Accumulation of copper and zinc in periphyton in response to dynamic variations of metal speciation in freshwater

Although the free ion activity model (FIAM) has been well-established in laboratory studies, there remains the need for field data in order to validate the applicability of this model in natural systems. The objective of this study was to investigate the response of copper and zinc accumulation in periphyton to short-term variations in metal concentration and speciation in freshwater. During heavy rain events, dissolved Cu in the Furtbach stream increased from 40 to 118 nM, while dissolved Zn increased from 45 to 147 nM due to the release of metals from contaminated sediments. Increases in free copper and free zinc ions in the water (from 10(-14) to 10(-11.5) M for Cu2+; from 1 to 15 nM for Zn2+) were observed during the onset of heavy rain events. Periphytic algae collected from artificial substrates had an intracellular copper content (0.2-2.8 micromol/g dry weight (dw)) that varied as a function of the exchangeable copper in the water (labile form) rather than the free Cu2+. Intracellular zinc content (1.5-8.0 micromol/g dw) was found to follow the same trend as the free zinc ion concentration. Adsorbed Cu and Zn on periphyton showed a very dynamic response to variations in dissolved metal concentration. Different concentrations of dissolved manganese during the two time periods may affect the accumulation of zinc and copper by competition for metal uptake.     

Model predictions of copper speciation in coastal water compared to measurements by analytical voltammetry

Trace metal toxicity to aquatic biota is highly dependent on the meta?s chemical speciation. Accordingly, metal speciation is being incorporated in to water quality criteria and toxicity regulations using the Biotic Ligand Model (BLM) but there are currently no BLM for biota in marine and estuarine waters. In this study, I compare copper speciation measurements in a typical coastal water made using Competitive ligand exchange-adsorptive cathodic stripping voltammetry (CLE-ACSV) to model calculations using Visual MINTEQ. Both Visual MINTEQ and BLM use similar programs to model copper interactions with dissolved organic matter-DOM (i.e., the Stockholm Humic Model and WHAM-Windermere Humic Aqueous Model, respectively). The total dissolved (<0.4 μm filter) copper concentration, [CuT] in the study sites ranged from <10 nM close to the open Baltic Sea to ca. 50 nM in the vicinity of a marina in the Stockholm Archipelago. The corresponding free copper concentration [Cu2+], measured by CLE-ACSV ranged from 10–13.2 M to 10–12.0 M for the reference and marina sites, respectively, whereas the corresponding [Cu2+] modeled calculations ranged from 10–12.5 M to 10–11.6 M. The low copper to DOM ratios (similar to 0.0004 mg Cu per mg DOC) in these coastal waters ensured that ambient dissolved copper was overwhelmingly chelated to strong Cu–binding ligands (12 < log KCuL1,Cu2+Cond >14). The modeled [Cu2+] could be fitted to the experimental values better after the conditional stability constant for copper binding to fulvic acid (FA) complexes in DOM in the SHM was adjusted to account for higher concentration of strong Cu-binding sites in FA.     

Effect of organic complexation on copper accumulation and toxicity to the estuarine red macroalga Ceramium tenuicorne: a test of the free ion activity model

Current water quality criteria (WQC) regulations on copper toxicity to biota are still based on total dissolved (<0.4 μm membrane filter) copper concentrations with a hardness modification for freshwaters. There are however ongoing efforts to incorporate metal speciation in WQC and toxicity regulations (such as the biotic ligand model-BLM) for copper and other metals. Here, we show that copper accumulation and growth inhibition of the Baltic macroalga Ceramium tenuicorne exposed to copper in artificial seawater at typical coastal and estuarine DOC concentrations (similar to 2-4 mg/L-C as fulvic acid) are better correlated to weakly complexed and total dissolved copper concentrations rather than the free copper concentration [Cu2+]. Our results using a combination of competitive ligand exchange-adsorptive cathodic stripping voltammetry (CLE-ACSV) measurements and model calculations (using visual MINTEQ incorporating the Stockholm Humic Model) show that copper accumulation in C. tenuicorne only correlates linearly well to [Cu2+] at relatively high [Cu2+] and in the absence of fulvic acid. Thus the FIAM fails to describe copper accumulation in C. tenuicorne at copper and DOC concentrations typical of most marine waters. These results seem to indicate that at ambient total dissolved copper concentration in coastal and estuarine waters, C. tenuicorne might be able to access a sizable fraction of organically complexed copper when free copper concentration to the cell membrane is diffusion limited.     

