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Environ. Sci. Technol. 2010, 44, 3580–3586

Validation of the Biotic Ligand Model in Metal Mixtures: Bioaccumulation of Lead and Copper Z H O N G Z H I C H E N , †,‡ L I N Z H U , * ,† A N D K E V I N J . W I L K I N S O N * ,‡ Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, College of Environmental Science and Engineering, Nankai University, Tianjin, China 300071, and De´partement de chimie, Universite´ de Montre´al, P.O. Box 6128, Succursale Centre-ville, Montre´al (QC), Canada H3C 3J7

Received January 31, 2010. Revised manuscript received March 23, 2010. Accepted March 25, 2010.

The biotic ligand model (BLM) has the potential to predict biological effects and bioaccumulation in metal mixtures. Pb and Cu uptake by the green alga Chlamydomonas reinhardtii have been quantified in single-metal exposures and in metal mixtures in order to test some of the key assumptions of the BLM. Stability constants for the interaction of the metals with biological uptake sites were determined from measured shortterm internalization fluxes. In the absence of competition, a value of 105.8 M-1 was obtained for Cu, while 105.9 M-1 was obtained for Pb. Competition experiments did not show a straightforward antagonistic competition as would be predicted by the BLM. Only at high Cu2+ concentrations (>1 µM) did Cu behave as a competitive inhibitor of Pb transport. Surprisingly, low concentrations of Cu2+ had a synergistic effect on Pb uptake. Furthermore, Cu uptake was independent of Pb when Cu concentrations were below 10-7 M. In order to explain the observed discrepancies with the BLM, membrane permeability and Cu transporter expression levels were probed. The expression of ctr2, a gene coding for a Cu transporter, increased significantlyinthepresenceofPb,indicatingthatbioaccumulation is much more dynamic than assumed in the equilibrium models.

Introduction In the environment, contamination rarely occurs for single metals and is invariably due to complex mixtures. Nonetheless, risk assessments are routinely based upon effects evaluations that have been obtained for single substances. Over the past few years, the biotic ligand model (BLM) has been proposed as a tool to quantitatively predict the manner in which water chemistry affects the speciation and biological availability of metals in aquatic systems (1-4). Because of its mechanistic facility in dealing with the interactions of components, the BLM approach has the potential to make significant advances to risk assessment metal mixtures. Yet unanswered is whether it is possible to predict the toxicity * Address correspondence to either author. Phone: +1-514 343 6741; fax: +1-514 343 7586; e-mail: [email protected] (K.J.W.). Phone: +86-22-2350 4202; fax: +86-22-2350 8936; e-mail: [email protected] (Z.L.). † Nankai University. ‡ Universite´ de Montre´al. 3580

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of metal mixtures using equilibrium constants that have been determined from the interactions of single metals at a given site of toxic action on the organism. A key point that remains to be verified is the extent that multiple metals interact with a singular site of action (dbiotic ligand), i.e. are metals (toxic or essential) taken up by one, several, or numerous biological uptake sites? If metals interact at a single uptake site (or biotic ligand), bioaccumulation will always decrease (or remain unchanged) in the presence of a second metal (5, 6). In contrast, toxicity assessments of metal mixtures have shown additive, synergistic, or antagonistic effects when compared to the exposure of a single metal (7). While the BLM has the capacity to model completely additive interactions by a two-site model and completely antagonistic interactions by a one-site model, synergism cannot be easily explained by equilibrium principles. Insight into the nature of the biotic ligands available for binding would appear to be a key for a better understanding of bioaccumulation and toxicity in order to improve models of trace metal interactions in metal mixtures. In this paper, we have evaluated some of the interactions between Pb and Cu uptake in order to estimate the nature and quantity of the metal uptake site(s). Lead and copper were selected because of their contrasting toxicity and essentiality. Pb is generally recognized to be toxic, while Cu can play a role in cellular metabolism at low concentrations and be toxic at higher concentrations. Results for Pb and Cu bioaccumulation by the unicellular green alga, Chlamydomonas reinhardtii, were discussed in the context of the BLM.

