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Consumption of periphyton is a potentially important route of metal exposure to benthic invertebrate grazers. The present study examined the bioaccumulation kinetics of dissolved and dietary Cd and Cu in five species of mayflies (class Insecta). Artificial stream water and benthic diatoms were separately labeled with enriched stable metal isotopes to determine physiological rate constants used by a biokinetic bioaccumulation model. The model was employed to simulate the effects of metal partitioning between water and food, expressed as the bioconcentration factor (BCF), as well as ingestion rate (IR) and metal assimilation efficiency of food (AE), on the relative importance of water and food to metal bioaccumulation. For all test species, the contribution of dietary uptake of Cd and Cu increased with BCF. For a given BCF, the contribution of food to the body burden increased with k(uf) , the metal uptake rate constant from food that combined variation in IR and AE. To explore the relative importance of water and diet exposure routes under field conditions, we used estimated site-specific aqueous free-ion concentrations to model Cd and Cu accumulation from aqueous exposure, exclusively. The predicted concentrations accounted for less than 5% of the observed concentrations, implying that most bioaccumulated metal was acquired from food. At least for the taxa considered in this study, we conclude that consumption of metal-contaminated periphyton can result in elevated metal body burdens and potentially increase the risk of metal toxicity., (Copyright © 2011 SETAC.)

In order to clarify the role of metallothioneins (MT) in copper (Cu) toxicity, this work aimed to assess the involvement of this protein in the accumulation and elimination strategies of Cu in the clam Ruditapes decussatus exposed to two sublethal concentrations (25 and 50 microgCul(-1)). The behaviour of MT in three different tissues of clams during the accumulation and depuration processes was also followed by gel-filtration chromatography to assess if Cu was bound to MT or to other cytosolic components. The 96 h LC50 for water-borne copper was 715 microgL(-1) in R. decussatus. The Cu accumulation pattern was dependent on Cu exposure concentrations. In clams exposed to 25 microgl(-1), total Cu accumulation in the three tissues increased linearly during the exposure period, while in those exposed to 50 microgl(-1) it followed the first order kinetic model. The greatest amount of Cu accumulated in all tissues is associated to the low molecular weight cytosolic fraction (>50%). The chromatographic assay indicated that Cu in the cytosolic fraction is bound to MT and MT levels increase with the increase of Cu exposure confirming the binding affinity of Cu to MT in all tissues. However, a smaller percentage of Cu seems to be bond to other ligands, such as GSH. Copper was exponentially eliminated (only studied in clams exposed to 25 microgl(-1)) and the estimated half-life was tissue dependent (9, 5 and 14 days for the gills, digestive gland and remaining tissues, respectively). Copper bound to the thermostable compounds was eliminated more quickly (t(1/2)=4-7 days) in all tissues than those bound to the thermolabile compounds (t(1/2)=7-18 days). Interestingly, MT is rapidly degraded (t(1/2)=7 and 18 days), suggesting that this protein is actively involved in the elimination of this metal, through the Cu-MT complex since MT and Cu are turning over simultaneously. Therefore, when Cu exposure is low, the clam can cope efficiently with the excess of Cu levels by increasing MT induction as well as rapidly eliminating this metal via the MT-Cu complex. Copper toxicity in the clam R. decussatus is associated to the limited capacity of MT induction at higher and environmental unrealistic Cu exposures especially in the gills and remaining tissues.

Biosorption of copper by Sphaerotilus natans in different conditions of ionic strength and pH was studied by performing sorption tests in batch and membrane reactors. Equilibrium batch tests evidenced the negative effect of ionic strength and the positive effect of pH on biosorption performances: the highest determined value for copper specific uptake, q, was about 60 mg/g at pH 6 and about 15 mg/g at pH 4. A competitive equilibrium model was successfully fitted to experimental data at different ionic strength levels to account for copper-sodium competition. In membrane reactor tests, experimental profiles of copper concentration in the permeate vs. time did not evidence a significant effect of ionic strength at low pH values (4 and 5). On the other hand a more remarkable effect of ionic strength on copper concentration in the permeate was observed at pH 6. Experimental profiles of continuous biosorption in the membrane reactors were successfully simulated by developing a dynamic model accounting for Cu-Na competition and for binding ability of cells fragments.

The toxic effects of Cd, Cu, and Zn on four different marine phytoplankton, Dunaliella tertiolecta, Prorocentrum minimum, Synechococcus sp., and Thalassiosira weissflogii, were examined by comparing the cell-specific growth rate, pulse-amplitude-modulated (PAM) parameters (maximum photosystem II quantum yield phiM and operational quantum yield phi'M, chlorophyll a content, and cellular metal concentration, over a 96-h period. The calculated no-observed-effect concentration (NOEC) based on both cell-specific growth rate and two PAM parameters (phiM and phi'M) were mostly identical. Thus, these PAM parameters and cell-specific growth rate were comparable in their sensitivities as the biomarkers for trace metal toxicity to marine phytoplankton. The cyanobacteria Synechococcus sp. was the most sensitive species among the four algal species tested because of its higher cell surface to volume ratio. The toxicity of the three tested metals followed the order of Cd > Cu > Zn based on the cellular metal concentration of the four algae at the NOEC. The cellular metal bioaccumulation followed the same Freundlich isotherm for each metal regardless of the algal species, indicating that the metal accumulation was a nonmetabolic process under high ambient metal concentrations and that the cell surface metal binding was comparable among the different species. For all the algae examined in our study, the bioaccumulation potentials of Cu and Zn were similar to each other, while the Cd bioaccumulation was much lower under environmentally realistic metal concentration.

A large acclimation experiment was performed with Daphnia magna in which two different copper bioavailability (as Cu2+) groups (N and M) were used. In the N group the cupric ion activity increased with increasing dissolved copper-acclimation concentration, while in the M group the ion activity decreased with increasing dissolved copper concentration. The activity of copper carbonates and hydroxides was up to an order of magnitude lower than the cupric ion activity. After five generations of acclimation, the acute copper sensitivity (mean +/- SD) of the N group ranged from 193 /- 24 to 296 +/- 50 microg Cu L(-1) and for the M group from 198 +/- 27 to 315 +/- 38 microg Cu L(-1) for daphnids acclimated to 1 and 100 microg Cu L(-1), respectively. The internal copper concentration of the acclimated daphnids also resulted in similar results between the two groups. Acclimation of the two daphnid groups for five consecutive generations to the three dissolved copper concentrations resulted in a shift in the optimal concentration range toward 1 microg Cu L(-1), using energy reserves as an endpoint. Our results suggest that copper acclimation and accumulation are related to the dissolved copper concentration of the culture medium, but not to the copper activity.