Comparison of copper speciation in estuarine water measured using analytical voltammetry and supported liquid membrane techniques

The supported liquid membrane (SLM) is a promising separation and preconcentration technique that is well-suited for trace metal speciation in natural waters. The technique is based on the selective complexation of metal ions by a hydrophobic ligand (carrier) dissolved in a water-immiscible organic solvent immobilized in a porous, inert membrane. This membrane separates two aqueous solutions: the test (or donor) solution and the strip (or acceptor) solution. The metal carrier complex is transported by diffusion across the membrane from the source to the strip solution where metal ions are back-extracted. The technique offers great potential to tune the selectivity by incorporating different complexing ligands in the membrane. A SLM was used to analyze the dissolved (<0.45 microm) copper speciation from two sites in the San Francisco Bay estuary; Dumbarton Bridge, [Cu]total approximately 27 nM, and San Bruno Shoals, [Cu]total approximately 23 nM. The sites were also characterized independently by differential pulse anodic stripping voltammetry (DPASV) using a Nafion-coated thin mercury film electrode (NCTMFE). The SLM employed 10 mM lasalocid, a naturally occurring carboxylic polyether ionophore, in nitrophenyl octyl ether (NPOE) asthe membrane complexing ligand, supported by a microporous, polypropylene, hydrophobic membrane. This is the first study where SLM technique has been compared with an independent speciation technique in marine waters. Results of copper speciation measurements from Dumbarton Bridge, a site in South San Francisco Bay where copper speciation has been well-characterized in previous studies using various voltammetric techniques, indicated that only about 3% (0.9 nM) of the total dissolved copper was SLM labile. The corresponding DPASV labile copper fraction was <0.4% (<0.1 nM) of total dissolved copper. The concentration of total copper binding ligands measured by the membrane technique was 471 nM as compared to 354 nM measured by DPASV, more than 1 order of magnitude higher than the total dissolved copper concentration. The SLM measurements were consistent with earlier copper speciation measurements that were made in South San Francisco Bay using other voltammetric stripping techniques.     

Measuring free metal ion concentrations in situ in natural waters using the Donnan Membrane Technique

Metal toxicity is not related to the total but rather to the free or labile metal ion concentration. One of the techniques that can be used to measure several free metal ion concentrations simultaneously is the Donnan Membrane Technique (DMT) in combination with the inductively coupled plasma-mass spectrometer (ICP-MS). However, free metal ion concentrations in natural waters are commonly below the detection limit of ICP-MS. We decreased the detection limit by making use of a ligand, and we developed a field DMT cell that can be applied in situ in natural waters. A kinetic approach can be used to calculate free metal ion concentrations when the equilibrium time becomes too large. The field DMT measured in situ in natural waters a free metal ion concentration ranging from 0.015% (Cu) to 13% (Zn) of a total metal concentration ranging from 0.06 nM (Cd) to 237 nM (Zn). The free metal ion concentrations were difficult to predict using an equilibrium speciation model, probably due to the uncertainty in the nature of the dissolved organic matter or the presence of other reactive colloids. It is shown that DMT can follow changes in the free metal ion concentration on times scales less than a day under certain conditions.     

