Estuarine, Coastal and Shelf Science 72 (2007) 420e431www.elsevier.com/locate/ecss

Two-dimensional determination of dissolved iron and sulfur species inmarine sediment pore-waters by thin-film based imaging.

Thau lagoon (France)

Didier Jezequel a,*, Roberta Brayner b, Edouard Metzger a, Eric Viollier a,Francois Prevot a, Fernand Fievet b

a Laboratoire de Geochimie des Eaux, Universite Paris 7 & IPGP, UMR CNRS 7047, 2, place Jussieu, 75251 Paris Cedex 05, Franceb ITODYS, Universite Paris 7, UMR CNRS 7086, 2, place Jussieu, 75251 Paris Cedex 05, France

Received 19 November 2006; accepted 23 November 2006

Available online 9 March 2007

Abstract

A device composed of a polyacrylamide gel thin-film (18 cm high, 5 cm wide and 0.4 mm thick) and a PVC (polyvinyl chloride) film wasused as a sediment probe to obtain iron and S(�II) sulfur dissolved species’ distributions in sediment pore waters. A porous protective membranewas set on top of hydrogel layer. Probes were deployed in May 2003 for 24e48 h in the superficial sediment of Thau lagoon (France), in a shell-fish farming area. The polyacrylamide gel layer was used as a DET (Diffusive Equilibration in Thin-films) device for 2D Fe(II) concentrationdetermination, and as the diffusive layer of a DGT-like (Diffusive Gradients in Thin-films) device for sulfur species study. The accumulationlayer of the DGT device consisted in a PVC film layer underneath the polyacrylamide layer. Iron determinations were performed by colorimetricmethods with Ferrozine and imaging technique. Image acquisitions were performed with a flatbed scanner. Fe(II) concentrations were deductedfrom densitometry analysis of the magenta zones (ImageJ software). The calibration curve was obtained by densitometry analysis of polyacryl-amide gel pieces which were equilibrated in known iron (II) concentration solutions. SFe distribution was performed but not quantified. Analysisof gray zones on the PVC layer provided a qualitative distribution of unidentified S(�II) dissolved compounds, related to H2S zone for whichinformation is obtained by classical methods (peeper and colorimetric measurements). EDX (X-ray energy dispersive spectrometry) and GIXR(Grazing-Incidence X-ray Diffraction) analyses of gray zone of this PVC layer provide evidence for FeS2 catalyzed precipitation onto this film.

Dissolved Fe(II) was mainly located near the sedimentewater interface (SWI), showing a nonhomogeneous layer about 10 mm thick. SmallFe rich domains appeared deeper in the sediment and likely confirm newest paradigms in the field of sediment biogeochemistry. S(�II) speciesare detected from 3 to 4 cm below the SWI, with a heterogeneous spatial distribution showing a burrow-like structure.� 2006 Published by Elsevier Ltd.

Keywords: hydrogen sulfide; iron; DET; DGT; colorimetric mapping; anoxic sediment; Thau lagoon

1. Introduction

Techniques based on diffusive hydrogels, either DET type(Diffusive Equilibration in Thin-films) or DGT type (DiffusiveGradients in Thin-films), among other applications, wereproposed as sedimentewater interface and pore water

* Corresponding author.

E-mail address: jezequel@ipgp.jussieu.fr (D. Jezequel).

0272-7714/$ - see front matter � 2006 Published by Elsevier Ltd.

doi:10.1016/j.ecss.2006.11.031

investigation tools with high spatial resolution (Davisonet al., 1991; Davison and Zhang, 1994; Davison et al.,2000). These sediment probes allow to obtain vertical concen-tration profiles of dissolved compounds, or at least reactivezones of the sediment where elements are released in porewaters. The spatial resolution is 100 mm when the gel layeris analyzed directly by spectroscopic microprobe, or by laserablation ICP-MS. The spatial resolution is 2 mm on con-strained DET from DGT Research Ltd. Classical tools suchas peepers (compartments filled with water and equilibrated

421D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

with natural waters) (Hesslein, 1976) or core samplings (withslicing and sample squeezing or centrifugation), offer at mosthalf centimeter resolution. The simplest gel system, con-strained DET, contains small volumes of agarose gel in smallcompartments. The agarose gel (about 98% in water) is equil-ibrated for 24 h in pore waters by diffusion of dissolved spe-cies through a porous protective membrane (against abouttwo to three weeks for classical peepers). Data obtained bythese probes correspond to 1D information, integrated forthe gel compartment area (1 mm high and 20 mm wide, so20 mm2) and for in situ deployment time.

In order to obtain a spatial representation of pore watercomposition over a short time period and to study small-scaleheterogeneity, a fast-response two-dimensional (2D) techniqueis required. Contributions of 2D probes are of great interest toimprove our knowledge of sediment biogeochemical dynam-ics. Several mapping devices are reported in the literature(see Viollier et al., 2003 for a brief review). They are mainlyoptical sensors, and planar optodes for dissolved oxygen dy-namic measurements are a typical example (Glud et al.,2001; Viollier et al., 2003). Polyacrylamide-based hydrogelthin-films represent an obvious possibility of two-dimensionalinvestigations in sediments. Fine slicing of either DET, afterimmobilization of metals by forming hydroxides in anNaOH bath (Shuttleworth et al., 1999), or Chelex basedDGT (as classically performed for 1D analysis (e.g. Foneset al., 2004) for a good 10� 1 mm2 resolution) representsa way to reach 2D distribution of several metals. However,the best reached 2D resolution is about 3� 3 mm2, and eventhough a little better resolution is possible, this tricky methodgenerates a huge number of samples (500 samples fora 3� 3 mm2 resolution for a 15� 6 cm2 probe, equivalent tothe surface of the probe presented in this paper).