Experimental Section Algal Cultures. Chlamydomonas reinhardtii (wild type CL 2010), a unicellular green alga, was employed in this study mainly because of the relative ease by which it is possible to culture and control its metabolic state, size, and surface area distribution. C. reinhardtii is also a useful species because of an existing complete knowledge of its genetic makeup (8). Algae were transferred from a week-old tris-acetatephosphate (TAP) (9) agar plate to a (4×) diluted TAP solution (I ) 10-2 M) (10). Cells were grown in an incubation chamber (Infors) at 20 °C under a 12:12 h light:dark regime using fluorescent lighting (80 µmol of photons m-2 s-1) and rotary shaking (100 rpm) until a density of (2-3) × 106 cells mL-1 was achieved, generally after 4 days. Cells were then diluted in fresh media to 1.0 × 105 cells mL-1 and once again allowed to attain logarithmic growth. Once cells attained midexponential growth [(1-3) × 106 cells mL-1], they were harvested by centrifugation (3700×g, 4 min) into 50 mL sterile (polypropylene) centrifuge tubes, resuspended in 10-2 M MES [2-(N-morpholino)ethanesulfonate, sodium salt, Sigma] at pH 6, centrifuged, harvested a second time, and then resuspended in 50 mL of a metal-containing 10-2 M MES media for the bioaccumulation experiments. The pH of the MES solution was adjusted using concentrated solutions of HNO3 (suprapur, Baker). Under these conditions, in the absence of added ligand, free metals (Pb2+, Cu2+) represented >96% of the total metal in solution. Preparation. All culture media and experimental solutions were sterilized (autoclave, 0.2 µm filtration) prior to use. All manipulations of algal cultures were performed under laminar flow conditions, and all bottle borders were flame sterilized. All exposure solutions were equilibrated 1 day prior to use. All polycarbonate materials were acid washed (minimum 24 h in 0.1% HNO3) and rinsed seven times with Milli-Q water (R > 18 MΩ cm; TOC < 2 µg L-1). Metal 10.1021/es1003457

