Integration of environmental signatures and omics-based approaches on the European flounder to assist with health assessment of estuarine ecosystems in Brittany, France.

This study aimed to develop a multidisciplinary approach to assess the ecological status of six moderate-sized French estuaries. For each estuary, we gathered geographical information, hydrobiological data, chemistry of pollutants and fish biology, including integration of proteomics and transcriptomics data. This integrative study covered the entire hydrological system studied, from the watershed to the estuary, and considered all the anthropogenic factors that can impact this environment. To reach this goal, European flounder (Platichthys flesus) were collected from six estuaries in September, which ensures a minimum residence time of five months within an estuary. Geographical metrics are used to characterize land use in each watershed. The concentrations of nitrite, nitrate, organic pollutants, and trace elements were measured in water, sediments and biota. All of these environmental parameters allowed to set up a typology of estuaries. Classical fish biomarkers, coupled with molecular data from transcriptomics and shotgun proteomics, highlighted the flounder's responses to stressors in its environment. We analysed the protein abundances and gene expression levels in the liver of fish from the different estuaries. We showed clear positive deregulation of proteins associated with xenobiotic detoxification in a system characterized by a large population density and industrial activity, as well as in a predominantly agricultural catchment area (mostly cultures of vegetables and pig breeding) mainly impacted by pesticides. Fish from the latter estuary also displayed strong deregulation of the urea cycle, most probably related to high nitrogen load. Proteomic and transcriptomic data also revealed a deregulation of proteins and genes related to the response to hypoxia, and a probable endocrine disruption in some estuaries. Coupling these data allowed the precise identification of the main stressors interacting within each hydrosystem.

Trace metal content from holopelagic Sargassum spp. sampled in the tropical North Atlantic Ocean: Emphasis on spatial variation of arsenic and phosphorus.

We document for the first time, the spatial distribution at basin scale (North tropical Atlantic Ocean) of As, P and trace metal (TM) concentrations in the three morphotypes belonging to the two holopelagic species Sargassum natans and S. fluitans and three morphotypes: S. natans VIII, S. natans I and S. fluitans III. These samples collected in the North equatorial current (NEC) and in the subtropical Sargasso Sea (sSS) (∼25°N, 60°W) were also compared to coastal samples collected downwind Guadeloupe Island and on the strand of Martinique (mangrove and beach). Along the studied zonal oceanic transect, the highest values of As (range 120-240 µg g-1, dry weight, dw) were found in the sSS area where primary production is highly limited by phosphorus. At these stations, the P content of Sargassum spp. was minimal (range 500-1000 µg g-1, dw) as well as the content in Cd and Zn known for their nutrient-like oceanic behaviors and distributions very similar to P. This illustrates for the first time in the natural environment, the higher bioaccumulation of arsenic in Sargassum spp. in P-limiting conditions which is due to the competition in the phosphate transporter between arsenate and phosphate. As compared to samples collected at sea, the Sargassum spp. collected in the strand of Martinique had (1) lower As concentrations (typical range 30-45 µg g-1, dw) and (2) much higher Al, Fe, Mn, Cr and Co concentrations, showing a certain ability of Sargassum spp. to be depurated of its As content in the coastal zone following competitive exchange with terrigenous metals.

An Integrated Biomarker Approach Using Flounder to Improve Chemical Risk Assessments in the Heavily Polluted Seine Estuary.

The objective of this study was to develop an integrative approach in ecotoxicology (from biomarkers to population genetics) to assess the ecological status of fish populations. Flounders (Platichthys flesus) collected after the spawning season in the heavily polluted Seine estuary were compared with the moderately polluted Bay of Douarnenez. The muscle energetic reserves were highly depleted in Seine vs. Douarnenez fish. The Seine fish displaying a reduced capacity to manage the oxidative stress and a higher energetic metabolism. An increase in the content of muscle membrane phospholipids (sphingomyelin, phosphatidylserine, free sterols) was detected in the Seine vs. Douarnenez fish. The data integration allowed to hypothesize relationships between membrane phospholipids, xenobiotic metabolism, bioenergetics, and antioxidant defence. The genetic diversity considering neutral markers was maintained in the heavily polluted Seine population compared with the Douarnenez population. Finally, we suggest that the high physiological cost of tolerance to toxicants in the Seine flounder population could compromise its capacity to respond in the future to an additional stressor like warming waters in shallow depth. Thus, this population could be submitted to an ecological risk.

Stream chemical dynamic and metal accumulation in a temperate watershed affected by agricultural practices (Penzé, NW France).