This study investigates the distribution of three trace elements (Cd, Cu and Zn) in tissues (exoskeleton, abdominal muscle and hepatopancreas) of two shrimp species (Penaeus merguiensis and Penaeus semisulcatus). Special emphasis in this study was placed on evaluating the effects of storage conditions on redistribution of the elements and moreover the potential roles of metallothionein in tissue redistribution. The specimens were collected from the northern part of Persian Gulf during September 2001 and January 2002. The concentrations of metals in the tissues were measured using an Inductively Coupled Plasma-Atomic Emission Spectrophotometer (ICP-AES) and an ICP-Mass Spectrometer (ICP-MS). Metallothionein levels were determined using Differential pulse polarography. Results show a similar hierarchy for Cd and Cu concentrations among different tissues as follows: hepatopancreas>exoskeleton>muscle; and for Zn, the hepatopancreas again contained the highest levels, though there were not any significant differences in Zn concentrations between muscle and exoskeleton. Significant differences were found between the species in the case of Cd and Zn concentrations. Cd and Zn levels in P. semisulcatus were markedly higher than in P. merguiensis. In general, there were highly significant differences among the storage conditions from accumulation the selected trace elements and metallothionein in both species point of view, but in most cases, no significant differences could be observed among the fresh, refrigerated and frozen specimens of P. semisulcatus. In regards to Zn and Cu accumulation in edible (muscle) tissues of P. merguiensis, optimum conditions for frozen storage is -30 degrees C as ice-frozen, and in terms of short-term storage, 1-day refrigeration is recommended. The redistribution of Zn and Cu during the storage period can be attributed to the role of metallothionein in their transport from organ(s) to muscle during storage. The variations in pH values measured in the muscle of P. merguiensis in different storage conditions showed a similar trend with metallothionein, Zn and Cu. In contrast, it appears that cadmium is transported from muscle to the other tissues. Exoskeleton is probably one of the recipient tissues of cadmium. The inter-elemental relationships were assessed for each species, separately. A significant positive relationship was observed between the concentrations of Zn and metallothionein in P. semisulcatus, whereas in P. merguiensis, Cd concentrations decreased significantly with increasing metallothionein. Highly significant differences between the sexes in Cu and Zn contents could be detected. The differences between the sexes in the levels of Zn and Cu were significantly greater in P. semisulcatus. Highly significant size-related differences were found in the levels of Cd, Cu and metallothionein in the selected tissues of the shrimp species. Comparison between the mean concentrations of the elements in muscle tissue and existing guidelines indicate that in nearly all cases, the concentrations were well below permissible levels for human consumption. The only exception was observed regarding mean values of Cd, which was somewhat greater than some of the recommended levels.

Biological methods for wastewater treatment are becoming more accepted all over the world. The method, which makes use of accumulating abilities of living cells, is called bioaccumulation. In contrast with it, biosorption takes advantage of sequestering capabilities of dry or dead cells, which is technologically advantageous. The differences between bioaccumulation and biosorption of copper from model solutions were studied in this work. Application of living algal cells has some disadvantages. Copper significantly damages the surface of living cells, which results in partial loss of cell-binding abilities and release of accumulated copper back into solution. The binding capacity of living cells is significantly lower than that of dead cells. There is also a possibility of desorption and reuse of biomass in case of biosorption.

Measurements of lethal effect concentrations (LC50) and bioaccumulation following water-only and whole-sediment exposures of the amphipod, Melita plumulosa, and the bivalve, Tellina deltoidalis, to copper, were combined with bioenergetic-based kinetic models of exposure pathways to explain causality in whole-sediment toxicity tests. For both organisms, lethal body concentrations (LBCs) were greater for water-only exposures than for sediment exposures and indicated that the rate of copper accumulation and/or the mode of toxicity of copper assimilated were different for dissolved and particulate phases. The net assimilation of copper, expressed as a lethal exposure concentration (LEC) that was independent of the postexposure copper efflux, was shown to better explain the observed toxicity. The LEC of copper was the same for both water-only and whole-sediment toxicity tests. It is predicted that, for each species, a large range of effect concentrations may be measured for sediments having the same total copper concentration. These are conditional effect concentrations, as their value will be determined by total copper concentrations, partitioning (Kd) relationships (sediment properties), organism physiology (uptake rates from waters, assimilation efficiencies from solids), and organism feeding behavior (feeding selectivity). The importance of these factors to the development of sediment quality guidelines for metals based on species sensitivity distributions is discussed.

The concentrations of lead, zinc, copper and cadmium accumulated by 12 emergent-rooted wetland plant species including different populations of Leersia hexandra, Juncus effusus and Equisetum ramosisti were investigated in field conditions of China. The results showed that metal accumulation by wetland plants differed among species, populations and tissues. Populations grown in substrata with elevated metals contained significantly higher metals in plants. Metals accumulated by wetland plants were mostly distributed in root tissues, suggesting that an exclusion strategy for metal tolerance widely exists in them. That some species/populations could accumulate relatively high metal concentrations (far above the toxic concentration to plants) in their shoots indicates that internal detoxification metal tolerance mechanism(s) are also included. The factors affecting metal accumulation by wetland plants include metal concentrations, pH, and nutrient status in substrata. Mostly concentrations of Pb and Cu in both aboveground and underground tissues of the plants were significantly positively related to their total and/or DTPA-extractable fractions in substrata while negatively to soil N and P, respectively. The potential use of these wetland plants in phytoremediation is also discussed.

Characterization of uptake and loss dynamics is critical to understanding risks associated with contaminant exposure in aquatic animals. Dynamics are especially important in addressing questions such as why coexisting species in nature accumulate different levels of a contaminant. Here we manipulated copper (Cu) stable isotopic ratios (as an alternative to radioisotopes) to describe for the first time Cu dynamics in a freshwater invertebrate, the bivalve Corbicula fluminea. In the laboratory, Corbicula uptake and loss rate constants were determined from an environmentally realistic waterborne exposure to 65Cu (5.7 microg L(-1)). That is, we spiked deionized water with Cu that was 99.4% 65Cu. Net tracer uptake was detectable after 1 day and strongly evident after 4 days. Thus, short-term exposures necessary to determine uptake dynamics are feasible with stable isotopes of Cu. In Corbicula, 65Cu depuration was biphasic. An unusually low rate constant of loss (0.0038 d(-1)) characterized the slow component of efflux, explaining why Corbicula strongly accumulates copper in nature. We incorporated our estimates of rate constants for dissolved 65Cu uptake and physiological efflux into a bioaccumulation model and showed that dietary exposure to Cu is likely an important bioaccumulation pathway for Corbicula.

The suitability of two polychaete worms, Australonereis ehlersi and Nephtys australiensis, and three bivalves, Mysella anomala, Tellina deltoidalis, and Soletellina alba, were assessed for their potential use in whole-sediment toxicity tests. All species except A. ehlersi, which could not be tested because of poor survival in water-only tests, survived in salinities ranging from 18 per thousand to 34 per thousand during the 96-hour exposure period. No mortality was observed in any of the species exposed to sediment compositions ranging from 100% silt to 100% sand for 10 days, thus demonstrating the high tolerance of the five species to a wide range of sediment types. All species showed decreased survival after exposure to highly sulfidic sediments in 10-day whole-sediment tests. In 96-hour water-only tests, survival decreased, and copper accumulation in body tissues increased with exposure to increasing copper concentration for all species except A. ehlersi, which again could not be tested because of its poor survival in the absence of sediment. S. alba and T. deltoidalis were the most sensitive species to aqueous copper (LC50s of 120 and 150 microg Cu/L, respectively). All species tested were relatively insensitive to dissolved zinc up to concentrations of approximately 1,000 microg/L. In addition and with the exception of N. australiensis, all species accumulated significant levels of zinc in their body tissues. Whole-sediment tests were conducted over a 10-day period with copper-spiked (1,300 microg/g) and zinc-spiked (4,000 microg/g) sediments equilibrated for sufficient time to ensure that pore water metal concentrations were well below concentrations shown to have any effect on organisms in water-only tests. Survival was decreased in the bivalves T. deltoidalis and S. alba after exposure to copper-spiked sediments, and all species-except T. deltoidalis, in which 100% mortality was observed-accumulated copper in their tissues. Exposure to zinc-spiked sediments significantly decreased the survival of only one species, T. deltoidalis. Both polychaetes appeared to regulate concentrations of zinc in their body tissues with no significant uptake of zinc occurring from the sediment phase. Of the five species assessed in this study, T. deltoidalis was found to be the most sensitive to copper- and zinc-contaminated sediments, and based on commonly used selection criteria (ASTM 2002a, ASTM 2002b, ASTM 2002c) is recommended for development as test species in whole-sediment toxicity tests., (Copyright 2004 Springer Science + Business Media, Inc.)