 2010 American Chemical Society

Published on Web 04/12/2010

speciation, free metal concentrations, and activities in the medium were determined by theoretical calculations using Visual MINTEQ version 2.5.3 using updated equilibrium constants from the NIST database (11). Bioaccumulation Measurements. In the first set of experiments, single-metal (Cu or Pb) biouptake experiments were performed in 350 mL of an experimental medium containing 10-6 M free metal, 10-5 M of Ca2+ added as the nitrate salt, and 10-2 M MES as the pH buffer (pH 6.0). Bioaccumulation was evaluated for exposures of 0, 10, 20, 30, 40, 50, and 60 min. For each time point, metal uptake was stopped by adding 5 mL of 10-2 M ethylenediaminetetraacetic acid (EDTA in 10-2 M MES at pH 6.0) to the exposure solutions (12). In subsequent experiments examining metal mixtures, the concentration of the first metal, Pb or Cu, was held constant at 5 × 10-8 M, 10-7 M, 5 × 10-7 M, or 10-6 M, while the concentration of the competing metal ion was varied from 1 × 10-8 M to 5 × 10-6 M. Bioaccumulation was quantified in short-term experiments (30 min) in order to decrease the likelihood of physiological changes in the algae (e.g., minimize cellular efflux) or physicochemical changes in the medium (e.g., decreasing metal concentrations, increasing complexation with extracellular ligands). As above, metal uptake was stopped by adding 5 mL of 10-2 M MES/ 10-2 M EDTA at pH 6.0. Controls included bioaccumulation experiments performed in the absence of competing ions and algae collected immediately prior to the exposures. Cellular Pb and Cu concentrations were determined following the digestion of the filtered (3.0 µm nitrocellulose, Millipore) EDTA/MES-washed algae using 0.3 mL of concentrated (65%) ultrapure HNO3 at 85 °C until solutions became colorless. Dissolved Pb and Cu were determined from a small volume of filtrate that was sampled prior to the EDTA wash. Total, dissolved, and cellular metal concentrations were determined by atomic absorption spectrophotometry (Varian AA240Z) or by inductively coupled plasma mass spectrometry (Thermo X7), depending on the concentration. For each experimental point, cell densities, sizes, and surface distributions were determined using a Coulter Multisizer 3 particle counter (50 µm orifice, Coulter Electronics). Determinations of Cellular Viability. The assay for cellular viability is based on the activity of dehydrogenase in metabolically active cells (13). In the presence of the electron coupling reagent, phenazine methosulfate (PMS; Sigma P9625), MTS {tetrazolium [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium]; Promega PRG1112} is reduced intracellularly by dehydrogenases to a formazan compound whose absorbance can be measured at 490 nm. Formazan production is thus proportional to the number of living cells. A previously described protocol (13) was followed closely except that a 4 h incubation (37 °C) was employed to allow color development. Cells were exposed to identical conditions as described above (bioaccumulation). Determinations of Membrane Permeability. Fluorescein diacetate (FDA; Molecular Probes, Inc.) was used as a probe of passive diffusion through biological membranes (14). The fluorescent product is formed following intracellular enzymatic hydrolysis such that the penetration of fluorescein diacetate is proportional to membrane permeability. The in vivo diffusion of 3 µM of the probe was determined after 30 min of contact with the algae in each of the exposure media. Gene Expression. After exposure to 30 mL of the experimental media, bioaccumulation was stopped as above by adding 3 mL of 10-2 M EDTA/10-2 M MES (pH 6.0). Cells were separated by centrifugation (4 min at 3700×g). The cell pellet was diluted in 700 µL of 10-2 M MES (pH 6.0) and transferred to an Eppendorf tube that was kept on ice until its centrifugation at 9500×g (13000 rpm, 4 min, 4 °C; accuSpin

FIGURE 1. Cellular Pb and Cu as a function of time (pH 6.0, [M]tot ) 10-6 M) for experiments performed with a single metal. Error bars represent standard deviations when larger than the symbol size (n > 2). Micro R, Fisher Scientific). Following removal of the supernatant, pellets were frozen on dry ice and stored at -80 °C until mRNA quantification. Gene expression changes were quantified using the QuantiGene 2.0 Reagent System (Affymetrix, Inc.). The QuantiGene 2.0 assay is a hybridizationbased assay performed on 96 well plates. The assay couples luminescent oligonucleotide probes to specific fragments of mRNA. An amplified luminescent signal is linearly proportional to the number of RNA molecules, in this case ctr2 (XM_001702418), that are present in the sample. Ctr2 is known to encode for a Cu transporter (15); its expression decreases when Cu levels are sufficiently high to satisfy cellular requirements. In order to take into account small variations of total mRNA quantities, samples were normalized to expression levels of the RPS26 housekeeping gene (ribosomal protein S26, component of cytosolic 80S ribosome, and 40S small subunit, XM_001691849), which is stably expressed under all of the evaluated experimental conditions. General Data Treatment and Analysis. At each time point, a mass balance was performed by taking into account total, dissolved, and cellular metal concentrations. Data were rejected if the sum of dissolved and cellular metals varied by >20% from the initial concentration. In order to take into account biological and analytical variability, duplicate samples were processed for each experimental condition, and experiments were repeated in triplicate using unique algal cultures. Data was presented as a function of measured (rather than nominal) metal concentrations. Michaelis-Menten constants were obtained from a nonlinear regression of the internalization fluxes against free ion concentrations using eqs S2 (single metal) or S3 (2 metals) of the Supporting Information.

