Comparison of Cd(II), Cu(II), and Pb(II) Biouptake by Green Algae in

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Environ. Sci. Technol. 2007, 41, 4172-4178

Comparison of Cd(II), Cu(II), and Pb(II) Biouptake by Green Algae in the Presence of Humic Acid CRISTINA LAMELAS AND VERA. I. SLAVEYKOVA* Environmental Biophysical Chemistry, Environmental Science and Technology Institute, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Station 2, 1015 Lausanne, Switzerland

The present study examines the role of humic acid, as a representative of dissolved organic matter, in Cd(II), Cu(II), and Pb(II) speciation and biouptake by green microalgae. Cellular and intracellular metal fractions were compared in the presence of citric and humic acids. The results demonstrated that Cd and Cu uptake in the presence of 10 mg L-1 humic acid was consistent with that predicted from measured free metal concentrations, while Pb biouptake was higher. By comparing Cd, Cu, and Pb cellular concentrations in the absence and presence of humic acid, it was found that the influence of the increased negative algal surface charge, resulting from humic acid adsorption, on cellular metal was negligible. Moreover, the experimental results for all three metals were in good agreement with the ternary complex hypothesis. Given that metal has much higher affinity with algal sites than humic acid adsorbed to algae, the contribution of the ternary complex to metal bioavailability was negligible in the case of Cd (II) and Cu (II). In contrast, the ternary complex contributed to over 90% of total cellular metal for Pb(II), due to the comparable affinity of Pb to algal sites in comparison with humic acid adsorbed to algae. Therefore, the extension of the biotic ligand model by including the formation of the ternary complex between the metal, humic acid, and algal surface would help to avoid underestimation of Pb biouptake in the presence of humic substances by green algae Chlorella kesslerii.

1. Introduction Humic substances represent an important fraction of dissolved organic matter (DOM) and influence numerous biogeochemical processes (1). They affect the speciation, transfer, and bioavailability of both trace metals (2) and hydrophobic organic pollutants (3). Humic substances are considered to protect aquatic microorganisms from heavy metal stress by reducing free metal ion concentrations and consequently decreasing their bioavailability (4-6). Nevertheless, experimental evidence exists concerning the controversial effect of humic substances on metal bioavailability (4, 6). Metal bioavailability corresponding to the free ion activity model (FIAM), and its recent development the biotic ligand model (BLM), was found for Cd and different algal species: Chlorella kesslerii (7), Chlamydomonas reinhardtii (8-10), * Corresponding author phone: ++41 21 693 63 31; fax: ++41 21 693 37 39; e-mail: [email protected]. 4172

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and Pseudokirchneriella subcapitata (10, 11). Growth inhibition by Cu consistent with the measured free copper concentrations was obtained for euryhaline algae Monochrysis lutheri (12) and embrio Xenopus loevis (13). Cu and Cd uptake in agreement with speciation measurement was also demonstrated for diatom Stephanodiscus hantzschili and chlorophyte Chlorella vulgaris (14). On the contrary, Al uptake by freshwater algae Chlorella pyrenoidosa (15) and juvenile Atlantic salmon Salmo salar (16) was overestimated, while Pb uptake by C. kesslerii in the presence of both humic and fulvic acids was underestimated by the FIAM (17, 18). Similarly Pb bioavailability to green algae C. vulgaris and diatom S. hantzschili in the presence of river-dissolved organic matter was higher then expected from speciation measurements (14). Moreover, Pb uptake by invertebrates and its toxicity increased in the presence of humic substances, even when compared to an equivalent total Pb concentration (19). Existing controversy is often related to the direct interactions between humic substances and algal surfaces in addition to the metal complexation in the medium (4, 5, 20). For example, due to their amphiphilic character, humic substances were reported to adsorb onto the algal surface and affect metal bioavailability (6, 9, 18, 21). This property seems to be microorganism, humic substance, and pH dependent (9, 21). Consequently, an enhancement of membrane permeability to both lipophilic metal complexes (e.g., Cd-diethyldithiocarbamate by algae P. subcapitata in the presence of Suwannee River humic acid (22)) and other lipophilic substances in the presence of aquatic humic and fulvic acids (23) was found. In addition, an increase of the negative charge on the algal surface and formation of a ternary complex between Pb, algae, and dissolved organic matter components were postulated to contribute to increased Pb uptake (17, 18). Nonetheless, it is currently unclear under what conditions the above direct interactions can significantly affect metal uptake by aquatic microorganisms and to what extent they can be considered generalized phenomena rather than exceptions. The main goal of the present study was to gain additional insight into the role of humic acid-algae interactions in metal bioavailability by comparing Cd(II), Cu(II), and Pb(II) bioavailability to green microalgae in the absence and presence of humic acid. Special attention was paid to two specific issues: (i) the increased negative algal surface charge in the presence of humic substances, and (ii) the possible formation of the ternary complex between metal, humic acid, and algae, and its significance with respect to metal bioupdate in the presence of dissolved organic matter.

