Ni Uptake by a Green Alga. 1. Validation of Equilibrium Models for

May 16, 2007 - University of Geneva, 30 Quai Ernest Ansermet,. 1211 Geneva 4 ... green alga Chlamydomonas reinhardtii were measured in the presence of...
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Environ. Sci. Technol. 2007, 41, 4258-4263

Ni Uptake by a Green Alga. 1. Validation of Equilibrium Models for Complexation Effects ISABELLE A. M. WORMS,† NALINI PARTHASARATHY,† AND K E V I N J . W I L K I N S O N * ,‡ CABE (Analytical and Biophysical Environmental Chemistry), University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland, and Department of Chemistry, University of Montreal, P.O. Box 6128, Succursale Centre-ville, Montreal, Canada H3C 3J7

Short term ( 18 MΩ cm, total organic carbon 1. Under the conditions that were employed here, L varied from 1.05 × 10-7 for 5 × 10-6 M NTA (inert complex) to 13.42 for 10-5 M DGA (labile complex).

Results and Discussion Ni Speciation. Based upon the thermodynamic calculations, in the absence of added ligand, Ni2+ accounted for >98% of the total Ni in the experimental solutions. These solutions were used to calibrate the ion exchange resins, giving a selectivity coefficient, λ, of 1.2 ( 0.3 g L-1. Validation was performed using the hydrophilic complexes. Experimental values were well correlated with calculated values with a coefficient of determination of 0.99 (data not shown). Neutral complexes could not be determined with this technique (39). Instead, the PLM technique was used to verify the formation of soluble lipophilic complexes in the presence of both 8-hydroxyquinoline and diethyldithiocarbamate (26). In the absence of a lipophilic ligand, no Ni was detected in the receiving cell of the modified PLM device, indicating that Ni2+ could not cross the lipophilic membrane separating the two half-cells. In contrast, in the presence of Ox or DDC, Ni concentrations in the strip solution increased linearly with time (Figure 1). For conditions in which Ni(Ox)20 represented 85% of the Ni in the source solution, the metal flux across the PLM was (2.02 ( 01) × 10-15 mol cm-2 s-1 while it was (8.58 ( 0.05) × 10-15 mol cm-2 s-1 for solutions containing 99% Ni(DDC)20. These fluxes corresponded to permeabilities of (2.97 ( 0.02) × 10-7 cm s-1 for the oxine complexes and (6.54 ( 0.03) × 10-7 cm s-1 for the DDC complexes. Ni Uptake. Initial uptake experiments were designed to verify the major assumptions of the equilibrium models, i.e., 4260

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FIGURE 1. Transport of Ni across the toluene-phenyl hexane supported PLM for a solution containing [Ni]tot ) (3.2 ( 0.1) × 10-8 M in the presence of 10-5 M oxine (b) or 5 × 10-6 M DDC (O) in 10-2 M NaMES, pH ) 6.0. Standard deviations are given when larger than the symbol size (n ) 2).