Results and Discussion Internalization of Single Metals. Following exposure of the algae to the experimental solutions, dissolved Pb or Cu concentrations decreased by e20% over the first few minutes of the experiment due primarily to adsorption to the cell wall (12). Subsequently, a small but constant internalization of the metals was observed, as demonstrated by the linear uptake curves shown in Figure 1 (internalization flux, Jint ) slope) and a small or negligible further decrease in dissolved metals. The observation of a linear uptake is consistent with the attainment of equilibrium between the external solution and the cell surface, a necessary condition of the BLM (4). The significant y-intercept that was consistently observed for Cu [(3.15 ( 0.4) × 10-11 mol cm-2] was due to the presence of Cu in the algal growth media (1.54 × 10-6 M total Cu). For experiments performed with a single exposure metal, internalization fluxes (Jint ) slopes) were about 1.5 times VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Logarithmic representation of internalization fluxes as a function of (a) [Cu2+] and of (b) [Pb2+] in the absence of competitor ion with a superimposed Michaelis-Menten plot. Representative constants are given in Table S1 of the Supporting Information. smaller for Pb (1.7 × 10-12 mol cm-2 min-1) than those for Cu (2.7 × 10-12 mol cm-2 min-1) (Figure 2). A linear increase of (log) Jint was observed as a function of the (log) free ion concentrations below 10-6 M (Cu: slope ) 0.88, r2 ) 0.94. Pb: slope ) 0.95, r2 ) 0.95). On a log-log graph, the slope of 1 implies that uptake was directly proportional to the free ion or surface bound metal concentrations (eq S1 of the Supporting Information) and that membrane permeability (Jint/[Mz+]) was constant [(4.5 ( 1.5) × 10-3 cm min-1 for Cu and (4.0 ( 1.0) × 10-3 cm min-1 for Pb]. In order to determine stability constants for the interaction of the metal with the transport sites, it is also necessary to perform experiments at (higher) concentrations where saturation occurs (i.e., near constant internalization fluxes above 10-6 M in Figure 2). When internalization fluxes were fitted with the MichaelisMenten equation (eq S2 of the Supporting Information), values of Jmax ) (8.4 ( 0.7) × 10-12 mol cm-2 min-1 and KM ) (1.5 ( 0.3) × 10-6 M were found for Cu, and Jmax ) (6.2 ( 0.2) × 10-12 mol cm-2 min-1 and KM) (1.2 ( 0.1) × 10-6 M were found for Pb (Table S1 of the Supporting Information). Reciprocal KM values, employed to determine stability constants for the interaction of Cu (105.8 M-1) and Pb (105.9 M-1) with biological transport sites were determined from the single-metal experiments and showed that the affinity was similar for both metal ions. The stability constant of 105.9 M-1 for Pb is similar to a previous value of 105.5 M-1 determined for Chlorella vulgaris under similar conditions (16). For Cu, the stability constant of 105.8 M-1 was of the same order of magnitude as previous values reported for C. reinhardtii: 106.7 M-1 for Cu uptake (17) and 105.6 M-1 for a 3582

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FIGURE 3. (a) Effect of Pb on Cu internalization fluxes. [Cu2+]: 5 × 10-8 M (4), 10-7 M (b), 5 × 10-7 M (3), 10-6 M (9). (b) Effect of Cu on Pb internalization fluxes. [Pb2+]: 5 × 10-8 M(4), 10-7 M (b), 5 × 10-7 M (3), 10-6 M (9). Lines represent calculations from eq S3 of the Supporting Information with KPb-Rcell ) 105.9 M-1 and K′Cu-Rcell ) 105.4 M-1 (r2 ) 0.59, 0.65, 0.65, and 0.45 for 5 × 10-8 M, 10-7 M, 5 × 10-7 M, and 10-6 M Pb2+, respectively). constant describing the competitive interaction of Cu with Cd uptake sites (10). The similarity of the Pb and Cu stability constants implies that in the presence of similar concentrations of both metals, about 50% reductions in each of the internalization fluxes would be expected if uptake occurs over a common transport site in contrast to no predicted reduction of uptake for transport over independent sites. This point will be examined in the next two sections. Influence of Pb on Cu Internalization. For the lowest Cu concentrations that were examined, Pb appeared to have little competitive effect on Cu internalization. Indeed, Cu uptake fluxes were almost nearly constant when in the presence of 5 × 10-8 to 5 × 10-6 M Pb2+ (b and 4 in Figure 3a). However, for the two highest Cu concentrations (3 and 9 in Figure 3a), a significant decrease in Cu internalization was observed, especially for Pb2+ concentrations exceeding 10-6 M. These observations cannot be explained by competitive equilibrium modeling, as found in the BLM (eq S3 of the Supporting Information). Indeed, given the observed competitive decrease of Cu internalization in the presence of Pb (>10-6 M Pb2+) at the high Cu exposures (5 × 10-7 and 10-6 M), decreasing Cu internalization fluxes would also be expected for the lower Cu exposures in the presence of competing ion (Pb2+) concentrations of 5 × 10-8 to 10-7 M. Influence of Cu on Pb Internalization. Increasing Cu consistently resulted in decreasing Pb internalization fluxes (Figure 3b). Nonetheless, as Pb decreased from 10-6 to 5×

