Back Accumulation of Diffusive Gradients in Thin-Films Devices with a Stack of Resin Discs To Assess Availability of Metal Cations to Biota in Natural Waters.

Determining species, concentrations, and physicochemical parameters in natural waters is key to improve our understanding of the functioning of these ecosystems. Diffusive Gradients in Thin-films (DGT) devices with different thicknesses of the resin or of the diffusive disc can be used to collect independent information on relevant parameters. In particular, DGT devices with a stack of two resin discs offer a simple way to determine dissociation rate constants of metal complexes from the accumulation of the target metal in the back resin disc. In this work, simple approximate expressions for the determination of the dissociation rate constant are reported and applied to a model Ni nitrilotriacetic complex as well as to Zn complexes in the Mediterranean Osor stream. Once the physicochemical parameters are known, one can plot the labile fraction of the metal complexes in terms of the thickness of the diffusion domain. These plots reveal a strong dependence on the nature of complexes as well as on the characteristics of the diffusion domain, and they are of high interest as predictors of availability to biota whose uptake is limited by diffusion.

Dissolution and Phosphate-Induced Transformation of ZnO Nanoparticles in Synthetic Saliva Probed by AGNES without Previous Solid-Liquid Separation. Comparison with UF-ICP-MS.

The variation over time of free Zn2+ ion concentration in stirred dispersions of ZnO nanoparticles (ZnO NPs) prepared in synthetic saliva at pH 6.80 and 37 °C was followed in situ (without solid-liquid separation step) with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping). Under these conditions, ZnO NPs are chemically unstable due to their reaction with phosphates. The initial stage of transformation (around 5-10 h) involves the formation of a metastable solid (presumably ZnHPO4), which later evolves into the more stable hopeite phase. The overall decay rate of ZnO NPs is significantly reduced in comparison with phosphate-free background solutions of the same ionic strength and pH. The effective equilibrium solubilities of ZnO (0.29-0.47 mg·L-1), as well as conditional excess-ligand stability constants and fractional distributions of soluble Zn species, were determined in the absence and presence of organic components. The results were compared with the conventional ultrafiltration and inductively coupled plasma-mass spectrometry (UF-ICP-MS) methodology. AGNES proves to be advantageous in terms of speed, reproducibility, and access to speciation information.

Metal (Pb, Cd, and Zn) Binding to Diverse Organic Matter Samples and Implications for Speciation Modeling.

This study evaluated the influence of dissolved organic matter (DOM) properties on the speciation of Pb, Zn, and Cd. A total of six DOM samples were categorized into autochthonous and allochthonous sources based on their absorbance and fluorescence properties. The concentration of free metal ions ( CM2+) measured by titration using the absence of gradients and Nernstian equilibrium stripping (AGNES) method was compared with that predicted by the Windermere humic aqueous model (WHAM). At the same binding condition (pH, dissolved organic carbon, ionic strength, and total metal concentration) the allochthonous DOM showed a higher level of Pb binding than the autochthonous DOM (84- to 504-fold CPb2+ variation). This dependency, however, was less pronounced for Zn (12- to 74-fold CZn2+ variation) and least for Cd (2- to 14-fold CCd2+ variation). The WHAM performance was affected by source variation through the active DOM fraction ( F). The commonly used F = 1.3 provided reliable CPb2+ for allochthonous DOMs and acceptable CCd2+ for all DOM, but it significantly under-predicted CPb2+ and CZn2+ for autochthonous DOM. Adjusting F improved CM2+ predictions, but the optimum F values were metal-specific (e.g., 0.03-1.9 for Pb), as shown by linear correlations with specific optical indexes. The results indicate a potential to improve WHAM by incorporating rapid measurement of DOM optical properties for site-specific F.

Extending the Use of Diffusive Gradients in Thin Films (DGT) to Solutions Where Competition, Saturation, and Kinetic Effects Are Not Negligible.