2. Experimental Section 2.1. Metal Biouptake by Microalgae Chlorella kesslerii. Cd(II), Cu(II), and Pb(II) biouptake by freshwater green algae Chlorella kesslerii (University of Toronto culture collection, UTCC 266) was determined. Uptake was examined in shortterm experiments (35 min), in order to reduce the effect of efflux, exudate production, or variable cell sizes and numbers. Free metal ion concentrations were varied in the range 10-9 to 10-5 M by preparing mixtures of 5 × 10-3 M citric acid or 10 mg L-1 humic acid and 10-7 to 10-4 M Cd(NO3)2, Cu(NO3)2, or Pb(NO3)2 in 10-2 M MES (2-[N-morpholino]ethanesulfonic acid) at pH ) 6.0. Because Cd or Pb uptake by algae followed the free metal ion model (8, 24), citric acid was used to buffer free metal ion concentration. The complexation of Pb(II), Cd(II), or Cu(II) by MES buffer was considered negligible (25, 26). 10.1021/es063102j CCC: $37.00

 2007 American Chemical Society Published on Web 05/04/2007

C. kesslerii was cultured in a standard algal growth medium (27) at 20 °C, with rotary shaking (100 rpm) and a 12:12 h light:dark regime. Cells were collected by gentle filtration when the mid-exponential algal growth phase was attained and washed with metal-free experimental medium. Algae were further re-suspended in the experimental medium containing a known quantity of metal and citric or humic acid. Final cell density in the medium was ∼8 × 106 to 1 × 107 cells mL-1. Aliquots of algal suspensions (10 mL) were filtered through 3.0-µm pore size regenerated cellulose filters and dissolved metal fraction was measured in the filtrate. Algae on the filter were then washed twice with 5 mL of 10-2 M EDTA (ethylenediaminetetraacetic acid) to remove metals adsorbed on the cell as previously optimized (28). Metal adsorbed on algae was determined in the filtrate. Intracellular fraction was measured following digestion of the cells on the filter with conc. HNO3 (suprapur, Baker) at 100 °C for 1 h. Metal concentration in the dissolved, adsorbed, and intracellular fractions was measured by inductively coupled plasma mass spectrometry (ICP-MS) (Hewlett-Packard 4500, Agilent Technologies, Palo Alto, CA) for each experimental run. Controls and corrections of filter blanks, chemicals used, and metal content in the cells from metal- free medium were performed. Cell densities, sizes, and surface distributions were determined with a Coulter Multisizer III particle counter (50-µm orifice) for each experimental run. Experiments were repeated at least twice, usually 3 times. 2.2. Determination of Free Metal Ion Concentration. Free cadmium, copper, and lead concentrations were measured by Cd2+-, Cu2+-, or Pb2+-selective electrodes, respectively, in each experimental medium before algal resuspension. A low detection limit Pb2+-selective electrode, with an optimized membrane composition, was applied for Pb2+ measurements in the presence of HA (17). Commercial electrodes (ThermoOrion Research ref nos. 9429 and 9448) were used to measure Cu2+ and Cd2+ concentrations. Details concerning experimental setup and calibration procedures can be found in refs 17 and 29. 2.3. Humic Acid Adsorption to Algae. Standard humic acid (HA) isolated from the Suwannee River was obtained from the International Humic Substances Society (Colorado School of Mines, Golden, CO) (30). Stock solution of humic acid (1 g L-1) was prepared in Milli-Q water and stored at 4 °C, and used for preparation of experimental medium containing 0-20 mg L-1 of HA. The quantity of humic acid adsorbed to the algae was determined as a difference between HA concentrations in the medium before and after contact with algae for 35 min. Humic acid concentrations in the medium were measured with a UV-vis spectrophotometer (Perkin-Elmer) at 280 nm. Preliminary experiments demonstrated that the HA adsorbed to 3.0-µm pore size regenerated cellulose filters used for algal isolation and that the amount of adsorbed HA increased with an increase in metal concentrations. Therefore to avoid an overestimation of humic acid adsorption to algae, a systematic control of humic acid losses in the absence of alga was performed. The results were corrected for the adsorption of humic acid to the filters. Control of the release of extracellular substances that could interfere with humic acid measurements was also performed. For Cu concentrations higher than 5 × 10-6 M, a release of exudates by Chlorella kesserii, absorbing at 280 nm, was detected. Therefore the Cu effect on humic acid adsorption was determined only for concentrations below 5 × 10-6 M. No exudates release was observed in the presence of high Pb(II) or Cd(II) concentrations. 2.4. Modeling of Metal Uptake and Humic Acid Adsorption to Algae. For a constant exposure time, metal uptake characteristics of C. kesslerii were determined by the fitting of intracellular (non-EDTA extractable) metal concentrations ({M}int) with the Michaelis Menten equation:

{M}int ) {M}int,max

KS,M[M2+]

(1)

1 + KS,M[M2+]

where {M}int,max is the maximal intracellular metal, [M2+] is the equilibrium free metal ion concentration, and KS,M is the average binding constant to the transport sites on the algal membrane. Similarly, for a given exposure time, the total cellular metal (intracellular plus adsorbed, {M}cell) was fitted by a hyperbolic equation, obtained by assuming that metal adsorbs simultaneously to two type of binding sites, but only one is followed by the internalization (31)

{M}cell ) {M}cell,max

KM-A[M2+]

(2)

1 + KM-A[M2+]

where {M}cell,max is the maximal cellular metal, and KM-A is the effective average binding constant of metal to algae, containing information about the metal adsorbed to both transport and adsorption sites and metal internalization. Given that no saturation was observed for HA adsorption to algae in the studied range of concentration, adsorbed HA concentrations were fitted by a linear regime Henry adsorption isotherm

{HA}ads ) KHA H [HA]

(3)

where {HA}ads is the humic acid adsorbed to algae, [HA] is the bulk humic acid concentration at equilibrium, and KHA H is the Henry adsorption constant. The quantity of total cellular metal ({M}cell,tot) in the system containing algae, metal, and humic acid was calculated as the sum of metal taken up by algae ({M}cell) and metal bound to humic acid adsorbed to algae ({M - HA}cell)

{M}cell,tot ) {M}cell + {M - HA}cell

(4)

The quantity of the metal bound to humic acid adsorbed to 2+ algae was calculated by the expression KHA H [HA]KM-HA[M ], where KM-HA is the binding constant of metal to HA. Therefore, it can be obtained that

{M}cell,tot ) {M}cell,max

KM-A[M2+] 1 + KM-A[M2+]

2+ + KHA H [HA]KM-HA[M ] (5)