FIGURE 4. Synergistic and antagonistic effects of Cu on Pb internalization fluxes after normalization by Pb concentration in the bulk medium. [Pb2+] were nominally 1 × 10-6 M, 5 × 10-7 M, 1 × 10-7 M, and 5 × 10-8 M. Error bars represent standard deviations from n (n ) 3 or 4) experiments. Treatments that do not share a common letter are significantly different from each other (p < 0.05). 10-8 M (20-fold decrease), the concentration required to cause decreases in Pb internalization would also have been expected to decrease by a similar amount (eq S3 of the Supporting Information). The expected shift in the inflection point of the internalization curves in Figure 3b (zone within rectangle) was not observed. Furthermore, a pronounced increase in the internalization fluxes was apparently observed in and around the inflection points in the same critical zone. Because Cu induced increases in the Pb bioaccumulation flux cannot be modeled by the biotic ligand model, the experiments were carefully repeated (Figure 4). From Figure 4, it was clear that as Cu increased in the exposure medium, Pb internalization fluxes actually increased except for the very highest concentration of competing ion. Indeed, for the highest Cu2+ concentrations, the constant determined for the binding of Cu to Pb transport sites, 105.4 M-1, was in reasonable agreement with that determined for the binding of Cu to Cu transport sites (i.e., single-metal studies, KCu-Rcell ) 105.8 M-1). Failure of the BLM at Low Cu Concentrations. At high Cu2+ concentrations, the effect of Cu on Pb internalization fluxes and the effect of Pb on Cu internalization could be explained by a single-site competitive equilibrium model such as the BLM. In contrast, at low [Cu2+], results could not be explained by equilibrium assumptions nor could the competition be modeled using a single uptake site. Three hypotheses were proposed to explain the observed discrepancies at low Cu2+: (i) Cu or the Cu/Pb mixtures had a beneficial or deleterious biological effect on the cells. (ii) Several Cu uptake sites were involved in the uptake. (iii) The Cu uptake site was highly regulated by the cell. Effects of Cu on Cell Viability and Cell Permeability. It was first necessary to verify whether the cells were stimulated by Cu at the low exposure concentrations or conversely whether the high concentrations caused cell morality or any sublethal effects. Under identical conditions to the bioaccumulation experiments, cell metabolism was estimated from the intracellular production of formazan, while membrane permeability was evaluated by measuring the penetration of fluorescein diacetate.

