Calculating equilibrium constants for metals and competing ions to the biological receptor in Hyalella azteca.

Dixon, G¹, U. Borgmann² and J. Schroeder1.

¹ University of Waterloo, Waterloo, ON,
² National Water Research Institute, Burlington, ON.

Binding constants for nickel and competing ions to Hyalella azteca were estimated as part of the development of a model to predict nickel toxicity. To estimate binding constants, adult Hyalella were exposed for seven days to nickel nitrate in three separate experiments that varied nickel, hydrogen or calcium concentrations while maintaining constant concentrations of the other two ions. Conditions of the exposures and the media used were identical in all experiments except for the concentration of the ion of interest. Nickel concentrations in tissue were related to concentrations of nickel, hydrogen and calcium in media by a Michaelis-Menton form of equation. Transformation of this equation allowed estimation of apparent binding constants for all three ions by linear regression, with X as the concentration of nickel, calcium or hydrogen in the medium and Y as the ratio of nickel in the medium to nickel in the tissue. However, binding constants estimated using this method were influenced by variability and possible outliers in the data set. To reduce the influence of data variability and outliers, apparent binding constants were recalculated using the median method. This method uses each possible combination of X and Y to estimate many values for each binding constant. The median value is then used as the estimated apparent binding constant. The relatively good fit of the estimates to the model demonstrated that biotic ligand modelling is possible in Hyalella and that the median method is suitable method for estimating binding constants.

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Subcellular partitioning of metals (Cd, Cu, Ni, Zn) in indigenous yellow perch collected along a polymetallic environmental gradient

Giguère, A., P.G.C. Campbell, L. Hare

Université du Québec, INRS-Eau, QC

This project is designed to test the prediction that relations between metal accumulation and metal-induced effects will be improved if the metal body concentrations are expressed not as total metals, but rather in terms of the metal's partitioning within particular target tissues (e.g., gills, liver, kidney). We are testing this prediction with indigenous fish (yellow perch: Perca flavescens) collected from 8 lakes with contrasting metal levels (especially Cd, Zn, and Ni) but similar trophic status.

Eight lakes were visited in the Rouyn-Noranda and Sudbury areas in June 2001. Water samples were collected by in situ dialysis in the epilimnion of each of these lakes to evaluate ambient metal levels. Juvenile perch (<10 g; N=120), a life stage that is exclusively planktivorous, were also collected in the 8 lakes. Trends in biomarkers (malondialdehyde, glutathione-peroxidase and glutathione-reductase) were quantified. Various cellular fractions of liver tissues were isolated by differential centrifugation (granules, nuclei and debris, larger organelles, microsomes, heat-sensitive and heat stable proteins) and metal levels were measured in each.

Metal subcellular partitioning results show a preferential accumulation of Cd and Cu in the subcellular fraction containing metallothioneins. An increase in the contribution of mitochondria and lysosomes to total Cd burden suggests that mitochondria might be a site of toxic effects. The two other studied metals, Zn and Ni, are more accumulated in the fraction containing heat-denatured proteins, which include many metallo-enzymes. Liver malondialdehyde concentrations and glutathione-reductase activity both decrease with increasing tissue Cu. Together, these results suggest an effect of high metallothionein levels on the concentrations of reactive oxygen species in the liver cells.

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Disruption of HPI axis in 1+ yellow perch (Perca flavescens) exposed to metals.

Gravel A.¹, P.G.C. Campbell ², and A. Hontela ¹.

¹-Département des Sciences biologiques, TOXEN Research Center, Université du Québec à Montréal, Montréal, Qc.
²-INRS-Eau, Université du Québec, Ste-Foy, Qc.