To improve the resolution and reduce the processing, di-rect analysis of the gel containing trapped metals, for exam-ple, has been done. Some attempts could be performed byPIXE gel analysis (Davison et al., 1991), but it is a time con-suming and expensive method. More recently, laser ablationmethod coupled to ICP-MS has been applied to a ChelexDGT layer (Fones et al., 2004), providing multi-elementalsemi-quantitative analysis at a 100 mm resolution. This tech-nique was applied previously as a quantitative analysis ofa layer of polyacrylamide with iminodiacetate as metals’binder, providing 1D profiles for Fe, Mn, Co, Cu and Ni(Motelica-Heino et al., 2003). These profiles were chosenalong a transect of a sulfide rich domain revealed by a 2Doptical analysis of a sulfur sensitive gel layer which wascombined in a single device with the previous metals’ fixinglayer (cf. below).

In fact, optical and colorimetric methods applied to poly-acrylamide hydrogel layers or other thin-films, are well adaptedto provide 2D information on various dissolved compoundspresent in pore waters. Colorimetric methods have provento be reliable, specific and in many cases very sensitive.After reagent addition to the hydrogel, coloration developsrapidly and can be recorded by a commercial flatbed scanneror a digital camera. In the case of hydrogen sulfide, direct

imaging acquisition (‘sulfur print’) in sediment can be obtainedwith a photographic silver-based paper, providing a semi-quantitative sulfide distribution (Lehmann and Bachofen,1999). Moreover, it became evident through the last decade,that biogeochemical interpretation of micro- or small-scalemeasurements faces the problem of spatial and temporal het-erogeneity (Viollier et al., 2003). It is, therefore, essential to ob-tain simultaneously several parameters for exactly the samesample, in order to avoid crosschecking difficulties of datafrom distinct devices or sampling time.

This point has been emphasized recently (Motelica-Heinoet al., 2003; Naylor et al., 2004), and, in order to overcomesediment heterogeneity sampling, authors propose a combinedDGT probe suitable for both sulfides and metals. In Nayloret al., 2004, the first film laid down onto the probe supportis a polyacrylamide thin film which contains AgI to trap sul-fides as Ag2S, as described previously (Teasdale et al.,1999). The second layer is a classical Chelex hydrogel suit-able for metals’ fixation, covered by a third layer of plainpolyacrylamide gel, providing the diffusional controlling ofDGT devices which allows concentration calculations. Aprotective porous membrane covers the last hydrogel layer.Metal concentration for Fe, Mn, Ni and Zn was obtained af-ter usual protocol (nitric acid elution and ICP-MS measure-ments) of 4� 10 mm gel piece. As the exposed gel windowwas 10 mm wide, this device does not allow 2D determina-tion of metals’ distribution. The gray coloration of the deeplayer, due to Ag2S after exposition in a sulfide environment,allows gray-scale images’ acquisitions with a classical scan-ner. Only 1D information of sulfides and metals was ex-ploited in this paper, but similar gel probes for sulfideswere described previously with a 2D quantitative determina-tion of either only SH2S data (Devries and Wang, 2003) orcombined 2D SH2S and 1D metals’ data (Motelica-Heinoet al., 2003).

The example reported here concerns 2D acquisition ob-tained simultaneously for dissolved iron Fe(II) distributionand sulfur S(�II) distribution at the sedimentewater interface(SWI), from 6 cm above the SWI to 12 cm in the sediment.These two components, both produced by diagenetic reactionsin organic matter mineralization, are considered of great inter-est to understand biogeochemical pathways in natural aquaticmedia. Their relatively high concentrations in marine pore wa-ters chemically influence several trace elements. For instance,iron cycle is linked to manganese cycle (Burdige, 1993), andhydrogen sulfide greatly influences some metals’ behaviorsby forming insoluble sulfides (ZnS, CdS.) (Morse, 1994;Morse and Luther III, 1999). FeS, as a relatively soluble sul-fide, plays a role in the distribution of more insoluble tracemetal sulfides and in pyritisation processes (Huerta-Diaz andMorse, 1992; Cooper and Morse, 1999).

Iron 2D distribution is obtained by a magenta-scale colora-tion with Ferrozine� of a polyacrylamide hydrogel layer work-ing as a DET, whereas sulfide 2D distribution is obtained bya gray-scale coloration of a PVC tape, working as a DGT-like device. The study site was located in Thau lagoon(France), in a shellfish farm zone area (C5 station, May 2003).

422 D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

2. Material and methods

2.1. Site description

Thau lagoon is located in the south of France (around N43�240, E 3�360). The surface area is 75 km2, the mean depth3.5 m and maximum depth 11 m (Deslous-Paoli et al., 1998).The salinity varies from 31 to 39 during the year, and tem-perature from 6 to 26 �C. Shellfish farming zones occupy20% of the surface, total production (mainly oysters Crassos-trea gigas, and mussels Mytilus galloprovincialis) reachingabout 22,000 tons a year. Shellfish are hanged on ropesfrom a support table, up to 1 m from the sediment floor.Scuba divers deployed the probes in May 2003 a few metersfrom a mussels’ table, C5 station (N 43� 25.9940 E 3�

39.6570), at 9 m depth. During this period (21e23 May),bottom water temperature was about 18 �C and dissolvedoxygen concentration was from 170 to 240 mmol L�1 (Ded-ieu et al., 2007). Porosity of the superficial sediment isaround 95%.

