On-field high-resolution quantification of the cobalt fraction available for bio-uptake in natural waters using antifouling gel-integrated microelectrode arrays.

We report the optimization, characterization, and validation of Adsorptive Square Wave Cathodic Stripping Voltammetry on antifouling gel-integrated microelectrode arrays for autonomous, direct monitoring of cobalt(II) metal species. Detection is accomplished by complexation with an added nioxime ligand. The limit of detection established for a 90 s accumulation time was 0.29 ± 0.01 nM in freshwater and 0.27 ± 0.06 nM in seawater. The microelectrode array was integrated in a submersible probe to automatically dose the complexing agent nioxime and realize an integrated sensing system. For the first time ever, the potentially bioavailable Co(II) fraction was determined in La Leyre River-Arcachon Bay continuum, enabling to evaluate the potential ecotoxicological impact of freshwater-carried Co(II) in the Arcachon Bay. The measured potentially bioavailable Co(II) concentrations were hazardous for aquatic biota along the continuum. The electrochemical Co(II) data were compared to ICP-MS data in various fractions to determine spatial Co(II) speciation.

On-Chip Antifouling Gel-Integrated Microelectrode Arrays for In Situ High-Resolution Quantification of the Nickel Fraction Available for Bio-Uptake in Natural Waters.

We aimed to monitor in situ nickel (Ni(II)) concentrations in aquatic systems in the nanomolar range. To achieve this, we investigated whether an analytical protocol for the direct quantification of cobalt (Co(II)) using adsorptive cathodic sweep voltammetry (Ad-CSV) on antifouling gel-integrated microelectrode arrays (GIME) we recently developed is also suitable for direct Ni(II) quantification. The proposed protocol consists of the reduction of the complex formed between Ni(II) (or Ni(II) and Co(II)) and nioxime adsorbed on the surface of the GIME-sensing element. The GIME enables to (i) avoid fouling, (ii) control the metal complex mass transport and, when interrogated by Ad-CSV, (iii) selectively determine the dynamic (kinetically labile Ni-nioxime) fraction that is potentially bioavailable. The nioxime concentration and pH were optimized. A temperature correction factor was determined. The limit of detection established for 90 s of accumulation time was 0.43 ± 0.06 in freshwater and 0.34 ± 0.02 nM in seawater. The sensor was integrated in a submersible probe in which the nioxime-containing buffer and the sample were mixed automatically. In situ field measurements at high resolution were successfully achieved in Lake Geneva during a diurnal cycle. The determination of the kinetically labile Ni-nioxime fraction allows one to estimate the potential ecotoxicological impact of Ni(II) in Lake Geneva. Additional Ni fractions were measured by ICP-MS and coupled to the in situ Ad-CSV data to determine the temporal Ni(II) speciation.

New insights into trace metal speciation and interaction with phytoplankton in estuarine coastal waters.

In coastal productive zones, phytoplankton activity may influence trace metal speciation and partitioning at short temporal scale. We coupled hourly in situ voltammetry quantification of the lead (Pb), cadmium (Cd), and copper (Cu) potentially bioavailable fractions, using an innovative submersible sensing probe (the TracMetal), to surface water sampling for the quantification of the targeted trace metals in the dissolved <0.2 µm and <0.02 µm fractions, suspended particles, and phytoplankton nets in the Gironde Estuary mouth. The in situ TracMetal monitoring reflected real-time dynamic Cd and Cu regeneration related to algal cells under post-bloom conditions as well as Pb remobilization due to photoreduction of colloids. The potentially bioavailable fraction consisted in 30, 30-50 and <10 % of the total dissolved fraction for Pb, Cd, and Cu, respectively, representing crucial ecotoxicological information. Metal bioconcentration factors using the dynamic fraction concentrations showed levels up to 107 for Cu in phytoplankton.

Speciation of Cu, Cd, Pb and Zn in a contaminated harbor and comparison to environmental quality standards.