Recently, we have reported that adult fish chronically exposed to metals in the environment exhibit an endocrine impairment characterised by a blunted cortisol secretion. Impaired cortisol secretion has also been detected in interrenal cells acutely exposed to Cd in vitro. The vulnerability of young fish to metal exposure has not been investigated so far, even though stability of fish populations depends on survival of young fish. Young of the year (YOY, body weight < 0.08g) and 1+ (body weight ~2.5g) yellow perch (P. flavescens) were captured in six lakes situated along a contamination gradient of Cd, Zn and Cu in the Abitibi mining region. They were subjected to a standardised stress test or an ACTH in vivo injection and sampled. Significantly higher Cd body burdens were detected in YOY and 1+ perch from contaminated lakes compared to reference lakes. Contaminated 1+ fish had an impaired capacity to respond to an acute stress, as measured by a lower total body cortisol level and a reduced mobilisation of glycogen reserves. Contamination did not have an effect on total body thyroid hormones levels, condition factor and hepato-somatic index (HSI). These results indicate that effects of environmental levels of metals are detectable after only one year of exposure in yellow perch. To compare the sensitivity of the interrenal cells to Cd in young and adult perch, the effects of a 60 min in vitro exposure on cortisol secretion were determined using a bioassay developed in our laboratory. Preliminary results of in vitro Cd exposure of interrenal cells from 1+ perch show that Cd inhibited ACTH-stimulated cortisol secretion in a dose-dependent manner. Cortisol secretion was inhibited at concentrations that did not affect cell survival. The LC50 and EC50 values of 1+ perch were similar to adult perch. The in vitro studies, combined together with field investigations will enable us to provide a mechanism-driven assessment of the impacts and risks associated with chronic environmental exposure to metals in young fish.

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Effect of ration quantity on tissue burden and cadmium toxicity in rainbow trout

Ho, C.G., K.A. Sloman, C.M. Wood, D.G. McDonald

While cadmium uptake and toxicity during chronic waterborne exposure has been extensively investigated in fish, the interaction of Cd exposure and ration quantity on growth, Cd accumulation and toxicity has received little attention Since water chemistry is known to influence Cd toxicity, it is possible that individual differences in growth rate may also affect toxicity. Therefore, the objective of the present study was to examine the relationship between growth, cadmium accumulation, and toxicity in individuals (identified through implanted PIT tags) by manipulating ration quantity. Fish in experiment 1 were fed 1% or 3% of their body weight daily and experiment 2 included an additional group that was fasted while being exposed to cadmium (~4 g/L). Overall toxicity was much greater in experiment 1 than 2 because of a difference in ambient temperature. 150C vs. 100C, consistent with previous findings (Eisler, 1971). In experiment 1, there was a sharp increase in acute Cd toxicity over 48h (80% vs. 13%) with the higher ration, and the survivors on the higher ration (sampled at 48h) accumulated a significantly higher level of cadmium in their kidney than fish fed a 1% ration. In experiment 2, at 10 0 C, mortality was much lower at the two ration levels; 18% by 18 days on the 3% ration vs. 8% on the 1% ration, while the fasted fish showed 63% mortality. In survivors, specific growth rate over the 18 day s of Cd exposure was proportional to ration (-1.2 ±0.13, 0.8 ±0.07 and 2.1 ±0.10% day -1 at 0, 1 and 3% ration, respectively). Good correlations between individual growth and cadmium accumulation in various tissues were also observed. Accumulation was inversely correlated with growth rate in the liver and kidney (r2= 0.49 and 0.54, respectively) but positively correlated (r2= 0.29) for the gill. Tissue Cd accumulation, however, was not indicative of mortality. Individuals with high growth rates show low levels of chronic Cd accumulation in target tissues but high levels of mortality. This study provides evidence that differences in growth rate can alter cadmium accumulation and toxicity. The results of this study suggest that risk assesssment of waterborne metals should look beyond tissue burdens and include nutritional factors and individual variation. This work was funded by NSERC Strategic Program, ICA, ILZRO, Falconbridge Ltd., Cominco Ltd., and the MITE Research Network.

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The importance of a diverse prey base for efficient energy transfers to fish in lakes recovering from metal contamination

Iles A., J. Rasmussen

McGill University, Department of Biology, Montreal, PQ

Fish living in metal-contaminated lakes are subject to the challenge of a simplified food web. A poorly understood component of food web theory is how the efficiency of energy transfer to top trophic compartments is affected by a reduction in the number of energetic pathways (due to species loss). It has been suggested that the trophic linkages that evolve over time in natural food webs may represent the most efficient pathways of energy transfer. If so, a reduction in biodiversity could compromise the amount of energy transferred to top trophic levels or the number of energetic pathways. Recent research has shown that a naturally diverse prey base is important for maintaining energy transfer to growing fish. Perch (Perca Flavescens) in healthy lakes with a diverse invertebrate community benefit from the energetic advantage of switching to progressively larger prey items as they grow until they are large enough to switch to piscivory. Simplified food webs caused by metal contamination reduce the efficiency of energy transfer to yellow perch and lead to energetic bottlenecks (poor growth) and stunting of the perch.