RATIONALE: Understanding the fate of metals in agricultural land is an important issue for agronomic sustainability. This study aimed at quantifying the export/retention of metals in a temperate watershed subject to important manuring activities. METHODS: The chemical composition of the Penzé stream was examined at high resolution during a 1-year study in 2012. After immediate on-site filtration, here demonstrated as necessary to avoid modification of the dissolved-particulate partition, the concentrations of 21 elements were determined using inductively coupled plasma (ICP) optical emission spectrometry and ICP mass spectrometry. This dataset was extended with the local atmospheric deposition of several metals (Cd, Cr, Cu, Pb, Ni and Zn) monitored on a monthly basis. RESULTS: Two groups were distinguished according to the evolution of the concentrations during floods. Some major cations (Na, Ca, Mg, Sr, K, Ba) and nitrate followed counter-clockwise hysteresis patterns originating from the dilution of the enriched groundwaters by surface waters. Conversely, Al, Fe, Mn, Ti, V, Cr, Co, Ni, Cu, Zn, Cd, Pb and U displayed high dissolved concentration increases at the early stage of floods due to washing out of the enriched soils. CONCLUSIONS: The comparison of stream output fluxes for the two main inputs for the watershed, i.e. atmospheric deposition and manure spreading, indicates that the vast majority of the Cu and Zn (>99 and 96%, respectively), mainly originating from pig manure, is accumulated in the watershed. The accumulation rates for other metals were >60% for Ni and Cr, >75% for As and >90% for Pb and Cd.

Dynamics and sources of reduced sulfur, humic substances and dissolved organic carbon in a temperate river system affected by agricultural practices.

Although reduced organic sulfur substances (RSS) as well as humic substances (HS) are widely suspected to play a role in, for example, metal speciation or used as a model of dissolved organic carbon (DOC) in laboratory studies, reports of their quantification in natural waters are scarce. We have examined the dynamics and sources of reduced sulfur, HS and DOC over an annual cycle in a river system affected by agricultural practices. The new differential pulse cathodic stripping voltammetry was successfully applied to measure glutathione-like compounds (GSHs), thioacetamide-like compounds (TAs) and the liquid chromatography coupled to organic detector to analyze HS and DOC at high frequency in the Penzé River (NW France). The streamflow-concentration patterns, principal components analysis and flux analysis allowed discrimination of the source of each organic compound type. Surprisingly, the two RSS and HS detected in all samples, displayed different behavior. As previously shown, manuring practice is the main source of DOC and HS in this watershed where agricultural activity is predominant. The HS were then transferred to the river systems via runoff, particularly during the spring and autumn floods, which are responsible of >60% of the annual flux. TAs had a clear groundwater source and may be formed underground, whereas GSHs displayed two sources: one aquagenic in spring and summer probably linked to the primary productivity and a second, which may be related to bacterial degradation. High sampling frequency allowed a more accurate assessment of the flux values which were 280 tC y(-1) for DOC representing 20 kg C ha(-1) y(-1). HS, TAs and GSHs fluxes represented 60, 13, and 4% of the total annual DOC export, respectively.

On the control of copper colloidal distribution by humic substances in the Penzé estuary.

In this study, we investigated the variations of colloidal Cu in a temperate macrotidal estuarine system (Penzé, NW France). The originality of this work resides on examining seven colloidal/dissolved fractions at seven different periods of the year whereas previous studies on estuaries generally considered two or three fractions and were focused on a unique survey. A high proportion of Cu (∼90%) was generally found as colloids (5 kDa-0.45 µm) throughout the salinity gradient with divergent size distributions being observed over the seasonal cycle. This consisted essentially in two contrasted periods, i.e. winter-spring with a greater association of Cu with high molecular weight (HMW) compounds (50 kDa-0.45 µm) and summer-autumn with Cu being found mainly as low molecular weight (LMW) forms (5-50 kDa). The comparison of Cu with humic substances (HS) data allowed to us to highlight the importance of the pedogenic refractory organic matter in controlling the concentrations and the size distribution of Cu in the estuary. In the mixing zone, Cu behaved conservative in autumn and winter but important additions of HMW compounds were observed in spring in the lower estuary as the result of particulate organic matter degradation in the sediment. Although HS appears to be the background chelators of Cu in the systems, the strong benthic inputs occurring in spring may be of different (biotic) origin and may be in part responsible for the higher association of Cu with HMW compounds.

Simple and simultaneous determination of glutathione, thioacetamide and refractory organic matter in natural waters by DP-CSV.