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FIGURE 2. Intracellular nickel (b), dissolved Ni (4), and the sum of adsorbed, internalized, and dissolved Ni (2) as a function of time for C. reinhardtii exposed to 3 × 10-7 M Ni2+ in the absence of a trace metal buffer. The solid line corresponds to the linear regression of intracellular Ni with time. internalization was rate-limiting, metal efflux was negligible, metal depletion in the bulk media was negligible. Indeed, dissolved nickel was rapidly consumed by the algae. Metal depletion of the bulk media was most important in the absence of metal buffer and for [Ni2+] < 10-6 M, decreasing by a maximum of 20% within 60 min (Figure 2, open triangles). At each time point, Ni was recovered ((10%) from the sum of adsorbed, intracellular, and dissolved metal (Figure 2, solid triangles). Over the short-term (max. 60 min) experiments, Ni internalization (i.e., non EDTA extractible Ni) was linear with time, although a small reduction in the internalization flux may have been observed near the end of the experiments (Figure 2, solid circles). Variations in the internalization fluxes and Ni depletion were subsequently minimized by using (metal) buffered solutions and a 30 min exposure. Ni Desorption and Efflux. The EDTA wash is an operational technique employed to distinguish between adsorbed and internalized metal that is based upon the assumption that in the presence of EDTA, desorption kinetics will be rapid while the elimination kinetics of a metal that has crossed a biological membrane (efflux) will be much slower (40, 41). Indeed, a significant proportion of the Ni was removed within first 5 min of the EDTA extraction (Figure 3). The subsequent, slowly desorbed fraction that was eliminated at longer rinse times can be attributed either to slow desorption of Ni or to Ni efflux. Experiments were not extended beyond 20 min to avoid deleterious effects of the EDTA on the algae. An efflux rate constant of keff ) (2.4 ( 0.7) × 10-2 min-1 was determined from a first-order exponential decay eq 6 for the points >1 minute: t t)1 [Ni]int ) [Ni]int × e-keff×t

(6)

Since EDTA has been shown to destabilize plasma membrane constituents, the decrease in internalized Ni found in the presence of EDTA could also be due to its perme-

FIGURE 3. Percentage of cellular Ni remaining as a function of time following the addition of 10-3 M EDTA. Cells were preincubated 30 min with 5 × 10-6 M Ni2+ at pH 6.0 in the absence (b) or presence (O) of 10-5 M Ca.

FIGURE 4. Logarithmic representation of Ni internalization fluxes as a function of [Ni2+] in the absence (O) and presence of diglycolic acid (b), citric acid (9), and NTA (1). The dashed line represents the calculated maximum diffusive flux for Ni2+. The solid line represents a Michaelis-Menten plot (eq 4) determined using KM ) (7.3 ( 1.2) × 10-6 M and Jmax ) (6.6 ( 0.2) × 10-13 mol cm-2 min-1. In all cases, standard deviations (n ) 3) are smaller than the symbol size. The coefficient of determination, r2, for values calculated from the Michaelis-Menten plot as a function of the experimental data was 0.99. abilization (42). However, in the case of Cd biouptake by the same algae, the efflux rate constant determined using the same protocol was 1 order of magnitude lower (4.2 ( 1.5) × 10-3 min-1 for a similar initial metal concentration (35). Since Ca decreased Ni loss (Figure 3, (43)) but not Pb or Cd losses, it is very likely that a strong Ni efflux rather than a slow Ni desorption was responsible for the decreasing cellular Ni. Ni Internalization Fluxes. Based upon the above results, great care was taken to avoid both metal depletion in the bulk media and significant cellular efflux in internalization experiments. For cells exposed to 10-8 M to 10-3 M Ni2+, internalization fluxes, Jint, were determined from the slopes of the intracellular Ni over the first 30 min of the exposure (Figure 2). Experimental internalization fluxes (data points, Figure 4) were 2 orders of magnitude lower than the theoretical maximum diffusion fluxes (dotted line calculated for DNi ) 5.57 × 10-6 cm s-1), a strong indication that internalization was rate-limiting under these conditions. The previous results are consistent with metal uptake being under a thermodynamic control with the implication that, below saturation of the uptake sites, biouptake will be directly proportional to [Ni2+] (i.e., in line with the BLM or FIAM). Indeed, in the absence of trace metal complexes, fluxes were directly proportional to [Ni2+] prior to attaining a constant value at high metal concentrations, i.e., above 10-6 M (open points, Figure 4). Based upon the fit of the Michaelis-Menten equation (solid line, r 2 ) 0.99), the results were consistent with a biological uptake that is mediated by a single transport pathway with a half saturation constant