Here we present preliminary evidence from an investigation into the importance of a diverse prey base for efficient energy transfer to top trophic levels in 15 lakes that are in various stages of recovery from metal contamination. Lactate dehydrogenase (LDH) levels were measured in the white muscle tissue of yellow perch and used as a proxy for fish activity. Comparisons with stomach contents and fish size at age data indicate higher growth rates and a greater number of diet shifts for perch in lakes that are in later stages of recovery. To date, all the available evidence suggests that lakes in later stages of recovery from heavy metal pollution have more diverse invertebrate communities and less-stunted perch than lakes that are still heavily affected by metal contamination

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Effects of dietary quality on the response to waterborne copper and cadmium exposure in trout: implications for Biotic Ligand Model-based risk assessment

Kamunde C., A. Matsuo, B. Baldisserotto, G. Pyle, D.G. McDonald, and C. M. Wood
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1

Acute toxicity of waterborne copper (Cu) and cadmium (Cd) results from the interference with homeostatic mechanisms for sodium (Na+) and calcium (Ca2+), respectively. Although the modifying effects of water quality factors such as pH, Ca2+, Na+, and dissolved organic matter on toxicity of these metals have been characterized, little is known about the effects of dietary quality factors on the response to waterborne metals exposure. We evaluated the effects of elevated dietary Na+ and Ca2+, respectively, during waterborne Cu and Cd exposures in rainbow trout to test the hypotheses that dietary Na+ and Ca2+ modulate the toxicity of these metals. Juvenile rainbow trout were fed diets containing elevated levels of Na+ as NaCl, and Ca2+ as CaCl2 for 7 days, followed by a challenge with 20 mg/L waterborne Cu (dietary Na+ exposed fish) and 50 mg/L Cd (dietary Ca2+ exposed fish). Unidirectional uptake rates of Na+, Ca2+, Cu, and Cd were assessed using direct flux measurements. Results obtained indicated that dietary Na+ pre-exposure reduced both waterborne Cu and Na+ uptake, while dietary Ca2+ reduced the uptake of waterborne Cd and Ca2+. We concluded that Na+ and Cu, and Ca2+ and Cd, respectively, share common uptake pathways at the gills. Binding of both Cu and Cd to the gills were reduced by dietary Na+ and Ca2+ pre-exposure, respectively, suggesting that dietary quality factors are important in determining metal accumulation to the gill, the primary target for acute toxicity. Metal binding to gills, under the Biotic Ligand Modeling (BLM) approach, is currently being developed as a tool for risk assessment. Because the BLM predicts the amount of metal bound to the gill that causes acute toxicity in a particular water quality, dietary quality parameters that modify metal binding to the gills could be important considerations in BLM-based risk assessment procedures. Ongoing studies are testing the hypothesis that fish under metal stress select diets rich in mineral content as a protective measure against toxicity of waterborne metals. (Supported by MITE-RN).

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Metals & aquatic communities: Interactions between metal exposure & fish diet

J. Kovecses, G. Sherwood and J.B. Rasmussen
McGill University, Department of Biology

Fish can accumulate metals from their diet, but there have been few investigations into how this exposure will change as fish diet changes. Benthic invertebrates are important prey items in the diets of many freshwater fish. As a result of living in metal-contaminated sediments, benthic invertebrates tend to accumulate high concentrations of metals in polluted systems. Over the course of growth, many fish switch from a planktivorous diet to a benthivorous diet. This switch may potentially increase their exposure to metals due to feeding on more contaminated prey. We collected invertebrates and perch (Perca flavescens) from lakes along a gradient of metal pollution in Rouyn-Noranda, QC., which has a long history of Cu smelting. Using stable isotope analysis, gut content analysis and metal analysis of perch livers, we determined how the perch exposure to metal changes as they switch from a planktivorous diet to a littoral, benthic diet. We show the proportion of benthivory in the diet is correlated to the concentration of metals in the liver of perch.

