DELU-R 02-003

D EL-SG-06-02

IN SITU VOLTAMMETRY AT DEEP-SEA HYDROTHERMAL VENTS by Donald B. Nuzzio, Martial Tailjefert, S. Craig Cary, Anna Louise Reysenbach and George W. Luther, ill

This work was sponsored, in qart, by the National Sea Grant College Program under Grant No. NA16RG0162-03 Project No. R/B-33!. Reprintedfrom American Chemical Society Symposmm Series ttt 1, EnvirOnmentalElectrochemistry: Analyses of TraceElement

Universityot DelawareNewark, Delaware 19716 ACKNOWLEDGEMENTS

This publication was supported,in part, by the National Sea Grant Coliege Program of the U.S. Department of Commerce's National Oceanic and Atmospheric Administration under NOAA Grant No. NA16FtG0162-03 Project No. R/B-33!. The views express herein do not necessarily reflect the views of any of those organizations.

Chapter 3 ln SituVoltammetry at Deep-Sea Hydrothermal Vents DonaldB.Nuzzio'*, Martial Taillefert, S.Craig Cary', AnnaLouise Reysenbach, and George W. Luther,III' 'AnalyticalInstrument Systems, Inc.,P.O. Box 458, Flemington, IVJ08822 CollegeofMarine Studies, University ofDelaware, 700Pilottown, Road, Lewes, DE 19958 'DepartmentofBiology, Portland StateUniversity, Portland, OR97201

Thereis a needto buildinstrumentation andsensors that can measurein situ chemicalchanges in dynamicenvironments. Hydrothermalvents are arguably the most dynamic aqueous systemson earth. The orifice of a ventapproaches 360 'C and spewsvast quantities of dissolvedhydrogen sulfide and iron intoambient seawater at 2 'C.These chemical species fuel incredibledeep-sea micro!biological communities, which may bea modelfor life onother planets, Here we describe an in situ submersibleanalyzer and electrodes for themeasurement of aqueouschemical species found near hydrothermal vents. A standardthree-electrode arrangement is controlled by a voltarnmetricanalyzer that is deployedfrom the deep-sea submersible,Alvin. Real time measurements fora variety of redoxspecies under flow conditions were made with a I00Iim Au/Hgsolid-state working electrode ata depthof2500 m. The solid-stateworking electrode was used to detect dissolved 02, S -II!,Fe II! andFeS,q molecular clusters, Our in situ data showthat significant changes canoccur in chemical speciation

40 C 2002American Chemical Society andanalyte concentration when waters are sampledand then measuredaboard ship.

Introduction

Voltammetric micro!electrode techniques have been used in a varietyof geochemistryand marine chemistry applications. Recently, in situ voltammetric measurementshave received increasing attention !. Bothdirect -4! andon- line -11, flow cell! type arrangementshave been used for trace metal and inajor chemical speciesdeterminations. However, voltamrnetricinstrumentation has only been deployed in shallow waters to date. There is a need to inake measurementsin the deep-sea,and this is particularlytrue of deep-sea hydrothermal vents that have an mcredible microbial and macrofaunal communityfueling itself via chemosynthesis2,13!, Changesin temperature canbe dramaticin this environmentand electrodes must be ableto respond preciselyin watersof lowerpH, high pressure, high temperature and high water flow rates.When high temperature waters mix with low temperaturewaters, their chemistriescan differ dramatically . Because organisms live in thesedynainic andextreme conditions, it is criticalto understandhow that chemistry drives or influences biology, In this paper we describe i n siru electrochemical instruinentationand the initial deployment of'it at 9 'N EastPacific Rise EPR; at a 2500meter water depth; 250 atmof pressure!and at GuaymasBasin, Gulf of California,2000 meter water depth 00 atmof pressure!.A companion paper in this volume ll! demonstratesthat current-concentrationcurves are affected by waterflow ratesbut not by pressure.At highflow ratesand reasonable scan rates - I V s '!,the current-concentration curvesbecome independent of water flow rate for the targetredox species. Deep-seahydrothermal vents can havehigh concentrationsof iron and sulfide.In this paperwe demonstratethe use of voltamrnetryto ineasureFe and S species.Using a solid-stategold amalgam Au/Hg! working electrode, we showthe simultaneousdetection and quantificationof severalsulfur and iron dissolvedspecies. In previouswork, dissolvedchemical species >, H~O~, Sg03,S', HS, I, Fe II!,Mn II!,organically complexed Fe III!,and FeS clusters! have been simultaneouslymeasured in situ in sedimentsand natural waters , 14,15!. 42