Adsorption and uptake of Cu by Emiliania huxleyi in natural seawater

Short-term kinetic experiments, carried out in natural coastal seawater (with predetermined background levels of trace metals and organic ligands, L) enriched with nitrate and phosphate, demonstrated that Emiliania huxleyi was able to uptake Cu very quickly. After 10 min of exposure (background Cu level in the inoculated cells: [Cu]total cellular = 9.3 x 10(-17) mol cell-1, [Cu]intracellular = 8.4 x 10(-17) mol cell-1, and [Cu]extracellular = 1.0 x 10(-17) mol cell-1) to a natural seawater which contained 29 nM total initial dissolved Cu concentration ([Cu]d) (29 nM [CuL] and 3.2 x 10(-13) M free Cu concentration, [Cu2+]) the intracellular Cu was already 28 x 10(-17) mol cell-1. This value corresponded to 85% of the intracellular metal observed in pseudoequilibrium conditions (after 24 h of exposure) and to 88% of the total metal sorption (adsorption plus uptake) after 10 min. In contrast, the external adsorption after 10 min was only 3.0 x 10(-17) mol cell-1 which corresponded to 60% of the extracellular metal in pseudoequilibrium conditions. Simultaneously occurred a very fast release of organic ligands (L) by E. huxleyi, the majority being identified by cathodic stripping voltammetry as glutathione. The production of exudates increased with both Cu concentration and exposure time. After 10 min of exposure, the production of exudates in a medium with 129 nM [Cu]d (72 nM [CuL] and 7.9 x 10(-13) M [Cu2+]) was 51 nM, about 42% of that observed in pseudoequilibrium. As the Cu complexes with the organic ligands present in the medium were very stable (logarithm of the conditional stability constant: 12.18 +/- 0.06) and the ligand concentration in the medium was relatively high (e.g. 123 nM CL in the medium with 129 nM initial [Cu]d after 10 min of exposure) most of the metal was organically bound in the medium.     

Comparison of copper speciation in coastal marine waters measured using analytical voltammetry and diffusion gradient in thin-film techniques

The diffusion gradient in thin-film hydrogel (DGT) probe is a promising tool for metal speciation work. Based on a passive sampling principle, it provides the potential for large data sets in complex regimes. DGT probes were deployed in waters characterized independently using competitive ligand exchange-adsorptive cathodic stripping voltammetry (CLE-ACSV). The CLE-ACSV used benzoyl acetone as the competitive ligand in discrete water samples collected during the deployment of the DGT probes. The DGT probes used a 15% polyacrylamide/0.4% bis-acrylamide cross-linker hydrogel and a Na-form of Chelex-100 to complex metal that fluxed into the probe through the hydrogel. Probes were deployed in locations characterized by the degree of pollution impact: the relatively pristine Vineyard Sound, MA, [Cu]total approximately 6 nM, small seasonally active harbors on Cape Cod, MA, [Cu]total = 12-64 nM, as well as a large polluted estuary, the Elizabeth River, VA, [Cu]total = 44-58 nM, and a large polluted port, San Diego Harbor, CA, [Cu]total = 23-103 nM. This is the first study where DGT probes have been compared with an independent speciation technique in marine systems and used to establish the diffusion coefficient of Cu-complexing ligands in situ. Results showed that the probes produced highly precise data sets, with substantial differences in copper accumulation between contaminated and pristine waters. Comparison of DGT results with CLE-CSV indicate that at least 10-35% of the organically complexed copper derived by CLE-ACSV measurements was DGT-labile. Diffusion coefficients (corrected to 25 degrees C) of organically complexed DGT-labile Cu through the hydrogel ranged from 0.77 x 10(-6) cm2 s(-1) in Vineyard Sound to 2.16 x 10(-6) cm2 s(-1) in the Elizabeth River estuary. Accumulation rates of copper were substantially higher in contaminated waters than in pristine waters, suggesting that the probes in their current form may be useful as tracking tools to detect episodic sources of contamination.     

