (e.g., Cd). However, the importance of metallothionein production relative to alternative strategies of metal detoxification, and its effectiveness in metal detoxification, remain largely unexplored in field situations. In the present study we explored metal-handling strategies in an adult benthic bivalve (Pyganodon grandis) and in juvenile yellow perch (Perca flavescens), exposed to Cd in their natural habitat. The two biomonitor species were collected from lakes located along a Cd concentration gradient. Ambient dissolved Cd concentrations were determined by in situ dialysis as a measure of metal exposure. Sub-cellular Cd partitioning was determined in target tissues (bivalve gills and digestive gland; perch liver) by differential centrifugation, and metallothionein was measured independently by a mercury-saturation assay in the bivalve tissues. Malondialdehyde concentrations were measured as a potential indicator of oxidative stress. Ambient dissolved Cd concentrations ranged from 0.06 to 0.57 nM in the nine lakes from which bivalves were collected, and from < 0.3 to 6.7 nM in the eight lakes from which yellow perch were sampled. Bioaccumulated Cd also varied from lake to lake, more so for the bivalve than for the yellow perch; the [Cd]max/[Cd]min ratios for the various tissues decreased in the order: bivalve gill Cd 28 > bivalve digestive gland Cd 18 > perch hepatic Cd 14. In the two lakes that were common to both the bivalve and perch studies, i.e. lakes Opasatica and Vaudray, accumulated Cd concentrations were consistently higher in the bivalve than in the perch. Cadmium-handling strategies were similar in the bivalve digestive gland and perch liver, in that Cd was mainly associated with the heat-stable protein (HSP) fraction. Furthermore, in these organs the contributions from the "mitochondria" and "lysosomes + microsomes" fractions were consistently higher than in the gill tissue. In the bivalve gill, the HSP fraction could only account for a small proportion (10+/-3%) of the total Cd burden, and the metal was instead largely sequestered in calcium concretions (58+/-13%). Along the Cd-exposure gradient, Cd detoxification appeared to be reasonably effective in the bivalve gill and digestive gland, as judged from the protection of the heat-denaturable protein (HDP) fraction. However, in both organs Cd concentrations did increase in potentially metal-sensitive organelles (mitochondria), and malondialdehyde concentrations increased along the exposure gradient in the gills (but not in the digestive gland). Cadmium detoxification seemed less effective in juvenile yellow perch. As total hepatic Cd increased, Cd concentrations increased in all sub-cellular fractions, including the HDP fraction that was well protected in the bivalve. The relative proportions of Cd in the various fractions did not vary appreciably along the exposure gradient and there was no evidence of a threshold exposure concentration below which sensitive metal pools were protected.     

Sub-cellular partitioning of metals (Cd, Cu, Zn) in the gills of a freshwater bivalve, Pyganodon grandis: role of calcium concretions in metal sequestration

Indigenous unionid molluscs, Pyganodon grandis, were collected from nine lakes in the Rouyn-Noranda area (Quebec, Canada) along a polymetallic concentration gradient (Cd, Cu, Zn). After excision, the gills were gently homogenised and the cellular compartments were separated by a differential centrifugation procedure that yielded the following particulate fractions: "nuclei + cellular debris", "mitochondria", "lysosomes + microsomes" and "granules". The supernatant remaining after the final ultracentrifugation step, i.e., the operationally-defined cytosol, was separated into a "heat-denaturable proteins" (HDP) fraction and a "heat-stable proteins" (HSP) fraction containing metallothionein (MT). The Cd, Cu and Zn content of each particulate and cytosolic fraction was determined and gill metallothionein was quantified independently by a mercury saturation assay. Cytosolic Cd concentrations were significantly related to the dissolved Cd concentrations at each site, but cytosolic Cu and Zn (essential metals) were not related to their respective ambient dissolved metal concentrations. Metallothionein concentrations increased along the metal contamination gradient and were related to cytosolic Cd (and Zn) in a concentration-dependent manner. However mass balance calculations showed that binding to metallothionein could only account for a small proportion of total gill metal ( approximately 10% Cd; approximately 3% Cu; approximately 1% Zn). Under these chronic exposure conditions, the three metals (Cd, Cu and Zn) were mainly located in calcium concretions present in the gills (respectively 58 +/- 13% of the total gill Cd, 64 +/- 6% of the total gill Cu and 73 +/- 6% of the total gill Zn). The overall contribution of granules to the total gill dry weight remained relatively constant among the different lakes, suggesting that lake-to-lake variations in granule synthesis were independent of the metal contamination gradient, i.e., these constituent elements of unionid gills act as non-inducible metal sinks at the cellular level. Metal concentrations increased proportionally in both the granules and the MT pool along the polymetallic gradient, suggesting a constant partitioning between these two compartments. Overall, despite an increase in Cd in the "mitochondria" fraction, metal sequestration mechanisms seem to be reasonably effective in detoxifying cadmium: in the cytosol, Cd concentrations in the potentially metal-sensitive HDP fraction remained relatively low and constant, even in specimens collected from the most contaminated lakes.     