The water column of harbors contains significant amounts of (priority) hazardous trace metals that may be released into coastal areas of high societal and economic interests where they may disturb their fragile equilibria. To deepen our understanding of the processes that influence the transport of the various metal fractions and allow for a more rigorous environmental risk assessment, it is important to spatially monitor the relevant chemical speciation of these metals. It is of particular interest to assess their so-called dynamic fraction, which comprises the dissolved chemical forms that are potentially bioavailable to living organisms. In this study this was achieved in the Genoa Harbor (NW Italy) for copper (Cu), lead (Pb), cadmium (Cd) and zinc (Zn) by applying a multi-method approach. For the first time in this system the dynamic fractions of the target metals (CuDyn, CdDyn, PbDyn, ZnDyn) were observed in real-time on-board by voltammetry using innovative electrochemical sensing devices. Trace metals in the operationally defined dissolved <0.2 µm and <0.02 µm fractions were equally quantified through sampling/laboratory-based techniques. The obtained results showed a clear spatial trend for all studied metals from the enclosed contaminated part of the harbor towards the open part. The highest CuDyn and CdDyn fractions were found in the inner part of the harbor while the highest PbDyn fraction was found in the open part. The proportion of ZnDyn was negligible in the sampled area. Small and coarse colloids were involved in Cu, Cd and Zn partitioning while only coarse colloids played an important role in Pb partitioning. The determined concentrations were compared to the Environmental Quality Standards (EQS) established by the EU and those determined by the Australia and New Zealand to trigger for 99 and 95% species protection values. The results of this work allow us to highlight gaps in the EQS for which metal concentration thresholds are excessively high or non-existent and should urgently be revised. They also reflect the need to quantify the potentially bioavailable fraction of hazardous trace metals instead of just their total dissolved concentrations. The data support the establishment of environmental quality standards and guidelines based on realistic risk assessment to protect aquatic life and resources and ultimately human health.

Global Analysis of the Zinc Homeostasis Network in Pseudomonas aeruginosa and Its Gene Expression Dynamics.

Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to counteract bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn homeostasis. Moreover, we identify PA2807 as a novel Zn resistance component in P. aeruginosa and highlight new regulatory links among Zn-homeostasis systems. Altogether, this work unveils a sophisticated and adaptive homeostasis network, which complexity is key in determining a pathogen spread in the environment and during host-colonization.

Advanced multichannel submersible probe for autonomous high-resolution in situ monitoring of the cycling of the potentially bioavailable fraction of a range of trace metals.

We report here on the development and application of a submersible, compact, low power consumption, integrated multichannel trace metal sensing probe (TracMetal). This probe is unique in that it allows high-resolution, simultaneous in-situ measurements of the potentially bioavailable (so-called dynamic) fraction of Hg(II), As(III), Cd(II), Pb(II), Cu(II), Zn(II). The TracMetal incorporates nanostructured Au-plated and Hg-plated gel-integrated microelectrode arrays. In addition to be selective to the fraction of metal potentially bioavailable, they offer protection against fouling and ill-controlled convective interferences. Sensitivities in the low pM for Hg(II) and sub-nM for the other target trace metals is achieved with precision ≤ 12%. The TracMetal is capable of autonomous operation during deployment, with routines for repetitive measurements (1-2 h-1), data storage and management, data computer visualization, and wireless data transfer. The system was successfully applied in the Arcachon Bay, to study the temporal variation of the dynamic fraction of the trace metals targeted. The in situ autonomous TracMetal measurements were combined with in situ measurements of the master bio-physicochemical parameters and sample collection for complementary measurements of the dissolved metal concentrations, organic matter concentrations and proxy for biological activities. The integration of all data revealed that various biotic and abiotic processes control the temporal variation of the dynamic fractions of the target metals (Medyn). The difference in the percentage of the dynamic forms of the metals studied and the short-term processes influencing their variation highlight the TracMetal potentiality as metal bioavailability-assessment sentinel to achieve comprehensive environmental monitoring of dynamic aquatic systems.

