Josep Galceran

The chronic toxicity of ZnO nanoparticles and ZnCl2 to Daphnia magna and the use of different methods to assess nanoparticle aggregation and dissolution

Abstract In this study, the effect of ZnO nanoparticles and ZnCl2 on growth, reproduction and accumulation of zinc in Daphnia magna was determined in a 21-day chronic toxicity test. A variety of techniques were used to distinguish the free zinc ion, dissolved, nanoparticle and aggregated zinc fraction in the Daphnia test medium. The results showed similar chronic effects on growth, reproduction and accumulation for the ZnO nanoparticles (EC10, 20, 50 reproduction: 0.030, 0.049, 0.112 mg Zn/l) and the ZnCl2 (EC10, 20, 50 reproduction: 0.014, 0.027, 0.082 mg Zn/l). A large fraction of the nanoparticles rapidly dissolved after introduction in the exposure medium. Aggregation of nanoparticles was also observed but within 48 h of exposure most of these ZnO aggregates were dissolved. Based on the combined dissolution kinetics and toxicity results, it can be concluded that the toxicological effects of ZnO nanoparticles at the chronic level can be largely attributed to the dissolved fraction rather than the nanoparticles or initially formed aggregates.

AGNES: A technique for determining the concentration of free metal ions. The case of Zn(II) in coastal Mediterranean seawater

Absence of Gradients and Nernstian Equilibrium Stripping (AGNES) is a recently suggested electroanalytical technique designed for the determination of the free concentration of heavy metals (such as Zn, Cd or Pb) which is here developed and applied to seawater samples. A key improvement for the implementation of AGNES with complex matrices is the development of a new blank, called the shifted blank (presented in this work for the first time), which can be applied to the same sample where the measurement is intended. The careful selection of the required parameters for the determination of the free Zn concentration (or activity) at the nanomolar level is described in detail. The methodology has been validated with a synthetic solution containing Zn and nitrilotriacetic acid (NTA) and then applied, as a first case, to two coastal seawater samples taken close to Barcelona and Tarragona (Catalonia, North-Eastern Spain) finding values in the range of 1-3nM, representing around 25% of total Zn. This technique can, in the near future, be crucial in helping to elucidate the role of the free zinc(II) concentration in natural waters.

Systematic investigation of the physicochemical factors that contribute to the toxicity of ZnO nanoparticles.

ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbeccos modified Eagles medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 μg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 μg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 μM of which only 0.45 μM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.

Evaluation of the Koutecký-Koryta approximation for voltammetric currents generated by metal complex systems with various labilities

Abstract The voltammetric response of metal complex systems with various labilities is analyzed by rigorous numerical simulation with the Finite Element Method of the time-dependent concentration profiles of the different species. The ensuing exact fluxes and the corresponding currents are compared to those derived from the Koutecký–Koryta (KK) approximation which assumes a discontinuous transition in the concentration profiles from non-labile to labile behavior. The results indicate a relatively far-reaching correctness of the KK approximation in the complete kinetic range from non-labile to labile complexes, as long as the kinetic flux is computed from the effective concentration of the complex in the reaction layer. Some approximate analytical expressions for this concentration are provided. The KK approximation is shown to be applicable for any metal-to-ligand ratio, provided that the thickness of the reaction layer is expressed in terms of the ligand concentration at the electrode surface.

Contribution of partially labile complexes to the DGT metal flux.

Penetration of complexes into the resin layer can dramatically increase the contribution of complexes to the metal flux measured with a DGT (diffusive gradients in thin films) sensor, but equations to describe this phenomenon were not available. Here, simple approximate analytical expressions for the metal flux, the lability degree and the concentration profiles in a DGT experiment are reported. Together with the thickness of the reaction layer in the gel domain, the effective penetration distance into the resin layer that would be necessary for full dissociation of the complex (λ(ML)) plays a key role in determining the metal flux. An increase in the resin-layer thickness (r) effectively increases the metal flux and the lability degree until r ≈ 3λ(ML). For the usual DGT configuration, where the thickness of the gel layer exceeds that of the resin layer, the complex is labile if r > (D(ML)/k(d))½, where D(ML) is the diffusion coefficient of the metal complex and k(d) its dissociation rate constant. A general procedure for estimating the lability of any complex in a standard DGT configuration is provided.

Reverse pulse polarography of labile metal + macromolecule systems with induced reactant adsorption: theoretical analysis and determination of complexation and adsorption parameters

A model for the interpretation of reverse pulse polarography (RPP) in metal macromolecular ligand systems is developed, including both adsorption of the macromolecule and the induced adsorption of the metal ion. The following basic assumptions are made: reversible charge transfer at a stationary planar electrode (static mercury drop electrode), labile complex, large excess of ligand compared with the total metal concentration, formation of a 1:1 complex and diffusion coefficients for ligand and complex species different from that of the free metal ion. Equations for the limiting current Ilim and for the RPP full wave are deduced for both linear and Langmuirian adsorption. It is found that adsorption does not modify the expressions for Ilim valid for the case without adsorption. Thus the stability constant K can be determined. In contrast, adsorption influences the shape and position of the RPP full wave. In the case of linear adsorption, an analytical expression has been deduced which relates the adsorption parameter KML1 to ΔE12 values, thus allowing the calculation of KML1 from RPP waves. This method has been applied to experimental results obtained for the Cd(II) polymethacrylate system.