Speciation of Cu and Zn in drainage water from agricultural soils

Inputs of copper and zinc from agricultural soils into the aquatic system were investigated in this study, because of their heavy agricultural usage as feed additives and components of fertilizers and fungicides. As the mobility and bioavailability of these metals are affected by their speciation, the lipophilic, colloidal and organic fractions were determined in drainage water from a loamy and a humic soil treated with fungicides or manure. This study therefore investigates the impact of agricultural activity on a natural environment and furthers our understanding of the mobility of metals in agricultural soils and aquatic pollution in rural areas. Marked increases in the total dissolved metal concentrations were observed in the drainage water during rain events with up to 0.3 microM Cu and 0.26 microM Zn depending on the intensity of the rainfall and soil type. The mobile metal fractions were of a small molecular size (<10 kD) and mainly hydrophilic. Lipophilic complexes originating from a dithiocarbamate (DTC) fungicide could not be observed in the drainage water; however, small amounts of lipophilic metal complexes may be of natural origin. Cu was organically complexed to > 99.9% by abundant organic ligands (log K 10.5-11.0). About 50% of dissolved Zn were electrochemically labile, and the other 50% were complexed by strong organic ligands (log K 8.2-8.6). Therefore very little free metal species were found suggesting a low bioavailability of these metals in the drainage water even at elevated metal concentrations.     

Nickel speciation and complexation kinetics in freshwater by ligand exchange and DPCSV

A technique of ligand exchange with DMG (dimethylglyoxime) and DPCSV was applied to determine Ni speciation in lake, river, and groundwater samples. The working conditions related to ligand-exchange equilibrium were optimized, and the ligand-exchange kinetics were examined. The observed pseudo-first-order rate, kobsd, was about 3 x 10(-5) (s-1) for Ni(DMG)2 complex formation with an excess of DMG (microM) over Ni (nM) at pH 7.1-7.7. The second-order exchange kinetic constants, kexch, were between 1.2 x 10(2) and 5.7 x 10(3) s-1 M-1 for ligand exchange of NiEDTA with DMG and between 5 x 10(2) and 7 x 10(3) s-1 M-1 for exchange of natural ligands with DMG in the freshwater samples under similar conditions. Ni ligand exchange between natural ligands and DMG occurred over days with half-lifes of 5-95 h. Total dissolved Ni concentrations in samples from various freshwater systems in Switzerland ranged from 4 nM in an oligotrophic lake to 30 nM in a small river affected by inputs from sewage effluents and agriculture. Free ionic Ni2+ concentrations were determined in the range of 10(-13)-10(-15) M (pNi = 12.2-14.7), indicating that more than 99.9% of dissolved Ni was bound by organic ligands with strong affinity (log K 12.1-14.9) and low concentrations (13-100 nM) at pH 7.2-8.2. Because of slow ligand-exchange kinetics, Ni speciation in natural waters may in many cases not reach equilibrium.     

Strong colloidal and dissolved organic ligands binding copper and zinc in rivers

The speciation or physicochemical form of copper and zinc in freshwater plays an important role in reactivity, bioavailability, and toxicity. Strong metal-binding ligands, which determine speciation, were detected by voltammetric methods, both anodic stripping voltammetry (ASV) and competitive ligand equilibration adsorptive stripping voltammetry (CLE-AdSV); the latter technique can detect nanomolar levels of extremely strong (log K' > 13) ligands. Through careful field site selection and the investigation of ultrafiltration permeate samples, natural organic ligands were measured with limited interferences of colloidal inorganic iron- and aluminum-based trace metal-binding phases. Furthermore, ultrafiltration allowed measurement of colloidal and dissolved ligands independently, and differences of ligand abundance and strength in different size classes are reported. For copper, ultrafilterable (<3 kDa) organic ligand site concentrations (expressed normalized to dissolved organic carbon) were on average 33% of the colloidal level, but ultrafilterable ligand log K' values were 0.5 log units stronger than those of the 0.4 microm filterable concentration. The ultrafilterable copper-binding ligand concentration showed a smaller variation across the rivers (25% rsd) than zinc-binding ligands (90% rsd). For all field sites and size fractions, strong ligand sites greatly exceeded metal concentrations; subsequently, equilibrium speciation modeling predict picomolar levels of free metal. Modeling also indicated that the very strong ligands (detected by CLE-AdSV) predominate, so modeling based solely on ASV data in freshwater may be inadequate. Competition experiments indicated that the very strong ligand sites are metal specific for copper and zinc.     