Experimental Methods

Chemicals and solutions

Chemicalsused in laboratoryexperiments were analyticalgrade from Fisher Scientific Co, Milli-Q quality Millipore! was usedfor all reagents.Laboratory measureinents were carried out in a 0.55 M NaC1 solution or in seawater, Mn II!, Fe II! and S -II! standardswere preparedfrom MnC12~ 4 H,O, ferrous ammoniuinsulfate, and NaqS9HqO. The mercuryplating solution was prepared as 0.1 N Hg NO> in 0.05N HNO,.

Electrodes

Goldamalgam PEEK" electrodes were made as described by Lutheret al ! by fixing 100 pm-diameter Au wire soldered to the conductor wire of a BNC cablewithin a bodyof 0.125"-diameterPEEK" tubing, which is commercially available as standardHPLC high-pressuretubing. The metal is fixed within the tubing with West System 105 epoxy resin and 206 hardener, A portion of the black outercoat andbraid of the BNC wire arereinoved to exposethe teflon shield and Cu conductor wire so that the Au wire soldered onto the Cu conductor canbe inserted into the PEEK ". Theepoxy is injectedinto the PEEK" tubing which containsthe gold wire that was previouslysoldered to the conductorwire of theBNC cable. Then the teflon is insertedinto the PEEK" tubing until the black coating of the BNC wire fits againstthe PEEK tubing, and the assembly is held with epoxy, which hasa moderatesetting time -1 hr!, and doesnot drain out the lower open side. On setting,the epoxyseals the tip andthe lower endcan be refilled with epoxy if necessary,Then the top end is coatedwith Scotchkote M! electricalcoating and Scotchfil M! electricalinsulation putty. PEEK" and high-purity epoxy fill permit the determinationof metal concentrations without risk of contamination,and at temperaturesas high as 150 'C. Pt counter and solid Ag/AgCI reference electrodes were made similarly but 500 lim diameterwire was usedfor each.These PEEK" electrodescould be usedas is or mated with standardHPLC fittings from Vpchurch,Inc for insertion into a flow cell //!. Once constructedthe working electrode Au! surfacewas sanded,polished and plated with Hg by reducingHg II! from a 0,1N Hg / 0.05 N HNO3solution, for 4 minutesat a potentialof -0.1 V, while purging with N2, The mercury/gold amalgam interface was conditioned using a 90-second -9 V polarization procedurein a 1 N NaOH solution 6!, The electrodewas then run in linear 43 sweep mode &orn -0.05 to -1.8 V versus a Saturated Calomel Electrode SCE! or Ag/AgCl electrodeseveral tiines in oxygenatedseawater to obtain a reproducible 02 signal. For DSVAlvin work, four Au/Hg electrodes can be controlled by the analyzer. The reference electrode was Ag/AgCl and the counter electrode was Pt wire, both of which weremounted on the basketof DSV Alvin so that they would not enter sulfidic waters 5!. For hydrothermal vent work, the Ag/AgCl reference was silver wire, which was oxidized in seawater at +9 V for 10 sec to form a AgCl coating. This electrode was used as a solid-state electrode in the seawatermedium =0.7! so that no pressureeffects on filling solutionswould hinder electrodeperformance. Peakpotentials of the analytesmeasured in situ and aboard ship were the same and similar to those for a saturated caloinel electrode SCE!. All laboratory and shipboardanalyses were carried out using an Analytical InstrumentSystems AIS! DLK-100A potentiostatcontrolled by a microcomputerusing software provided by the manufacturer.A DLK-SUB-I was used for allinsituwork see below!.