Lability and mobility effects on mixtures of ligands under steady-state conditions

Analytical solutions for the steady-state flux arriving at an active surface from a mixture (in which one active species reacts with non-active ligands in the medium) can be helpful in a variety of problems: voltammetric techniques, heterogeneous processes in reactors, toxic or nutrient uptake, techniques of diffusive gradients in thin films (DGT), etc. Under any geometry that sustains steady-state, a convenient combination of the reaction–diffusion equations leads to a simpler formulation of the problem for arbitrary diffusivities of the species and arbitrary rate constants of the first-order conversion between the active species and the non-active species. The resulting problem can be characterised in terms of a list of dimensionless parameters involving the kinetic and mobility properties of each species. A lability degree for each 1∶1 complex in terms of the surface concentrations leads to: (i) a lability criterion specific for each complex in the mixture and (ii) the assessment of the relative contribution of each complex to the resulting flux. Semi-infinite spherical diffusion (as in the Gel Integrated MicroElectrode, GIME, biouptake modelling of micro-organisms, etc.) is specifically considered and some consequences of its full analytical solutions are discussed.

Key Role of the Resin Layer Thickness in the Lability of Complexes Measured by DGT

Analysis of the dynamic features of diffusion gradients in thin film devices (DGT) indicates that the penetration of complexes into the resin layer dramatically increases their lability. This should be taken into account when interpreting DGT measurements in terms of the dynamics of solution speciation. The experimental accumulation of Cd by DGT sensors in Cd-NTA systems confirmed these theoretical analyses. A computational code, which allows a rigorous digital simulation of the diffusion-reaction processes in the gel and resin layers, was used to model the results and to demonstrate the effect of the complex penetration into the resin layer on the lability degree. These findings suggest that DGT renders all complexes much more labile than if the resin-diffusive gel interface was considered as a perfect planar sink, explaining why DGT often measures a high proportion of the metal in a natural water. This information is relevant since some studies have stressed the importance of labile complexes as a source of bioaccumulated metal.

Determination of free Zn2+ concentration in synthetic and natural samples with AGNES (Absence of Gradients and Nernstian Equilibrium Stripping) and DMT (Donnan Membrane Technique)

The determination of free Zn(2+) ion concentration is a key in the study of environmental systems like river water and soils, due to its impact on bioavailability and toxicity. AGNES (Absence of Gradients and Nernstian Equilibrium Stripping) and DMT (Donnan Membrane Technique) are emerging techniques suited for the determination of free heavy metal concentrations, especially in the case of Zn(2+), given that there is no commercial Ion Selective Electrode. In this work, both techniques have been applied to synthetic samples (containing Zn and NTA) and natural samples (Rhine river water and soils), showing good agreement. pH fluctuations in DMT and N(2)/CO(2) purging system used in AGNES did not affect considerably the measurements done in Rhine river water and soil samples. Results of DMT in situ of Rhine river water are comparable to those of AGNES in the lab. The comparison of this work provides a cross-validation for both techniques.

Humic acid complexation to Zn and Cd determined with the new electroanalytical technique AGNES

Environmental context. Humic substances are complex mixtures that play an important role in trace metal bioavailability in soils and aquatic environments. The bioavailability of a metal depends on what chemical forms, or species, it is in. We need to know how much of the metal is present as a free metal ion in solution, and how much is bound up in complexes with humic acids, for example. This work reports the complexation of Cd and Zn to humic acids by means of a simple and robust technique, AGNES (absence of gradients and Nernstian equilibrium stripping). Abstract. AGNES (absence of gradients and Nernstian equilibrium stripping), an emerging electroanalytical technique specifically designed for the determination of the free concentration of heavy metals in aqueous solutions, is here implemented to characterise the binding of CdII and ZnII to a soil humic acid. A set of metal titration experiments were performed by adding Cd or Zn to a purified humic acid (Aldrich) at pH 4, 5, 6 and 7 and measuring the free metal concentration by AGNES. The application of a program with two potential steps along the deposition stage allows for the reduction of the deposition time in the humic titration. The polyelectrolytic effects of the macromolecular ligand were taken into account through the Donnan model. Data free of electrostatic effects were reasonably described by the NICA isotherm, which accounts for heterogeneity, considering just a monomodal distribution (because of the range of pH covered). The obtained affinity parameters indicate a similar strength for Zn and Cd binding to the purified humic acid.