2.2. Gel and PVC probes

A glass plate (25 cm high, 8 cm wide and 3 mm thick) sup-ported the sediment probe (Fig. 1). A piece (18 cm height and5 cm width) of polyvinyl chloride (PVC) adhesive white film(brand: Carrefour� 5-m long roll) was taped on one face.

250 mm

80 mm

a b c d

Fig. 1. Schematic representation of the combined DETeDGT probe. The let-

ters correspond to the following layers: a, glass plate 3 mm thick; b, PVC

S(�II) sulfur sensitive film; c, polyacrylamide hydrogel layer for dissolved

iron determination; d, 0.2 mm PVFD porous membrane, affixed to the glass

plate with tape on the borders (not shown).

The PVC surface was then covered by a polyacrylamidethin-film, prepared as follows (all reagents were from Pharma-cia Biotech�): SOLUTION 1: 5 mL of acrylamide solution(30% w) was mixed with 1.5 mL of bis-acrylamide solution(2% w). SOLUTION 2: 130 mL of ammonium persulfate solu-tion (10% w) and 30 mL of TEMED were mixed with 3.5 mLof deionized water (Millipore Milli-Q� system). Solutions 1and 2 were mixed together and immediately poured ontoa glass plate, within a plastic frame of 18� 5 cm (internal di-mensions) and 0.3 mm thickness. The exact thickness is ob-tained by pressing the mixture with a Plexiglas cover plate.Polymerization was sufficient after 30 min at room tempera-ture. The top plate was removed, and a 0.2 mm pore sizehydrophilic polyvinylidene fluoride (PVDF) membrane (Dura-pore� Millipore) was laid down the polyacrylamide gel inorder to transfer the hydrogel onto the PVC layer of theprobe.

The hydrogel is finally covered with a PVDF porous mem-brane and fixed with a waterproof PVC tape. A space of 1 cmwas left between the thin-film and the tape edge, in order toallow gel expansion due to further hydration. The PVDF mem-brane constitutes a permeable layer for dissolved compounds,a protective layer from sediment particles, and maintains thehydrogel on the support. The whole probe was stored in natu-ral seawater for gel hydration during one day, and this solutionwas stripped with nitrogen bubbling at least 4 h before deploy-ment in the sediment.

Furthermore, a piece of polyacrylamide hydrogel (same sizeas the previous one) is assigned to Fe(II) colorimetric develop-ment after back-equilibration (see below). It is first rinsed in de-ionized water for 2 h, then equilibrated in a 10�2 mol L�1

Ferrozine (C20H14N4O6S2 Aldrich) and 0.1 mol L�1 ammo-nium acetate (p.a. Merck) solution for one day. We call it, inthe text below, the reactive hydrogel.

Probes, carried underwater in their deoxygenated waterbag, were deployed into the sediment by scuba divers (1/3of the height probe was in the water column, 2/3 in the sed-iment), and left 24e48 h for equilibration with natural waters.The gel layer can be considered like a two-dimensional DETfor dissolved compounds such as iron. Probes were treatedwithin 15 min after retrieval: the PVDF membrane was rap-idly discarded and the reactive hydrogel was laid downonto the gel probe. A glass plate was placed on the top ofthe gel layers and was fixed with tape to the probe glassbackside in order to handle the complete device and to limitevaporation from hydrogels. After 5 min, the characteristicmagenta coloration of FerrozineeFe(II) complex was devel-oped. The flat assembly gel probeþ reactive gelþ glass platewas scanned (HP Scanjet 3570c) about every 10 min. Thecalibration of the color development is explained in the fol-lowing paragraph.

For the next step, the reactive gel layer was removed andreplaced by a PVDF membrane wetted with a Ferrospectral�

Merck reagent, allowing total dissolved iron detection (this re-agent contains Ferrozine and thioglycolic acid as a reducingagent for Fe(III)). After a few minutes, a glass plate replacedthis membrane. After 10 min, this new setting developed new

423D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

magenta spots and was scanned (see conditions above). Nocalibration was attempted for this method.

2.3. Iron (II) calibration method

In order to quantify the concentration of dissolved iron inthe gel probe, the coloration of the magenta zones has to becompared to colored standard gels obtained in the same con-ditions. Four standard solutions of Fe(II) were prepared froma Mohr salt (NH4)2Fe(SO4)2$6H2O (Prolabo R.P. Normapur)0.25 mol L�1 solution acidified with sulfuric acid, by dilutionwith deaerated Milli-Q water. Concentration standard rangedfrom [Fe(II)]¼ 25 to [Fe(II)]¼ 200 mM (10�6 mol L�1).Small pieces (2.5� 4 cm) of polyacrylamide gel, preparedas described above, were set onto a microscope glass plate,with white PVC tape underneath, and covered with thePVDF membrane. These mini-probes were put in standardsolutions (100 mL) for 12 h and deionized water to measurethe method blank. After gel equilibration, each standardprobe was treated as the sediment probe was. The five stan-dard gels were set together and scanned. Intensity of coloredzones of the DET was treated by ImageJ 1.31 software (Ras-band, 2003): images were decomposed into primary colorsred, green and blue intensities, each being converted ingray-scale image (computer-imaging densitometry). For pro-file extraction, color was analyzed from a transept of about0.15 mm width.