A multigeneration acclimation experiment was performed with Daphnia magna exposed to copper to assess possible changes in tolerance and to establish the optimal concentration range (OCEE) of this species. The hypothesis was tested that as the bioavailable background concentration of an essential metal increases (within realistic limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. During 18 months the daphnids were exposed to six different, environmentally relevant, copper background concentrations ranging between 0.5 and 100 microg Cu L(-1) (7 x 10(-15) and 3.7 x 10(-9) M Cu2+). An increase in acute (effect concentration resulting in 50% immobility: 48-h EC50) and chronic copper (effect concentration resulting in 50% or 10% reproduction reduction: 21-d EC50, 21-d EC10) tolerance was observed with increasing exposure concentration. The 48-h EC50 increased significantly from 204 +/- 24 microg Cu L(-1) to 320 +/- 43 microg Cu L(-1). A nonsignificant change from 48.0 (47.9-48.0) microg Cu L(-1) to 78.8 (66.3-93.6) microg Cu L(-1) was noted in the chronic toxicity assays. The optimal concentration range was assessed using different biological parameters (i.e., net reproduction [R0]), energy reserves (Ea), body length measurements, filtration rates, and body burdens. After three generations of acclimation the OCEE ranged between 1 and 35 microg Cu L(-1) (2 X 10(-14) to 80 x 10(-12) M Cu2+). Body burden measurements revealed an active copper regulation up to 35 microg Cu L(-1) (80 pM Cu2+). It can be concluded that acclimation of D. magna to copper does occur in laboratory experiments, even at realistic copper background concentrations (10(-11) - 10(-9) M Cu2+). However, it is suggested that this phenomenon is of less importance in the context of regulatory risk assessments. An optimal copper concentration range for D. magna was observed between 1 and 35 microg Cu L(-1) (10(-14) - 10(-11) M Cu2+), indicating that copper deficiency can occur in routine laboratory cultures.

Previous work has suggested that the ratio of copper residues to zinc in the gills of rainbow trout may indicate short-term exposure to increased levels of waterborne copper. However, the effect of exposure to a combination of increased copper and zinc concentrates in the water column was unknown. We exposed rainbow trout to 8 +/- 2 microg L(-1), 40 +/- 2 microg L(-1) and 90 +/- 9 microg L(-1) of waterborne copper and 21 +/- 3 microg L(-1), 129 +/- 40 microg L(-1), and 202 +/- 40 microg L(-1) of waterborne zinc in a 2-factor experiments and gill copper and zinc residues were examined. Other gill parameters analyzed included the concentrations of calcium, magnesium, sodium, and potassium, liver copper and zinc concentrations and plasma copper, calcium, sodium, and potassium are also reported here. Copper residues in the gill filaments were significantly higher in the highest level of copper exposure (high Cu, 4.06 microg g(-1); low Cu 2.41 microg g(-1); 0 Cu 2.01 microg g(-1); P = 0.001), whereas no differences were seen in zinc concentrations at any treatment level. Gill sodium and plasma calcium concentrations were also decreased at the highest waterborne copper concentrations. Although copper-zinc ratios in the gills were significantly different between the highest and lowest copper treatments (P = 0.002, F = 6.59), copper-sodium and copper-magnesium ratios were more sensitive to waterborne copper exposure (P = 0.001, F = 17.91 and P = 0.002, F = 15.45, respectively). These copper-metal ratios may be better indicators of copper loading in the water column.

The free ion activity model (FIAM) has already been confirmed under laboratory conditions for many trace metals but has still to be validated under natural conditions where the presence of natural organic ligands influences metal speciation and bioavailability. The goal of this study was to test if the FIAM is followed under natural conditions by measuring copper and zinc speciation as well as metal accumulation in periphyton. Periphyton was exposed in microcosms to natural river water with different added concentrations of copper (25-258 nM) or zinc (18-501 nM) and additions of a synthetic ligand (NTA). Free Cu2+ was in the range of 10(-16.5)-10(-14.5) M and Zn(2+) was in the range of 0.7-8.7 nM, as measured by competitive ligand exchange coupled with cathodic/anodic stripping voltammetry. Other metal complexes were either measured or computed. Bioaccumulation of zinc in periphyton appeared to be controlled by the free zinc ion concentration, confirming the FIAM. In contrast, bioaccumulation of copper was controlled by weakly complexed copper (including Cu2+ plus inorganic and weak organic complexes), which is in disagreement with the FIAM, and appears to be caused by limitation of copper diffusion due to very low free Cu2+ occurring in natural environments.

Although the differential responses of stream taxa to metal exposure have been exploited for bioassessment and monitoring, the mechanisms affecting these responses are not well understood. In this study, the subcellular partitioning of metals in operationally defined metal-sensitive and detoxified fractions were analyzed in five insect taxa. Samples were collected in two separate years along an extensive metal contamination gradient in the Clark Fork River (MT, USA) to determine if interspecific differences in the metal concentrations of metal-sensitive fractions and detoxified fractions were linked to the differences in distributions of taxa relative to the gradient. Most of the Cd, Cu, and Zn body burdens were internalized and potentially biologically active in all taxa, although all taxa appeared to detoxify metals (e.g., metal bound to cytosolic metal-binding proteins). Metal concentrations associated with metal-sensitive fractions were highest in the mayflies Epeorus albertae and Serratella tibialis, which were rare or absent from the most contaminated sites but occurred at less contaminated sites. Relatively low concentrations of Cu were common to the tolerant taxa Hydropsyche spp. and Baetis spp., which were widely distributed and dominant in the most contaminated sections of the river. This suggested that distributions of taxa along the contamination gradient were more closely related to the bioaccumulation of Cu than of other metals. Metal bioaccumulation did not appear to explain the spatial distribution of the caddisfly Arctopsyche grandis, considered to be a bioindicator of metal effects in the river. Thus, in this system the presence/ absence of most of these taxa from sites where metal exposure was elevated could be differentiated on the basis of differences in metal bioaccumulation.