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Metal dynamics in juvenile yellow perch - a caging study

Kraemer, L., P.G.C. Campbell, L. Hare

INRS-ETE, Ste-Foy, QC

A pilot-scale enclosure experiment was used to determine the feasibility of using habitat-swap experiments to study metal (Cd, Cu and Zn) uptake and elimination dynamics in juvenile yellow perch (Perca flavescens). Measurements of metal exchange rates between organisms and their environment should allow us to predict response times to changes in ambient metal concentrations. Cages were constructed (1.7m diameter, 2.5m deep) and placed in Lake Ontario (reference lake) and Lake Dufault (contaminated lake) in the Rouyn-Noranda area in northwestern Quebec. The cages were suspended from floating rafts (4 cages per raft, 3 rafts per lake). Juvenile yellow perch were collected from Lake Opasatica and Lake Dufault and were either transplanted to cages (25-30 per cage) within that lake (caged controls) or reciprocally transplanted (transplanted fish). In addition to these samples, indigenous yellow perch were also collected from both Lake Dufault and Lake Opasatica in July and August, representing non-caged controls. Due to the extremely high mortality observed with yellow perch from Lake Dufault (almost 100% mortality observed after 3-4 days for both the transplanted and control fish), it was not possible to collect data on metal depuration. However, sufficient numbers of Lake Opasatica fish survived to allow us to study metal uptake dynamics. Tissues from gills, liver, kidney and gastrointestinal tract were collected over a period of 30 days. Preliminary results suggest that after 30 days, the gills of transplanted fish had cadmium levels which approached those of indigenous fish from the contaminated lake. In contrast, copper and zinc demonstrated much slower rates of net accumulation in the gills. In the liver, the rates of Cd, Cu and Zn accumulation were much slower than the gills, with only a slight increase observed after 30 days of transplantation.

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The in vitro effect of cadmium on cortisol secretion by interrenal cells from Rainbow trout (Oncorhynchus mykiss) and Yellow perch (Perca flavescens).

Lacroix A. and A. Hontela

Département des Sciences biologiques, TOXEN Research Center, Université du Québec à Montréal, Montréal, Qc.

It is well established that cortisol secretion of fish is impaired by chronic environmental exposures to heavy metals. Field studies have shown that Yellow perch (Perca flavescens) inhabiting metal-contaminated lakes exhibit impaired cortisol secretion, in vivo and in vitro. Moreover, an in vitro study demonstrated that ACTH and dbcAMP-stimulated cortisol secretion by interrenal cells of Rainbow trout (Oncorhynchus mykiss) was significantly compromised by exposure to cadmium (Cd). There is evidence that these two species have a different sensitivity to environmental contaminants since it is recognized that Rainbow trout is one of the most sensitive fish and since Yellow perch is an ubiquitous fish in metal-contaminated lakes. The present study was designed to test the hypothesis that the effect of Cd on in vitro cortisol secretion is different in these two species. The purpose of the present study was to compare the in vitro effect of Cd on cortisol secretion by interrenal cells of Rainbow trout and Yellow perch, to assess the sensitivity of interrenal steroidogenesis in these two teleosts. Interrenal cells were exposed to Cd (0.01mM - 10mM) for 60 min, then stimulated with ACTH, the main secretagogue to the cortisol synthesis, dbcAMP, an analog of cAMP, an important second messenger in the signalling pathway for cortisol synthesis, or with pregnenolone, a cortisol precursor. The results show that Cd inhibited ACTH-stimulated cortisol secretion in a dose-dependent manner in both fish. However, the EC50s, concentration resulting in 50% inhibition, were significantly different. The Rainbow trout appears to be more sensitive (EC50 = 0.09 mM) than the Yellow perch (EC50 = 0.26 mM). On the other hand, in vitro LC50s, concentration reducing by 50% the cells viability, were not significantly different (O. mykiss LC50 = 15.8 mM and P. flavescens LC50 = 9.8 mM). These results demonstrated that Cd has a higher endocrine-disrupting potential in trout than in perch. In both species, Cd affected the ACTH, dbcAMP and pregnenolone-stimulated cortisol secretion the same way. The results show that the pregnenolone maintained the cortisol secretion until the viability was impaired. These results confirm that, for both species, Cd interferes in the signalling pathway of cortisol synthesis in a step prior to the pregnenolone formation. Data provided by the present study revealed important differences in vulnerability of interrenal steroidogenesis between Rainbow trout and Yellow perch. Mechanistic studies are important for a better understanding of the links between exposure and effects.