2.4. Adhesive tape characterization

Grazing-Incidence X-Ray Diffraction (GIXR) measure-ments were performed using a D8-Bruker X-ray diffractom-eter equipped with Soller slits (6¼ 0.5�) in a rockingcurve mode. GIXR patterns were recorded using a Cu Ka ra-diation. The scanning was carried out in a continuous modeand the counting time was 5 s per step of 0.05� (2q) during12 or 60 h.

For scanning electron microscopy (SEM), adhesive tapesamples were coated with gold in a Balzers Union SCD 40sputter-coater and analyzed on a Cambridge Stereoscan 120instrument at an accelerating voltage of 20 kV.

X-ray energy dispersive spectrometry (EDX) was per-formed using an (EDXD) EDAX system equipped with a superultra thin window (SUTW) connected to a JEOL JSM 6100scanning electron microscope operating at 25 kV. Atomiccompositions (%) were obtained with Genesis software. Sam-ples were coated with carbon for EDX analysis (carbon coaterCressington 208 Carbon).

X-ray photoelectron spectroscopy (XPS) was performed ina Thermo VG Scientific Sigma Probe spectrometer equippedwith a monochromatic Al Ka X-ray source (1486.6 eV) wasused at a spot size of 400 mm. The pass energy was set at100 and 40 eV for the survey and the high-resolution spectra,respectively. The step size was 1 eV for the survey spectrumand 0.2 eV for the high-resolution spectra. Charge compensa-tion was achieved with a flood gun of 2.6 eV electrons usingstandard procedures for this spectrometer.

3. Results and discussion

3.1. A e Dissolved iron distribution

Fig. 2 corresponds to a sulfur and iron 2D probe, scanned32 min after reaction with Ferrozine. The magenta zones cor-respond to rich dissolved iron (II) ones. They are locatedmainly near the sedimentewater interface (SWI). Further-more, small spots (less than 1 mm2) are visible in the sediment,from about 3 cm depth and deeper. Magenta coloration in-creases with time, particularly for the SWI zone, except inthe case of three larger spots (one on the left of the gel probe,two on the right). Not surprisingly, outlines of spots becamemore and more fuzzy with time, and small details were erased,due to lateral diffusion of dissolved species in the polyacryl-amide layer. Fig. 3a shows enlargements of the right spots:accurate shapes are vanishing with time. Note that the 10 minscan picture corresponds to about 20e25 min after the gelprobe retrieval from sediment.

Fig. 2. Image of a 2D probe corresponding to 32 min of reaction time with the

Ferrozine gel layer. In magenta, dissolved iron (II) distribution (mainly near

the sedimentewater interface); in gray: sulfur distribution (located from about

3 to 4 cm below the SWI and downward). Arrows #1 to #3 show three rich iron

zones located at the SWI.

424 D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

(b)

(a)

10 min. 20 min. 32 min. 42 min. 73 min.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 100 200 300

10 min.

20 min.

32 min.

42 min.

73 min.

Estimated [Fe(II)] µM

estimated in situ shape

dep

th (

cm)

Fig. 3. (a) Enlargements of the right spots (#2 and #3) shown in the previous picture, with time evolution (from 10 to 73 min of reaction time between the retrieved

gel and the reactive gel). Dashed line in the first image corresponds to the profile presented in (b); (b) iron concentration profiles with depth for the central magenta

spot of the 10 min reaction time scan.

Calibration curves were determined by computer-imagingdensitometry of color images (RGB), and of each primarycolor (Red, Green or Blue) derived image. Only the green con-tribution is exploited, because it leads to a more sensitive andlinear response versus iron concentrations (sensitivity order:green curve> RGB colors curve> red curve> blue curve).Image acquisitions were performed 11, 27, 78 and 209 min af-ter affixing the Ferrozine gel onto the standard gel. Except forthe first one, for which response was about 20% lower, colorintensity was stable with time. For [Fe(II)] determinations,the 32 min probe image and the 27 min standard imageswere used (for which calibration coefficients were: greenunits¼�0.3523� [Fe(II)]þ 201.28; R2¼ 0.9951; n¼ 4;[Fe(II)] in mM).

Fig. 4 shows [Fe(II)] profiles through the SWI obtained bysoftware treatment along a vertical section of the central zoneof the probe. Average of adjacent profiles (0.4 mm space) re-duces the noisy curves obtained for one pixel (0.13 mm) widetransect. Dissolved Fe(II) concentrations show a peak-like dis-tribution and reach about 75 mM just below or very near theSWI (the exact position is inaccurate because of a non planarsediment surface). Peak extension is about 10 mm above andbelow the interface (smaller extensions were found dependingon the position of the vertical section; data not shown). These

characteristics are compatible with other data obtained byclassical DET (constrained DET from DGT Research Ltd.filled with agarose gel, with a 2 mm step vertical 1D resolu-tion) for the same station and the same period. The concentra-tion reaches more than about 300 mM in the right magenta spot(Figs. 2 and 3).

Fig. 5 shows total dissolved iron distribution for the samesample, after Ferrospectral� addition. Magenta colorationwas more intense, and some new spots appeared below theiron rich layer near the SWI. They could correspond to smallFe(III) rich domains released by bacterial activity, or to smalldomains where re-oxidation of initial Fe(II) was very rapid af-ter the retrieval of the probe. However, coloration could be dueto dust contamination or to a greater sensibility of this reagent;no calibration was attempted.