For ecological risk assessment, the additive model may be used to empirically predict toxic mixture effects. Detailed toxicity tests were performed to determine whether effects of mixtures of copper-cadmium and copper-carbendazim on Caenorhabditis elegans were similar to the effects of the individual compounds. Effects on the course of reproduction, the length of the juvenile period, the length of the reproductive period, and body length were analyzed. Dose-response data were compared to the additive model and tested for four deviation patterns from additivity: No deviation, synergistic/antagonistic deviation, dose ratio-dependent deviation, dose level-dependent deviation. During the exposure, the cadmium-copper effect on reproduction changed from a synergistic, to a dose ratio-dependent deviation from additivity. More cadmium in the mixture decreased the toxicity and more copper increased the toxicity. The effect of copper-carbendazim on reproduction was synergistic at low dose levels and antagonistic at high dose levels and independent of time. Mixture effects on the juvenile and reproductive period were similar to single component effects. It was concluded that the observed time-dependence of toxic interactions was small and that interactions on the timing of reproduction were not found. The additive model underestimated mixture effects on reproduction and body length.

Trace metal contamination in urban aquatic ecosystems in Hawaii is a significant problem, especially in terms of Cu, Pb, and Zn. These trace metals are linked to automobile usage. An in-depth study was designed to determine the influence of road sediments and storm sewers on bioavailable (0.5 M HCl) trace metal concentrations in bed sediments of Nuuanu stream, Oahu. Lead was the most enriched trace metal in the watershed. Compared to baseline Pb concentrations of <3 mg/kg, road sediments averaged 186 mg/kg, with a maximum value of 3140 mg/kg. Stream bed sediments had average Pb values of 122 mg/kg, with a maximum of 323 mg/kg. Al-normalized enrichment ratios (ERs) for the <63 microm fraction indicated that the watershed was significantly polluted in the lower, urbanized reaches, with maximum ER values of 560 and 94 for Pb in road sediments and stream sediments, respectively. Median ER values for Cu, Pb, and Zn in stream sediments were 2, 36, and 5, respectively. Rainfall events prior to sediment sampling masked any influence that storm sewer outlets might have had on the localized spatial distribution of metals associated with bed sediments. However, there was a general pattern of increasing trace metal concentrations downstream as the fluvial network traversed residential areas and commercial, highly trafficked areas in the lower portions of the watershed.

Lake ecosystems are vulnerable to heavy-metal pollution. Fish samples are considered as one of the most indicative factors, in freshwater systems, for the estimation of trace metals pollution potential. Lake Pamvotis (NW Greece) is a typical Mediterranean ecosystem of great importance in regard to biodiversity and to aesthetic value. The fish species found most commonly in the lake are Cyprinus carpio, Silurus aristotelis, Rutilus ylikiensis, and Carassius gibelio. The aim of the present study is to evaluate the level of contamination of two essential heavy metals (copper and zinc) appearing at high concentrations in lake water in the above four fish species. Metal concentrations were measured by atomic absorption spectroscopy in three different tissues (muscle, liver, gonads) in order to assess the fish contamination. A two-factor analysis of variance, based on the procedure of general linear models, was employed in which fish species (four levels) and fish tissue (three levels) were examined for potential influence on Cu and Zn concentrations. Differences between level means per factor were treated using Tukey's multiple comparisons of means. The study showed that C. carpio and R. ylikiensis presented the highest metal content. Tissues analysis revealed that liver and gonads accumulated the highest levels of Cu and Zn. Metal concentration in the edible part of the examined fish (muscle) were in the safety-permissible levels for human consumption.

Enteromorpha species are widely used as biomonitors of copper (Cu) contamination in coastal waters, but the effects of Cu at the subcellular level and possible mechanisms of metal resistance are scarcely known. To contribute to the understanding of the Cu accumulation process in macroalgae species, we exposed adult individuals of Enteromorpha flexuosa to 50, 250, and 500 microg Cu/L in seawater for 5 days under controlled conditions. Thereafter, gross photosynthesis rate (GPR) and metal accumulation were measured. Conventional transmission electron microscopy (CTEM) and energy-dispersive X-ray analysis (EDXA) were used to study the metal accumulation process at the cellular level. Treatments with 250 and 500 microg Cu/L were observed to cause an inhibition of the GPR in the algae. Cu accumulation in samples exposed to 500 microgCu/L was 5284+/-561 microg Cu/g (dry wt), whereas in control samples (no Cu addition), accumulation was 9+/-1 microg/g. In cells of plants undergoing the 50 microg Cu/L treatment, the cytoplasm was clearly vacuolated. Thickening of cell walls and increase of relative number of starch granules and of lipid bodies were the main cellular changes observed in plants exposed to 250 and 500 microg Cu/L. EDXA of algae cells after 250 and 500 microg Cu/L exposure detected Cu mainly in vacuole precipitates. Cu was also detected in chloroplasts and in some epiphytic bacteria. It was concluded that E. flexuosa did not avoid penetration of Cu into the cytoplasm and consequently its toxic effects in concentrations of 250 and 500 microg Cu/L. Precipitates containing Cu in vacuoles should be related to a metal immobilization mechanism, minimizing the Cu toxicity for cells. The epiphytic bacteria can act as a biofilter diminishing the availability of free Cu(+2) for algae accumulation.

Stormwater wetlands are created to retain water from storms and snow melt to reduce sediment, nutrient, and contaminant pollution of natural waterways in metropolitan areas. However, they are often a source of attractive habitat to wetland-associated wildlife. In this study of 12 stormwater wetlands and a larger, older reference site, elevated concentrations of zinc and copper were found in sediments and carcasses of 8-day-old red-winged blackbird (Agelaius phoeniceus) nestlings inhabiting stormwater sites. Although nesting success in the stormwater wetlands was comparable to national averages, sediment zinc concentrations correlated with clutch size, hatching success, fledgling success, and Mayfield nest success, suggesting that the nestlings may have been stressed and impaired by elevated zinc. This stress may have been direct on the nestlings or indirect through effects on the availability of food organisms.

This paper reports a study into the deployment of a metal adsorbent in situ to estimate bioavailable Cd(II) in marine waters. Eight adsorbents were screened in the laboratory to test their ability to accumulate Cd(II) from deionised water and artificial seawater, and an oxidised activated carbon was selected for further investigation. The adsorption isotherm at Cd(II) concentrations 0.16-38 microgl(-1) and at salinity 15 followed the Freundlich equation. The adsorbent was contained in nylon bags (pore size 35 microm) and dialysis tubes (membrane pore size 2 nm) to produce deployable devices and to investigate the effect of housing material on Cd(II) accumulation. The devices were tested in the laboratory and deployed at four field sites for up to 3 weeks. The adsorbent in the nylon bags reached equilibrium towards the end of this period and the measured contents were in good agreement with expected contents predicted from known seawater Cd(II) concentrations and the adsorption isotherm. The dialysis tubes accumulated significantly lower amounts of Cd(II) than the nylon bags, probably due to an initial lag as Cd(II) diffused into the dialysis bag and due to biofouling which reduced diffusion. The relationship between concentrations of Cd(II) accumulated by the mussels (indicating the bioavailable Cd(II) fraction) and the devices at different field sites could be described by the Freundlich model. The goodness of fit of this relationship was better for the dialysis tubes than the nylon bags. The adsorbent in the nylon bags may have collected small particles from seawater which affected the Cd(II) analysis. Both devices demonstrate potential as indicators of the relative bioavailable fraction of Cd(II) to Mytilus edulis in marine waters.