DGT (Diffusion Gradients in Thin films) was designed to sample trace metals in situ at their natural concentrations. The setup and the experimental deployment conditions were established to allow interpretation of a linear accumulation of metal with time, using a simple expression based on a steady-state flux under perfect sink conditions. However, the extension of DGT to a wide range of analytes and its use under varied conditions has shown that, in some situations, these conditions are not fulfilled, so that accumulations with time are nonlinear. Previously, when such curvature was observed, concentrations in solution could not be reliably calculated. Here, we present fundamentally derived equations that reproduce the time accumulation for three situations: (i) kinetic limitations in the binding to the resin, (ii) saturation or equilibrium effects, or (iii) non-negligible competitive effects. We show how the accumulations can be quantified, in terms of the required kinetic and thermodynamic constants, and provide practical guidance for their use to obtain reliable estimates of solution concentrations. Solutions containing Mg or Mn, where all three situations can prevail, are used as examples. Calculated concentrations show reasonable agreement with the experimentally known values and with the results of a numerical model of the system, significantly improving the estimations based on perfect sink conditions. Such an approach opens up the possibility of using DGT more widely in challenging systems and allows DGT data to be interpreted more fully.

Accumulation of Mg to Diffusive Gradients in Thin Films (DGT) Devices: Kinetic and Thermodynamic Effects of the Ionic Strength.

Availability of magnesium is a matter of concern due to its role in many environmental and biological processes. Diffusive Gradients in Thin Films (DGT) devices can measure Mg availability in situ. This work shows that Mg accumulation in water largely increases when ionic strength (I) decreases. This phenomenon can be explained from (i) the increase of both the association equilibrium (K) and rate (ka,R) constants for the reaction between Mg cations and resin sites, and (ii) the growing contribution of the partitioning of Mg cations at the resin-gel interface, as I decreases. Two theoretical models that take into account electrical interactions among Mg cations, background electrolyte, and resin sites can successfully be used to determine ka,R and K at each I. Both models yield similar ka,R values, which fulfill an expression for the kinetic salt effect. For freshwater (with a typical salinity of 10 mM and circumneutral pH), the binding of Mg is so fast and strong that the simplest perfect-sink DGT expression can be helpful to predict (overestimation lower than 5%) the accumulation in solutions with Mg concentrations up to 1 mM whenever the deployment time is below 9 h. Perfect sink conditions can still be applied for longer times, in systems with either a lower I or a lower Mg concentration.

Determination of the Free Metal Ion Concentration Using AGNES Implemented with Environmentally Friendly Bismuth Film Electrodes.

Ex situ plated Bi film electrodes (Bi-FE) have been employed, for the first time, to measure the free concentration of Pb(II) in aqueous solutions using absence of gradients and Nernstian equilibrium stripping (AGNES) with stripping chronopotentiometry (SCP) quantification. The amount of deposited Pb°, below a certain threshold, follows a Nernstian relationship with the applied potential. This threshold can be interpreted as the frontier of transition from surface deposition to solid (bulk) formation of Pb°. AGNES with Bi-FE yielded a very good detection limit (3σ) for Pb(II) of 6.0 × 10(-9) M with an applied gain of 398 and a deposition time of 400 s. The ability of the Bi film electrode to perform speciation measurements was demonstrated for Pb(II)-PSS and Pb(II)-IDA systems. The measured values with the Bi-FE were in good agreement with the values obtained using the Hg film electrode and/or the values reported in the literature.

Measurement of metals using DGT: impact of ionic strength and kinetics of dissociation of complexes in the resin domain.

As the measurement of metals by DGT (diffusion gradients in thin films) in low salinity media has been controversial, a thorough study of the impact of ionic strength (I) is timely. DGT accumulations of Cd, Co, and Ni in the presence of NTA at pH 7.5 with I in the range from 10(-4) to 0.5 M were obtained. An observed decrease in the metal accumulation as the ionic strength of the system decreased is partially explained by the electrostatic repulsion between the negatively charged resin domain and the dominant negatively charged complex species M-NTA. This electrostatic effect reduces the complex penetration into the resin domain, especially for nonlabile complexes, which do not fully dissociate in the gel domain. Analytical expressions, based on the Donnan model, were able to quantify these electrostatic effects. Additionally, the data indicate that the kinetic dissociation constant of M-NTA complexes in the resin layer is higher than Eigen predictions, suggesting a ligand-assisted dissociation mechanism. As the ionic strength decreases, the rate of reaction in the resin layer decreases due to the repulsion between the negatively charged resin sites and the complex species. This decrease contributes to the decrease in metal accumulation. These novel, previously unconsidered, effects of ionic strength and the ligand-assisted dissociation mechanism in the resin domain will affect DGT measurements made in freshwaters and soils.

