Interaction between Cu and Thiols of Biological and Environmental Importance: Case Study Using Combined Spectrophotometric/Bathocuproine Sulfonate Disodium Salt Hydrate (BCS) Assay.

Considering the biological and ecological importance of Cu-thiol interactions and the discrepancies in previous research, this study focuses on Cu interactions with biologically and ecologically relevant thiols: glutathione (GSH), L-cysteine (L-cys), 3-mercaptopropionic acid (MPA), and thioacetic acid (TAA) in aqueous solution. The addition of Cu(II) to a thiol-containing solution led to a rapid reduction of Cu(II) and the formation of a Cu(I)-thiol complex. The mechanism of Cu(II) reduction and Cu(I) complex formation as well as the kinetics of Cu(I) oxidation strongly depend on the structural properties of the individual thiols investigated. The reducing power of the investigated thiols can be summarized as follows: L-cys ≅ GSH > MPA > TAA. The reaction order, with respect to Cu(I) oxidation, also changes over the time of the reaction course. The deviation of the reaction kinetics from the first order with respect to Cu(I) in the later stages of the reaction course can be attributed to a Fenton-like reaction occurring under low thiol concentration conditions. At high Cu:thiol ratios, in the case of GSH, L-cys, and MPA, the early stage of the reaction course is characterized by high Cu(I) stability, most likely as a result of Cu(I) complexation by the thiols present in excess in the reaction mixture.

Vanadium(IV) and vanadium(V) complexation by succinic acid studied by affinity capillary electrophoresis. Simultaneous injection of two analytes in equilibrium studies.

The interaction of V(IV) and V(V) with succinic acid was investigated by affinity capillary electrophoresis (ACE) in aqueous acid solutions at pH values 1.5, 2.0 and 2.4, and different ligand concentrations. V(IV) and V(V) form protonated complexes with succinic acid ligand at this pH range. The logarithms of the stability constants, measured at 0.1 mol L-1 (NaClO4/HClO4) ionic strength and 25 °C, are logß111=7.4 ± 0.2 and logß122=14.1 ± 0.5 for V(IV), and logß111=7.3 ± 0.1 for V(V), respectively. The stability constant values, extrapolated to zero ionic strength with the Davies equation, are logß°111=8.3 ± 0.2 and logß°122=15.6 ± 0.5 for V(IV) and logß°111=7.9 ± 0.1 for V(V). The application of ACE to the simultaneous equilibria of V(IV) and V(V) (injection of two analytes) was also attempted. When the results were compared with those obtained when introducing only one analyte in the capillary, using the traditional version of the method, similar stability constants and precision are obtained. The possibility of studying two analytes simultaneously decreases the time needed for the determination of the constants; this feature is especially valuable when working with hazardous materials or when only small quantities of ligand are available.

Investigation of thiol compounds (L-cysteine, thioacetic acid and ethanethiol) with V(V) and V(IV) using combined spectroscopy and chromatography.

The interactions of V(V) and L-cysteine, thioacetic acid and ethanethiol were studied in aqueous solution using chromatographic and spectral analysis. The chromatographic determination of V(V) and V(IV) species in the presence of thiols was enabled by inducing the ligand exchange reaction with EDTA as the competing ligand. Analytical setup allowed investigation of the possible redox and structural transformations of V(V) in the presence of thiols used over a wide pH range. Obtained data strongly suggest that the reduction of V(V) is proton catalyzed in case of L-cysteine and thioacetic acid. In the case of ethanethiol, the reduction did not seem to be proton dependent, as no reduction was observed above pH = 2. Thus, reduction was inhibited by the deprotonation of L-cysteine and thioacetic acid, with L-cysteine being the strongest reducing agent of V(V), followed by thioacetic acid and finally ethanethiol. Apart from structural thiol properties, the reduction reaction seems to be influenced by the aqueous V(V) speciation due to the observed nonlinear kinetics. In the case of all investigated thiols, the formation of V(V)-thioester intermediate species was an essential step for V(V) reduction. The structural properties of the V(IV)-thiol complexes were also found to be pH-dependent.

Chromatographic and spectrophometric studies of vanadate (+V) reduction by 3-mercaptopropionic acid.