3. Results and Discussion To better understand different interactions in the ternary systems of interest, each of the binary systems containing algae and humic acid, algae and metal, or metal and humic acid was studied. Obtained conditional binding constants were further used to model Cd(II), Cu(II), and Pb(II) uptake to Chlorella kesslerii in the presence of humic acid. Model estimations were compared to the experimentally obtained cellular and intracellular concentrations as measures for metal bioavailability. Hypotheses concerning the electrostatic and ternary complex contributions were further tested. 3.1. Metal Complexation by Humic Acid. Free metal ion concentrations were measured by the ion selective electrodes with the aim of determining the conditional binding constant for metal-humic acid complexes under the studied conditions (pH ) 6.0 and I ) 5 × 10-3 M). The proportion of complexed metal increased with decreasing total metal concentrations. For a constant concentration of HA (10 mg L-1), [Cd]tot/[Cd2+] decreased from 9 to 1.3 when the total Cd concentration was varied between 10-7 M and 5 × 10-5 M. In the case of Cu, [Cu]tot/[Cu2+] ranged from 85 to 7, when VOL. 41, NO. 11, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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the total concentration increased from 3 × 10-7 to 10-5 M. The experimentally obtained conditional stability constants for Cu-HA complexes KCu-HA varied between 107.3 and 105.2 for a degree of site occupation θ ) [CuHA]/[HA] ranging from 10-2 to 10-0.6 and assuming 4.9 mmol g-1 of carboxyl groups (30) and 80% deprotonation at pH of 6.0. Under the similar conditions, Pb-humic acid complexes were less stable, with KPb-HA ) 106.6 and 104.4 for a degree of site occupation θ ranging from 10-2.7 to 10-0.35. The lowest conditional stability constants were determined for Cd-HA complexes: KCd-HA decreased from 104.2 to 103.4 for θ range from 10-3 to 10-0.9. In summary, for similar metal to Suwannee River humic acid ratios, the conditional binding constants decreased in the order KCu-HA > KPb-HA > KCd-HA in agreement with the Irving-Williams series (32). Obtained binding constants were further used to evaluate both the quantity of metal bound to humic acid adsorbed to algae and free metal ion concentrations in the medium, assuming that humic acid on the algal surface and in the bulk exhibit similar complexing properties. 3.2. Humic Acid Adsorption to Chlorella kesslerii. The amount of humic acid adsorbed to algae ({HA}ads) increased linearly by increasing bulk humic acid concentration from 2 to 20 mg L-1 (Figure 1 in the Supporting Information). The -3 cm was Henry adsorption constant KHA H of (8.7 ( 0.3) × 10 determined. The obtained value was comparable to that estimated for fulvic acid binding to C. kesslerii (KFA H ) 9 × 10-3 cm) under similar experimental conditions (pH ) 6.0 and I ) 5 × 10-3 M) (18). In contrast, KHA H was lower than that -2 estimated for algae Chlorella sp. at pH 4.0 (KHA H ) 5 × 10 HA cm) and for two other green algae C. reinhardtii (KH ) 3 × -2 cm) (21), which 10-1 cm) and S. subspicatus (KHA H ) 1 × 10 is in agreement with the lower humic acid adsorption to algae at higher pH. Although not yet very well understood, the adsorption mechanism of humic substances to microalgae is assumed to involve electrostatic and hydrophobic interactions and/or the formation of hydrogen bonds (9, 21). Hydrogen bond formation between covalently bound OH groups on algae and carboxylic groups of fulvic acid was suggested (9). The interaction between the hydrophobic domain of humic substances and hydrophobic moieties of lipids and proteins in the cell walls was also considered to contribute to the fulvic acid binding to algae (33). 3.3. Metal Bioaccumulation in the Presence of Citric Acid. Cellular (adsorbed plus intracellular) and intracellular metal quantities increased when the free metal concentration in the medium containing mixtures of metal and citric acid was augmented for Cd(II), Cu(II), and Pb(II). The increase of bulk free Cd concentrations from 10-9 to 10-7 M resulted in a linear increase (slope of 1) in both cellular and intracellular Cd (Figure 1a) in agreement with the FIAM. Likewise, Cd uptake fluxes by algae C. reinhardtii followed FIAM in the presence of different synthetic ligands, including citric acid. In contrast, in the presence of citric acid intracellular Cd uptake by algae Selenastrum capricornutum was underestimated by FIAM, because of the accidental transport of the Cd-citric acid complex across the membrane (34). Cellular cadmium tended to saturation at Cd2+ larger than approximately 5 × 10-6 M, while intracellular Cd reached constant values at lower Cd2+ (e.g., [Cd2+] > 5 × 10-7 M). The existence of a single saturation plateau supported the hypothesis that Cd internalization occurs predominantly via a single transporter. The amount of maximal cellular metal after 35 min of exposure ({Cd}cell,max) was higher than that of maximal intracellular Cd, as could be expected from the multiple binding sites on the algal surface. The effective conditional binding constant corresponding to the total cellular Cd (KCd-A) was estimated by eq 2 to be 105.6 M-1 4174

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FIGURE 1. Logarithmic representation of cellular (adsorbed + intracellular, squares), and intracellular (circles) metal as a function of free metal concentration in the presence of citric acid (full symbols) or 10 mg L-1 HA (open symbols) at pH 6.0, exposure time ) 35 min. Solid and dashed lines correspond to the fit by the eqs 1 and 2 of intracellular and cellular metal respectively, obtained in the presence of citric acid. Error bars represent the standard deviations from 2-4 experiments: (a) Cd(II), (b) Cu(II), and (c) Pb(II). (Table 1). This value was approximately 20 times lower than that corresponding to the Cd binding sites of the membrane (KS,Cd ) 106.9 M-1), since it takes into account both metal adsorbed on the algae and intracellular metal. Obtained binding constants were in general agreement with those reported for Cd binding by different organisms as reviewed in ref 6. For example, conditional binding constants for Cd to membrane transport sites KS,Cd of 106.2 M-1 were estimated for the green algae C. reinhardtii at pH ) 7.0 (8). KS,Cd of 106.7 M-1 was obtained for C. kesslerii by measuring the internalization fluxes at pH ) 6.0 (7). Slightly higher values were determined for other microalgae P.

