In situ electrochemical assessment of cytotoxicity of chlorophenols in MCF-7 and HeLa cells.

An in situ electrochemical method was used to assess the cytotoxicity of chlorophenols using human breast cancer (MCF-7) and cervical carcinoma (HeLa) cells as models. On treatment with different chlorophenols, the electrochemical responses of the selected cells, resulting from the oxidation of guanine and xanthine in the cytoplasm, indicated the cell viability. In addition, the in situ in vitro electrochemical method was further compared with the traditional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Although similar cytotoxicity data were obtained from both methods, the effective concentrations of chlorophenols that inhibited 50% cell growth (EC50 values) from the electrochemical method were only slightly lower than those from the MTT assay. These results indicate that the in situ in vitro electrochemical method paves a simple, rapid, strongly responsive, and label-free way to the cytotoxicity assessment of different chlorophenol pollutants.

A novel electrochemical method to evaluate the cytotoxicity of heavy metals.

There is an ongoing search to develop techniques for detection of heavy metals which are highly toxic and can cause damaging effects even at very low concentrations. In this present study, we report a label-free electrochemical method based on the direct voltammetric response of human cervical carcinoma (HeLa) cells on a highly sensitive graphene modified electrode. Five heavy metals were tested with the method and the results were validated by the traditional methyl tetrazolium (MTT) assay. The results revealed that the most toxic metal was Cr, followed by Cd, Cu, Pb and Zn. A good correlation between the two methods was observed. This work will be beneficial in providing a novel monitoring method to detect hazardous pollutants in the field of environmental toxicology.

Metal flux through consuming interfaces in ligand mixtures: boundary conditions do not influence the lability and relative contributions of metal species.

In a mixture of metal ions and complexes, it is difficult to predict ecological risk without understanding the contribution of each metal species to biouptake. For microorganisms, the rate of uptake (internalization flux) has not only a major influence on the total metal flux but also on the bioavailability of the various metal species and their relative contributions to the total flux. In this paper, the microorganism is considered as a consuming interface, which interacts with the metal ion, M, via the Michaelis-Menten boundary conditions. The contribution of each metal complex to the overall metal flux, in relation to its lability, is examined for a number of important boundary parameters (the equilibrium constant K(a) of metal with transport sites, internalization rate constant k(int) and total transport sites concentration {R}(t)). Computations were performed for Cu(II) complexes, in a multicomponent culture medium for microoganisms. For a one-ligand system, results were acquired using rigorous mathematical expressions, whereas approximate expressions, based on the reaction layer approximation (RLA) and rigorous numerical computations (computer codes MHEDYN and FLUXY), were employed for ligand mixtures. Under the condition of ligand excess, as often found in the natural environment, the relative contribution of each metal species to the total flux is shown to be independent of the boundary conditions. This finding has important implications, including an improved basis for relating the analytical signals of dynamic metal speciation sensors to metal bioavailability.

Quantifying diffusion in a biofilm of Streptococcus mutans.

In biofilms, diffusion may limit the chemical activity of nutrients, toxic compounds, and medicines. This study provides direct, noninvasive insight into the factors that will most effectively limit the transport of antibiotics and biocides in biofilms. Self-diffusion coefficients have been determined for a number of fluorescent probes in biofilms of Streptococcus mutans using fluorescence correlation spectroscopy. The effects of probe size and charge and the roles of biofilm pH, ionic strength, and heterogeneity were studied systematically. The relative diffusion coefficients (D in the biofilm divided by that in water) decreased with increasing probe size (3,000-molecular-weight [3K], 10K, 40K, 70K, and 2,000K dextrans). Studies using variably charged substrates (tetramethylrhodamine, Oregon Green, rhodamine B, and rhodamine 6G) showed that the self-diffusion coefficients decreased with an increasing negative charge of the fluorescent probes. No significant effect was observed for changes to the ionic strength (10⁻4 to 10⁻¹ M) or pH (4 to 9) of the biofilm. Biofilm heterogeneity was responsible for variations of ca. one order of magnitude in the diffusion coefficients.

Interfacial metal flux in ligand mixtures. 3. Unexpected flux enhancement due to kinetic interplay at the consuming surface, computed for aquatic systems.