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 (Dulbecco's modified Eagle's 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.

Release of indium from In2O3 nanoparticles in model solutions and synthetic seawater.

Indium oxide (In2O3) nanoparticles (NPs) are used in electronic devices, from which indium (as its nanoparticulate form or as other generated chemical species) can be released to natural waters. To assess for the impacts of such releases (e.g. toxic effects), information on the kinetics and thermodynamics of the In2O3 dissolution processes is key. In this work, the evolution with time of the dissolution process was followed with the technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping) by measuring the free indium concentration ([In3+]). AGNES can determine the free ion concentration in the presence of nanoparticles without a prior separation step, as shown in the case of ZnO nanoparticles, a procedure that is more accurate than the typical sequence of centrifugation+filtration+elemental analysis. Excess of indium oxide NPs were dispersed in 0.1 mol L-1 KNO3 at various pH values ranging from 2 to 8. Additional dispersions with bulk In2O3 at pH 3 or NPs in synthetic seawater at pH 8 were also prepared. The temperature was carefully fixed at 25 °C. The dispersions were continuously stirred and samples were taken from time to time to measure free indium concentration with AGNES. 180-day contact of In2O3 to solutions at pH 2 and 3 was not enough to reach equilibrium. The dissolution of the NPs at pH 3 was faster than that of the bulk (i.e. non nanoparticulate) material. Equilibrium of the NPs with the solution was reached at pH 4 and 5 in KNO3 and at pH 8 in seawater, in shorter times for higher pH values, with free indium concentrations decreasing by a factor of 1000 for each increase in one pH unit. The solubility products of In(OH)3 and In2O3 were compared. Equilibration of NPs with synthetic seawater took <18 days, with an average free [In3+] (up to 196 days) of 1.03 amol L-1.

Limits of the linear accumulation regime of DGT sensors.

A key question for the practical application of DGT (Diffusive Gradients in Thin films) as dynamic sensors in the environmental monitoring of trace metals is the influence of pH and dissolved ligands over the linear accumulation regime. Protons compete with metal ions for the binding to the DGT resin sites at relatively low pH, whereas high affinity dissolved ligands compete with resin sites for the binding of metals. Any of the two phenomena can lead to a departure from the linear accumulation regime and an underestimation of the actual species concentration in solution. These effects are studied here through numerical simulation of the diffusion-reaction processes in both gel and resin domains using a detailed chemical model of metal ions and protons interacting with resin sites. Results were tested successfully against experimental data of the Cd-NTA representative system. Charts to delimitate the range of experimental conditions (pH, ligand concentration and strength) where the linear accumulation regime prevails, can be helpful for designing sampling strategies in field conditions. For example, it is foreseen that perturbations of linear regime within 10 h of deployment are negligible above pH 5 and weak complexation (log K' < 0) or above pH 7 and strong complexation (log K' < 3), where K' is the effective stability constant. These plots can also be approximately used for partially labile systems whenever the time is replaced with the product lability degree times t.

Kinetic mixture effects in diffusion gradients in thin films (DGT).

The penetration of complexes into the resin domain of the DGT devices has a large influence on the lability degree of these complexes, since the reaction layer (the layer where there is net dissociation) extends from the diffusive gel into the resin domain. Numerical simulation shows that, typically, the contribution to the metal accumulation from dissociation of complexes inside the resin domain is dominant. As a consequence, in excess of ligand, the influence of the ligand concentration on the lability degree is much reduced, in comparison with this effect in the voltammetric sensors. The presence of a mixture of ligands leads to parallel complexes that mutually influence their lability degrees. In general, the interaction between the complexes has an impact on the lability degree of each one, but the total metal accumulation is less sensitive due to cancellation (mutually opposite effects of a couple of complexes). This result paves the way to predict the metal accumulation from the lability degree available for each complex in a single ligand system. Maximum discrepancies of 10% have been found in these predictions which can still be reduced if thicker resin gels are used.

A new tool for the determination of humic substances in natural waters: Pulsed voltammetry approach.