FIGURE 5. Nickel internalization fluxes, Jint, as a function of Ni2+ measured by ion exchange for Chlamydomonas reinhardtii (white symbols) and Chlorella kesslerii (black symbols). Fluxes were determined from a 40 min exposure to solutions containing either 10-7 M Ni and 5-20 mg L-1 of HA (squares) or 5-20 mg L-1 FA (triangles) or for solutions containing 5 × 10-9 - 10-6 M Ni and 1-25 mg L-1 of FA (diamonds). For both alga, control experiments were performed using 10-5 M citrate (circles). The solid line represents the Michaelis-Menten plot obtained previously for C. reinhardtii in the absence of HS (i.e., Figure 2). When predicted Jint values are plotted against measured values, slope ) 0.91 ( 0.05, r 2 ) 0.97 for C. reinhardtii and slope ) 1.09 ( 0.05, r 2 ) 0.98 for C. kesslerii. Standard deviations (n ) 4) are shown when larger than the symbol size. KM ) 10-5.1 M (KNi-Rs ) 1/KM ) 105.1 M-1) and a maximum internalization flux, Jmax ) 6.61 × 10-13 mol cm2 s-1. Hydrophilic Complexes. Internalization fluxes were also evaluated for [Ni2+] ranging from 2.5 × 10-10 M to 10-3 M in the presence of three well-defined hydrophilic Ni complexes (NTA, citrate, or diglycolic acid) for which complex labilities, with respect to the biouptake process, ranged from inert (NTA, citrate) to labile (DGA) (see Materials and Methods). In all the cases, ligand concentrations were in large excess (g10×) compared to the total concentration of Ni. In the presence of Ni complexes, internalization fluxes were directly proportional to the concentration of free nickel (Figure 4, solid points), without any significant change in the Michaelis-Menten parameters (KM ) (9.9 ( 0.9) × 10-6 M, Jmax) (6.9 ( 0.2) × 10-13 mol cm-2 min-1; Student t test, P < 0.02) as compared to those obtained in the absence of the complexes. The dependence of the internalization flux on the free ion concentration is strong evidence that equilibrium was attained between the metal in the bulk solution and that adsorbed to biological transport sites. The results, while somewhat expected, are not in complete agreement with previous work on Ni. For example, in polluted waters the growth rate of P. subcapitata was shown to decrease as the quantity of labile Ni complexes (free + inorganic complexes) increased (21). In that case, Ni speciation was obtained by CLEM (competing ligand exchange method) using exchange with a Chelex 100 resin and quantification by atomic absorption. Humic Substances. Given the above results, our working hypothesis was that humic substances would reduce biouptake in direct proportion to the concentration of free ion. To have a medium that was as realistic as possible and to limit cellular efflux (41, 43), 10-5 M Ca2+ was added to the experimental media. Furthermore, two alga (C. reinhardtii, C. kesslerii) were examined to see if the conclusions could be generalized for organisms with very different internalization fluxes. Indeed, Ni internalization fluxes by C. kesslerii were 2 orders of magnitude smaller than those observed for C. reinhardtii, in good agreement with results obtained previously for Cd (23, 44). For C. kesslerii, control experiments were run in the same medium but in which [Ni2+] was varied in the presence of citrate. In the presence of the humic substances, Ni biouptake by C. reinhardtii could be fitted on the basis of the Michaelis-Menten parameters that were obtained above (Figure 5, only data obtained below saturation VOL. 41, NO. 12, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Effect of lipophilic complexes on Ni permeability (PNi) for the green alga Chlamydomonas reinhardtii. Permeability was evaluated on the basis of calculated [Ni2+] (black bars) or calculated [NiL] (white bars) and normalized by PNi for the control solution. Grey bars indicate PNi values, calculated on the basis of the predominant species, determined in the presence of 8.12 × 10-5 M Mg2+. Experiments were performed using 4.3 × 10-8 M Ni and 10-5 M Ox or 5 × 10-6 M DDC in 10-2 M NaMES at pH 6. are presented). For both algal species, Ni internalization fluxes were linearly related to the concentration of free ion determined by IET, independent of the experimental protocol. The above result was in very good agreement with the biotic ligand or free ion activity (i.e., equilibrium) models. In contrast, in the presence of humic substances, the uptake of trace metals has been shown to increase with respect to predictions made on the basis of equilibrium concentrations of free ion (e.g., 11, 13). This result has been attributed to the adsorption of natural organic matter (NOM) on the biological surface with a subsequent modification of cell surface charge (10, 11, 13) or membrane permeability (10, 45, 46); or to the formation of a bioavailable ternary complex on the cell surface (13). The latter explanation is consistent with results that show that in the presence of organic matter, Cd internalization was predicted by simple equilibrium considerations (23, 47, 48). For C. reinhardtii, results for Ni biouptake are consistent with those obtained for Cd biouptake by the same alga (i.e., good predictive ability of [Mz+] in both cases). For C. kesslerii, the lack of increase in Ni bioaccumulation in the presence of humic substances (this study) strongly suggested that previously observed increases in Pb uptake were not due to an increased surface charge nor to an overall increase in membrane permeability (PNi ) JNi/[Ni2+]) but rather to the formation of a bioavailable ternary complex (NOM-Pb-Rs; 13). The precise role of NOM will therefore depend on the nature of the NOM, the biological surface, and the trace metal being examined. Lipophilic Complexes. While the role of hydrophilic ligands on trace metal uptake has been reasonably well studied, experiments examining the role of lipophilic complexes are more rare. In the presence of 8-hydroxyquinoline (Ox) and diethydithiocarbamate (DDC), biouptake (permeability) was greater than could be predicted on the basis of [Ni2+] alone (Figure 6, black bars). When Ni permeability was calculated on the basis of the predominant species in solution, Ni decreased in the order Ni2+ ≈ Ni(DDC)20 > Ni(Ox)20 (Figure 6, white bars). The presence of hardness ions such as Mg2+ generally decreases Ni2+ permeability (49) but is expected to have little effect on hydrophobic transport. In this case, metal fluxes were 15.5× faster for DDC complexes and 1.6× faster for Ox complexes (Figure 6, gray bars), in good agreement with results obtained for the diatom Thalassiosira weissflogii (16). For both lipophilic complexes, transport across the biological membrane was much more affected than transport across the permeation liquid membrane, likely due to the complex nature of the biological membrane, including the effects of cations on membrane rigidity (factors not seen for the PLM). Given their larger 4262