Formazan production was completely abolished, as expected, when the cultures were exposed to lethal concentrations of 2% formaldehyde. A significant reduction in cell viability was also revealed by the formazan assay when the cells were exposed to high concentrations of either Pb or Cu (Figure 5a,b). In the simultaneous presence of both metals, viability reductions compared to the control were only observed at high Cu concentrations. No significant differences were observed between controls and cells in the low Cu exposures, suggesting that Cu had neither a stimulatory nor a toxic effect in the critical low Cu concentration range. Although viability decreases (20-30%) at the high Cu concentrations could potentially contribute to the observed decrease in Pb uptake due to a reduction in viable cell numbers, it is unlikely to explain the observed 35-50% reduction in Pb internalization fluxes (Figure 4). Across a given Cu concentration in the Pb2+ exposure solutions, no significant differences in cell viability were observed (ANOVA, p > 0.05; Figure 5b). Documented toxicological effects of Cu include a disruption in the plasma membrane integrity associated with increased permeability to cations and an increase in H+adenosine triphosphatase activity (20, 21). Indeed, Vranken et al. have suggested that synergisms among toxic compounds may result, in part, from an overall increased permeability of the plasma membrane (22). Nonetheless, neither Pb nor Cu appeared to increase membrane permeability under the conditions examined in this study (Figure 5c,d). The only significant permeability reductions were observed for the highest metal concentrations and were consistent with the observed decrease in metal uptake at these concentrations. How Many Cu Uptake Sites Were Observed? The results show that no competitive effect of Pb at low Cu concentrations (Figure 3a) could be explained by the presence of two Cu uptake sites: a Pb-independent, high affinity site and a Pb-dependent, low affinity site. Indeed, such an interpretation would be consistent with genetic and biochemical studies performed on a number of model eukaryotes in which the existence of two types of transmembrane copper transporters VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Viability of the cells as given by (a,b) the formazan production of viable cells and (c,d) permeability of Chlamydomonas reinhardtii to fluorescein diacetate (FDA). White bars represent control exposures: (a,c) [Pb2+] ) 0 M and (b,d) [Cu2+] ) 0 M. Hatched bars represent from left to right: (a,c) [Pb2+] ) 10-7 M and [Pb2+] ) 10-6 M and (b,d) [Cu2+] ) 10-7 M and [Cu2+] ) 10-6 M. Statistically significant differences (student t test) are indicated by * (p < 0.05) or ** (p < 0.01). have been established (18, 19), including the copper transporting P-type ATPases that function to distribute copper within the cell (23) and the CTR family of copper transporters (24, 25). Indeed, Page et al. (15) have indicated that Chlamydomonas has a high-affinity copper uptake system that is induced under conditions of copper deficiency and repressed under conditions of copper sufficiency. This system appears to consist of a cell surface Cu(II) reductase that is more active in copper-deficient cells than in copper-replete cells. The Cu uptake curve in Figure 2a does not unambiguously suggest that a second Cu transporter is being employed by the algae. Indeed, the presence of a second plateau on the uptake curve would typically be indicative of a second Cu transporter. Furthermore, the near overlap of the two internalization curves at low Cu (two lower curves in Figure 3a), suggest that the high affinity transporter would have been operating close to its Jmax. In that case, the estimated Jmax value of (9.3 ( 1.3) × 10-13 mol cm-2 min-1 (Figure 3a) does appear to be consistent with a small plateau in the Michaelis-Menten plot of the Cu internalization fluxes found at (9.4-A3.0) × 10-13 mol cm-2 min-1 (Figure 2a). Although two transport sites are not required to explain the data in Figure 2a, calculations that assume two transport sites with Jmax values of 8.4 × 10-12 mol cm-2 min-1 (Table S1 of the Supporting Information) and 9.3 × 10-13 mol cm-2 min-1 (Figure 3a) result in Cu stability constants of 106.9 and 105.8 M-1. Such an explanation is consistent with the presence of a low affinity, passive transporter operating at higher Cu concentrations and a high affinity, active transporter controlling uptake at low Cu concentrations. Controls on Cu Uptake by the Cell. From Figure 4, it is clear that as Cu increased in the exposure medium, Pb internalization fluxes actually increased except for the very highest concentration of competing ion. For Chlamydomonas, it has been reported previously that ctr2, which codes for a high-affinity copper transporters, is transcriptionally regulated by copper (15). In the absence of Pb, low concentrations of Cu were sufficient to suppress ctr2 expression (3-fold repression, Figure 6). However, when 10-7 M Pb2+ was also present in the exposure media, significant 3584