Humic substances (HS) in natural waters can be determined with a new, simple and sensitive method based on their influence on the background current in a differential pulse - adsorptive cathodic stripping voltammetry. The proposed method, termed PB-HS (pulsed background - humic substances) is discussed in detail, including its application in natural samples from the Krka River estuary. The method was additionally compared with absorbance measurements as well as with the typical electrochemical HS quantification in natural waters based on HS complexation with molybdenum (Mo). A good correlation between methods was observed, with PB-HS showing slightly better sensitivity to humic compounds than classical spectrophotometry. Higher HS concentrations measured with the Mo-method may be due to the enhanced hydrophobicity reached at pH 2 that is required by the method. Advantages of the proposed PB-HS method, compared to existing voltammetric methods for HS quantification, are that it does not require any reagent addition (except buffer) and that it can be used at the natural pH of water as well as in a wide salinity range, which is crucial for its application in estuarine waters.

Kinetic signatures of metals in the presence of Suwannee River fulvic acid.

This work provides new information on the dissociation kinetics of metal-fulvic acid (FA) complexes. Diffusive gradients in thin-film (DGT) devices deployed in solutions containing metals and 30 mg L(-1) Suwannee River FA at pH 5 and 7, at two different metal-to-ligand ratios, were used to estimate an apparent diffusive boundary layer (ADBL) thickness at the gel-solution interface. The discrepancy between the ADBL thickness measured for metals that are known to dissociate from complexes quickly (e.g., Cd) and that of other trace metals was exploited to calculate the rate of complex dissociation. When the ADBL thickness is plotted for a suite of metals, a "kinetic signature" is created. There was a clear kinetic signature at pH 7, with substantial kinetic limitation for Cu, Pb, and Ni and none for Cd, Co, and Mn (i.e., Cu-, Pb-, and Ni-FA complexes dissociated more slowly). At pH 5, the kinetic signature was less distinct, due in part to slow association kinetics of Mn, and possibly Cd and Co, with the resin. The good sensitivity of the method to small changes in dissociation kinetics was able to show that the dissociation of most metal-FA complexes is sufficiently fast to not limit the DGT measurement.

Lability criteria in diffusive gradients in thin films.

The penetration of metal complexes into the resin layer of DGT (diffusive gradients in thin films) devices greatly influences the measured metal accumulation, unless the complexes are either totally inert or perfectly labile. Lability criteria to predict the contribution of complexes in DGT measurements are reported. The key role of the resin thickness is highlighted. For complexes that are partially labile to the DGT measurement, their dissociation inside the resin domain is the main source of metal accumulation. This phenomenon explains the practical independence of the lability degree of a complex in a DGT device with respect to the ligand concentration. Transient DGT regimes, reflecting the times required to replenish the gel and resin domains up to the steady-state profile of the complex, are also examined. Low lability complexes (lability degree between 0.1 and 0.2) exhibit the longest transient regimes and therefore require longer deployment times to ensure accurate DGT measurements.

Direct determination of free Zn concentration in samples of biological interest.

The speciation of essential metal ions in biological fluids, such as blood plasma and serum, is of fundamental importance to understand the homeostasis of these elements. The activity of metal ions such as Zn2+ in extracellular media is thought to affect their interaction with membrane-bound transporters, and thus is critical for their cellular uptake. Previous approaches to determine "free" Zn2+ (i.e. the hexa-aquo ion) are based on separation by either chromatography or ultrafiltration, or on metallochromic dyes. However, both types of approach are prone to affect the relevant equilibria. These drawbacks can be circumvented with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping), since it can measure free zinc concentration without perturbing the sample speciation. Here, a Bovine Serum Albumin (BSA) + Zn synthetic mixture and Fetal Bovine Serum (FBS) are analyzed as proof of concept. Adsorption of BSA on the surface of the Hanging Mercury Drop Electrode (HMDE), despite the advantage of its renewal, is so intense that it blocks appropriate attainment of the required equilibrium, and only estimations of [Zn2+] can be derived. In contrast, a rotating disc electrode with a thin mercury film deposited on it (TMF-RDE) is advantageous because of its small volume and enhanced mass transfer. Protein adsorption can be prevented by covering the TMF-RDE with Nafion. A free Zn concentration [Zn2+] = 2.7 nmol L-1 was found at pH 7.0, total Zn 20 µµmol L-1 and BSA 600 µµmol L-1. A sample of FBS with fixed pH 7.2 (MOPS 0.08 mol L-1) yielded [Zn2+] = 0.25 nmol L-1. This methodology opens the way to free metal concentration determinations in biological fluids.

