Liesbeth Verheyen

Metal Complexation Properties of Freshwater Dissolved Organic Matter Are Explained by Its Aromaticity and by Anthropogenic Ligands

Dissolved organic matter (DOM) in surface waters affects the fate and environmental effects of trace metals. We measured variability in the Cd, Cu, Ni, and Zn affinity of 23 DOM samples isolated by reverse osmosis from freshwaters in natural, agricultural, and urban areas. Affinities at uniform pH and ionic composition were assayed at low, environmentally relevant free Cd, Cu, Ni, and Zn activities. The C-normalized metal binding of DOM varied 4-fold (Cu) or about 10-fold (Cd, Ni, Zn) among samples. The dissolved organic carbon concentration ranged only 9-fold in the waters, illustrating that DOM quality is an equally important parameter for metal complexation as DOM quantity. The UV-absorbance of DOM explained metal affinity only for waters receiving few urban inputs, indicating that in those waters, aromatic humic substances are the dominant metal chelators. Larger metal affinities were found for DOM from waters with urban inputs. Aminopolycarboxylate ligands (mainly EDTA) were detected at concentrations up to 0.14 μM and partly explained the larger metal affinity. Nickel concentrations in these surface waters are strongly related to EDTA concentrations (R2=0.96) and this is underpinned by speciation calculations. It is concluded that metal complexation in waters with anthropogenic discharges is larger than that estimated with models that only take into account binding on humic substances.

Diffusion limitations in root uptake of cadmium and zinc, but not nickel, and resulting bias in the Michaelis constant

It has long been recognized that diffusive boundary layers affect the determination of active transport parameters, but this has been largely overlooked in plant physiological research. We studied the short-term uptake of cadmium (Cd), zinc (Zn), and nickel (Ni) by spinach (Spinacia oleracea) and tomato (Lycopersicon esculentum) in solutions with or without metal complexes. At same free ion concentration, the presence of complexes, which enhance the diffusion flux, increased the uptake of Cd and Zn, whereas Ni uptake was unaffected. Competition effects of protons on Cd and Zn uptake were observed only at a very large degree of buffering, while competition of magnesium ions on Ni uptake was observed even in unbuffered solutions. These results strongly suggest that uptake of Cd and Zn is limited by diffusion of the free ion to the roots, except at very high degree of solution buffering, whereas Ni uptake is generally internalization limited. All results could be well described by a model that combined a diffusion equation with a competitive Michaelis-Menten equation. Direct uptake of the complex was estimated to be a major contribution only at millimolar concentrations of the complex or at very large ratios of complex to free ion concentration. The true Km for uptake of Cd2+ and Zn2+ was estimated at <5 nm, three orders of magnitude smaller than the Km measured in unbuffered solutions. Published Michaelis constants for plant uptake of Cd and Zn likely strongly overestimate physiological ones and should not be interpreted as an indicator of transporter affinity.

Labile complexes facilitate cadmium uptake by Caco-2 cells

The Free Ion Activity Model (FIAM) predicts that metal uptake in biota is related to the free ion activity in the external solution and that metal complexes do not contribute. However, studies with plants have shown that labile metal complexes enhance metal bioavailability when the uptake is rate-limited by transport of the free ion in solution to the uptake site. Here, the role of labile complexes of Cd on metal bioavailability was assessed using Caco-2 cells, the cell model for intestinal absorption. At low Cd(2+) concentration (1 nM), the CdCl(n)(2-n) complexes contributed to the uptake almost to the same extent as the free ion. At large Cd(2+) concentration (10 μM), the contribution of the complexes was much smaller. At constant Cd(2+) concentration, Cd intake in the cells from solutions containing synthetic ligands such as EDTA increased as the dissociation rate of the cadmium complexes increased, and correlated well with the Cd diffusion flux in solution measured with the Diffusive Gradient in Thin Films technique (DGT). The Cd intake fluxes in the cells were well predicted assuming that the specific uptake is limited by diffusion of the free Cd(2+) ion to the cell surface. Our results underline that speciation of Cd has a major effect on its uptake by intestinal cells, but the availability is not simply related to the free ion concentration. Labile complexes of Cd enhance metal bioavailability in these cells, likely by alleviating diffusive limitations.

Natural dissolved organic matter mobilizes Cd but does not affect the Cd uptake by the green algae Pseudokirchneriella subcapitata (Korschikov) in resin buffered solutions

Natural dissolved organic matter (DOM) can have contrasting effects on metal bioaccumulation in algae because of complexation reactions that reduce free metal ion concentrations and because of DOM adsorption to algal surfaces which promote metal adsorption. This study was set up to reveal the role of different natural DOM samples on cadmium (Cd) uptake by the green algae Pseudokirchneriella subcapitata (Korschikov). Six different DOM samples were collected from natural freshwater systems and isolated by reverse osmosis. In addition, one (13)C enriched DOM sample was isolated from soil to trace DOM adsorption to algae. Algae were exposed to standardized solutions with or without these DOM samples, each exposed at equal DOM concentrations and at equal non-toxic Cd(2+) activity (∼4 nM) that was buffered with a resin. The DOM increased total dissolved Cd by factors 3-16 due to complexation reactions at equal Cd(2+) activity. In contrast, the Cd uptake was unaffected by DOM or increased maximally 1.6 fold ((13)C enriched DOM). The (13)C analysis revealed that maximally 6% of algal C was derived from DOM and that this can explain the small increase in biomass Cd. It is concluded that free Cd(2+) and not DOM-complexed Cd is the main bioavailable form of Cd when solution Cd(2+) is well buffered.

Labile synthetic cadmium complexes are not bioavailable to Pseudokirchneriella subcapitata in resin buffered solutions.

The Free Ion Activity Model (FIAM) predicts that cadmium (Cd) uptake by organisms is identical for solutions with the same free Cd(2+) concentration and inorganic composition. Clear exceptions to the FIAM have been shown for Cd uptake by plant roots, periphyton and human cells where labile Cd complexes increase bioavailability and which has been attributed to their role in enhancing Cd diffusion towards the uptake cells. Here, we assessed the role of labile Cd complexes on Cd uptake by algae, for which diffusion limitations should be less pronounced due to their smaller size. Long-term (3 days) Cd uptake by the green algae Pseudokirchneriella subcapitata was measured in resin buffered solutions with or without synthetic ligands and at three Cd(2+) ion activities (pCd 8.2-5.7). The free Cd(2+) activity was maintained during the test using a metal-selective resin located in the algal bottles. Total dissolved Cd increased up to 35-fold by adding the synthetic ligands at constant Cd(2+) activity. In contrast, Cd uptake by algae increased maximally 2.8 fold with increasing concentration of the synthetic ligands and the availability of the complexes were maximally 5.2% relative to Cd(2+) for NTA and CDTA complexes. It is concluded that labile Cd complexes do not greatly enhance Cd bioavailability to the unicellular algae and calculations suggest that Cd transport from solution to these small cells is not rate limiting.