The technique of diffusive gradients in thin films (DGT) has been shown to be a promising tool to assess metal bioavailability in soils under laboratory conditions. In this study we used DGT to investigate the resupply kinetics of Cu and Zn under in-situ conditions in a polluted lysimeter soil and compared the results with laboratory measurements using undisturbed soil cores at defined water contents as well as homogenized soil samples. Results differed considerably between these treatments, although the same soil material was used in all experiments. A small pool of rapidly available Zn was found in the field but not in the homogenized soil. Soil solution pH and dissolved metal concentrations also varied significantly between the soil treatments. In addition, we compared the DGT-measured effective concentration with the uptake of Cu and Zn into the shoots of Lolium perenne (Ryegrass) under the same three types of conditions, i.e., field, soil cores, and homogenized soil. A close relationship was found which was not linear but could be described by a saturation-type behavior. L. perenne is a metal excluder plant, and thus, metal accumulation is limited by translocation of metals from roots to shoots. DGT predicted plant metal uptake much better than the soil solution concentration or pH. The results of this study suggest that DGT may be successfully used under field conditions to study the kinetics of metal resupply. Plant metal concentrations were not well predicted in all cases by the effective concentration CE under field conditions. Some plants took up considerably more metals than estimated by CE. Variations in metal uptake independent of their bioavailability can be caused by local variations in microsite conditions, e.g. light, temperature, water, and nutrients. To some degree, such indetermination has to be expected as an inherent feature of the system and the concept of bioavailability.

The effects of metal mixture (Cd+Cu) versus single-metal exposure on total MT response and bioaccumulation were investigated in the freshwater bivalve Dreissena polymorpha. A two-month exposure period, including two levels of contamination, was chosen for each of the two metals: 5, 10 microg/L for Cu, and 2, 20 microg/L for Cd, with mixtures of, respectively, 5 microg/L Cu+2 microg/L Cd, 5 microg/L Cu+20 microg/L Cd, 10 microg/L Cu+2 microg/L Cd, and 10 microg/L Cu+20 microg/L Cd. Total MT contents were assessed by an Ag-saturation method, and metals contents were determined by atomic absorption spectrometry. Results at the whole-organism level showed a significant and early increase of total MT biosynthesis after exposure to Cd. This increase was significantly correlated with Cd bioaccumulation. By contrast, Cu did not modify total MT response, and mussels limited Cu bioaccumulation. The mixture either did not influence or only weakly influenced metal accumulation and MT response to Cu and Cd after long-term exposure. Our results suggest that the form of MT existing in D. polymorpha was not Cu-inducible. This could limit the use of MT in D. polymorpha as a biomarker of heavy metal pollution in freshwater ecosystems.

The cadmium (II) and copper (II) adsorption properties of chemically pretreated biomass of red marine alga Gracilaria fisheri were investigated. Batch equilibrium experiments showed that the maximum adsorption capacity values of the pretreated biomass for cadmium and copper were 0.63 and 0.72 mmol g(-1), respectively. The equilibrium data fitted well to the Langmuir isotherm model. The adsorption capacity increased as pH increased and reached a plateau at pH 4.0. The cadmium and copper uptake rates were rapid with 90% of the biosorption completed within 30 minutes. The presence of light metal ions (Na+, K+, Mg2+ and Ca2+) in solution had an insignificant effect on cadmium and copper sorption capacity. These findings indicate a positive potential for the biosorbent development with effective heavy metal removal capacity in the presence of light metal ions in waste streams by using the biomass of plentifully available red marine algae.

Bioaccumulation of cadmium, copper, and zinc was examined in common ragworms Hediste diversicolor from control (Bay of Somme, Blackwater) and metal-rich (Seine estuary, Boulogne harbor, Restronguet Creek) sites in France and the United Kingdom. The degree of exposure in the field was assessed by considering both total concentrations in superficial sediment and the quantities of metals which may be released in vitro at different pH levels. Among the three contaminated sites, release of the three metals was not detectable in Boulogne harbor, in correlation with limited enhancement of the metal concentrations in the common ragworms from this site. Even at those sites where zinc could be released in vitro from the sediment, zinc concentrations were not enhanced in common ragworms, in agreement with previous findings indicating that the body content of this metal is regulated in H. diversicolor. At all the studied sites, bioaccumulated zinc was mainly in cytosolic form. The distribution of cadmium and copper varied according to the origin of the common rag-worms, the insoluble fraction increasing with the degree of contamination (cadmium in the Restronguet Creek, copper in the Seine estuary, and even more in Restronguet Creek). In the cytosolic fraction, metals were partly linked to cytosolic heat-stable thiolic compounds (CHSTC) with molecular masses (5-6 kDa and about 12 kDa) consistent with metallothionein-like proteins (MTLP). Metal-binding to MTLP varied with the degree of contamination and with the metal studied. In contrast to many invertebrates, the presence of metal-binding CHSTC (MM about 2 kDa) other than MTLP seems to be a peculiar feature of H. diversicolor.

Sewage sludge is a source of organic matter and nutrients, but a major obstacle for its recycling is that the municipal wastewater sludge has low but significant levels of contaminants. This investigation, on the acute toxicity of copper in sewage sludge, was conducted with three organisms, Daphnia magna, Lemna minor and Raphanus sativus (seeds). The toxicity of the leakage water from sewage sludge spiked with CuSO(4) was studied for 64 days. The toxicity increased during the first 8-16 days and then started to decrease. The first increase in toxicity was due to ammonia, but after 32 days, a dose-related effect of copper was found. After 64 days, L. minor had an EC50 of 3800 mg Cu/kg dw for 7 days growth inhibition, a LOEC of 3200 mg Cu/kg dw and a NOEC of 1600 mg Cu/kg dw. D. magna had an EC50 of 18100 mg Cu/kg dw (24-h immobility) and a NOEC of 12800 mg Cu/kg dw. Root elongation of R. sativus was reduced at 25600 mg Cu/kg dw. Both for Daphnia and Lemna, the pH of the leakage water had an effect of the toxicity. This means that chemical speciation and bioavailability is very important for the hazard assessment of copper in sludge and soil.

A gram-negative bacterium Pseudomonas putida 5-x with high Cu2+ accumulating capability was isolated from electroplating effluent in Kwun Tong, Hong Kong. The pretreated cells without superficial layer-capsule, isolated cell envelopes and the separated peptidoglycan layer materials were obtained from fresh P. putida 5-x cells, their Cu2+ adsorption capacities and properties were compared with that of the fresh cells. Pretreatment by 0.1 mol L(-1) HCl enhanced Cu2+ adsorption capacity due to the degradation of cell superficial layer-capsule of P. putida 5-x cells. Isolated cell envelopes possessed five times more Cu2+ adsorption capacity than that of fresh intact cell. The Cu2+ adsorption of separated peptidoglycan layer materials indicated that the peptidoglycan layer only played 10-15% part of the Cu2+ adsorption capacity, and implied other cell surface components such as outer membrane or inner membrane might play an important role in such high Cu2+ binding of the cell envelopes. The adsorption process of fresh cells, pretreated cells and cell envelopes of P. putida 5-x could be described with Freundlich isotherm, while the adsorption of Cu2+ by separated peptidoglycan layer materials was better described with Langmuir isotherm.