The reduction of vanadate (+V) in the presence of 3-mercaptopropionic acid was studied using a chromatographic method for the determination of vanadate (+V) versus vanadyl (+IV) species. Ion chromatography was combined with spectrophotometric investigation of the absorption properties of the solution. The chromatographic method for the separation of vanadate (+V) and vanadyl (+IV) was carried out with an anion exchange column. In the initial reaction mixture containing vanadate (+V) and 3-mercaptopropionic acid, ethylenediaminetetraacetic acid - EDTA was added in an excessive amount relative to the concentration of reactants in the solution. After the ligand exchange reaction, the added EDTA terminates the reduction, allowing redox speciation in the solutions. A strong pH dependence of the reduction rates in the investigated solution was observed. The vanadate reduction seems to proceed in 2 steps: 1) formation of the intermediate vanadate (+V)-thioester; 2) reduction reaction and formation of the vanadyl (+IV)-thiol complex. The obtained results strongly suggest that the reaction of vanadate (+V)-thioester formation is proton catalyzed. It was observed that the overall reduction rates are pH dependent due to the complex vanadate (+V) solution speciation and changes in the ionic form of 3-mercaptopropionic acid.

Rhenium Distribution and Behavior in the Salinity Gradient of a Highly Stratified Estuary and Pristine Riverine Waters (The Krka River, Croatia).

The abundance and distribution of dissolved Re (DRe) were determined in the freshwater part of the Krka River (Croatia), which drains a karst landscape, and in the salinity gradient of its highly stratified estuary. Due to the low DRe concentration, a batch procedure consisting of a pre-concentration step using an anion exchange resin (Dowex) and analysis of DRe in 8 M HNO3 eluate using high-resolution inductively coupled plasma mass spectrometry (HR ICP-MS) was applied. Due to potentially inconsistent recoveries, which ranged from 60 to 87%, quantification was performed using the isotope dilution technique (ID). DRe concentrations in the Krka River increased downstream, from 6.2 pM at the spring site to 11.9 pM upstream of the estuary region. Weathering of the surrounding carbonate lithology is assumed to be the source of the natural Re. Two specific anomalies were registered: a strong increase in DRe concentration due to anthropogenic input near the town of Knin (27.5 pM) and a decrease at a downstream site caused by subsurface input of freshwater from the Zrmanja River, resulting in a relatively low DRe concentration (8.5 pM). In the estuarine region, a near-conservative behavior of DRe was found in the salinity gradient of the upper surface layer, with DRe concentrations ranging from 18 to 38 pM. Anthropogenic input was suspected within the estuarine segment near the urban area, causing a small positive deviation from the conservative line. In the bottom seawater layer, a minor decrease in DRe concentration in the most upstream estuarine regions was apparent, implying weak scavenging of Re.

Uranium isotopes as a possible tracer of terrestrial authigenic carbonate.

The concentration and isotopic composition of uranium (δ238U, 234U/238U activity ratio) in combination with traditional isotopes (δ18O, δ13C) were examined as potential tracers of authigenic carbonate formation in a karst aquifer. The U concentration and 234U/238U activity ratios in the tufa-precipitating sections of two connected karst rivers (Krka and Zrmanja, Croatia) decreased downstream in water and in precipitated carbonate due to active self-purification processes, i.e. adsorption of isotopically lighter U(VI) on mineral particles, sedimentation and co-precipitation with carbonate. The isotopic composition of carbonate in tufa mostly resembled the 234U/238U activity ratio and the δ238U values of dissolved U in water but was also affected by the presence of detrital carbonate flushed into the river from soil and weathered bedrock. This interpretation was supported by the δ18O and δ13C values of tufa, which were shifted out of equilibrium with river water and dissolved in organic carbon and in their isotopic signature, which showed the presence of lithic carbonate. Large fluctuations of the δ238U values of water, leachable U (eluted in acetic acid buffered with Na-acetate) and residual U fraction could not be fully explained by available data due to the overlapping U isotopic signatures of leachable (mainly carbonate) and residual fractions of soil, bedrock and tufa. Therefore, a long-term, systematic, seasonal and event-based observation of the isotopic composition of dissolved and suspended particulate U in water is necessary. Nevertheless, the U isotopes were found to have the potential to be used as identifiers of authigenic carbonate and the storage of CO2 in terrestrial river sediments, to improve knowledge on fluxes within local and global biogeochemical carbon cycle.

Redox Speciation of Vanadium in Estuarine Waters Using Improved Methodology Based on Anion Exchange Chromatography Coupled to HR ICP-MS System.