Field Experiments

For hydrothermal vent work, two working electrodes as well as a therinocouplesensor and tubing that lead into a flow cell inlet and a discrete syringesampler system were placedin a sensoror wandpackage 5! that canbe held by a manipulator arm! of Alvin, The wand was held over the vent orifice and areas along the length of vent chimneys. These latter areas are termed diffuse flow becausewater temperaturescan rangefrom 8 to 125 'C and do not emanate&om the vent orifice. A flow cell 1,15! was fixed in the submersible's basket that was bathed by waters at 2 'C. A submersible electrochemical analyzer DLK-SUB I! from Analytical InstrumentSystems, Inc. was used for data collection see below!. The electrochemicalpackage was deployed during cruises to 9 'N East Pacific Rise May, 1999! and GuaymasBasin, Gulf of California January 2000!. Separatediscrete sampleswere taken with a gas tight syringe sampler 1,16! from the samewaters for comparisonwith the flow cell measurements. The discrete sampleswere measuredaboard ship by voltammetry 1!. We typically madethree to five replicatemeasurements per samplewith the flow cell system,Electrodes were calibrated at different temperature7! as well as for flow 1!. 44

Voltammetry

The three-electrodeconfiguration working, reference and counter! was used to determine the concentrationof the speciespresent in natural waters. Linear sweepvoltammetry LSV!, cyclic voltammetry CV! and squarewave voltammetry SQW! were used for analyses, The following conditions were generallyapplied during the LSV and CV scans:scan rate = 200, 500 or 1000 mVs ',scan range = -0,1to -1.75V, equilibrationtime = 5 s, Squarewave voltammograinswere conducted under the sameconditions with a pulseheight of 24 mV, I mV scanincrement and 50 mV s ' scanrate. To preventmemory effects,caused by the accumulationof sulfideand inetal species on themercury surface,conditioning stepswere appliedto the working electrodeas per Brendel andLuther l7!. To reoxidizemetals Mn, Fe!that are reduced at theamalgam, a potential of -0.1 V was applied over 10-30 secondsbefore eachscan. When sulfidewas present,conditioning at 0.9 V for 10 s wasemployed since the metals and sulfide are not electroactiveat that potential l4, 17!. All LSV and CV scansshown in thefigures below are actual data; i.e., no softwaresmoothing routinesare usedto enhancethe quality,

Results i~t:Discussion

Instrumentation

A schematicof the analyzer,electrodes and sensorpackage system is providedin Figure1. The DLK-SUB-I is an electrochemicalanalyzer built by AnalyticalInstrument Systems, Inc, Theinstrument is designedto performall of the standardvoltamrnetric analyses that would be availablein a shipboardor land-basedlaboratory. The following standardvoltammetric techniques are possibleusing the DLK-SUB-1:linear sweep, cyclic, normal pulse, differential pulse,square wave. Chronoamperometry as well as strippingtechniques for monitoringtrace levels of analytescan also be performed.This versatility providesgreat flexibility to the researcherfor in situ experiments. The instrumentutilizes the 24 V DC power availablefrom DSV Alvin for its power source. All waterproof connectionswere made with connectorsfrom Impulse,Inc. and the aluminumhousing meter length; 20 cm outside diameter!of theinstruinent was rated for operationaf full oceandepth -6000 rn; 600 atm!. The instrumentis a completestand-alone package capable of being deployedfor long periodsof time from Alvin, or remotelyoperated vehicles ROV! with tethersup to 1500meters without the need for signalamplification. Figure1. Schematic of theanalyzer, electrodes and cable communication through the hull of Alvin. Thevoltammetry hardware is linked to an IBM compatible computer inside the housing.The internal computer communicates with anothercomputer throughthe hull of Alvin via a 15-mRS 232 cable and is controlledby an operator,who can reprogram waveforrns to respondto theradically different environmentsfound at vents. A separate1-meter cable is used to make connectionswith the working, counter, and reference electrodes and the pressure housing.This cable has four inputs for workingelectrodes that can be selected oneat a timevia a multiplexer!,one input for the counterelectrode and another for thereference electrode, Another input for groundingthe reference electrode from the submersibleinsured signal integrity. The electrode wand is constructed of Delrinand has a stainlesssteel handle so that the manipulator arm! of Alvin canhold and deploy the electrodes without breaking them.

Hydrothermal vent measurements

Themajor aqueous species, which are found near hydrothermal vents, react atthe Au/Hg electrode according to thefollowing electrode reactions 7,18!.

Oz + 2 H' + 2 e m H>O> -0.30 V a! HzOz+2H+ + 2e m HzOz -1.30 V lb! HS H>S!+ Hg -+ HgS+ H'+ 2 e ads.onto Hg <-0.60 Va! HgS+ H'+2 e e+HS+ Hg --0,60 V b! FeS~+ 2e + H' m Fe Hg!+ HS -1.1 V ! Fe" + Hg + 2e ++ Fe Hg! -1,43V ! 46 6e-8

4e4

2e-8

-1.5 -1.0

Volts vs Ag/Agcl

FigureZ, Five LSV scans 00m V s'! of ambientdeep-seawater at 2500 m depthand 2 'C. Ozis the only major species present.