This study investigates the mechanisms of copper tolerance of a population of black-banded rainbowfish (Melanotaenia nigrans) (Richardson). The population has been exposed to elevated copper concentrations for over 40 years, due to leachate from the Rum Jungle uranium/copper mine. At the time of collection the 96 h EC(50) of exposed [E] fish was 8.3 times higher than that of reference [R] fish. The bioconcentration of 64/67Cu in fish was used to investigate the mechanism of copper tolerance in E fish. Both E and R fish were exposed to low (L(Cu), 30 microg Cu l(-1)) and elevated (E(Cu), 300 microg Cu l(-1)) copper concentrations for 24 and 48 h, respectively. Radioactivity was measured at seven or eight time points in four tissue sections: head (including gills, heart and brain), internal organs (including gastrointestinal tract, liver, kidneys and gonads), muscle and whole body. One-compartment bioconcentration models were fit to data and compared using an F-test. Copper concentrations in all tissue sections were significantly (P<0.05) less (up to 50%) in E fish compared with the respective tissue sections of R fish when exposed to both L(Cu) and E(Cu). The exception was copper accumulation in the internal organs, which was not significantly different between E and R fish exposed to E(Cu). The mechanism of copper tolerance was concluded to be reduced copper uptake in the gills, rather than increased binding or elimination. Allozyme electrophoresis was performed to determine if genetic selection had occurred in the E fish population. Allozyme frequencies at the AAT-1 and GPI-1 loci were significantly (P<0.05) different between E and R fish. Heterozygosity was reduced in E fish compared with that of R fish. Collectively these results suggest that genetic selection may have occurred in the E fish population. Consequently, the selection of allozymes less sensitive to copper may be another mechanism of copper tolerance of E fish. This is the first study on the mechanisms of copper tolerance in a wild fish population that has been exposed to elevated copper concentrations. These findings aid the understanding of metal tolerance in fish and emphasise the importance of sample selection and its implication for toxicity testing.

The spatial and seasonal variation of total and subcellular distribution of Cd, Cu, and Zn was followed in different tissues (gills, digestive gland, and remaining tissues) of the clam Ruditapes decussatus collected along a metal contamination gradient in the Ria Formosa lagoon (southern Portugal) and compared with metallothionein (MT) concentrations.Total metal concentrations decreased according to the sequence digestive gland > gills > remaining tissues for Cd, digestive gland approximately gills > remaining tissues for Cu and gills > digestive gland > remaining tissues for Zn. MT concentrations in these tissues decreased according to the same sequence observed for Cd. In all the tissues, the highest subcellular concentration was in the cytosol for Cd and Cu and in the pellet for Zn. Among the three metals, Cd concentrations showed the most evident spatial variation. In all tissues, total and subcellular Cd concentrations decreased from the inner parts of the lagoon toward the ocean. However, no significant spatial or seasonal variation occurred in clam tissues for the other two metals, though marginal elevated Cu concentrations were observed in the inner parts of the lagoon. Therefore, Cu subcellular distribution in clam tissues was not significantly altered by Cu changes in the lagoon and are the baseline levels for normal metabolism of this clam population. The fact that total Zn concentrations remained unchanged both spatial and seasonal suggested that these clams regulate Zn in their tissues. In the three tissues, MT bind most significantly to Cd and Cu, while Zn, although binding to MT, is preferably bound to other ligands. MT concentrations showed the same spatial and seasonal variation of Cd and were significantly related with total and heat-treated cytosolic Cd in all tissues. For Cu a significant relationship between MT and total or cytosolic Cu was only observed in the remaining tissues. No relationship was observed between MT and total or cytosolic Zn concentrations. Metals and MT concentrations increased with the increase in the condition index for the gills and the digestive gland and decreased from the remaining tissues.Cd concentrations in the gills increased only in the heat-treated cytosolic fraction while Zn in this fraction decreased. Thus Cd concentrations in this tissue displaced Zn from the MT-fraction, leading to a modification of the soluble/insoluble Zn ratio once total Zn concentrations remained unchanged. This modification reflects a perturbation in the normal metabolism in this tissue due to the excess of Cd present. With the exception of the gills, Zn subcellular distribution in the other two tissues was similar among sites and season. The model that describes the relationship between MT, metals, and weight in the gills, digestive gland and remaining tissues also indicates that Cd was the only metal that influence MT synthesis significantly in all the tissues. The induced and/or existent MT was sufficient to bind free Cd ions present in the cells, preventing any damage to cellular metabolism in this clam population. Therefore, MT in the gills and digestive gland of R. decussatus can be used as an early warning signal for Cd exposure and are a useful biomarker to assess the toxicological status of this population in the Ria Formosa lagoon.

A two-dimensional HPLC system, tandemly coupled to an ICP-MS, has been used to study copper accumulation and turnover in the visceral complex cytosol of the gastropod, Littorina littorea. Animals were exposed for 8 weeks to NTA-buffered seawater containing stable isotopic 65Cu and then transferred to media containing stable isotopic 63Cu. The free ion activity of each isotope was maintained at 10(-11) M. Size exclusion (SE) HPLC showed Cu associated with haemocyanin (HC) and metallothionein-like (MT) proteins in two ligand pools with apparent molecular weights of >300 kDa and approximately 17 kDa, respectively. The MT pool was inducible by Cu, could assimilate the metal from both intrinsic and extrinsic sources and showed a higher rate of Cu accumulation and turnover than the HC pool. The induction of this pool also caused the sequestration and cytosolic redistribution of Zn, Cd, Pb, Mn and Co. Further fractionation of the MT pool by ion-exchange (IE) HPLC revealed that the Cu was associated with a single, major isoform of the protein that was Cu inducible and also bound trace quantities of Zn and Pb. A number of additional metal containing proteins were also resolved by IE. the most prominent of which also bound Pb, Mn and minor quantities of Zn. The significance of these findings in metal homeostasis and detoxification is discussed.

Cd, Zn and Cu levels were determined in sediments and Crangon crangon from 9 sites in the Severn Estuary/Bristol Channel during winter 1999. Metal levels in both shrimp and sediments varied significantly between sites and were related to proximity of input and/or sediment type. In the upper Estuary, Cd levels in shrimp were 100x higher than other reported values whereas sediment Cd contamination was comparable. It is suggested that high Cd levels in shrimp are due to the high inputs and enhanced bioavailability of metal during winter.

Changes in Prochilodus scrofa gill tissue and in blood responses were investigated after 96-h copper exposure and transference to clean water. Gill damage was characterized by epithelial lifting, cell swelling, pavement, chloride and mucous cell proliferation, and blood vessel anomalies. Restoration of gill structure was slow, with no tissue improvements in the first 2 days in clean water. From the 7th to the 15th day, the recovery of gill tissue began to become evident, with complete recovery occurring on the 45th day in clean water. Hematocrit, red blood cells, and hemoglobin concentration showed a significant increase after copper exposure, remaining high until the 7th day after transference to clean water. Plasma Na(+) and Cl(-) concentration decreased significantly and K(+) increased significantly after copper exposure and, on the 7th day in clean water, plasma ions showed no significant difference from those in control fish. Gill tissue restoration took longer than the recovery of blood parameters, possibly implying extra energy needs, which may be critical, depending on the fish's life cycle., ((c) 2002 Elsevier Science (USA).)