Data obtained with the gel probe presented here concerningdissolved iron distribution in the pore waters of the coastal ma-rine sediment of C5 Thau station are in good agreement withclassical views of diagenetic reactions occurring in such amedia. They confirm other data obtained for the same siteby other techniques (classical peepers with a centimeter verti-cal resolution, and DET peepers with a 2 mm resolution e cf.Metzger et al., 2007), showing dissolved iron just below theSWI, with a sharp peak (maximum concentrations observed

425D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

between 50 and 100 mM), and decreasing some millimetersdeeper. Dissolved ferrous iron in pore waters is a by-productof microbiologically mediated reactions. The iron peak shapedistribution just below the SWI is interpreted as a diageneticsequence involving Fe(II) production by anaerobic mineraliza-tion of OM followed by immobilization of iron by reactionwith H2S diffusing upward from the deeper sediment. TheC5 station is located near a shellfish farming area (Mytilus gal-loprovincialis mussels), which produces large organic matter(OM) deposition, mainly represented by shellfish faeces(POC varies from 6 to 5% from superficial sediment to 5 cmdepth) (Elbaz-Poulichet et al., 2005). Aerobic degradation ofOM and re-oxidation of reduced compounds from anaerobicreactions lead to the total consumption of dissolved oxygenwithin the first 2 mm of sediment. Only a small part (20e30%) of O2 uptake corresponds to aerobic respiration (Dedieuet al., 2007).

Dissolved ferrous iron would also be produced by second-ary diagenetic reaction such as 2Fe(OH)3 (s)þH2Sþ 4CO2 /2Fe2þþ S� þ 4HCO3

� þ 2H2O. The biogeochemical descrip-tion and interpretation of this site are detailed in Metzgeret al. (2007).

There are evidences for heterogeneous distribution near theSWI, and also for the presence of small iron rich environmentsdeeper in the sediment. They could correspond to micronicheswith specific bacterial activities, depending on the OM supplyand iron-oxyhydroxide distribution.

0 20 40 60 80 100

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

dep

th (

cm)

[Fe(II)] µM

SWI

Fig. 4. Iron (II) concentration profiles obtained by software treatment of a ver-

tical section of the central zone of the DET, around the SWI (location of the

SWI is not well defined). Spatial vertical space between both profiles is only

0.4 mm. In bold black: mean values of the two previous profiles.

Note that for classical peepers with water filled compart-ments, surface of exchange with pore waters is about 2.5e5 cm2, depending on the peeper design, and 20 mm2 forconstrained DET (DGT Research Ltd.). So concentrationdata correspond to an integrated signal, not directly compara-ble to our 2D recordings. On the other hand, the 2D probegives information with a spatial resolution that could be com-pared with microelectrode data (voltammetric electrodes, goldtype, for example (Brendel and Luther, 1995)), which concerna very small domain around the electrode tip (surface corre-sponding to about twofold the external diameter of microelec-trode, i.e. typically 100 mm). Information from suchmicroelectrodes is sometimes not directly comparable with in-tegrated methods (core samples, peepers or constrained DET),especially in nonhomogeneous sediment (as it generally is).2D probes could help for comparison and interpretation ofdata from different techniques, but one has to keep in mindthat temporal resolution is usually different: several days orweeks for classical peepers, hours for gel peepers and minutesto an hour (for a complete profile) for microelectrodes.

It is clear also as shown in Fig. 3 that lateral diffusion ofdissolved compounds in the gel layer after retrieval representsa limitation for the analysis of very small details. Fig. 3b pres-ents vertical profiles of the central spot: the Fe(II) peak waswidening with time. For this reason, the concentration profilethrough the SWI, shown in Fig. 4, obtained about 45 min after

Fig. 5. Total dissolved iron distribution revealed by a mixed reactant (Ferro-

zine and iron reductant).

426 D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

probe retrieval, corresponds to underestimated gradients andconcentration values. This is the cost of non-constrained pla-nar DET used with no fixing reaction, but, nevertheless, ifthe gel layer is treated just after retrieval from sediment andscanned within less than 30 min, a reasonable millimetric pre-cision can be obtained from this method. Moreover, temporalseries of scanned images could be modeled to reach initial‘true’ concentrations and profile shape. Thus, the in situpeak broadness in Fig. 3 can be estimated from experimentaldata assuming a first order law for peak broadening. However,data from planar probes should be considered with care be-cause of (1) the perturbation of the sediment when the probeis deployed and (2) the presence of the probe can modify dis-persion coefficient near the SWI (Viollier et al., 2003). Datacorresponding to water just above the sediment may be af-fected by these perturbations, and for these reasons and be-cause no other value obtained from a different method isavailable, we do not present here any iron flux values.

The 2D gel colorimetric technique is much faster and lessexpensive than direct gel analysis by PIXE (Proton InducedX-ray Emission) (e.g., Davison et al., 1991). Previous studiesfrom our laboratory confirmed the possibility to measure ironand manganese in a 2D polyacrylamide DET by PIXE, afterimmobilization of these elements by the precipitation of hy-droxides and gel drying (lacustrine pore waters, Aydat lake,France e unpublished results), but acquisition of one datapoint took about 30 min (10� 20 mm2 spot analysis e PierreSue Laboratory, CEA Saclay, France), several hours, therefore,for a complete profile and much more for a map. The mainlimitation of colorimetric technique is the mono-elementalanalysis: it requires one gel layer per compound.