The electrochemicalpackage was first testedin cold watersaway from high temperaturevents and high flow rates. Thus, no significant change in signal could be expected.Figure 2 shows five LSV scans of Oi in ambient 2 'C seawaternear the vent site at 2500 meter water depth. The electrochemical analyzer and electrode package shows excellent reproducibility. The 0> concentrationis 46.6+ 3.3 pM consistentwith previousresults 9!, The packagewas also tested 0.5 meter above a vent orifice where the temperaturewas 25'C. At the orifice the temperaturewas 360 'C as measuredby a second thermocouple without electrodes attached. The electrode measurements were thus performed at a safe distance from the orifice, Black smoke from iron monosulfide and pyrite precipitation was observed emanatingfrom the vent orifice, Figure3A displayssignals for only free H~S,FeS,molecular clusters and total S -2! [SAys= Z FeS+ HiS]. 0, is not detected.The reproducibility of thesemeasurements indicates that the vent had constantchemical output at this time as hot iron and sulfide rich vent waters mix with cold ambient seawater. There was no double peak detectednear the free sulfide peak 4,20!, which indicatesthat polysulfideswere not being formed quickly underthese conditions, This is not surprisingbecause the pH is approximately5 measuredaboard ship! at this location abovethe vent orifice, Sulfide oxidation is quite slow at pH < 6 Zl!, We note that one scan in the sequence! showed a cutoff of the SAys signal that is due to a fluctuation in power from the submersible.Figure 38 shows the concentration versus time plot from five consecutive CV scans. Although a standardfor FeSclusters is not available,the difference between freeH,S andtotal S -2![SAys] is an indicatorof theFeS,q concentration 1!. 2e-6

1e-6

0

-1 e-6 -1.5 -1.0 -0.5 Volts vs AQCI! 500

4GO

~ o 3PG

200

a 100

0 30 60 90 120 150 180 time sec! Figure3. A! C Vscans 000 m Vs'! 0.5meter above a ventorifice at 25"C. The asteriskindicatesthat noise from thesubmersible cutoff the Says signal; B! time course of the data. 48

In contrast Figure 4A shows a CV measurement in the tube of the polychaete,Alvinella pompjeana. During measurementat a scanrate of 200 mV s', thetemperature was 80 + 20 'C. Thisdramatic fluctuation in temperature inakes it impossible to measureconcentrations, but the voltMnmogramclearly indicatesthat only FeSqqFe II! and total S -2! are measurable.Thus, free HqS wasbound with Fe + asFeS~ molecular clusters. A samplewas also drawn into theflow cell at 2 'C!, andanalyzed with anotherelectrode under no flow. Figure 4B showsthat only a signalfor total S -2! is presentin the flow cell. Thesedata indicate that the chemicalspeciation probably due to precipitation of FeS! was changed as the waters were cooled. The current also changed two orders of magnitude&om Figure 4A to 4B becauseof the dramatictemperature change. We attribute the noise in Figure 4B to electricalnoise from the subinersible.The flow cell data correspondsto a concentrationof 250 p.M.A discretesample was also taken and analyzedlater aboard ship by voltaminetry Figure 4C! using purge and trap methods/,22!. The concentrationwas found to be 139 pM and indicates that further precipitation of FeS and FeSi occurred or that H>S was liberatedfrom solution. Figure 4C showsa small free H>Ssignal indicatingthat some dissociationof FeS occurredwhen the samplewas brought onboard ship where the temperaturewas 20 'C. In addition, the smaller S+vssignal indicates that sulfide was lost on sampling and transport to the lab. These data clearly show that chemical speciation and analyte concentrationcan change with sampling and storageand that real time dataare essentialin understandingwhat chemistryan organismactually experiences. For Alvinellapompj eana the Fe ' binds sulfide as FeS in its habitat, and detoxifiesthe free sulfide for the organisin 5!. Additional reasonsfor the changesshown in Figure 4A to 4C are that the syringesfor discretesainples are not subsampledfor measurementsfor up to six hours after collection allowing for reaction or loss of sulfide if the gas tight syringes malfunction. The samples from these syringes are placed into a voltarnmetriccell at one atmospherepressure for measurement,which could lead to a changein the equilibriumfor FeS eq, 5! froin that at 2500meter water depth 50 atmospheres!.Also, it takes approximatelytwo minutes to fill the syringes during sampling. During that time the temperaturecan fluctuate drainatically with significant chemical changes,and the manipulator arm! of Alvin has beenobserved to move during sampling.