The interactions of sublethal waterborne copper exposure and social dominance behavior were examined in juvenile rainbow trout. Dominance hierarchies were determined between pairs of fish by behavioral observations and among groups of 10 fish by the use of passive integrated transponder (PIT) tagging equipment. The present study is one of the first to utilize this novel PIT tag method for behavioral assessment. Feeding behavior was quantified by placing a PIT tag recorder at the entrance to the feeding area. Linear dominance hierarchies were documented based on these observations of feeding behavior. Dominance hierarchies established in control water were not altered by exposure to 30 microg/L of copper; however, physiological responses of fish to sublethal concentrations of copper were related to social rank. Subordinate fish exhibited a higher accumulation of copper in both gill and liver tissue. Subordinates of paired fish were also shown to have a higher uptake of sodium than dominant fish, and the uptake of sodium was correlated with uptake of copper from the water. Therefore, within a population of fish, it cannot be assumed that individuals of different social status will exhibit the same physiological responses to the presence of copper.

Metals are transferred into the food web by ground-dwelling organisms, among others. This study aimed to identify the most important factors that determine the bioavailability of heavy metals to the spider Pirata piraticus living in the intertidal sediments of the Scheldt estuary (Flanders, Belgium). At five locations, which represent a varying degree of metal contamination and salinity, the superficial layer of sediments was characterised for physico-chemical properties and heavy metal (Cd, Cu, Zn) content and extractability. Spiders were sampled at the same locations and analysed for Cd, Cu and Zn. Higher Cd, Cu and Zn contents were found in spiders on sites with lower total metal contents in the sediment. These sites were closest to the river mouth and were characterised by a higher salinity. Significant, positive correlations were found between the chloride content of the sediments and the Cd, Cu and Zn content of P. piraticus. Similarly, a strong relationship was observed between the ratio of exchangeable Cd and Zn to the total cation exchange capacity and the contents of these elements in P. piraticus. These field data indicated that salinity, cation exchange capacity and exchangeable metal contents were of most importance in determining bioavailability of heavy metals in these intertidal sediments.

Preset larval Mercenaria mercenaria were exposed to nominal concentrations of 1 (control) to 495 microg Cu/L in artificial seawater and monitored for mortality, activity, development, and metamorphosis in sealed 30-mm plastic petri plates containing 1.5 ml of artificial seawater or toxicant solution. The plastic petri plates sorbed only about 2.6 microg/L at any dose and allowed direct observation of larval clams under a light microscope for a period of two weeks; control survivorship was in excess of 60% at 400 h. The dose-response curve for mortality for clams exposed to copper and fed Isochrysis galbana was characterized by survival similar to or better than controls at doses of 5 and 14 microg Cu/L, while doses of 7 and > or = 29 microg Cu/L exhibited mortality greater than controls. Values of lowest concentration at which 50% of the organisms died (LC50) were 62.4, 21.2, and 11.7 microg Cu/L, and the lowest observed adverse effect concentration values of 57, 29, and 29 microg Cu/L were determined at 48, 96, and 192 h, respectively. In contrast, activity, as judged by swimming, exhibited a typical exponentially decreasing response at these same concentrations. Experiments on the uptake of dissolved copper by I. galbana confirmed literature reports that these algae concentrate copper. Ingesting copper-containing algae was demonstrated to be a source of copper toxicity for larval clams.

Carp were exposed to copper adsorbed on kaolin particles at various concentrations (0-2.4 mg/L), and net accumulation due to elevated adsorbed copper in the surrounding water was observed. Copper speciation in the water and fish gill microenvironment was modeled in the presence and absence of kaolin using a chemical speciation program (MINTEQA2). The adsorption affinity constants of kaolin for copper at various pH values used in the speciation calculations were experimentally determined, and the quantitative relationship between the affinity constant and pH was modeled. Copper accumulations in fish gills exposed to kaolin-adsorbed copper was then observed. The results indicate that desorption occurred in the fish gill microenvironment due to both mucus competition of copper and slight increase in water pH. Furthermore, the available copper species increased as a result of desorption, causing a net accumulation of Cu by the gills.

Experiments were conducted comparing the individual removals of cadmium and copper from water via biosorption using Scenedesmus abundans, a common green algae, to removal in a multi-component system to determine competitive effects, if any, between the metals. The goal was to characterize the biological treatment of water contaminated with heavy metals using live aquatic species. In addition, experiments were performed to measure cell viability as a function of metal concentration and also to compare metal removal using living species to that using nonliving ones. It was shown that, while both living and nonliving S. abundans removed cadmium and copper from water, living algae significantly outperformed nonliving algae. Further, in characterizing biosorption by three concentrations of live S. abundans, capacity curves were created comparing the metal biosorbed per mass algae to the initial metal concentration in solution. The algae concentration was not a factor in the biosorption of either metal individually, such that the capacity of the algae for the metal increased with decreasing algae concentration. At the lowest algae concentration considered, competitive effects were observed at copper and cadmium concentrations above 4 mg/l each. At the highest algae concentration considered, no competitive effects were observed in the range of cadmium and copper concentrations studied (1-7 mg/l). It was concluded that biological treatment of heavy metal contaminated water is possible and that at adequately high algae concentrations, multi-component metal systems can be remediated to the same level as individual metals.

Adsorption and uptake of Cu2+ by Chlorella vulgaris were distinguished by extracting the surface-bound Cu2+ with EDTA. The uptake of Cu2+ followed Michaelis Menten kinetics. The maximum rate of Cu2+ uptake (0.362fmolcell(-1) h(-1)) was obtained at pH 6.0. The rate of Cu2+ uptake was greater for cultures in the exponential phase of growth, and increased with a rise in temperature from 6 to 25 degrees C, thus pointing towards an active mechanism. The maximum number of Cu2+ binding sites was 3.245 fmol cell(-1) at pH 4.5. Adsorption of Cu2+ was strongly pH-dependent thereby indicating that the number and nature of metal binding sites on the cell surface change with changing chemistry of the solution. Unlike uptake, the adsorption remained unaffected by small changes in temperature. Older cultures displayed a higher Cu2+ adsorption capacity than the exponentially growing ones thus suggesting generation of new and/or additional Cu2+ binding sites on older cells of C. vulgaris. By pH titration, the cation-exchange capacity of Chlorella, measured in terms of H+/ Na+ exchange, was about 17 fmol cell(-1) at pH 10.5. Negligible cation exchange capacity at and below pH 5.0 indicated that ion exchange was not the sole mechanism of Cu2+ adsorption by Chlorella. The uptake and adsorption of Cu2+ were inhibited by 100 microM of various cations including other heavy metal ions. The general concept that cations competitively inhibit accumulation of metals in living organisms does not hold for C. vulgaris. Non-competitive, uncompetitive and mixed inhibition of Cu2+ uptake and adsorption by various cations were more common than competitive inhibition.

Carp were exposed to a synthetic solution with varied concentrations of Cu-EDTA and constant levels of other copper species and the amount of copper accumulated in the fish gills was measured. The distributions of copper species in both the bulk solution and the fish gill microenvironment were modeled using MINTEQA2. Under experimental conditions without EDTA, Cu(OH)2 was the most dominant species in the bulk solution while Cu-mucus dominated the gill microenvironment. When Cu-EDTA was added to the solution, copper accumulation in the fish gills increased significantly, suggesting a change in copper speciation. The amount of copper accumulated in the fish gills correlated positively to the concentration of Cu-mucus, rather than to free copper.