Although reduced sulphur substances, such as thiol compounds, contain extremely reactive functional groups in the cell, and influence metal speciation and solubility, very few techniques have been developed to quantify such substances in natural waters. In this paper we present a novel method that allows for the simultaneous identification and quantification of glutathione (GSH), thioacetamide-like compounds (TA), and refractory organic matter (ROM) by differential pulse cathodic stripping voltammetry (DP-CSV). Organic compounds are initially deposited on a mercury drop electrode at 0.000 V, pH 1.95, in the presence of ~200 nmol L(-1) Mo(VI), and then stripped, creating reduction peak currents at specific potentials. Using a 60-s deposition time, limits of detection (LODs) are 1 nmol L(-1), 81 nmol L(-1) and 14 µg C L(-1) for GSH, TA and ROM, respectively. By increasing the deposition time to 300 s, LOD is decreased to 0.2 nmol L(-1), 22 nmol L(-1) and 2 µg C L(-1), respectively. This method has a number of advantages in terms of its rapidity, low cost, and relative simplicity (due to the lack of derivatization and pre-concentration steps) and is also an effective method for simultaneously analysing GSH, TA and ROM in water. When not mixed in solution, GSH, L-cysteine and N-acetyl-L-cysteine, as well as TA-like compounds and thiourea, can be detected and identified by measuring their peak potential and standard addition, due to the acidic pH, which also allows for a longer preservation of the filtered sample. The new method described in this paper was tested along an entire river-seawater gradient of the Aulne Estuary (Brittany, France) to assess its capability in terms of determining these natural organic compounds in various surface waters.

Sulfide determination in hydrothermal seawater samples using a vibrating gold micro-wire electrode in conjunction with stripping chronopotentiometry.

A rapid electrochemical stripping chronopotentiometric procedure to determined sulfide in unaltered hydrothermal seawater samples is presented. Sulfide is deposited at -0.25 V (vs Ag/AgCl, KCl 3M) at a vibrating gold microwire and then stripped through the application of a reductive constant current (typically -2 µA). The hydrodynamic conditions are modulated by vibration allowing a short deposition step, which is shown here to be necessary to minimize H(2)S volatilization. The limit of detection (LOD) is 30 nM after a deposition step of 7s. This LOD is in the same range as the most sensitive cathodic voltammetric technique using a mercury drop electrode and is well below those reported previously for other electrodes capable of being implemented in situ.

Speciation of dissolved copper within an active hydrothermal edifice on the Lucky Strike vent field (MAR, 37 degrees N).

The objective of this study was to determine the concentrations of different fractions of dissolved copper (after filtration at 0.45 microm) along the cold part of the hydrothermal fluid-seawater mixing zone on the Tour Eiffel edifice (MAR). Dissolved copper was analyzed by stripping chronopotentiometry (SCP) after chromatographic C(18) extraction. Levels of total dissolved copper (0.03 to 5.15 microM) are much higher than those reported for deep-sea oceanic waters but in accordance with data previously obtained in this area. Speciation measurements show that the hydrophobic organic fraction (C(18)Cu) is very low (2+/-1%). Dissolved copper is present mainly as inorganic and hydrophilic organic complexes (nonC(18)Cu). The distribution of copper along the pH gradient shows the same pattern for each fraction. Copper concentrations increase from pH 5.6 to 6.5 and then remain relatively constant at pH>6.5. Concentrations of oxygen and total sulphides demonstrate that the copper anomaly corresponds to the transition between suboxic and oxic waters. The increase of dissolved copper should correspond to the oxidative redissolution of copper sulphide particles formed in the vicinity of the fluid exit. The presence of such a secondary dissolved copper source, associated with the accumulation of metal sulphide particles, could play a significant role in the distribution of fauna in the different habitats available at vents.

Dissolved and particulate metals (Fe, Zn, Cu, Cd, Pb) in two habitats from an active hydrothermal field on the EPR at 13 degrees N.

The distribution of Fe, Cu, Zn, Pb, Cd between the dissolved (<2 microm) and the particulate (>2 microm) fractions was measured after in-situ filtration in two hydrothermal habitats. The total metal concentration ranges exhibit a clear enrichment compared with the seawater concentration, accounting for the hydrothermal input for all the metals considered. Iron is the predominant metal (5-50 microM) followed by Zn and Cu. Cd and Pb are present at the nM level. At the scale studied, the behavior of temperature, pH and dissolved iron is semi-conservative whereas the other dissolved and particulate metals are characterized by non-conservative patterns. The metal enrichment of the >2 microm fraction results from the settlement and accumulation of particulate matter close to the organisms, acting as a secondary metal source. The enrichment observed in the dissolved fraction can be related to the dissolution or oxidation of particles (mainly polymetallic sulfide) or to the presence of small particles and large colloids not retained on the 2 microm frit. SEM observations indicate that the bulk particulate observed is characteristic of crystalline particles settling rapidly from the high temperature smoker (sphalerite, wurtzite and pyrite), amorphous structures and eroded particles formed in the external zone of the chimney. Precipitation of Zn, Cu, Cd and Pb with Fe as wurtzite, sphalerite and pyrite is the main process taking place within the area studied and is semi-quantitative. The distribution of the dominant observed fauna has been related to the gradient resulting from the dilution process, with the alvinellids worms colonizing the hotter and more variable part of the mixing zone, but also to the metallic load of the mixing zone. Dissolved and particulate metal concentrations are therefore necessary abiotic factors to be studied in a multiparametric approach to understand the faunal distribution in hydrothermal ecosystems.