A new approach to improve the accuracy of DGT (Diffusive Gradients in Thin-films) measurements in monitoring wells.

The Diffusive Gradients in Thin-films (DGT) technique represents an ideal tool for monitoring water quality of inorganic species in systems with a high flow such as rivers, streams, lakes and seas. However, in low-flow systems (non-turbulent waters), the influence of a diffusive boundary layer (DBL) formed on the surface of the DGT device has been observed, which can lead to erroneous measurements by DGT. Therefore, the use of DGT in wells for groundwater monitoring is still very limited until now. In this sense, the present study evaluates the applicability of the DGT technique in non-turbulent and low-flow water systems. We propose a new way to calculate the DBL with the objective to carry out a robust DGT analysis in environmental monitoring wells. For this purpose, DGT devices with different diffusive gel thicknesses were deployed in an experimental set-up simulating a groundwater monitoring well. A DBL thickness (for each element) was calculated from the slopes of the linear regressions between the DGT accumulated mass of metal and the deployment time (4, 8, 12, 24 and 48 h) for each of the two diffusive gel thicknesses. The mean DBL thickness (averaging the individual DBL thicknesses calculated from the slopes) was 0.06 cm. The concentrations of the analysed elements were corrected with this DBL with the result that the metal concentrations measured by DGT improved and were highly approximated to their actual total values in this non-complexing medium.

Effective concentration signature of Zn in a natural water derived from various speciation techniques.

The uptake of nutrients or toxicants by different organisms in aquatic systems is known to correlate with different fractions of the nutrient's or toxicant's total concentration. These fractions can be provided by different analytical techniques, from which the better correlation is expected to be found for those with a characteristic length comparable to that in the considered organism uptake. An effective concentration signature can be built up with the concentration values associated to the availability (i.e. fluxes in dynamic techniques) of the nutrient or toxicant measured by various analytical techniques with different characteristic lengths. Here, this new representation was obtained for the pool of Zn complexes in the Mediterranean stream Riera d'Osor (Girona, Catalonia, Spain) with a suite of four analytical techniques. Absence of Gradients and Nernstian Equilibrium Stripping (AGNES) and Polymer Inclusion Membrane (PIM) devices provided the free Zn concentration. Linear Anodic Stripping Voltammetry provided a labile fraction (defined here as cLASV, higher than the free concentration), related to the diffusion layer scale. Diffusion Gradients in Thin-films provided higher labile fractions (known as DGT concentrations, cDGT) connected to the different characteristic lengths of different configurations (e.g. one or two resin discs) longer, in any case, than that corresponding to LASV. The combination of the information retrieved by the techniques allowed to quantify lability degrees of the pool of Zn complexes and to build up the effective concentration signature for this water.

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.

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.

Availability of metals to DGT devices with different configurations. The case of sequential Ni complexation.

The analytical technique DGT (Diffusive Gradients in Thin-films) is able to gain access to a wealth of information by carefully interpreting accumulation data from passive samplers with different configurations (i.e. different thicknesses of its constituent layers). A set of DGT devices were simultaneously deployed in solutions of Ni and nitrilotriacetic acid (NTA) of different concentrations to measure the availability of Ni in these solutions. Accumulations indicate that the availability of Ni depends on both the thickness of the resin and the thickness of the diffusive gel. In both cases, the lability degree increases as the thickness increases. As the formation of successive complexes (such as Ni(NTA)2) proceeds, the availability of the metal decreases, which is quantitatively explained by reducing the formulation to a case with only one complex, but with an effective dissociation rate constant that decreases as the concentration of NTA increases. Simple analytical expressions are reported to quantify the lability degree in the different DGT configurations. These results indicate that a set of different DGT devices can characterize the availability of a cation in a natural sample with uptake processes at different spatial or time scales. Alternatively, and from a more fundamental point of view, information on speciation, mobilities and labilities of the species present in natural samples can be obtained with a set of DGT configurations.