Understanding the processes controlling metal biouptake in a mixture of ligands is a requirement for making predictions on dynamic risk assessment in ecotoxicology. In ligand mixtures, the metal uptake flux, due to the dissociation of non labile complexes, can be significantly enhanced by the presence of ligands forming labile complexes, even when the proportions of the latter are very small in the bulk solution. The flux enhancement results from a peculiar kinetic interplay, at the interface, between the labile and non labile species, which influences the lifetime of free metal ion and the reaction layer thickness. An extension of the concept of reaction layer, to multiligand systems, is used here, to explain the physicochemical basis of this flux enhancement and to compute the flux of trace metals in a simple way. This phenomenon is exemplified with four environmentally relevant systems including: (i) simple ligands (Pb-NTA-diglycolate; Cu-OH(-)-CO3(2-)), (ii) fulvic substances (Cu-fulvics-citrate), and (iii) aggregate complexants (Pb-aggregate-CO3(2-)). These examples are typical cases in laboratory experiments, in natural freshwaters and in soil-waters. They show that the flux enhancement effect may occur with all major inorganic and organic environmental complexants. It may be ubiquitous in natural waters or in biological systems and might play an important role in biouptake of toxic or vital metals.

Interfacial metal flux in ligand mixtures. 1. The revisited reaction layer approximation: theory and examples of applications.

Understanding the physical chemical behaviors of each metal species in a solution containing a mixture of ligands is a prerequisite, e.g., for studying metal bioavailability or making predictions on dynamic risk assessment in ecotoxicology. For many years, the reaction layer concept has been used fruitfully due to its simplicity for understanding and making predictions on diffusion/reaction processes. Until now, it has been applied mainly to solutions containing one ligand. Here, we reconsider the fundamentals of this approach and extend it to multiligand systems. It is shown that each metal complex has its own reaction layer (so-called composite reaction layer), which results from the interplay of this particular complex with all the other complexes. Moreover, it is shown that the overall metal flux can be computed by assuming the existence of one single fictitious equivalent reaction layer thickness for the whole of the complexes. This equivalent reaction layer is a mathematical combination of all the composite reaction layers. Simple analytical equations are obtained, which make it possible to readily interpret the role of the various types of metal species in a mixture. The revisited reaction layer approach, denoted as the reaction layer approximation (RLA), is validated by comparing the total metal flux, the individual fluxes of each metal species, and their concentration profiles computed by the RLA with those obtained by a rigorous mathematical approach. The examples of Pb(II) in a modified Aquil medium and of Cu(II) in solutions of nitrilotriacetic acid and N-(2-carboxyphenyl)glycine are treated in detail. In particular, an original result is obtained with the Cu/NTA/N-(2-carboxyphenyl)glycine system, namely an unexpected flux enhancement is observed, which is specific to solutions with ligand mixtures. The corresponding physicochemical mechanism is not readily understood by the rigorous mathematical (either numerical or analytical) solutions due to their involved combination of parameters. On the other hand, we show that, due to the simplicity of the RLA concept, the RLA facilitates elucidation of the physicochemical mechanism underlying complicated processes.

Metal flux in ligand mixtures. 2. Flux enhancement due to kinetic interplay: comparison of the reaction layer approximation with a rigorous approach.

The revisited reaction layer approximation (RLA) of metal flux at consuming interfaces in ligand mixtures, discussed in the previous paper (part 1 of this series) is systematically validated by comparison with the results of rigorous numerical simulations. The current paper focuses on conditions under which the total metal flux is enhanced in the ligand (and complex) mixture compared to the case where the individual fluxes of metal complexes are independent of each other. Such an effect is exhibited only in ligand mixtures and results from the kinetic interplay between the various complexes with different labilities. It is exemplified by the Cu/NTA/N-(2-carboxyphenyl)glycine system (see part 1 paper), in which we show that the flux due to the less labile complex (CuNTA) is increased in the presence of a ligand (2-carboxyphenyl)glycine) that forms labile Cu complexes, even when the latter is in negligible proportion in the bulk solution. This paper first explains how the so-called composite and equivalent reaction layer thicknesses computed by RLA can be determined graphically from the concentration profiles of free metal and its complexes, as obtained by rigorous calculations. This approach allows comparison between the latter and RLA predictions. Comparison between these reaction layer thicknesses is then done using the chemical system mentioned above. The mechanism of flux enhancement with this system is studied in detail by following the change of the concentration profiles and reaction layer thicknesses with the increase of concentration of the ligand forming labile complexes. The mechanism of flux enhancement is well explained by the RLA and is validated by the concentration profiles obtained by rigorous numerical simulations. Based on this validation, the RLA is used to predict the conditions of the individual complex labilities and degree of complexation required to get flux enhancement in a two-ligand system. Due to compensation effects between kinetic and thermodynamic factors, a maximum flux enhancement is observed in a specific range of ratios of the lability indices of the two complexes. Flux enhancement might play a significant role in metal uptake in environmental or biological systems and should be considered in data interpretation.