TABLE 1. Maximal Cellular ({M}cell,max) and Intracellular ({M}int,max) Metal Concentrations (mol cm-2) and Respective Conditional Binding Constant (M-1) at pH ) 6 and I ) 5 × 10-3 M, 35 min Exposure Time, Obtained from the Experiments in the Presence of Citric Acid metal Cd (II) Cu (II) Pb (II) a

{M}cell,max (2.6 ( 0.8) × (1.6 ( 0.3) × 10-11 a (1.2 ( 0.2) × 10-9 b (6.1 ( 0.5) × 10-10

Values corresponding to high-affinity binding sites.

{M}int,max

KM-A

10-10

10-11

105.6 108.6 a 104.8 b 105.2 b

(2.1 ( 0.6) × (1.3 ( 0.4) × 10-11 a (1.9 ( 0.7) × 10-10 b (1.5 ( 0.5) × 10-11

KS,M 106.9 109.1 a 105.4 b 10 5.5

Values corresponding to low-affinity binding sites.

subcapitata KS,Cd ) 107.6 at pH ) 5.0 in the nanomolar range of Cd concentrations (10). More complex was the interaction between Cu(II) and algae. As for Cd(II), the increase of free copper in the medium resulted in an increase in both cellular and intracellular copper concentrations. In contrast to Cd, however two saturation plateaus were observed (Figure 1b). The first corresponded to the medium free copper concentration Cu2+ in the range 10-8 to 10-7 M, and the second was obtained at [Cu2+] above 10-5 M. The experimental data for intracellular copper were well fitted by two binding sites with the Michealis Menten equation, which permitted determination of Cu binding characteristics corresponding to the two binding sites (Table 1). This finding is in agreement with literature data for Cu uptake by other green algae Scenedesmus subspicatus (33), where a similar biphasic uptake system has been demonstrated. However, conditional stability constants were higher (KS,Cu1 ) 1013.2 M-1 and KS,Cu2 ) 1011.6 M-1), and could be related to the lower free Cu concentration used. The obtained results contrasted to a single uptake system with KS,Cu ) 106.9 M-1 obtained from Cu toxicity studies with the tropical freshwater alga Chlorella sp. (35). Similar to Cd, the total cellular and intracellular Pb concentrations in the presence of citric acid were directly related to the free lead ion concentrations with a single saturation plateau above 10-5 M Pb2+ (Figure 1c). Under the studied conditions, in the whole concentration range the adsorbed lead was higher than the intracellular, confirming the restrained internalization of this toxic metal. The obtained values for the binding constants corresponding to total cellular and intracellular Pb are given in Table 1. They were in good agreement with those calculated from the internalization fluxes in the presence of different synthetic ligands (KS,Pb ) 105.5 M-1) (24). Overall, the results from biouptake studies revealed that in the presence of citric acid, the binding affinity of the metal to algae decreased in the order: Cu2+ . Cd2+ > Pb2+ for metal concentrations below 10-7 M and Cd2+ > Cu2+ ≈ Pb2+ for metal concentrations above 10-7 M . 3.4. Metal Bioaccumulation in the Presence of Humic Acid. Metal loading of Chlorella kesslerii was reduced significantly in the presence of 10 mg L-1 of humic acid in comparison to a system without humic acid. This reduction was more pronounced for Cu than for Cd, as could be expected from the higher binding affinity of Cu to humic acid. Moreover, intracellular and cellular Cd or Cu in the presence of 10 mg L-1 humic acid were superimposed on the experimental points corresponding to equivalent free metal ion concentrations, obtained in the presence of citric acid (Figures 1a and b, full symbols), while those determined for Pb were shifted to higher values (Figure 1c, full symbols). These results were in agreement with previous studies on Cd uptake by different green algae (7, 8, 10) or Cu toxicity to algae Monochrysis lutheri (12) as well as Pb biouptake in the presence of humic substances (17-19). Comparison of the biouptake of the three divalent metal ions Cd(II), Cu(II), and Pb(II) in the presence of 10 mg L-1 HA allowed testing as to whether or not the enhancement