3.2. B e Sulfur distribution

The second information afforded by this imaging method isthe distribution of some sulfur species, corresponding to thegray coloration of the PVC tape which is visible showingthrough the polyacrylamide layer (Fig. 2). Actually data fromclassical peeper samplings (cf. Metzger et al., 2007) showedthat hydrogen sulfide (colorimetric determination of SH2S con-centrations by the methylene blue method, Spectroquant�

14779 Merck kit) appears in the C5 station sediment at about4e5 cm depth. At this depth, no more dissolved iron is detectedneither by peeper (classical or DET) samplings nor by 2D probes(except small domains for this latter device). The gray limit ap-pears about 3e4 cm below the SWI, in good agreement withclassical SH2S determinations, but it is not necessarily parallelto the sedimentewater interface. Except for a 5 mm transitionzone between white and gray, the bottom of the PVC film ap-pears uniformly gray (such a color saturation is observed inAgI-DGT; (cf. Teasdale et al., 1999)). Furthermore, a lack ofcoloration with a finger shape is present on the left part. No cal-ibration was attempted for this technique. The following partpresents some morphological and structural characterizationof unexposed white and exposed gray PVC tapes. From thesetechniques, we try to understand the role of adhesive tape iniron sulfide formation as well as the crystalline phase obtained.

Moreover, it must be stressed that the gray coloring was not ob-served on every kinds of PVC tapes used. Indeed, various brandsof PVC tapes may contain different additives. Their character-ization was undertaken in order to emphasize the key featurethat is related to the gray development. These results contributeto improve the new method for 2D determination of dissolvediron and sulfur species in marine sediment pore waters bythin-films presented in this paper and may be to extend thismethod to others sulfur species.

The crystalline phases present in adhesive tape samples be-fore and after immersion into Thau lagoon sediment werecharacterized by Grazing-Incidence X-Ray Diffraction(GIXR) measurements (Fig. 6). For unexposed white sample(Carrefour�, 5-m long tape roll), PVC polymer presents(Fig. 6a) an amorphous phase (large peak between 15 and26�(2q)) but TiO2 (rutile), CaCO3 (calcite) and sodium acetatephases were also observed. These phases correspond to themineral load of the adhesive tape. After immersion intoThau lagoon superficial sediment for 24 h and developmentof a gray coloration, a slightly GIXR diffractogram evolutionwas noted (Fig. 6b). First of all, a considerable relative inten-sity decrease of Na acetate/PVC ratio was observed. This

********

*

TiO2 (rutile)

Sodium acetate

CaCO3 (calcite)

TiO2 (rutile)

Sodium acetate

CaCO3 (calcite)

10 20 30 40 50 60 70

2θ

10 20 30 40 50 60 70

2θ

(a)

FeS2

(b)

Inte

nsity

(a.

u.)

Inte

nsity

(a.

u.)

Fig. 6. GIXR patterns of adhesive tape films: (a) PVC based white tape (Car-

refour� 5 m roll tape); (b) gray PVC film (same type) obtained after immer-

sion into Thau lagoon sediment.

427D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

behavior could be attributed to partial surface lixiviation dueto the dissolution of Na acetate in the pore waters.

Moreover, in addition to PVC and mineral load, smallerpeaks were also observed that may be assigned to pyrite aniso-tropic phase (FeS2; JCPDS 88-2293). Many different types ofiron sulfide minerals are formed in association with sulfate-reducing bacteria but they show none of the characteristicsof a controlled biomineralization process (Mann, 2001).Most of these products arise from the reaction of metabolicproducts such as H2S with Fe2þ species in the surrounding en-vironment. Colorimetric determination by the methylene bluemethod shows that H2S appears in the C5 station sediment atabout 4e5 cm depth that promotes FeS2 formation hypothesis.It is important to remember that C5 station in Thau lagoon islocated near a shellfish farming area that is very rich in organicmatter deposition and it is very difficult to identify all compo-nents by GIXR technique.

XPS analysis was used to characterize the chemical structureand composition of adhesive tape films before and after immer-sion into Thau lagoon. For untreated films, Na 1s, O 1s, N 1s,C 1s, Cl 2p and Si 2p peaks were observed. There is some degreeof surface contamination by adventitious hydrocarbons,nitrogen and silicon. Nitrogen was originated from polymersupport and polyacrylamide gel residue whereas silicon wasoriginated from a glass plate used as a support. After immersion,the same elements were detected but a significant concentrationdecrease (counts/s) was observed (not shown). This phenome-non emphasizes the hypothesis of partial surface lixiviation ob-served by GIXR analysis. It is worth noting that titanium fromTiO2 (rutile), calcium from CaCO3 (calcite), iron and sulfurousspecies were not detected. XPS analysis is only a surface char-acterization technique and the limit of electron penetration isabout 5 nm. The fact is that this adhesive tape presents a multi-layer film structure and TiO2, CaCO3, iron and sulfurous speciesmay be deeper than 5 nm.

Fig. 7 shows the key XPS core-level regions for these sam-ples. The C 1s region of untreated film (Fig. 7a) was fittedwith three components centered at 285.71, 286.36 and287.47 eV assigned to CeC/CeCl (in PVC network) andO]CeO/O]CeO� species, respectively (Moulder et al.,1992). The latter has a contribution of carboxylates from acetateanions, which are counterbalanced by Naþ cations that was alsodetected (Fig. 7c). For gray sample (Fig. 7b) the C 1s peak wasalso fitted with three components centered at 285.69, 286.43 and287.52 eV assigned to CeC/CeCl and trace of O]CeO/O]CeO� species, respectively (Moulder et al., 1992). In thiscase, a decrease of carbon concentration was observed. Conse-quently Naþ cations were almost disappeared (Fig. 7d).