In Situ Voltammetric Sensor of Potentially Bioavailable Inorganic Mercury in Marine Aquatic Systems Based on Gel-Integrated Nanostructured Gold-Based Microelectrode Arrays.

The development and field validation of newly designed nanostructured gold-plated gel-integrated microelectrode (Au-GIME) arrays applied to the direct in situ square wave anodic stripping voltammetry (SWASV) quantification of the potentially bioavailable inorganic mercury (Hg(II)) species in the coastal area are presented. The Au-GIME consists of arrays of 100-500 interconnected iridium (Ir)-based microdisks that are electroplated with renewable Au nanoparticles (AuNPs) or Au nanofilaments (AuNFs) and covered with an agarose gel. The gel protects the sensor surface from fouling and ensures that mass transport of analytes toward the sensor surface is by pure diffusion only and therefore independent of the ill-controlled convective conditions of the media. The responses of these sensors to direct SWASV measurements of inorganic Hg(II) at near-neutral pH were investigated first in synthetic media and then in UV-irradiated marine samples. The analytical responses were found to be correlated to the number of interconnected microelectrodes and the morphology of the nanostructured Au deposits and independent of the media composition for chloride concentration ≥0.2 M (salinity S ≥ 13) and pH ranging from 7 to 8.5. The AuNF-GIMEs have detection and quantification limits at a low pM level, fulfilling the requirement of sentinel tools for real-time monitoring of the dynamic fraction of Hg(II) in coastal area. The AuNF-GIMEs were incorporated in an in-house advanced multichannel sensing probe for remote in situ high-resolution trace metal monitoring. Field evaluation and validation were successfully performed as a part of a field study in Arcachon Bay (France), from which environmental data are presented. This work marks the first time that an autonomous electrochemical sensing probe successfully measures Hg(II) and its hourly temporal variation in situ without chemical modification of the sample.

In Situ Detection of Macronutrients and Chloride in Seawater by Submersible Electrochemical Sensors.

A new submersible probe for the in situ detection of nitrate, nitrite, and chloride in seawater is presented. Inline coupling of a desalination unit, an acidification unit, and a sensing flow cell containing all-solid-state membrane electrodes allows for the potentiometric detection of nitrate and nitrite after removal of the key interfering ions in seawater, chloride and hydroxide. Thus, the electrodes exhibited attractive analytical performances for the potentiometric detection of nitrate and nitrite in desalinated and acidified seawater: fast response time ( t95 < 12 s), excellent stability (long-term drifts of <0.5 mV h-1), good reproducibility (calibration parameter deviation of <3%), and satisfactory accuracy (uncertainties <8%Diff compared to reference technique). The desalination cell, which can be repetitively used for about 30 times, may additionally be used as an exhaustive, and therefore calibration-free, electrochemical sensor for chloride and indirect salinity detection. The detection of these two parameters together with nitrate and nitrite may be useful for the correlation of relative changes in macronutrient levels with salinity cycles, which is of special interest in recessed coastal water bodies. The system is capable of autonomous operation during deployment, with routines for repetitive measurements (every 2 h), data storage and management, and computer visualization of the data in real time. In situ temporal profiles observed in the Arcachon Bay (France) showed valuable environmental information concerning tide-dependent cycles of nitrate and chloride levels in the lagoon, which are here observed for the first time using direct in situ measurements. The submersible probe based on membrane electrodes presented herein may facilitate the study of biogeochemical processes occurring in marine ecosystems by the direct monitoring of nitrate and nitrite levels, which are key chemical targets in coastal waters.

SCHeMA EU Project Summer School Report (Bilbao June 16 - 17, 2016).

This conference report describes the training activities that took place in the frame of the Integrated in Situ Chemical MApping probe (SCHeMA) summer school organized from the 14th to the 16th of June 2016 in Bilbao (Spain).

Direct arsenic(III) sensing by a renewable gold plated Ir-based microelectrode.