of negative algal surface charge (17, 18) in the presence of humic substances could affect metal biouptake. In fact, for the same humic acid concentrations, the modifications of the algal surface charge, due to humic acid adsorption, should be the same for divalent ions Cd(II), Cu(II), and Pb(II). For example, under the studied conditions the electrophoretic mobility value increased from -2.6 × 10-8 to -3.05 × 10-8 m2 V-1 s-1 when 10 mg L-1 HA was present (17). This value corresponded to more negative algal surface potential that would result in increased surface concentrations of Cd2+, Cu2+, or Pb2+ in comparison to those in the bulk solution. Based on this electrostatic consideration, a similar increase in the cellular Cd, Cu, or Pb would be expected. This contrasted to the experimental observation that no increase in the uptake for Cd(II) and Cu(II) was observed in the presence of HA for comparable free metal concentrations (Figure 1 full symbols). Thus the increase in the negative surface charge seems to play an insignificant role in the uptake of these metals under the studied conditions. Moreover, no increase in Cd bioaccumulation by algae P. subcapitata for a similar increase in the algal surface charge was found (9, 10), further supporting the idea that the changes in algal surface charge play a limited role in the uptake of these metals. The formation of a ternary complex between Pb-humic acid and algae and its contribution to the biological uptake were explored as a reasonable possibility to account for enhanced Pb internalization observed in the presence of different components of dissolved organic matter in our previous study (17). In the present work, the potential contribution of ternary complex to metal bioavailability was further evaluated for Cd(II) and Cu(II) and compared to the biouptake of Pb(II). In essence, metal bound to microalgae in the presence of humic acid is postulated to be the sum of the metal bound to the algae and metal bound to the humic acid adsorbed to algae (eq 5). Depending on the relative contribution of these two fractions to the metal uptake, the increase or not in cellular metal concentration could be observed. To highlight the contribution of the ternary complex to metal bioavailability, two limiting cases will be considered. The first corresponds to the case where the metal has much higher affinity to algal sites than to the humic acid adsorbed to algae. Thus for given free metal ion concentration and prior to the saturation: {M}cell,max KM-A . KHA H KM-HA[HA]. In this case, the contribution of the ternary complex to metal bioavailability will be negligible and measured cellular concentrations will be consistent with BLM model predictions. On the other hand, for {M}cell,max KM-A , KHA H KM-HA[HA], the ternary complex would be expected to contribute to cellular metal. The obtained values of the conditional binding constant for metal-humic acid, humic acid-algae, and metal-algae (see above sections) were further employed to predict cellular metal concentrations by assuming that the ternary complex contributed to uptake. Within the whole concentration range {Cd}cell,max KCd-A was much greater than KHA H KCd-HA[HA], and therefore no contribution of the ternary complex could be expected. Indeed, more than 90% of the cellular Cd would be expected to bind VOL. 41, NO. 11, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Humic acid adsorbed to algae ({HA}ads (M)) as a function of total metal concentration ([HA]ini ) 10 mg L-1, I ) 5 × 10-3 M, pH ) 6.0: Cd (1), Cu(9), and Pb (b)).

FIGURE 2. Comparison of experimentally determined cellular metal in the presence of 10 mg L-1HA over free metal concentrations (b) to model estimation (lines). Representation of total quantity of metal bound to algae estimated by eq 5 (---); metal bound to algae (s); metal bound to HA adsorbed to algae (‚‚‚); (a) Cd(II), (b) Cu(II), and (c) Pb(II). Parameters used to make the model metal algae interaction -6 L cm-2 ; K taken from Table 1. KHA Cd-HA varied from H ) 8.7 × 10 104.2 to 10 3.4 M-1 for Cd concentrations between 10-7 and 10-5 M Cd; KCu-HA varied from 107.2 M-1 for 10-7 M Cu to 105.2 M-1 for 10-5 M Cu. KPb-HA varied from 106.2 M-1 for 5 × 10-7 M Pb to 104.5 M-1 for 5 × 10-5 M Pb. Data for the metal uptake in the presence of HA are taken from Figure 1. to algae, while the fraction of ternary complexes is estimated to represent between 7 and 10% of the total cellular metal. On the contrary, in the case of Pb(II), {Pb}cell,maxKPb-A was always much lower than KHA H KPb-HA[HA], thus a major contribution of the ternary metal complex to bioavailability should be expected. In the presence of 10 mg L-1 HA, the fractions of ternary complexes are estimated as being approximately 95% and 57% at 10-9 and 10-6 M [Pb2+], 4176