In order to know the surface morphology of the adhesivetape films, scanning electron microscopy analysis (SEM)was employed (Fig. 8). The unexposed sample presents a slightsurface roughness. On this surface, a lot of particles with var-ious size distributions were observed (Fig. 8a). On the otherhand, after in situ immersion, the surface film presentsa high roughness with an etch pit distribution on the whole sur-face tape (Fig. 8b). To simulate Thau lagoon conditions, an ad-hesive tape sample was immersed in NaCl aqueous solution

(10 g L�1) during 24 h (Fig. 8c). Like Thau sample, the simu-lated sample presents same etch pits’ formation on whole sur-face tape but the roughness is close to initial one. Thisexperimental simulation proves that sodium acetate particleswere dissolved in water solution leaving, in their place, holesfrom about 1e10 mm in diameter. These results are in agree-ment with GIXR and XPS analyses. It is important to considerthat iron and sulfurous species could be deposited into theseholes, or that the presence of pores allows dissolved speciesto reach a reactive surface able to promote the precipitationof FeS2 and other phases.

In order to confirm the presence of iron and sulfurous speciesinside the film, the composition of the samples was quantified byEDX measurements with accelerating voltage of 25 kV (Fig. 9).In this case, the limit of electron penetration is about 3 mm. Foruntreated sample, C, O, Ni, S, Cl, Ca and Ti peaks were detected(Fig. 9a). Ca, Ti and O peaks were assigned to TiO2 and CaCO3

species and C and Cl to PVC based white tape. A trace of sulfu-rous compounds was also observed. Finally, for in situ immersedsample, besides previous elements, Mg, Al, Si, K and Fe peakswere also detected, and S signal was notably higher. Fe/S atomicratio was 0.4, which confirms the presence of at least two sulfu-rous compounds (FeS2 and an unidentified sulfurous com-pound). FeS2 detection is in agreement with GIXR analysis.According to XPS and EDX analyses, iron and sulfur specieswere not detected on the surface tape but between 5 nm and3 mm, close to TiO2 rutile phase.

To try to understand the mechanism of iron and sulfur cap-ture, two other kinds of adhesive white tapes were immergedin Thau lagoon in the same conditions. To the great astonish-ment, no gray coloring was observed. For 25-m long tape rollCarrefour� sample, GIXR analysis shows the amorphousphase of PVC, CaCO3 (calcite) but a TiO2 anatase crystallinephase was identified instead of rutile one (not shown). Sodiumacetate was not detected. The surface morphology of this sam-ple was also studied before and after simulation in NaCl10 g L�1 aqueous solution (not shown). Contrary to Thau sam-ple, no etch pits were observed after immersion in NaCl solu-tion. For Plasto� white tape, rutile and calcite were detectedbut not Na acetate (GIXR measurements; not shown).

Furthermore, an orange PVC tape (Plasto� adhesive tape)developing a gray coloration in C5 pore waters was analyzed(GIXR measurements; not shown): again it contains both cal-cite and rutile (but this last in low proportion), and also sodiumacetate.

Furthermore, other observations were noted concerningPVC films:

(a) white PVC tape (Carrefour� 5 m):- coloration is increasing with time up to the saturation

level of gray,- coloration is very stable with time (at least two years); it

is not removed by acid attack (one drop of 65% nitricacid, for example),

- coloration is inhibited or strongly limited when the PVCsurface is covered by transparent Scotch tape (paperrepair type),

428 D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

0

50000

100000

150000

200000

279280281282283284285286287288289290291292293294295296297298

Binding Energy (eV)

-20000

-10000

0

C1s

C1sB

C

0

20000

40000

60000

80000

100000

279280281282283284285286287288289290291292293294295

Binding Energy (eV)

-15000

-10000

-5000

0

C1s

C1s

E

F

14000

15000

16000

17000

18000

1066106710681069107010711072107310741075107610771078107910801081

Binding Energy (eV)

-1500

-1000

-500

0

Na1s

Na1s

6200

6300

6400

6500

6600

6700

6800

1066106710681069107010711072107310741075107610771078107910801081

Binding Energy (eV)

-500

-400

-300

-200

-100

0

Na1s

Na1s

(b)

(c)

(d)

(a)

Res

idua

lsR

esid

uals

Res

idua

lsR

esid

uals

Counts / s

Counts / s

Counts / s

Counts / s

Fig. 7. XPS core-level regions for key elements in adhesive tape films: (a) C 1s region from PVC based white tape (Carrefour� 5 m roll tape); (b) C 1s region from

gray PVC film (same type); (c) Na 1s region from PVC based white tape; (d) Na 1s region from gray PVC film (same type).

429D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

- coloration is not obtained in Na2S solution even after 48 h,but light gray coloration is observed after 24 h whendissolved iron is added in the sodium sulfur solution (thisspike produced immediately a black FeS suspension);

(b) miscellaneous:- pure PVC film, made from evaporation of dissolved

polyvinyl chloride high molecular weight (Selectophore�

Fluka) in tetrahydrofuran, does not induce gray colora-tion ascribed to sulfurous iron in the same sedimentwhere the Carrefour� PVC tape turned dark gray,

Fig. 8. Scanning electron micrographs of adhesive tape films: (a) unexposed

PVC based white tape (Carrefour� 5 m roll tape); (b) after immersion into

Thau lagoon; (c) after immersion in NaCl solution (10 g L�1).