Dissolved iron analysis in estuarine and coastal waters by using a modified adsorptive stripping chronopotentiometric (SCP) method.

An electrochemical method based on adsorptive stripping chronopotentiometry (SCP) with a rotating mercury film electrode has been developed for the determination of dissolved iron (III) at subnanomolar concentrations in estuarine and coastal waters. The detection limit was 0.11 nM after adsorption time of 60s. Compared to the other chronopotentiometric methods available for dissolved iron measurement in natural and estuarine waters, the procedure described here exhibits a 15-fold better sensitivity. Therefore, it allows one to accurately quantify concentrations commonly found in estuarine and coastal waters. Moreover, by using the speciation scheme proposed by Aldrich and van den Berg (Electroanalysis 10 (1998) 369), several forms could be measured, i.e. reactive iron (Fe R) and reactive iron (III) (Fe(III) R), or estimated, i.e. complexed iron (Fe C) and reactive iron (II) (Fe(II) R). The method described here is reliable, fast, inexpensive and compact. It was applied successfully to the study of the chemical speciation of dissolved iron along the salinity gradient of the Aulne estuary (Brittany-France).

A stripping chronopotentiometric (SCP) method with a gold film electrode for determining inorganic arsenic species in seawater.

An electrochemical method based on stripping chronopotentiometry (SCP) with a gold film electrode has been developed for determining arsenic in seawater. The detection limits were 0.053 ppb (0.71 nM) and 0.022 ppb (0.29 nM) for total inorganic As (As(T)) and As(III) after deposition times of 60 and 150 s, respectively. Compared to other stripping chronopotentiometric methods that use a gold macroelectrode to perform measurements of arsenic in seawater, the procedure described here exhibits better sensitivity and a fourfold shorter deposition time. Among the SCP methods, our procedure had proven its ability to analyse arsenic(III) in seawater. It therefore allows the concentrations of the various arsenic inorganic species in seawater--i.e. As(T), As(III) and As(V)--to be analysed. The proposed method is reliable, inexpensive and compact. It was successfully applied to the study of arsenic speciation along the salinity gradient of the Penzé estuary (NW France).

Determination of dissolved iron(III) in estuarine and coastal waters by adsorptive stripping chronopotentiometry (SCP).

An adsorptive stripping chronopotentiometric (SCP) method has been developed for quantification of dissolved iron in estuarine and coastal waters. After UV-digestion of filtered samples the Fe(III) ions in non-deoxygenated samples were complexed with solochrom violet RS (SVRS). The complexes were then accumulated by adsorption on the surface of a mercury-film electrode. The stripping step was performed by applying a constant current of -17 microA. Sensitivity and detection limit were 15 ms nmol(-1) L (270 ms microg(-1) L) and 1.5 nmol L(-1) (84 ng L(-1)), respectively, for 60-s electrolysis time. Compared with the only other chronopotentiometric method available for measurement of iron in natural waters, our procedure is fifty times more sensitive in a quarter of the electrolysis time. It therefore enables detection of the concentrations currently found in estuarine and coastal waters. The method was successfully used to study the behaviour and seasonal variations of dissolved iron in the Penzé estuary, NW France.

Measurement of total selenium and selenium(IV) in seawater by stripping chronopotentiometry.

We developed a stripping chronopotentiometric method (constant current stripping analysis, CCSA) with a mercury film electrode for selenium quantification in seawater. A sensitivity and detection limit of 222 ms ng(-1) l and 4 ng l(-1) (50 pM), respectively, were accomplished for a 3-min electrolysis time. Compared to the other chronopotentiometric methods available for a single selenium measurement only in natural waters, our procedure exhibits a ten times better sensitivity. It, therefore, allows one to reach the current concentration thresholds found in coastal and oceanic waters (30-200 ng l(-1)). Moreover, a simple change in operating conditions enables one to also quantify Se(IV), a toxic dissolved species. With respect to the other electrochemical methods of current use, our procedure is beneficial because of its ease-of-use: it needs neither degassing step, nor catalyser.