In-situ speciation of Ni and Zn in freshwaters: comparison between DGT measurements and speciation models

The technique of DGT (diffusive gradients in thin films) was used for the first time to measure in situ the distribution of Zn and Ni between inorganic species and complexes with fulvic and humic acids in natural waters. With DGT, metals are bound to a resin embedded in a layer of hydrogel after diffusive transport through an adjacent layer of hydrogel. The metal concentrations in the waters can be quantified using simple diffusion equations. By using devices with hydrogels of different pore size, large and small complex species were discriminated. Inorganic species diffuse freely through all gels, but larger organic complexes with fulvic and humic acids diffuse less freely through more restricted gels (gels with smaller pore size). Systematic differences between DGT devices containing gels of different pore size were obtained. Their calibration for the diffusion of fulvic and humic complexes allowed calculation of the concentrations of labile inorganic (Zn, 34.6 +/- 2.5 nM; Ni, 23.5 +/- 0.9 nM) and labile organic (Zn, 43.1 +/- 2.9 nM; Ni, 11.2 +/- 0.7 nM) complexes. The concentration of Zn measured by anodic stripping voltammetry in samples returned to the laboratory lay between the DGT-measured inorganic concentration and the total dissolved concentration, consistent with partial measurement of organic complexes of Zn. The speciation model WHAM successfully predicted the species distribution of Ni, Zn, and Cu, provided that competitive binding by Fe(III) was considered. Using the speciation models WHAM and ECOSAT, free ion activities of Zn and Ni were calculated from (1) the total inorganic species measured by DGT and (2) the total dissolved species and dissolved organic carbon. The calculations confirmed the good model predictions of metal-humic binding but highlighted problems with default databases used for the speciation of inorganic components.     

Analysis of copper binding in the ternary system Cu2+/humic acid/goethite at neutral to acidic pH

Binding of heavy metal and actinide ions to natural colloids, such as humic substances (HSs) and metal (hydr)-oxides, plays an important role in the ecotoxicological behavior of these ions. Several thermodynamic models have been constructed to predict the speciation of these ions in metal/HS or metal/oxide binary systems. However, in natural environments the adsorption of HSs on oxides can influence the binding of target metals, leading to deviation from the additivity of calibrated binary models. In this study binding of copper (Cu2+) to the purified Aldrich humic acid (PAHA)/goethite complex in the neutral to acidic pH region was investigated by measuring Cu2+ binding isotherms. The measured isotherms were compared with the results obtained for the binary systems under similar conditions. The comparison revealed that Cu2+ binding in the ternary system is enhanced with respect to the sum of Cu2+ binding in the corresponding binary systems. From the analysis of the charging behavior of the adsorbed PAHA as well as the smeared-out potential profile near the PAHA/goethite interface, the increase of Cu2+ binding to the complex was mainly attributed to the decrease of proton competition to the functional groups of the adsorbed PAHA and the change of the electrostatic potential in the vicinity of the goethite surface.     