Metal flux and dynamic speciation at (bio)interfaces. Part III: MHEDYN, a general code for metal flux computation; application to simple and fulvic complexants.

Metal flux at consuming interfaces (e.g., sensors or microorganisms) is simulated in environmental multiligand systems using a new numerical code, MHEDYN (Multispecies HEterogeneous DYNamics), based on the lattice Boltzmann method. The attention is focused on the computation of the maximum flux of Cu(II), that is, the flux controlled by diffusion-reaction in solution, irrespective of processes occurring at the interface. In parts III and IV of this series, three types of typical environmental complexants are studied: (a) simple ligands (OH- and C03(2-)), (b) fulvic or humic substances including many sites with broadly varying rate constants, and (c) aggregates including a broad range of sizes and diffusion coefficients. Part III focuses on computations in the presence of simple ligands and fulvic/humic substances separately, and part IV discusses the case of aggregate complexes alone and the mixtures of all ligands in typical natural waters. These papers describe the dynamic contribution of the various types of sites for fulvic and aggregate Cu(II) complexes for the first time. Whenever possible, the metal fluxes computed by MHEDYN are compared with those given by another code, FLUXY, based on a fully different mathematical approach, and very good agreement between these codes is obtained. In all cases, MHEDYN computes the concentration profile of each complex and its time evolution, as well as the steady-state flux and the corresponding contribution of each complex to the flux. The metal fluxes can be computed at a planar consuming surface such as an organism or a sensor surface, in presence of an unlimited number of complexation reactions of the metal M, and for any metal/ligand concentration ratio, with values of the physicochemical parameters ranging over many orders of magnitude.

Metal flux and dynamic speciation at (bio)interfaces. Part IV: MHEDYN, a general code for metal flux computation; application to particulate complexants and their mixtures with the other natural ligands.

Metal flux at consuming interfaces (e.g., sensors or microorganisms) is simulated in environmental multiligand systems using a new numerical code, MHEDYN (Multispecies HEterogeneous DYNamics), based on the lattice Boltzmann method. The attention is focused on the computation of the maximum flux (i.e.,the flux controlled by diffusion-reaction in solution) of Cu(II). Part III described flux computation in the presence of simple ligands and fulvic/humic substances. This paper (Part IV) discusses the case of metal complexes formed with aggregates including a broad range of sizes and diffusion coefficients and their mixture with simple and fulvic ligands under typical natural water conditions. This paper describes the dynamic contribution of the various size classes of aggregate Cu(II) complexes for the first time. In two typical waters containing mixtures of ligands, the contribution of aggregates is found to be small, whereas that of fulvics may play a major role, even under pH conditions where the lability of their Cu(II) complexes is low. These results point out the great usefulness of MHEDYN for dynamic speciation in very complex mixtures. In all cases, MHEDYN enables us to compute the concentration profile of each complex and itstime evolution, as well as the steady-state flux and the corresponding contribution of each complex to the flux. Thus, MHEDYN should be very useful for comparing theoretical predictions with experimental measurements of metal bioavailability or of dynamic sensor response in a complete aquatic medium.

Metal flux and dynamic speciation at (bio)interfaces. Part I: Critical evaluation and compilation of physicochemical parameters for complexes with simple ligands and fulvic/humic substances.

In the computation of metal flux in aquatic systems, at consuming surfaces like organism membranes, diffusion processes of metal ions, ligands, and complex species, as well as the kinetic and thermodynamic aspects of their chemical interactions, must be considered. The properties of many natural ligands, however, are complicated (formation of successive complexes for simple ligands, polyelectrolytic properties and chemical heterogeneity for macromolecular ligands, large size distribution and fractal structure for suspended aggregates). These properties should be properly modeled to get the correct values of the chemical rate constants and diffusion coefficients required for flux computations. The selection of the most appropriate models and parameter values is far from straightforward. This series of papers discusses the various models and compiles the parameters needed for the three most important types of complexants found in aquatic systems: the small, simple ligands, the fulvic and humic compounds, and the colloidal "particles" or aggregates. In particular, new approaches are presented to compute the rate constants of metal complex formation, with both fulvics/humics and particles/aggregates. The method to include the site distribution of fulvics/humics and the size distribution of particles/aggregates in metal flux computation at consuming interfaces is also discussed in detail. These models and parameters are discussed critically and presented in the same framework, forthe computation of metal flux in presence of any of the above complexants or mixtures. Such parameters, largely spread in the literature, are gathered here and selected specifically for environmental applications. The focus in Part I of the series is on simple ligands and fulvic/humic compounds. Part II deals with particulate and aggregate complexants.

