Sensor development for routine prediction of metal biouptake in freshwaters and soil solutions

Results are presented of application of several sensors for trace metal speciation analysis in freshwater systems. All techniques were used simultaneously during a multi-partner field campaign. The techniques are compared with respect to their performance and to the metal species concentrations (Cu, Zn, Cd and Pb) detected. The analytical sensors comprise: - Diffusion gradients through thin-film gels (DGT); - In situ voltammetry using gel-impregnated microelectrodes (GIME-VIP, Gel Impregnated Microelectrode Voltammetric In situ Profiling system); - Stripping chronopotentiometry (SCP); - Permeation liquid membranes (PLM); - Donnan membrane technique (DMT); - Competitive ligand-exchange / stripping voltammetry or adsorption stripping voltammetry (CLE-SV or CLE-AdSV). All methods were found to be applicable in both hard-waters and soft-waters, although in some cases problems with detection limits were encountered at the low total concentrations in pristine waters. The detected Cu, Cd and Pb concentrations decreased in the order DGT > GIME-VIP > PLM > DMT, in agreement with the theoretical expectations. Methods involving in situ measurements (GIME-VIP) or in situ exposure (DGT, DMT and PLM) appear to be appropriate in avoiding artifacts of sampling and sample handling. This result will be submitted for publication in the international peer reviewed journal, Environmental Science & Technology. It is useful for any end user that is concerned with measurement of trace metals in freshwaters, e.g. other researchers, water-monitoring agencies.

Guidelines are presented for extraction protocols to distinguish between surface-bound and internalised metal, thus facilitating interpretation of bioaccumulation data. The approach is based on the assumption that equilibrium is rapidly attained between the metal in solution and that adsorbed to biological surface sites and that internalisation is the slow rate-determining step. Distinction between adsorption and internalisation can thus be made on the basis of the kinetics of the reactions. A washing agent should rapidly (within a few minutes) desorbs metal from the surface of the organism, whilst not accessing the internalised metal within this time frame. The utility of several washing agents has been assessed: at pH 6, EDTA was found to be acceptable in most cases. General guidelines for extraction protocols are: - The washing agent should form metal complexes with high stability and be added in excess over the molar concentration of metal adsorbed on the biological surface. - The concentration of washing agent must be optimised to limit biological effects within the timeframe of the extraction, e.g. modification of membrane permeability. - The presence of metal efflux must be verified.

A user friendly computer program for the calculation of internalisation fluxes and bio-uptake amounts under diffusion and linear or langmuirian adsorption to one or more kind of sites summarising all the simulation work developed within BIOSPEC project is being produced. This result is still in developmental stage.

A protocol (recipe) has been established for preparation of synthetic solutions that simulate the major ion composition of hard- and soft- freshwaters. These solutions provide a convenient basis for, e.g. testing of sensors in a well-defined, yet environmentally relevant medium, and for comparative inter-laboratory studies. The generalised strategy allows a synthetic solution of any given freshwater to be prepared.

An EXCEL function is being developed that will recover the binding curve of a labile ligand from voltammetric titration data. Published approximate expressions to recover the binding curve of a heterogeneous macromolecular ligand (which leads to fully labile complexes with a metal ion) will be embedded as EXCEL functions. The limiting currents or the shifts of the half-wave potentials in a voltammetric titration experiment will be the user-inputs within the typical framework of an EXCEL spreadsheet. This format will be of special interest to researchers non-specialised in numerical computations and avoids numerical simulation of transport processes at any ligand-to-metal ratio.

This result presents a test of the predictive capabilities of two commercially available equilibrium speciation models by comparison with measurements of trace metal species in situ in a soft-water river, a hard-water lake and a hard-water stream. Diffusive gradients in thin films (DGT) and voltammetry at a gel-integrated microelectrode (GIME) were used to measure dynamic species. The Donnan membrane technique (DMT) and hollow fibre permeation liquid membrane (HF-PLM) were used to measure free ion activities. Speciation calculations used WHAM 6, incorporating Humic Ion Binding Model VI and Visual MINTEQ incorporating NICA-Donnan. Predictions of dominant species using the two models agreed reasonably well, even when colloidal oxide components were considered. Measurements of Cd, Cu and Ni using DGT and GIME agreed reasonably well with each other and with model predictions of the metal transported through the gel layer. Some lower values for GIME were attributed to it having a shorter time window that excluded some species with slower rates of dissociation. For the Pb in soft-water with high dissolved organic carbon DGT and GIME measurements agreed well, but were an order of magnitude lower than model predictions, which appeared to under estimate colloidal Pb. Model predictions of the free ion activity of Ni agreed well with DMT measurements. Predictions of the free ion concentrations of Cu and Pb using Visual MINTEQ were substantially lower than those made using WHAM 6, but both predictions were substantially less than the values using DMT and HF-PLM, which were in good agreement. The results highlight the need to test further model predictions of free ion activities in natural waters rather than in test solutions containing isolated humic substances. The limiting conditions under which equilibrium models may be applicable for freshwaters are identified. The result is useful for any end user concerned with predicting trace metal speciation in freshwaters, e.g. other research, water quality control agencies.

A user-friendly computer program is available for the calculation of fluxes and currents towards a planar or spherical consuming interface (analytical sensor or organism). It is applicable to a medium with a homogeneous ligand or a mixture of ligands for any ligand-to-metal ratio under labile or non-labile conditions. A rapid analytical solution can be obtained for the flux, even in the presence of many metal complexes, irrespective of their dissociation and diffusion rates. The approach offers an approximation of a more rigorous (time consuming) numerical solution. This tool will assist end-users or researchers interested in exploiting a dynamic approach to metal speciation analysis and bio uptake by facilitating optimal design of experiments, and in the subsequent analysis of the analytical results.

The case of transient bio-uptake under linear adsorption has been analytically solved. The codes for this calculation are currently available in Mathematica, which is a highly standard commercial application. In its present form, the code can be used by non-specialists to perform computations by simply "cutting and pasting" the pieces of code into a Mathematica file. Researchers working in bio-uptake studies can use this code to check their transient fluxes and to discriminate among parameters that cannot be solved by steady-state fluxes alone. The code allows checking of accumulated amounts, and recovery of bio-uptake parameters. The code can also be used to assess the impact of the predicted maximum in the plot of amount of adsorbed species to be internalised versus time. A more sophisticated user-interface in the form of a compact presentation as a notebook including input/output of data and a package that will perform all the calculations is currently under development.

A dynamic approach to metal ion speciation analysis in aquatic ecosystems is essential for development of a rigorous basis for predictions of bioavailability and for reliable risk assessment strategies. A given sensor for speciation analysis is characterised by an effective response time that defines the metal species it is able to measure, i.e. its kinetic window. We present a common dynamic interpretation framework, based on rigorous flux expressions, for a suite of sensors that span a range of response times. The coherence and complementarily of the attainable information is assessed by measurements on well-defined metal-NTA systems. Interpolation from a kinetic spectrum of speciation analysis information is proposed as a practical strategy for addressing questions of bioavailability. This result has been submitted for publication in the international peer-reviewed scientific journal, Environmental Science & Technology. The information will allow any interested end users (e.g. other researchers, water quality monitoring agencies, etc.) to apply our approach.

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