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FIGURE 6. Gene expression of ctr2 exposed to 0 M Pb (O) and 10-7 M Pb (b). Gene expression values were normalized to the housekeeping gene RPS26. Data are normalized to the control containing no metals and are presented as the mean ( standard error. The dotted line represents the normalized expression of the control. increases in ctr2 expression levels were observed (Figure 6). For a constant Pb2+ concentration, ctr2 expression levels decreased with increasing Cu2+. While no competition was observed between Cu and Pb for the high affinity transporter, it is possible to assume that the increased expression of ctr2 in the presence of Pb shifted Cu biouptake to the high affinity site, which could have resulted in the observed increased internalization of Pb (via the low affinity site). Such an interpretation would be consistent with the increasing Pb internalization (synergism) observed in Figures 3b and 4. Furthermore, the active nature of the uptake (i.e., metal control on transporter expression) clearly demonstrates that the biouptake process for the two metals cannot be simply described by equilibrium modeling. Environmental Relevance. Underlying the BLM is an equilibrium model that assumes either a completely additive interaction for competing metals (two independent sites or

biotic ligands) or a completely antagonistic one (one biotic ligand). If the transport sites on the algae were in simple equilibrium with the bulk solution, the addition of competitors should have resulted in a predictable decrease of Cu or Pb uptake (and the resulting effects). This was clearly not observed. On the contrary, for the two metals examined here, two transporters were required in order to properly account for the observed biouptake results: a high affinity active Cu transporter that may have been regulated by the ctr2 gene and a low affinity passive transporter where Cu2+ and Pb2+ interacted competitively. The high affinity transporter appeared to be under a feedback control from Pb2+ and Cu2+. A synergistic role of Cu has been reported previously for the bioaccumulation of Pb and other metals by different aquatic organisms (26, 27) and bacteria (28). For example, a similar effect has been reported by Komjarova and Blust (29) who showed that the addition of Cu resulted in complex nonlinear variations in gill and whole body Pb uptake rates in zebrafish (Danio rerio). Because Cu interferes with sodium uptake (3), they suggested that the competition of Cu and Pb for Na+ and Ca2+ channels may have contributed to a complex Pb uptake pattern in the presence of Cu. Algae also have a number of transport systems that are sensitive to their external surroundings (30). Cu is an essential trace metal. Essential trace metals are often highly regulated in order to avoid situations of deficiency and overload (31). Because toxic metals can also be internalized by the transport systems available for nutrients, it is essential to understand the cellular regulation of these transport systems when interpreting effects. A schematic representation of the uptake of Pb and Cu is provided in Figure S1 of the Supporting Information with the caveat that the schema has been greatly simplified in order best represent the data collected here (limited concentration range, limited gene probing, etc.). Because metals are often taken up by more than one transport system and because each system may predominate under different physicochemical conditions, the assumption that BLM binding sites are independent of one other is necessarily an oversimplification. Such complex interactions may explain a number of the exceptions that have been observed to the BLM (27, 32). Given that the BLM is more concerned with toxicity (or biological effects) than bioaccumulation, the superposition of toxicity results on top of this already complex scenario would appear to be difficult. Nonetheless, if one assumes that biological effects result from accumulated metal, the above results should be useful in better understanding the complexity of metal-metal interactions and should help facilitate the development of more rigorous tools for their prediction. It will be necessary to keep these limitations in mind when applying the BLM to natural waters containing multiple contaminants.

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Acknowledgments The authors gratefully acknowledge the support of the NSERC MITHE Research Network. A complete list of the sponsors is available at http://www.mithe-sn.org/sponsors.cfm. Z.C. is supported by the China scholarship Council. ICP-MS analysis was run at INRS-ETE; their technical assistance is greatly appreciated.

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Supporting Information Available

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BLM theory as related to bioaccumulation, constants obtained from the Michaelis-Menten plots of uptake fluxes, and a schematic representation of Pb and Cu biouptake. This information is available free of charge via the Internet at http://pubs.acs.org.

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