An improved methodology was developed for V redox speciation in estuarine waters using a hyphenated technique consisting of ion chromatograph (IC) with an anion exchange column and a high-resolution inductively coupled plasma mass spectrometer (HR ICP-MS). This approach enables the direct determination of V(V), whereas reduced species (mainly V(IV)) are calculated by subtracting V(V) concentrations from the measured total V concentration. Based on the "on-column" V(V) chelation mechanism by EDTA, with the eluent composed of 40 mmol L-1 ammonium bicarbonate, 40 mmol L-1 ammonium sulphate, 8 mmol L-1 ethylenediaminetetraacetic acid and 3% acetonitrile, the method was successfully used for analyses of V redox speciation in samples taken in the vertical salinity gradient of the highly stratified Krka River estuary. Due to the matrix effects causing different sensitivities, a standard addition method was used for V(V) quantification purposes. The limit of detection (LOD) was also found to be matrix related: 101.68 ng L-1 in the seawater and 30.56 µg L-1 in the freshwater. Performed stability tests showed that V redox speciation is preserved at least 7 days in un-treated samples, possibly due to the stabilization of V-reduced species with natural organic matter (NOM). The dominant V form in the analysed samples was V(V) with the reduced V(IV) accounting for up to 26% of the total dissolved pool. The concentration of V(IV) was found to correlate negatively with the oxygen concentration. Significant removal of dissolved V was detected in oxygen depleted zones possibly related to the particle scavenging.

Experimental Confirmation of Isotope Fractionation in Thiomolybdates Using Ion Chromatographic Separation and Detection by Multicollector ICPMS.

Molybdenum 98Mo/95Mo isotope ratios are a sediment paleo proxy for the redox state of the ancient ocean. Under sulfidic conditions, no fractionation between seawater and sediment should be observed if molybdate (MoO42-) is quantitatively transformed to tetrathiomolybdate (MoS42-) and precipitated. However, quantum mechanical calculations previously suggested that incomplete sulfidation could be associated with substantial fractionation. To experimentally confirm isotope fractionation in thiomolybdates, a new approach for determination of isotope ratios of individual thiomolybdate species was developed that uses chromatography (HPLC-UV) to separate individual thiomolybdates, collecting each peak and analyzing isotope ratios with multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). Using commercially available MoO42- and MoS42- standards, the method was evaluated and excellent reproducibility and accuracy were obtained. For species with longer retention times, complete chromatographic peaks had to be collected to avoid isotope fractionation within peaks. Isotope fractionation during formation of thiomolybdates could be experimentally proven for the first time in the reaction of MoO42- with 20-fold or 50-fold excess of sulfide. The previously calculated isotope fractionation for MoS42- was confirmed, and the result for MoO2S22- was in the predicted range. Isotopic fractionation during MoS42- transformation with pressurized air was dominated by kinetic fractionation. Further optimization and online-coupling of the HPLC-MC-ICPMS approach for determination of low concentrations in natural samples will greatly help to obtain more accurate species-selective isotope information.

Chronoamperometric study of elemental sulphur (S) nanoparticles (NPs) in NaCl water solution: new methodology for S NPs sizing and detection.

BACKGROUND: Elemental sulfur (S) persists in natural aquatic environment in a variety of forms with different size distributions from dissolved to particulate. Determination of S speciation mainly consists of the application of chromatographic and electrochemical techniques while its size determination is limited only to the application of microscopic and light scattering techniques. S biological and geochemical importance together with recent increases of S industrial applications requires the development of different analytical tools for S sizing and quantification. In recent years the use of electrochemical measurements as a direct, fast, and inexpensive technique for the different nanoparticles (NPs) characterization (Ag, Au, Pt) is increasing. In this work, electrochemical i.e. chronoamperometric measurements at the Hg electrode are performed for determination of the size distribution of the S NPs. RESULTS: S NPs were synthesized in aqueous medium by sodium polysulphide acidic hydrolysis. Chronoamperometric measurements reveal the polydisperse nature of the formed suspension of S NPs. The electrochemical results were compared with dynamic light scattering measurements parallel run in the same S NPs suspensions. The two methods show fairly good agreement, both suggesting a log-normal size distribution of the S NPs sizes characterized by similar parameters. CONCLUSIONS: The preliminary results highlight the amperometric measurements as a promising tool for the size determination of the S NPs in the water environment.