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Is there any relationship between critical tissue burden of cadmium and cadmium toxicity in comparing two disparate fish species?

S. Niyogi, Wood C.M. and McDonald D.G.

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1.

Water quality characteristics of natural waters can greatly influence the survivability of fish species to cadmium. In this study we compared the effect of hardness on cadmium toxicity between rainbow trout (Oncorhynchus mykiss), a model species on which the Biotic Ligand Model (BLM) is basically developed and yellow perch (Perca flavescens), an endemic species to metal contaminated sites in the Sudbury region of Northern Ontario. The acute cadmium tolerance (in terms of median lethal time or LT50) was evaluated in these two species at two different cadmium concentrations (50 & 200 mg/L) at both soft and hard water under static condition with 100% renewal after every 24 hours. Yellow perch was found to be 2-3 times and >8 times more tolerant to cadmium in soft and hard water respectively. Cadmium burden analysis in gills and whole body at the time of death in two species showed greater level of cadmium accumulation in yellow perch than rainbow trout although the apparent rate of uptake was lower in the former. A short term (3 hour) in-vivo cadmium gill binding experiment at different waterborne calcium concentration revealed lower cadmium uptake and greater inhibitory effect of calcium on cadmium binding in yellow perch than in rainbow trout, but these differences did not adequately explain the difference in cadmium toxicity. Moreover, although hard water had a significant inhibitory effect (presumably a calcium effect) on cadmium uptake in both species compared to soft water, it had a much smaller protective effect against cadmium toxicity in rainbow trout than in yellow perch. These observations led us to conclude that between two species, there is probably no relationship between critical cadmium tissue burden and mortality. This implies that the acute cadmium BLM developed in rainbow trout will need modifications to explain toxicity in yellow perch and perhaps other species.

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An in vitro approach for modelling branchial copper binding in rainbow trout.

L.N. Taylor, Baker, D.W., McDonald, D.G., Wood, C.M.

McMaster University, Hamilton, ON.

The biotic ligand model for copper takes into account the influence of water chemistry parameters on the metal fraction available for binding to fish gills. Such influences include pH, the concentration of copper, complexation by anions (e.g. HCO3-, OH- and DOM), and competition by cations (e.g. Ca2+ and H+). The amount of metal that results on the gill surface has been related to, and hence used to predict, acute copper toxicity (DiToro et al., 2000).
The main objective of this study was to characterize, in detail, the individual effects of water chemistry (Ca2+, Na+, dissolved organic matter (DOM), pH, alkalinity) on the rapid binding of copper to the gill surface of rainbow trout. An in vitro gill binding assay, modified from Reid and McDonald (1991), was utilized. In this assay individual gill arches were exposed for 5 min to 64Cu labelled copper solutions ranging from 0.02 to 0.16 M in water chemistries reflecting the full range of fresh water values for the Great Lakes. The gills displayed saturable Cu binding within this Cu range but gill Cu binding was completely unaffected over the full range of calcium, sodium and alkalinity concentrations used. Only low pH (pH 4.0) and commercial DOM (Aldrich humic acid at 3 mg C/L) altered copper binding to rainbow trout gills in vitro. These findings were consistent with the results of geochemical modelling of our water chemistry (using MINEQL+, Version 4.5), which showed that H+ and DOM affected the free cupric ion concentration. However, DOM (up to 80 mg C/L) was only able to reduce Cu on the gills by 50%. We hypothesize that in the range of 0.02 to 0.16 M Cu there are two high affinity Cu binding sites on the gills, one having a substantially higher affinity for copper than DOM. The absence of a calcium effect on gill copper binding was in accord with in vivo evidence that calcium primarily acts to alter the physiology of the gill binding sites through acclimatory processes, rather than through competitive interactions. This work was funded by N.S.E.R.C. Strategic Program, ICA, ILZRO, Falconbridge Ltd., Cominco Ltd., and the MITE Research Network.

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