- FeS or sulfur deposits were observed onto other plasticmaterials when deployed in sulfide lake waters; thiscoloration can be temporary (Plexiglas pieces), or perma-nent (PVC pieces),

- calcite and anatase powders, each spread over a supportand covered by a PVDF membrane in order to be usedas sediment probes, did not reveal any gray colorationafter in situ immersion in C5 sediment.

Another possibility to explain the presence of FeS2 islinked to the way of deployment of the probe: when intro-duced vertically in the sediment, small amounts of iron fromthe upper layers of sediment can be swept in depth. In thezone where hydrogen sulfide is present, formation of FeS orFeS2 can occur and its deposition is induced onto the PVC sur-face. Due to a somewhat homogeneous distribution of FeS2 onthe PVC tape at depth (more than 10 cm below the SWI), thisexplanation can be ruled out.

In conclusion, the hypothesis that the gray coloration of thisbrand of PVC film is linked to the presence of S(�II) sulfur inpore waters is nevertheless highly probable. It can be summa-rized as follows: the mineral load (probably rutile) of PVCtape would act as a catalyser of an iron sulfide phase, frompore water dissolved species (Fe2þ, HS� or H2S, FeS com-plex.). Another component of the PVC tape, the plasticizer,could also play a role in gray coloration development, butthere is no evidence of this hypothesis. Dissolved speciesrather than colloids are supposed to react in these conditions,because the gray coloration appears even through a hydrogelpolyacrylamide layer. Contact between catalyser and dissolvedcompounds is provided by high porosity, i.e. by pores in thePVC matrix, obtained by sodium acetate crystals’ dissolution.Tapes showing a gray coloration in the aforementioned condi-tions (where dissolved FeS at low concentration could be themain reactive species from the pore waters) all contain rutile,calcite and sodium acetate. Further investigations have now tobe performed to validate or to dismiss the darkening reactionhypothesis.

The PVC tape and the diffusive gel layer could work asa DGT for a dissolved S(�II) compound, but only qualitativedata are presented in this paper and a calibration procedure hasto be done. Unlike AgI-based films for which photoreductionof AgI to Ag occurs at light, or photographic paper, this tape isnot photo-sensible and its use is much more easier than AgI-DGT. Nevertheless, this latter method is well described andthe coloration is unambiguously due to dissolved sulfides,mainly H2S and HS� (Teasdale et al., 1999; Devries andWang, 2003; Motelica-Heino et al., 2003; Naylor et al.,2004). The 2D probe used in this illustration shows the hetero-geneity of sulfur distribution in the C5 Thau sediment: the sul-fur limit is not parallel to the SWI, a lack of coloration isobserved (which could correspond to a macro-organism bur-row). Such a spatial heterogeneity, including bacterial micro-niches or induced by the rhizosphere, for example, has beendescribed in either marine (Motelica-Heino et al., 2003) or la-custrine (Shuttleworth et al., 1999; Devries and Wang, 2003)environments.

430 D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

Fig. 9. EDX measurements of adhesive tape films: (a) PVC based white tape (Carrefour� 5 m roll tape); (b) gray PVC film (same type) obtained after immersion

into Thau lagoon sediment.

4. General conclusion

The sediment pore water probe presented in this paper repre-sents an easy and fast to use tool for both dissolved iron andsulfur determinations with two-dimensional informationadvantages. This technique allows a semi-quantitative deter-mination for iron by comparison of color intensity withstandards, but sulfur identification and quantification are to beimproved. The true nature of the S(�II) compound binding by

the PVC tape has to be cleared up, and this device should becompared to the well described AgI-based DGT devices,which are supposed to trap SH2S and polysulfide (Nayloret al., 2004).

An important possibility of this combined probe is to obtainsimultaneously two sets of data for two different compounds atexactly the same location in the sediment. Examples of theThau lagoon pore waters show great heterogeneity in the dis-tribution of both the studied compounds. These illustrate why

431D. Jezequel et al. / Estuarine, Coastal and Shelf Science 72 (2007) 420e431

one can meet some difficulties in combining data from differ-ent techniques working with different scales (spatial and prob-ably temporal scales), mainly between microelectrodes and 1Ddialysis methods.

This method could be developed for other compounds forwhich a colorimetric reaction can be applied. In this way, alka-linity should react with a specific reagent (Podda and Michard,1994; Sarazin et al., 1999), orthophosphate with classical mo-lybdate reagent, hydrogen sulfide could be analyzed by themethylene blue method, sulfate by precipitation of white sulfatebarium over a black background. These probes will have to useseveral gel layers to be treated separately, depending on the spe-cific reagent. All the operations should be done within minutesor less than half an hour to limit lateral and vertical diffusion andloss of information. A relative positioning system between thedifferent gel layers should be used to recombine differentimages.

Acknowledgements

This study was supported by PNEC (MICROBENT pro-gram) and IFREMER. Authors thank J.-L. Guillou (IFREMERSete) for the field support, J. Knoery (IFREMER Nantes), andM.-J. Vaulay (ITODYS, Universite Paris 7) for EDX analysis,C. Mangeney (ITODYS, Universite Paris 7) for XPS measure-ments, and J.-J. Bourrand (LGIS, Universite Paris 7), M. Can-tou (Universite Montpellier II) and S. Devanne for divingassistance.

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