Bioavailability of Cd to the common carp, Cyprinus carpio, in the presence of humic acid

The uptake of cadmium by the common carp, Cyprinus carpio, was studied in chemically defined freshwater in the absence and presence of commercial humic acid. This was done to evaluate whether the cadmium uptake by carp in the presence of humic acid was related to the ambient Cd2+ -ion activity or whether the complexed metal also contributed to the uptake. Uptake of Cd during a 3-h period of exposure was used as a measure of the biological availability of the metal. The uptake rate data for Cd in total fish and gills obtained in the absence (control) and presence (treatment) of humic acid were analyzed using a Michaelis-Menten model for mediated transport. The Michaelis-Menten parameters KM and Vmax obtained in the control and the treatment experiment were compared for each of the two investigated carp compartments (total fish and gills). The model parameter estimates for Cd uptake by total carp in the treatment experiment (KM = 0.41 +/- 0.11 micromol l(-1); Vmax = 0.66 +/- 0.13 micromol kg(-1) h(-1)) were not significantly different from the model parameters in the control experiment (KM = 0.34+/-0.06 micromol l(-1); Vmax = 0.58+/-0.07 micromol kg(-1) h(-1)) on the basis of Welch's approximate t-test. Similarly, the Michaelis-Menten model parameter estimates for Cd uptake by carp gills in the treatment experiment (KM = 0.15 +/- 0.06 micromol l(-1); Vmax = 5.14 +/- 1.07 micromol kg(-1) h(-1)) were not significantly different from the model parameters derived from the control experiment (KM =0.27 +/- 0.09 micromol l(-1); Vmax = 7.63+/-1.38 micromol kg(-1) h(-1)). This indicated that the Cd uptake rate by total carp and in carp gills in the presence of commercial humic acid followed the measured variations in Cd2+ -ion activity as predicted by the control experiment.     

Influence of long-term exposure to dietary cadmium on growth, maturation and reproduction of goldfish (subspecies: Prussian carp Carassius auratus gibelio B.)

The influence of long-term exposure of goldfish to dietary cadmium (Cd) on its accumulation in tissues, growth, ovarian development, luteinizing hormone (LH) secretion and a response to hormonal stimulation of spawning were evaluated. The study was conducted on four groups of females for the period of 3 years, from the age of 10 weeks to second spawning. Four doses of Cd were applied in the feed: 0 (control group), 0.1, 1 and 10 mg Cd g(-1) of feed (wet weight). The highest dose of Cd (10 mg g(-1)) inhibited growth and caused several behavioural effects. In contrast, lower dose of Cd (1 mg g(-1)) stimulated fish growth. The doses of Cd from 0.1 to 1 mg Cd g(-1) did not influence ovarian development. The gonado-somatic index (GSI) and histological analysis of ovaries showed no differences in ovarian development between the control group and the groups receiving these doses of Cd. However, in the group receiving the highest Cd dose, GSI decreased. This was associated with persistent, long-lasting elevation of plasma LH levels. Ovulation did not occur in this group. Injections of salmon GnRH-analogue (sGnRHa) alone or with domperidone (a dopamine receptor antagonist) in sexually mature fish caused an increase of LH levels in all groups, although in the group fed with the highest Cd dose the effect was weaker than in the other groups. After the first spawning season, a negative effect of lower Cd doses (0.1 and 1mg Cd g(-1)) on ovarian recrudescence (rebuilding of ovaries) and on the response to the consecutive hormonal stimulation of spawning was observed (lower number of ovulating females). There was a significantly higher content of Cd in the livers of fish than in their muscles. The results of hormonal stimulation of spawning and histological analysis of ovaries suggest that in goldfish cadmium acts mainly at the level of ovary rather than on the pituitary gland. We suppose that in the natural environment cadmium present in the feed can play an important role in the accumulation of this element in fish tissues and can influence vital physiological processes.     