Samples of Atlantic salmon (Salmo salar), saithe (Pollacius virens), blue mussel (Mytilus edulis), brown seaweed (Ascophyllum nodosum) and sediment were collected from six different fish farms. Five of the farms used net pens treated with copper-containing coatings, whereas one farm did not use copper-containing coating (this was used as a reference location). Samples of muscle, liver and gills of Atlantic salmon and saithe, blue mussel and brown seaweed were freeze dried, homogenised, wet digested and analysed for copper by flame atomic absorption spectrometry. The results showed no significant differences in copper concentrations among the samples from the different locations. The copper contents of some of the samples appeared to be in the upper part of the normal concentration range. From a nutritional point of view, the use of copper-coatings on net pens did not affect the quality of the seafood products either within, or around the net pen.

Acid mine drainage from abandoned mines and acid mine pit lakes is an important environmental concern and usually contains appreciable concentrations of heavy metals. Because sulfate-reducing bacteria (SRB) are involved in the treatment of acid mine drainage, knowledge of acute metal toxicity levels for SRB is essential for the proper functioning of the treatment system for acid mine drainage. Quantification of heavy metal toxicity to mixed cultures of SRB is complicated by the confounding effects of metal hydroxide and sulfide precipitation, biosorption, and complexation with the constituents of the reaction matrix. The objective of this paper was to demonstrate that measurements of dissolved metal concentrations could be used to determine the toxicity parameters for mixed cultures of sulfate-reducing bacteria. The effective concentration, 100% (EC100), the lowest initial dissolved metal concentrations at which no sulfate reduction is observed, and the effective concentration, 50% (EC50), the initial dissolved metal concentrations resulting in a 50% decrease in sulfate reduction, for copper and zinc were determined in the present study by means of nondestructive, rapid physical and chemical analytical techniques. The reaction medium used in the experiments was designed specifically (in terms of pH and chemical composition) to provide the nutrients necessary for the sulfidogenic activity of the SRB and to preclude chemical precipitation of the metals under investigation. The toxicity-mitigating effects of biosorption of dissolved metals were also quantified. Anaerobic Hungate tubes were set up (at least in triplicate) and monitored for sulfate-reduction activity. The onset of SRB activity was detected by the blackening of the reaction mixture because of formation of insoluble ferrous sulfide. The EC100 values were found to be 12 mg/L for copper and 20 mg/L for zinc. The dissolved metal concentration measurements were effective as the indicators of the effect of the heavy metals at concentrations below EC100. The 7-d EC50 values obtained from the difference between the dissolved metal concentrations for the control tubes (tubes not containing copper or zinc) and tubes containing metals were found to be 10.5 mg/L for copper and 16.5 mg/L for zinc. Measurements of the turbidity and pH, bacterial population estimations by means of a most-probable number technique, and metal recovery in the sulfide precipitate were found to have only a limited applicability in these determinations.

The effects of physicochemical conditions, such as pH, water hardness, flow rates and natural organic substances on the sensitivity of Ceriodaphnia dubia to the toxic effects of copper were investigated using static bioassay cups and specially designed flow-through bioassay chambers. We found that C. dubia was very sensitive to pH changes and the total copper LC50 values of C. dubia neonates increased by 15-fold as the pH increased from pH 7 to 10. It was also observed that the LC50 values increased sharply upon increasing the water hardness value to 2.4 meq. In addition, increasing flow rates from zero to 50 mL hr(-1) also increased its sensitivity to copper, which was possibly due to hydrodynamic stress. The presence of natural organic substances (humic acid and dissolved organic matter) and suspended particles decreased the toxic effect of copper. This significant decrease in the toxicity of copper in the presence of natural organic materials can be explained by a reduction in the free ion concentration due to complexation. Furthermore, we observed that the kinetics of copper interactions with natural organic materials are a significant factor in the toxic effect of copper and that the acute LC50 values increased with increasing reaction time between solubilized copper and water-borne organics.

This study was conducted to: (1) assess the role of wetland vegetation in the removal of oxygen demand and nitrogen under tropical conditions, (2) estimate the uptake of nitrogen and copper by wetland plants and (3) investigate the speciation of Cu in wetland media among four operationally defined host fractions, namely exchangeable, carbonate, reducible and organically bound. Four laboratory-scale wetland units, two free-water-surface (FWS) and two subsurface-flow (SF) with one of each planted with cattails (Typha augustifolia), were fed with primary-treated sewage and operated at nominal retention times of 0.6-7 days. The influent and effluent BOD/COD and nitrogen concentrations were monitored to assess the performance of the wetland units for various mass loading rates. At the end of the study, all cattail plants were harvested and analyzed for total Kjeldahl nitrogen (TKN). Four other wetland units, which were identical to the first four, were fed with domestic wastewater spiked with copper in increasing concentrations. Copper speciation patterns in the sand layer were determined at the end of the study. The results showed that wetland vegetation did not play an important role in oxygen demand removal but were capable of removing about 22% and 26% of the nitrogen input in the FWS and SF wetland units, respectively. Mass balance analysis indicated that less than 1% of copper introduced was taken up by the cattails. Copper speciation patterns in the sand media showed that the exchangeable fraction contributed 30-57% and 63-80% of the nonresidual copper in the planted and unplanted FWS wetlands, respectively. For SF units, the percentages were 52-62% and 59-67%, respectively. This indicates that large amount of copper in the media were potentially remobilizable.

Short-term kinetic experiments, carried out in natural coastal seawater (with predetermined background levels of trace metals and organic ligands, L) enriched with nitrate and phosphate, demonstrated that Emiliania huxleyi was able to uptake Cu very quickly. After 10 min of exposure (background Cu level in the inoculated cells: [Cu]total cellular = 9.3 x 10(-17) mol cell-1, [Cu]intracellular = 8.4 x 10(-17) mol cell-1, and [Cu]extracellular = 1.0 x 10(-17) mol cell-1) to a natural seawater which contained 29 nM total initial dissolved Cu concentration ([Cu]d) (29 nM [CuL] and 3.2 x 10(-13) M free Cu concentration, [Cu2+]) the intracellular Cu was already 28 x 10(-17) mol cell-1. This value corresponded to 85% of the intracellular metal observed in pseudoequilibrium conditions (after 24 h of exposure) and to 88% of the total metal sorption (adsorption plus uptake) after 10 min. In contrast, the external adsorption after 10 min was only 3.0 x 10(-17) mol cell-1 which corresponded to 60% of the extracellular metal in pseudoequilibrium conditions. Simultaneously occurred a very fast release of organic ligands (L) by E. huxleyi, the majority being identified by cathodic stripping voltammetry as glutathione. The production of exudates increased with both Cu concentration and exposure time. After 10 min of exposure, the production of exudates in a medium with 129 nM [Cu]d (72 nM [CuL] and 7.9 x 10(-13) M [Cu2+]) was 51 nM, about 42% of that observed in pseudoequilibrium. As the Cu complexes with the organic ligands present in the medium were very stable (logarithm of the conditional stability constant: 12.18 +/- 0.06) and the ligand concentration in the medium was relatively high (e.g. 123 nM CL in the medium with 129 nM initial [Cu]d after 10 min of exposure) most of the metal was organically bound in the medium.