Metal flux and dynamic speciation at (bio)interfaces. Part II: Evaluation and compilation of physicochemical parameters for complexes with particles and aggregates.

The computation of metal flux in aquatic systems at consuming surfaces like organism membranes must consider the diffusion processes of metal ions, ligands, and complex species, as well as the kinetic and thermodynamic aspects of their chemical interactions. Many natural ligands, however, have complicated properties (formation of successive complexes for simple ligands, polyelectrolytic properties and chemical heterogeneity for macromolecular ligands, large size distribution and fractal structure for suspended aggregates). These properties should be properly modeled to get the correct values of the chemical rate constants and diffusion coefficients required for flux computations. The selection of the most appropriate models and parameter values is far from straightforward. In this series of papers, models and compilations of parameters for application to the three most important types of complexants found in aquatic systems, the small, simple ligands, the fulvic and humic compounds, and the colloidal "particles" or aggregates, are discussed. In particular, new approaches are presented to compute the rate constants of metal complex formation for both fulvics/humics and particles/aggregates. A method to include the site distribution of fulvics/ humics and the size distribution of particles/aggregates in metal flux computation at consuming interfaces is also discussed in detail. These models and parameters are discussed critically and presented in a single consistent framework, applicable to the computation of metal flux in presence of any of the above complexants ortheir mixtures. Part I of the series focuses on simple ligands and fulvic/ humic compounds. Part II deals with particulate and aggregate complexants.

Roles of dynamic metal speciation and membrane permeability in metal flux through lipophilic membranes: general theory and experimental validation with nonlabile complexes.

The study of the role of dynamic metal speciation in lipophilic membrane permeability in aqueous solution requires accurate interpretation of experimental data. To meet this goal, a general theory is derived for describing 1:1 metal complex flux, under steady-state and ligand excess conditions, through a permeation liquid membrane (PLM). The theory is applicable to fluxes through any lipophilic membrane. From this theory, fluxes in the three rate-limiting conditions for metal transport are readily derived, corresponding, namely, to (i) diffusion in the source solution, (ii) diffusion in the membrane, and (iii) the chemical kinetics of formation/dissociation of the metal complex in the interfacial reaction layer. The theory enables discussion of the reaction layer concept in a more general frame and also provides unambiguous criteria for the definition of an inert metal complex. The theoretical flux equations for fully labile complexes were validated in a previous paper. The general theory for semi- or nonlabile complexes is validated here by studying the flux of Pb(II) through PLMs in contact with solutions of Pb(II)-NTA and Pb(II)-TMDTA at different pHs and flow rates.

Roles of metal ion complexation and membrane permeability in the metal flux through lipophilic membranes. Labile complexes at permeation liquid membranes.

The various physicochemical factors that influence the flux of carrier-transported metal ions through permeation liquid membranes (PLM) are studied systematically. Understanding PLM behavior is important (i) to optimize the application of PLM as metal speciation sensors in environmental media and (ii) because PLM may serve as bioanalogical devices that help to elucidate the environmental physicochemical processes occurring at the surface of biological membranes. Diffusion of free and complexed metal ions in solution, as well as diffusion of the metal carrier complex in the membrane, is considered. The respective roles of diffusion layer thickness, ligand concentration, complex stability, carrier concentration, and membrane thickness are studied experimentally in detail and compared with theory, using various labile complexes, namely, Pb(II)-diglycolate, Cu(II)-diglycolate, and Cu(II)-N-(2-carboxyphenyl)glycine. Conditions where either membrane diffusion or solution diffusion is rate limiting are clearly discriminated. It is shown in particular, that, by tuning the carrier concentration or membrane thickness, either the free metal ion concentration or the total labile metal species are measured. PLM can thus be used to determine whether models based on the free ion activity in solution (such as BLM or FIAM models) are applicable to metal uptake by microorganisms in a real natural medium.