Interaction between accumulation of cadmium selenium in the tissues of turbot Scophthalmus maximus

The interaction between accumulation of waterborne cadmium and selenite in juvenile turbot, Scophthalmus maximus, was investigated in the laboratory. Intestine, kidney and liver of turbot exposed to 150 μg Cd 1⁻¹ accumulated cadmium linearly with time over 5 wk at rates of 0.50, 0.014 and 0.11 μg Cd g⁻¹ dry wt. d⁻¹, respectively. Gills, skin and muscle reached steady-state cadmium levels of ca. 15, 0.8 and 0.25 μg Cd g⁻¹ after 1–3 wk of exposure. Plasma and erythrocytes reached steady-state concentrations of ca. 0.1 and 1–2 μg Cd ml ⁻¹ after 1–2 wk of exposure. Exposure to 105 μg Se-SeO₃²⁻1⁻¹ did not consistently alter selenium concentrations in gills, skin, liver, muscle and erythrocytes of juvenile turbot. Kidneys accumulated selenium linearly (0.029 μg Se g⁻¹ dry wt. d⁻¹) with time over 5 wk, while intestine reached a steady-state level after 2 wk. Selenium concentrations in the plasma were maintained close to the ambient level throughout the exposure time. Concurrent exposure to selenite augmented cadmium accumulation rates in gills, kidney and liver and reduced cadmium accumulation in intestine and erythrocytes; cadmium accumulation in spleen, skin, muscle and plasma was not affected. Concurrent exposure to cadmium depleted erythrocytes and partly skin of selenium and reduced accumulation of selenium in kidney and plasma, whereas selenium accumulation patterns in gills, intestine, liver, muscle and spleen were not affected by exposure to cadmium.     

In vitro characterization of cadmium and zinc uptake via the gastro-intestinal tract of the rainbow trout (Oncorhynchus mykiss): Interactive effects and the influence of calcium

An in vitro gut sac technique was employed to study whether Cd and Zn uptake mechanisms in the gastro-intestinal tract of the rainbow trout are similar to those at the gills, where both metals are taken up via the Ca transport pathway. Metal accumulation in surface mucus, in the mucosal epithelium, and transport into the blood space were assayed using radiolabelled Cd or Zn concentrations of 50micromolL(-1) in the luminal (internal) saline. Elevated luminal Ca (10 or 100mmolL(-1)versus 1mmolL(-1)) reduced Cd uptake into all three phases by approximately 60% in the stomach, but had no effect in the anterior, mid, or posterior intestine. This finding is in accordance with recent in vivo evidence that Ca is taken up mainly via the stomach, and that high [Ca] diets inhibit Cd accumulation from the food specifically in this section of the tract. In contrast, 10mmolL(-1) luminal Ca had no effect on Zn transport in any section, whereas 100mmolL(-1) Ca stimulated Zn uptake, by approximately threefold, into all three phases in the stomach only. There was no influence of elevated luminal Zn (10mmolL(-1)) on Cd uptake in the stomach or anterior intestine, or of high Cd (10mmolL(-1)) on Zn uptake in these sections. However, high [Zn] stimulated Cd transport into the blood space but inhibited accumulation in the mucosal epithelium and/or mucus-binding in the mid and posterior intestine, whereas high [Cd] exerted a reciprocal effect in the mid-intestine only. We conclude that Cd uptake occurs via an important Ca-sensitive mechanism in the stomach which is different from that at the gills, while Cd transport mechanisms in the intestine are not directly Ca-sensitive. Zn uptake does not appear to involve Ca uptake pathways, in contrast to the gills. These results are discussed in the context of other possible Cd and Zn transport pathways, and the emerging role of the stomach as an organ of divalent metal uptake.     

Tissue-specific cadmium and metallothionein levels in freshwater crab Sinopotamon henanense during acute exposure to waterborne cadmium

The freshwater crabs Sinopotamon henanense were exposed to different concentrations of waterborne cadmium (Cd). The relationship between tissue-specific Cd accumulation and metallothionein (MT) induction was investigated using the Cd saturation assay and atomic absorption spectrophotometry method. The results showed that Cd accumulation rose significantly in all tissues studied after Cd exposure, and the Cd accumulation level in various tissues followed the following order: gill > hepatopancreas > muscle > ovary. MT levels were clearly tissue-specific after Cd exposure. Hepatopancreas was found to have the highest MT level, followed by the gill, muscle, and ovary. In conclusion, the results indicated although Cd exposure clearly resulted in MT induction, its synthesis does not correlate with Cd accumulation in the later stage of Cd exposure. The calculated ratios of actual Cd to theoretical maximum Cd-MT in the hepatopancreas were <1.0 under acute waterborne Cd at all sampling points, indicating that the hepatopancreas had much greater Cd-binding potential of MT than the gill, muscle, or ovary. It is clear from our results that a positive correlation was shown between MT induction and Cd accumulation both in hepatopancreas and gill. Therefore, MT induction can be considered as a biomarker for acute waterborne Cd pollution.     