Ion-pair chromatography coupled to inductively coupled plasma-mass spectrometry (IPC-ICP-MS) as a method for thiomolybdate speciation in natural waters.

Molybdenum precipitates preferentially under reducing conditions; therefore, its occurrence in sediment records is used as an indicator of paleoredox conditions. Although thiomolybdates (MoO4-xSx(2-) with x = 1-4) supposedly are necessary intermediates in the process of molybdenum precipitation under anoxic conditions, there is no information about their abundance in natural environments, because of a lack of element-specific methods with sufficiently low detection limits. Here, we optimized ion-pair chromatographic separation for coupling to an inductively coupled plasma-mass spectrometry detector (IPC-ICP-MS). 2-Propanol (10%-25% gradient) replaced the previously used acetonitrile (25%-75%) as the solvent, to reduce the carbon load into the plasma. In synthetic solutions, formation of thiomolybdates was found to occur spontaneously in the presence of excess sulfide and the degree of thiolation was highest at pH 7. Excess hydroxyl led to a transformation of thiomolybdates to molybdate. Under acidic to neutral conditions, precipitation of molybdenum and hydrolysis of tetrathiomolybdate were observed. Flash-freezing was found to be suitable to stabilize tetrathiomolybdate, with <4% transformation over more than two months. High ionic strengths matrices (>2 mM) negatively affected the detection of molybdate, which eluted mainly in the dead volume, but had no negative effect on higher thiolated molybdates. Detection limits were ∼10 nM. With the newly developed IPC-ICP-MS method, thiomolybdates were found to form spontaneously in euxinic marine waters after adding a molybdate spike and occur naturally in sulfidic geothermal waters.

Community shift from phototrophic to chemotrophic sulfide oxidation following anoxic holomixis in a stratified seawater lake.

Most stratified sulfidic holomictic lakes become oxygenated after annual turnover. In contrast, Lake Rogoznica, on the eastern Adriatic coast, has been observed to undergo a period of water column anoxia after water layer mixing and establishment of holomictic conditions. Although Lake Rogoznica's chemistry and hydrography have been studied extensively, it is unclear how the microbial communities typically inhabiting the oxic epilimnion and a sulfidic hypolimnion respond to such a drastic shift in redox conditions. We investigated the impact of anoxic holomixis on microbial diversity and microbially mediated sulfur cycling in Lake Rogoznica with an array of culture-independent microbiological methods. Our data suggest a tight coupling between the lake's chemistry and occurring microorganisms. During stratification, anoxygenic phototrophic sulfur bacteria were dominant at the chemocline and in the hypolimnion. After an anoxic mixing event, the anoxygenic phototrophic sulfur bacteria entirely disappeared, and the homogeneous, anoxic water column was dominated by a bloom of gammaproteobacterial sulfur oxidizers related to the GSO/SUP05 clade. This study is the first report of a community shift from phototrophic to chemotrophic sulfide oxidizers as a response to anoxic holomictic conditions in a seasonally stratified seawater lake.

Inshore capture-based tuna aquaculture impact on Posidonia oceanica meadows in the eastern part of the Adriatic Sea.

Mapping and monitoring of the seagrass Posidonia oceanica in the eastern (Croatian) part of the Adriatic Sea since 2004 indicates a significant decline in meadow density in an area impacted by inshore capture-based tuna aquaculture. The density and overall condition of P. oceanica meadows impacted by tuna farms near Fulija Islet was compared to two reference sites (Iz Island and Mrtovnjak Islet). The factors with the most significant influence on P. oceanica meadows were found to be the input of organic matter originating from the cages, as well as high epiphyte biomass caused by nutrient enrichment. Significant differences in nutrient concentrations were found between the sites impacted by tuna farms (Fulija Islet) and the control stations. Shoot density of the P. oceanica meadows decreased at the stations in close vicinity to the tuna farm, which suggests that the tuna farm activity strongly affected the surrounding meadows.

Electrochemical and colorimetric measurements show the dominant role of FeS in a permanently anoxic lake.