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respectively. For Cu, 70-75% of the cellular Cu was directly associated to the high or low affinity sites of algae, while less than 30-25% was bound to the humic acid adsorbed to algae. Therefore no significant contribution of the ternary metal complex to Cu biouptake should be expected. Indeed, a good agreement between experimentally determined cellular Cd, Cu, or Pb in the presence of humic acid and the above model estimations was obtained (Figure 2). Although the uptake of Pb by C. kesslerii in the presence of humic acid was higher than that expected from data in the absence of ligands (17), a good fit between experimental results and model predictions was obtained for cellular Pb in the presence of 10 mg L-1 HA. (Figure 2c) The above model consideration explicitly assumes that the metal binds to humic acid adsorbed to algae (e.g., formation of the complex: M-HA-algae). This could be valid only on the condition that the adsorption of the humic acid does not significantly modify the interaction between algae and metal and conversely that the metal does not affect humic acid adsorption (e.g., by bridging). To explore the latter assumption, the effect of Cu(II), Cd(II), and Pb(II) on humic acid adsorption was compared. For the same concentration of metal-humic acid complex, if metal bridging is the prevailing mechanism, it could be expected that the amount of humic acid adsorbed to the algal surface should be greater for the metal with higher binding affinity to algae and will decrease in the following order: Cu(II) > Cd(II) > Pb(II). Contrary to that expectation, experimental observations revealed that humic acid adsorbed to the algal surface was independent of the presence or absence of increasing concentrations of Cu, Cd, or Pb in the range 10-8 to 10-5 M (Figure 3). The lower proportion of metal-humic acid complexes (M-HA) as compared with the free humic acid might hide the effect. Only for total lead concentrations greater than 10-5 M did the concentration of the Pb-HA prevail and an increase of the humic acid adsorption was determined. Although no such tendency was observed (Figure 3), it is difficult to draw conclusions because of the indirect manner of determining HA adsorption and because of exudate release in the presence of high Cu concentrations. In any case in the presence of environmentally relevant metal concentrations, no increase in humic acid adsorption on C. kesslerii could be expected. In conclusion, results obtained in the present study suggest that in addition to complexation of the metal in the medium, humic acid adds supplementary binding sites to the algal surface. The contribution of those binding sites (e.g., by ternary complex formation) to metal biouptake by algae, and therefore an underestimation of metal bioavail-

ability by models such as the biotic ligand model, could be expected to be greater under the following conditions: (i) high quantity of humic acid adsorbed onto algal surface; (ii) relatively high affinity of the metal to humic acid; or (iii) relatively low affinity of the metal to algae. These results imply that a further extension of the biotic ligand model by including the formation of a ternary complex would improve its site-specific predictive capacity, especially in the case of dissolved organic carbon-rich surface waters. To account for the ternary complex, only one additional parameter, the conditional binding constant of the humic acid to algae, needs to be added to the “classic” BLM. This extension of predictive capacity would help to resolve the existing controversy in literature data concerning metal bioavailability in the presence of dissolved organic matter. It is believed that the obtained results and their implications represent a step toward the improvement of understanding concerning the interactions of metal, dissolved organic matter, and algae in natural waters and the establishment of site-specific water quality criteria.

Acknowledgments Warm thanks are extended to the Swiss National Science Foundation PP002-102640 for providing funding directly related to this work. The technical assistance of Michel Martin in ICP-MS measurements is highly appreciated. Stimulating discussions with K. Dedieu, E. Alasonati, R. Hajdu, I. Xifra Olive´, and Z. Szigeti Al Gorani were very helpful in improving the manuscript.

Supporting Information Available Data concerning humic acid adsorption to algae versus bulk humic acid concentration obtained by UV-vis spectrophotometry. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review December 31, 2006. Revised manuscript received March 21, 2007. Accepted March 29, 2007. ES063102J