Modeling complexometric titrations of natural water samples

Complexometric titrations are the primary source of metal speciation data for aquatic systems, yet their interpretation in waters containing humic and fulvic acids remains problematic. In particular, the accuracy of inferred ambient free metal ion concentrations and parameters quantifying metal complexation by natural ligands has been challenged because of the difficulties inherent in calibrating common analytical methods and in modeling the diverse array of ligands present. This work tests and applies a new method of modeling titration data that combines calibration of analytical sensitivity (S) and estimation of concentrations and stability constants for discrete natural ligand classes ([Li]T and Ki) into a single step using nonlinear regression and a new analytical solution to the one-metal/two-ligand equilibrium problem. When applied to jointly model data from multiple titrations conducted at different analytical windows, it yields accurate estimates of S, [Li]T, Ki, and [Cu2+] plus Monte Carlo-based estimates of the uncertainty in [Cu2+]. Jointly modeling titration data at low-and high-analytical windows leads to an efficient adaptation of the recently proposed "overload" approach to calibrating ACSV/CLE measurements. Application of the method to published data sets yields model results with greater accuracy and precision than originally obtained. The discrete ligand-class model is also re-parametrized, using humic and fulvic acids, L1 class (K1 = 10(13) M(-1)), and strong ligands (L(S)) with K(S) > K1 as "natural components". This approach suggests that Cu complexation in NW Mediterranean Sea water can be well represented as 0.8 +/- 0.3/0.2 mg humic equiv/L, 13 +/- 1 nM L1, and 2.5 +/- 0.1 nM L(S) with [CU]T = 3 nM. In coastal seawater from Narragansett Bay, RI, Cu speciation can be modeled as 0.6 +/- 0.1 mg humic equiv/L and 22 +/- 1 nM L1 or approximately 12 nM L1 and approximately 9 nM L(S), with [CU]T = 13 nM. In both waters, the large excess (approximately 10 nM) of high-affinity, Cu-binding ligands over [CU]T results in low equilibrium [Cu2+] of 10(-14.5 +/- 0.2) M and 10(-13.3 +/- 0.4) M, respectively.     

Biotic ligand model, a flexible tool for developing site-specific water quality guidelines for metals

The biotic ligand model (BLM) is a mechanistic approach that greatly improves our ability to generate site-specific ambient water quality criteria (AWQC)for metals in the natural environment relative to conventional relationships based only on hardness. The model is flexible; all aspects of water chemistry that affect toxicity can be included, so the BLM integrates the concept of bioavailability into AWQC--in essence the computational equivalent of water effect ratio (WER) testing. The theory of the BLM evolved from the gill surface interaction model (GSIM) and the free ion activity model (FIAM). Using an equilibrium geochemical modeling framework, the BLM incorporates the competition of the free metal ion with other naturally occurring cations (e.g., Ca2+, Na+, Mg2-, H+), togetherwith complexation by abiotic ligands [e.g., DOM (dissolved organic matter), chloride, carbonates, sulfide] for binding with the biotic ligand, the site of toxic action on the organism. On the basis of fish gill research, the biotic ligands appear to be active ion uptake pathways (e.g., Na+ transporters for copper and silver, Ca2+ transporters for zinc, cadmium, lead, and cobalt), whose geochemical characteristics (affinity = log K, capacity = Bmax) can be quantified in short-term (3-24 h) in vivo gill binding tests. In general, the greater the toxicity of a particular metal, the higher the log K. The BLM quantitatively relates short-term binding to acute toxicity, with the LA50 (lethal accumulation) being predictive of the LC50 (generally 96 h for fish, 48 h for daphnids). We critically evaluate currently available BLMs for copper, silver, zinc, and nickel and gill binding approaches for cadmium, lead, and cobalt on which BLMs could be based. Most BLMs originate from tests with fish and have been recalibrated for more sensitive daphnids by adjustment of LA50 so as to fit the results of toxicity testing. Issues of concern include the arbitrary nature of LA50 adjustments; possible mechanistic differences between daphnids and fish that may alter log K values, particularly for hardness cations (Ca2+, Mg2+); assumption of fixed biotic ligand characteristics in the face of evidence that they may change in response to acclimation and diet; difficulties in dealing with DOM and incorporating its heterogeneity into the modeling framework; and the paucity of validation exercises on natural water data sets. Important needs include characterization of biotic ligand properties at the molecular level; development of in vitro BLMs, extension of the BLM approach to a wider range of organisms, to the estuarine and marine environment, and to deal with metal mixtures; and further development of BLM frameworks to predict chronic toxicity and thereby generate chronic AWQC.     