We aim to determine arsenic(III) in natural aquatic systems in the nanomolar range and at natural pH. In view of a future application of a gel integrated electrochemical detection approach to reduce fouling and to control mass transport, we introduce here a microelectrode capable of quantifying As(III) that consists of a gold plated Ir-based microelectrode (Au-IrM). The key advantage of this approach is the ability to renew the Au layer by electrochemical control for better robustness in the field. The microsensor was electrochemically characterized by Square Wave Anodic Stripping Voltammetry. The obtained results demonstrate that the stripping peaks exhibit reproducible linear calibration curves at pH 8 for As(III) concentrations from 10 to 50 nM and from 1 to 10 nM, using 3 and 36 min preconcentration times, respectively. The interference by copper and chloride is negligible for an As : Cu concentration ratio of 1 : 20 and a chloride concentration of 0.6 M typically found in seawater. The gold layer exhibits a lifetime of 7 days. The measurements are reproducible over time for a given gold layer (RSD < 9%) and between renewed layers (RSD ≤ 12.5%). While this work forms the basis for further progress on gel coated microelectrode arrays, As(III) detection in freshwater samples was successfully demonstrated here.

Potentiometric sensing array for monitoring aquatic systems.

Since aquatic environments are highly heterogeneous and dynamic, there is the need in aquatic ecosystem monitoring to replace traditional approaches based on periodical sampling followed by laboratory analysis with new automated techniques that allow one to obtain monitoring data with high spatial and temporal resolution. We report here on a potentiometric sensing array based on polymeric membrane materials for the continuous monitoring of nutrients and chemical species relevant for the carbon cycle in freshwater ecosystems. The proposed setup operates autonomously, with measurement, calibration, fluidic control and acquisition triggers all integrated into a self-contained instrument. Experimental validation was performed on an automated monitoring platform on lake Greifensee (Switzerland) using potentiometric sensors selective for hydrogen ions, carbonate, calcium, nitrate and ammonium. Results from the field tests were compared with those obtained by traditional laboratory analysis. A linear correlation between calcium and nitrate activities measured with ISEs and relevant concentrations measured in the laboratory was found, with the slopes corresponding to apparent single ion activity coefficients γ(*)(Ca(2+)) = 0.55(SD = 0.1mM) and γ(*)(NO(3)(-)) = 0.75(SD = 4.7µm). Good correlation between pH values measured with ISE and CTD probes (SD = 0.2 pH) suggests adequate reliability of the methodology.

Environmental Sensing of Aquatic Systems at the University of Geneva.

Aquatic environments are complex living systems where biological and chemical constituents change rapidly with time and space and may exhibit synergistic interactions. To understand these processes, the traditional approach based on a typically monthly collection of samples followed by laboratory analysis is not adequate. It must be replaced by high-resolution autonomous in situ detection approaches. In our group at the University of Geneva, we aim to develop and deploy chemical sensor probes to understand complex aquatic systems. Most research centers around electrochemical sensing approaches, which involves: stripping voltammetry at gel-coated microelectrode arrays for direct measurements of bioavailable essential or toxic trace metals; direct potentiometry for the measurement of nutrients and other species involved in the nitrogen and carbon cycles; online desalination for oceanic measurements; the development of robust measurement principles such as thin layer coulometry, and speciation analysis by tandem electrochemical detection with potentiometry and dynamic electrochemistry. These fundamental developments are combined with instrument design, both in-house and with external partners, and result in field deployments in partnership with environmental researchers in Switzerland and the European Union.

Direct optical carbon dioxide sensing based on a polymeric film doped with a selective molecular tweezer-type ionophore.