Accumulation of nitrate in the tissues of Penaeus monodon following elevated ambient nitrate exposure after different time periods.

Penaeus monodon (10.56 +/- 0.71 g) exposed individually to 0.026 (control), 0.774, 3.646, 7.004, 21.234 and 36.079 mM nitrate in 25 ppt seawater were examined for nitrate accumulation in hemolymph, gill, eyestalk, heart, foregut, midgut, hepatopancreas and muscle after 1, 3, 6, 12 and 24 h, respectively. Concentration of nitrate in the tissues increased directly with ambient nitrate concentration and exposure time except for muscle. Following 24-h exposure to 3.646 mM nitrate, nitrate concentrations measured in the muscle (0.202 micromol g(-1)), hepatopancreas (0.330 micromol g(-1)), heart (0.527 micromol g(-1)), foregut (0.632 micromol g(-1)), gill (0.927 micromol g(-1)), hemolymph (0.946 micromol ml(-1)), eyestalk (1.214 micromol g(-1)), and midgut (1.529 micromol g(-1)), respectively. The concentration of muscle nitrate was the lowest, and the concentration of midgut nitrate was the highest among the tissues tested. P. monodon following exposure to ambient nitrate at 21.234 and 36.079 mM, nitrate accumulation measured in the muscle (0.854 and 0.980 micromol g(-1)), hepatopancreas (1.139 and 1.552 micromol g(-1)), heart (1.468 and 1.879 micromol g(-1)), foregut (2.195 and 3.341 micromol g(-1)), gill (2.398 and 3.325 micromol g(-1)), hemolymph (3.327 and 3.948 micromol ml(-1)), eyestalk (3.461 and 4.264 micromol g(-1)) and midgut (3.343 and 5.239 micromol g(-1)), and never reached a plateau after 24 h.     

Xanthate effects on cadmium intracellular distribution in rainbow trout (Oncorhynchus mykiss) gills

The uptake of ¹⁰⁹cadmium through perfused rainbow trout gills in the presence of xanthates was studied, and the subcellular distribution of cadmium in perfused gill tissue was determined. Pnenol absorption was also studied because xanthates form hydrophobic Cd complexes with a log P_{octanol/water} similar to that of phenol.

1. Xanthate concentrations higher than 10⁻⁵ M increased the rate of cadmium transfer through the gills and cadmium retention in gill tissue. Cadmium was present as a hydrophobic complex at this and higher xanthate concentrations.

2. A redistribution of cadmium from metallothionein to high molecular weight cadmium binding fractions occurred in the presence of 10⁻⁴ M xanthate.

3. The rate of phenol transfer across the gill epithelium was much higher than the rate of cadmium transfer regardless of whether xanthate was present. The rate of phenol transfer stabilized much faster than the rate of cadmium transfer irrespective of whether xanthate was present, indicating that different uptake mechanisms were involved.

We conclude that in the presence of xanthate concentrations higher than 10⁻⁵ M cadmium is taken up as a hydrophobic Cd(xanthate)₂ complex by the epithelial cells. Within the cell the complex dissociates, and the metal ion is bound to intracellular cadmium-binding ligands. The metal is probably translocated through the basolateral membrane as a free ion.     

Cadmium, zinc and the uptake of calcium by two crabs, Carcinus maenas and Eriocheir sinensis

The uptake of dissolved cadmium and zinc by crustaceans can usually be explained by the passive process of facilitated diffusion involving a transport protein in the membranes of permeable surfaces. Cadmium ions will also enter via uptake routes for calcium, given the similar size of the two free ions. This study has investigated the interaction of cadmium (and comparatively zinc) and calcium uptake in two crabs that show different permeability responses to changes in salinity, with consequently different effects on the uptake of cadmium and zinc with salinity change. Ca uptake rates in Carcinus maenas decreased in reduced salinity (33-15) with the decreased Ca concentration of the medium and increased if the Ca concentration was increased at salinity 20. It is concluded that Ca uptake over the salinity range 33-15 is via apical Ca channels in gill ionocytes, passively down an electrochemical gradient. The Ca uptake rate of Eriocheir sinensis showed no significant decrease over the salinity range 33-10 (probably because of the small differences in an already low Ca uptake rate in this crab against a background of inter-individual variability), but decreased significantly at salinity 5. Added calcium increased the Ca uptake rate of E. sinensis at salinities 15 and 5, supporting the interpretation that Ca uptake in gills is typically passive via apical Ca channels. Cadmium (but not zinc) inhibited calcium uptake in both crabs at 15 salinity, indicating sharing of Ca channels by Cd, but not at salinity 5 (E. sinensis only) when Ca may be taken up into gill ionocytes by another (active?) physiological process.