Chemical speciation of dissolved Cu, Ni, and Co in a contaminated estuary in southwest Spain and its influence on plankton communities

Four surveys of the Huelva Estuary in southwest Spain and its sources, the Tinto and the Odiel Rivers, were carried out between 1996 and 1998. The surveys investigated the impact of metalliferous mining of sulfide-rich ores in the catchment area on metal speciation, metal concentrations in a macrophyte, and phytoplankton diversity and abundance. Chemical speciation measurements in the lower Tinto Estuary showed that metals were predominantly electrochemically labile (> 99% of total dissolved Cu, Co, and Ni at 10 microM Cu, 424 nM Co, and 500 nM Ni, S = 28). Concentrations of Cu complexing ligands and free cupric ions [Cu2+] in the Gulf of Cádiz ranged between 5.3 and 38 nM and 0.2-7.9 pM, respectively, with conditional stability constants of the ligands of log K'(CuL) = 11.7-12.6. At enhanced dissolved Cu concentrations in the lower Huelva Estuary, Cu complexing ligands were saturated with Cu, resulting in nanomolar [Cu2+], which increased upstream. Metal tissue concentrations of the macrophyte Blindingia marginata were high, and a clear relationship between dissolved labile Cu and macrophyte tissue Cu concentrations was observed. A low biodiversity was observed in the Huelva system (Shannon-Wiener indices (H) typically < 0.2). Nevertheless, the maximum biomass was observed in the lower Tinto Estuary, which showed high labile metal and nutrient concentrations and a low biodiversity (H < 0.02), thereby suggesting adaptation through evolutionary processes of the phytoplankton community to the harsh conditions.     

Thiol and metal contents in periphyton exposed to elevated copper and zinc concentrations: a field and microcosm study

Phytochelatins are metal-binding polypeptides produced by algae under metal exposure. The aim of this study was to investigate the effects of metal concentration variations in natural systems on periphyton at the biochemical level by analyzing its intracellular thiol content, in particular phytochelatins. To that purpose, two field campaigns were conducted in a stream subject to an increase of dissolved metal concentrations (particularly Cu and Zn) during rain events, which results in an increase of their accumulation in periphyton. At background metal concentrations, several thiols were detectable in periphyton, namely, glutathione (GSH), gamma-glutamylcysteine (gammaGluCys), phytochelatins (PC2), and some unidentified thiols, U1 and U2. Glutathione and gammaGluCys contents were found to vary independently of the rain, as well as U1 and U2, whereas the phytochelatin content increased during the rain events. To investigate whether Cu or Zn may be responsible for this increase, microcosm experiments were carried out with natural water enriched with Cu, Zn, and Cd separately, and Cu and Zn in combination. In this study, GSH, PC2, and U1 were also detected, but not gammaGluCys. An increase in accumulated Cu content did not induce any changes in thiol content, whereas an increase of the Zn content induced a decrease in GSH content and an increase in phytochelatin content. Zinc rather than Cu may thus induce a phytochelatin content increase in periphyton in the field studies. Addition of Cu and Zn in combination also induced an increase in phytochelatin content. Cadmium was found to be the most effective inducer, with the production of larger phytochelatins (PC3-4). This study is the first one to report changes in thiol content in periphyton in response to an increase of the metal concentration in natural freshwaters.     

Interpretation of in situ speciation measurements of inorganic and organically complexed trace metals in freshwater by DGT