A novel optical method for the determination of CO(2) concentration in aqueous and gaseous samples of plasticized PVC film is presented. The detection principle makes use of a direct molecular recognition of the carbonate ion by a molecular tweezer-type ionophore, which has previously been demonstrated to exhibit excellent carbonate selectivity. The carbonate ion is extracted together with hydrogen ions into a polymeric film that contains the anion exchanger tridodecylmethylammonium chloride, a lipophilic, electrically charged, and highly basic pH indicator, which is used for the readout in absorbance mode, in addition to the lipophilic carbonate ionophore. According to known bulk optode principles, such an optical sensor responds to the product of the carbonate ion activity and the square of hydrogen ion activity. This quantity is thermodynamically linked to the activity of carbon dioxide. This allows one to realize a direct carbon dioxide sensor that does not make use of the traditional Severinghaus sensing principle of measuring a pH change upon CO(2) equilibration across a membrane. A selectivity analysis shows that common ions such as chloride are sufficiently suppressed for direct PCO(2) measurements in freshwater samples at pH 8. Chloride interference, however, is too severe for direct seawater measurements at the same pH. This may be overcome by placing a gas-permeable membrane over the optode sensing film. This is conceptually confirmed by establishing that the sensor is equally useful for gas-phase PCO(2) measurements. As expected, humid air samples are required for proper sensor functioning, as dry CO(2) gas will not cause any signal change. The sensor showed acceptable response times and good reproducibility under both conditions.

In situ evaluation of cadmium biomarkers in green algae.

In situ measurements provide data that are the highly representative of the natural environment. In this paper, laboratory-determined biomarkers of Cd stress that were previously identified for the green alga Chlamydomonas reinhardtii, were tested in two French rivers: a contaminated site on the Riou Mort River and an "uncontaminated" reference site on the Lot River. Transcript abundance levels were determined by real time qPCR for biomarkers thought to be Cd sensitive. Transcript levels were significantly higher (>5 fold) for organisms exposed to the contaminated site as compared to those exposed at the uncontaminated site. Biomarker mRNA levels were best correlated to free Cd (Cd(2+)) rather than intracellular Cd, suggesting that they may be useful indicators of in situ stress. The paper shows that biomarker expression levels increased with time, were sensitive to metal levels and metal speciation and were higher in the "contaminated" as opposed to the "reference" site.

Decrease of labile Zn and Cd in the rhizosphere of hyperaccumulating Thlaspi caerulescens with time.

By using a rhizobox micro-suction cup technique we studied in-situ mobilization and complexation of Zn and Cd in the rhizosphere of non-hyperaccumulating Thlaspi perfoliatum and two different Thlaspi caerulescens ecotypes, one of them hyperaccumulating Zn, the other Zn and Cd. The dynamic fraction (free metal ions and small labile complexes) of Zn and Cd decreased with time in the rhizosphere solution of the respective hyperaccumulating T. caerulescens ecotypes, and at the end of the experiment, it was significantly smaller than in the other treatments. Furthermore, the rhizosphere solutions of the T. caerulescens ecotypes exhibited a higher UV absorptivity than the solution of the T. perfoliatum rhizosphere and the plant-free soil. Based on our findings we suggest that mobile and labile metal-dissolved soil organic matter complexes play a key role in the rapid replenishment of available metal pools in the rhizosphere of hyperaccumulating T. caerulescens ecotypes, postulated earlier.

In situ monitoring of the diurnal cycling of dynamic metal species in a stream under contrasting photobenthic biofilm activity and hydrological conditions.

The diurnal evolution of the dynamic fraction, i.e., the potentially bioavailable fraction, of Cd, Cu, and Pb in a small river impacted by mining and smelting waste was studied in situ, under contrasting biofilm activity and hydrological conditions, using an automated voltammetric analyzer. The in situ, near real-time measurements revealed persistent dynamic metal species diurnal cycles. These cycles were affected mainly by the biochemical conditions rather than hydrological conditions. The data obtained from the in situ measurements, coupled with complementary laboratory analyses, revealed that various processes control the diurnal dynamic metal species cycles in the studied site; the trends of the diurnal cycles of the dynamic metal species can be different from those observed for the dissolved metal species measured in filtered samples. Moreover, the dynamic fraction of a given cationic metal can show diurnal cycles with opposite trends depending on the environmental conditions. All these findings highlight the interest and importance of automated, continuous measurements of specific relevant environmental metal fractions, compared to punctual weekly or monthly traditional sampling strategies of total dissolved metal analysis, to allow more appropriate water quality control and reliable assessment of metal ecotoxicological impact.