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permeability in complex media, even a small amount of hydrophobic complex could have a disproportionate effect on biouptake and potential toxicity in natural waters. Nonetheless, other than the determination of a few hydrophobic pesticides or siderophores (14, 50), few data are currently available on the environmental prevalence of hydrophobic metal complexes, mainly due to a lack of suitable analytical techniques (51). Environmental Implications. A number of regulatory agencies still routinely employ total or “dissolved” metal concentrations to set maximum acceptable levels for effluents or pollutant point sources. Based on the work presented here, it would appear that predictions of Ni bioavailability would greatly benefit from BLM or FIAM based models and (equilibrium based) measurements of Ni speciation. The presence of hydrophilic complexes and complexes with humic substances reduced bioaccumulation of Ni in direct proportion to the concentration of the complexes (i.e., bioaccumulation directly related to [Ni2+]). Since the presence of hydrophobic ligands increased bioaccumulation, their quantification and behavior in natural waters should be an area of concern. Among other areas for future research, the role of (hydrophobic) engineered nanoparticles should be carefully considered.

Acknowledgments We thank Dr. M. Martin for ICP-MS measurements as well as J.P. Lander and D. Wahida for technical assistance with the ion exchange technique. The Canadian Natural Sciences and Engineering Council and the ECODIS project (European Commission’s 6th framework program, subpriority 6.3 “Global Change and Ecosystems”, contract 518043) are acknowledged for providing funding for this work.

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Received for review December 20, 2006. Revised manuscript received March 2, 2007. Accepted March 12, 2007. ES0630339

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