The dynamic speciation technique, diffusive gradients in thin-films (DGT), has been used in freshwater to determine simultaneously, from a single set of in situ measurements, (1) the equilibrium distribution of metal ions between simple inorganic complexes and larger organic complexes and (2) information on the rates of dissociation of these complexes. DGT devices with different diffusion layer thicknesses (0.3, 0.54, 1.34, and 2.14 mm) were used to estimate the in situ dissociation kinetics. Information on the species distribution was obtained by using two types of gel, which allow relatively free (polyacrylamide, APA) and more retarded (restricted, RES) diffusion of the metal complexes. The full theoretical basis of the technique is developed and applied to in situ measurements of Mn, Fe, Co, Ni, Cu, Cd, and Pb in a pristine river (Wyre, U.K.), with high DOC(15mg L(-1)), assuming that organic complexes are dominated by fulvic acid. These first DGT measurements that do not rely on assumptions about complex lability or the distribution of species, are compared to total dissolved measurements, previously reported speciation calculations and measurements using alternative speciation techniques. Examination of calculation consistency suggests that the effective mean diffusion coefficients of metal complexes with organic matter under in situ conditions may be larger than those measured in the laboratory using extracted fulvic acid.     

Chemical speciation of Zn,Cd, Cu,and Pb in pore waters of agricultural and contaminated soils using Donnan dialysis

Knowledge of trace metal speciation in soil pore waters is important in addressing metal bioavailability and risk assessment of contaminated soils. Numerous analytical methods have been utilized for determining trace metal speciation in aqueous environmental matrixes; however, most of these methods suffer from significant interferences. The Donnan dialysis membrane technique minimizes these interferences and has been used in this study to determine free Zn2+, Cd2+, Cu2+, and Pb2+ activities in pore waters from 15 agricultural and 12 long-term contaminated soils. The soils vary widely in their origin, pH, organic carbon content, and total metal concentrations. Pore water pM2+ activities also covered a wide range and were controlled by soil pH and total metal concentrations. For the agricultural soils, most of the free metal activities were below detection limit, apart from Zn2+ for which the fraction of free Zn2+ in soluble Zn ranged from 2.3 to 87% (mean 43%). Five of the agricultural soils had detectable free Cd2+ with fractions of free metal ranging from 59 to 102% (mean 75%). For the contaminated soils with detectable free metal concentrations, the fraction of free metal as a percentage of soluble metal varied from 9.9 to 97% (mean 50%) for Zn2+, from 22 to 86% (mean 49%) for Cd2+, from 0.4 to 32.1% (mean 5%) for Cu2+, and from 2.9 to 48.8% (mean 20.1%) for Pb2+. For the contaminated soils, the equilibrium speciation programs GEOCHEM and WHAM Model VI provided reasonable estimates of free Zn2+ fractions in comparison to the measured fractions (R2 approximately 0.7), while estimates of free Cd2+ fractions were less agreeable (R2 approximately 0.5). The models generally predicted stronger binding of Cu2+ to DOC and hence lower fractions of free Cu2+ as compared with the observed fractions. The binding of Cu2+ and Pb2+ to DOC predicted by WHAM Model VI was much strongerthan that predicted by GEOCHEM.     

Species kinetics and heterogeneous reactivity of dissolved Cu in natural freshwaters

Using high specific activity 64Cu2+ as radiotracer, the distribution kinetics among Cu species were established in natural organic-rich freshwaters under steady-state conditions, i.e., with minimal disturbance of existing equilibria. Study sites with contrasting suspended particulate matter (SPM) characteristics were investigated. Our analytical protocol allowed the differentiation between the following Cu species: SPM associated Cu, dissolved reactive (free and labile) Cu, and organically complexed Cu. The data obtained were successfully evaluated by compartmental analysis, which showed the importance of organically complexed Cu in freshwaters, and the dominant role of the interactions between organically complexed Cu and SPM in a SPM-rich water. The kinetic 54Cu measurements indicated that the attainment of equilibrium between dissolved reactive and organically complexed Cu took ca. <1-2 h, and 4-15 h for the interaction between dissolved organically complexed and SPM associated Cu. The kinetic study was augmented by voltammetric measurements of the dissolved (stable) Cu equilibrium speciation conditions in the natural waters. These measurements showed that the waters contained very low cupric ion concentrations (10(-12)-10(-15) M), with more than 99.9% of the dissolved Cu complexed by strong organic ligands (conditional stability constants: 10(13.4)-10(15.4)).