Remote in situ voltammetric techniques to characterize the biogeochemical cycling of trace metals in aquatic systems.

The contamination of aquatic ecosystems by natural and anthropogenic metals has lead to a need to better characterize their impact in the environment. To a large extent, the fate and the (eco)toxicity of these elements in aquatic systems are related to their chemical speciation, which may vary continuously in space and time. Detailed measurements of the fraction of specific metal species or groups of homologous metal species and their variation as a function of the bio-physicochemical conditions of the natural media are thus of prime importance. To determine these metal fractions as well as redox chemical species regulating their distribution (dissolved oxygen, sulfides, iron and manganese oxides), new analytical tools capable of performing in situ, real-time monitoring in both water columns and sediments with minimum perturbation of the media are required. This paper reviews the challenges associated with metal speciation studies, and the progress made with state of the art voltammetric techniques to measure the speciation of metals in situ. More specifically, it summarizes the specific conceptual, analytical, and technical criteria that must be considered and/or fulfilled to develop rugged, field deployable, non-perturbing sensors and probes. Strategies used to satisfy these criteria are presented by describing the up-to-date most advanced voltammetric sensors, mini-/micro-integrated analytical systems, and submersible equipments developed for in situ measurements of trace metals and main redox species in aquatic systems. The spatial and temporal resolutions achieved by these news tools represent a significant advantage over traditional laboratory techniques, while simultaneously remaining cost effective. The application of these tools to aquatic systems is illustrated by several examples of unattended and remote in situ monitoring and/or profiling in water columns and sediments.

Comparison of analytical techniques for dynamic trace metal speciation in natural freshwaters.

Several techniques for speciation analysis of Cu, Zn, Cd, Pb, and Ni are used in freshwater systems and compared with respect to their performance and to the metal species detected. The analytical techniques comprise the following: (i) diffusion gradients in thin-film gels (DGT); (ii) gel integrated microelectrodes combined to voltammetric in situ profiling system (GIME-VIP); (iii) stripping chronopotentiometry (SCP); (iv) flow-through and hollow fiber permeation liquid membranes (FTPLM and HFPLM); (v) Donnan membrane technique (DMT); (vi) competitive ligand-exchange/stripping voltammetry (CLE-SV). All methods could be used both under hardwater and under softwater conditions, although in some cases problems with detection limits were encountered at the low total concentrations. The detected Cu, Cd, and Pb concentrations decreased in the order DGT > or = GIME-VIP > or = FTPLM > or = HFPLM approximately = DMT (>CLE-SV for Cd), detected Zn decreased as DGT > or = GIME-VIP and Ni as DGT > DMT, in agreement with the known dynamic features of these techniques. Techniques involving in situ measurements (GIME-VIP) or in situ exposure (DGT, DMT, and HFPLM) appear to be appropriate in avoiding artifacts which may occur during sampling and sample handling.

Model predictions of metal speciation in freshwaters compared to measurements by in situ techniques.

Measurements of trace metal species in situ in a softwater river, a hardwater lake, and a hardwater stream were compared to the equilibrium distribution of species calculated using two models, WHAM 6, incorporating humic ion binding model VI and visual MINTEQ incorporating NICA-Donnan. Diffusive gradients in thin films (DGT) and voltammetry at a gel integrated microelectrode (GIME) were used to estimate dynamic species that are both labile and mobile. The Donnan membrane technique (DMT) and hollow fiber permeation liquid membrane (HFPLM) were used to measure free ion activities. Predictions of dominant metal species using the two models agreed reasonably well, even when colloidal oxide components were considered. Concentrations derived using GIME were generally lower than those from DGT, consistent with calculations of the lability criteria that take into account the smaller time window available forthe fluxto GIME. Model predictions of free ion activities generally did not agree with measurements, highlighting the need for further work and difficulties in obtaining appropriate input data.