Recent publications have shown that the anodic reaction between FeS and Hg can be used for electrochemical detection of colloidal and particulate FeS in natural waters. Anodic waves that were recorded around -0.45 V (vs Ag/AgCl) in model solutions correspond to the electrochemical transformation of nanoparticulate FeS to HgS. Here, as a further step, the proposed approach is tested on anoxic, sulfidic, and iron-rich samples of a meromictic freshwater lake (Lake Pavin, France). Based on new and more comprehensive work on FeS electrochemistry in model and anoxic Lake Pavin samples, a new interpretation is given for previously recorded voltammetric signals in sulfide and iron rich environment, usually designated FeS(aq), and its role in controlling solubility of different FeS phases. A comparison of the depth profiles of S(-II) measured by voltammetry and the methylene blue method showed that the majority of S(-II) is in the form of FeS. In the monimolimnion layer, thermodynamic calculations based on total Fe(II) and S(-II) concentration, measured by ferrozine and the methylene blue method, predict precipitation of FeS with log K(s) values between -3.6 and -3.8, very close to mackinawite's K(s) value. In the upper part of the same layer, precipitation of greigite is predicted. It is shown that modification of a Hg electrode by surface-formed FeS has a significant influence on voltammetric Fe(II) determination, since reduction of Fe(II) under such conditions occurs both on bare (-1.4 V) and on FeS modified Hg surfaces (-1.1 V); Fe(II) may be underdetermined when only the -1.4 V peak is measured.

Accumulation mechanism for metal chalcogenide nanoparticles at Hg0 electrodes: copper sulfide example.

Mercury electrodes preconcentrate metal chalcogenide nanoparticles effectively, enabling their detection at submicromolar concentrations (as Sigma chalcogenide) by adsorptive cathodic stripping voltammetry. Understanding the unique behavior of nanoparticle analytes during preconcentration is critical for lowering detection limits and for quantification. A multistep mechanism is proposed on the basis of accumulation experiments with polydisperse copper sulfide (CuxS) nanoparticles. Particles first diffuse and adsorb at the Hg0 surface. When both the electrode and particles have negative surface potentials, this process resembles charge-impeded coagulation, obeying the Schulze-Hardy rule at various electrolyte strengths. Consequently, accumulation rates are surprisingly sensitive to electrolyte concentration. Choosing accumulation potentials where the electrode and particles have opposite surface potentials greatly improves collection efficiency, especially for the smallest particles. After adsorption, particles undergo transformations. One product is a more stable (harder to reduce) form of CuxS, interpreted to consist of adclusters or adlayers. A very significant (approximately 0.3 V) negative shift in reduction potential results from this transformation. Loss of analyte to at least one nonelectroactive product is also observed. Loss is greatest for the smallest particles and is sensitive to choice of accumulation potential. To improve accumulation efficiency, accumulation potentials more positive that the potential of zero charge of Hg electrodes are advantageous but care must be taken to remove dissolved chalcogenides under these conditions in order to avoid artifacts.

Voltammetric characterization of metal sulfide particles and nanoparticles in model solutions and natural waters.

Voltammetric scans in sulfidic natural waters often reveal reduction peaks in the range -0.9 to -1.35 V versus Ag/AgCl. These peaks have been attributed to iron sulfide complexes or clusters. However, sols containing CuS nanoparticles now also are known to produce reduction peaks in this range. Here we investigate the voltammetric behavior of two additional metal sulfides at the Hg electrode in 0.55 M NaCl + 0.03 M NaHCO3 electrolyte, pH=8.5. We show that Pb and Hg sulfides, either as suspended powders or as precipitated nanoparticles, also yield cathodic peaks between -0.9 and -1.35 V, similar to peaks obtained with CuS and FeS. For precipitated nanoparticles, the position and shape of these reduction peaks change with ageing. Freshly formed nanoparticles produce less negative reduction peaks than aged nanoparticles. Peaks from aged nanoparticles often consist of two or more superimposed reduction peaks. When all other experimental parameters are held constant, the amount of nanoparticle analyte accumulated on the electrode increases with the amount of ageing (< or = 1 h). Addition of EDTA or acidification followed by purging can be used to distinguish PbS nanoparticles and Fe sulfide clusters from CuS and HgS nanoparticles or from colloidal S. This test was applied to interpret -0.9 to -1.35 V reduction peaks observed in two meromictic lakes. In conjunction with other evidence, this test suggests that FeS clusters are present in one case whereas colloidal S is present in the other. Interpreting -0.9 to -1.35 V voltammetric peaks observed in sulfidic natural waters requires caution, but these peaks are potentially rich sources of information about trace metal speciation.