Fe" + H>Sm FeS,q+ 2 H' ! 49

2e-5 1e-5-1.0

-0.5 4e-7

2e-7

-1.5 -1.0 -0.5

2e-6 1e-6-1.5-1.0 -0.5 Volts Ag/AgCI! Figure4. A! CV00 mVs! scantaken in thetube of thepolychaete at 80 +20 'C; B:C Vscans 000 m Vs'! taken of a samplepumped into a flow cell at Z "C; C!CV scans 00 mVs'!taken of asubsample aboardship at20 "C. 50

Conclusions

Realtime, in situmeasurements in oceanic hydrothermal vent environments wereaccomplished with a submersibleelectrochemical analyzer and PEEK" Au/Hgelectrodes fordissolved OFe", FeS~,free H,S and total sulfide S~vs = ZFeS,q+H,S!. The electrochemical instrumentation isrobust towork in extreme environmentsand is commerciallyavailable. The electrodes are designed to operateattemperatures upto 150'C. Our observations demonstrate thatreal timein situvoltainmetric analysis provides a moreprecise measure of chemical speciationand a measureof the chemical concentration of analytes that emanate from vents.In contrast,oxidation and precipitation reactions during sample retrievaland handling of discretesamples can result in anunderestimate ofthese concentrationsand in the actualchemical speciation.

Acknowledgments

Theauthors would like to thanktheir own lab groups, R, Lutz, T, Shank,the DSVAlvin pilots and the crew of the R/V Atlantis for assistance, This study was partiallyfunded by grants from the Office of Sea Grant / NOAA NA16RG0162- 03!,NSF OCE-9714302 andSBIR-9760571! andNASA NAS13-0013!.

References I, Taillefert,M.; Luther, III, G.W.; Nuzzio, D. B. Electroanal, 2000, 12, 401- 412, 2. Tercier,M-L.; Buffle, J.; Zirino, A,; DeVitre, R. R.Anal. Chirn Acta 1990, 23 7, 429-437. 3. Tercier,M-L.; Buffle, J.; Graziottin, F. Electroanal. 1998 10, 355-363. 4. Tercier,M-L.; Belmont-Hebert, C.;Buffle, J. Environ. Sci. Technol., 1998, 32, 1515-1521. 5, Herdan,J' Feeney, R., Kounaves, S.P.; Flannery, A. F.;Storment, C.W.; Kovacs,G. T. A.; Darling, R. B, Environ. Sci. Technol., 1998, 32, 131-136. 6. Luther,III, G.W,; Reimers, C,E.; Nuzzio, D. B.; Lovalvo, D. Environ. Sci. Technol. 1999, 33, 4352-4356, 7. DeVitre, R, R.;Buffle, J.; Perret, D.; Baudat, R. Geochim.Cosmochim. Acta. 1988, 52, 1601-1613. 8. Lieberman,S.H.; Zirino, A. Anal.Chem, 1974, 46, 20-23. 9. Martinotti,W.; Queirazza,G.; Realini,F.; Ciceri,G. Anal.Chim. Acta. 1992, 261, 323-334. 51

10. Newton, M. P.; van den Berg, C. M. G. Anal Chim Acta 1987, 199, 59-76. 11. Luther, III, G. W.; Bono, A.; Taillefert, M; Cary, S. C. In Electrochemical Methods for the Environmental Ana/yses of Trace Element Biogeochemistry, Taillefert, M.; Rozan, T., Eds. American Chemical Society Symposium Series; American Chemical Society: Washington, D. C,, 2001, this voluine, Chapter 4, 12 Cavanaugh,C, et al, Science1981, 213, 340-342. 13 Felbeck, H, Science 1981, 213, 336-338. 14 Luther,III, G. W.; Glazer,B. T.; Hohman,L.; Popp, J. I.; Taillefert, M.; Rozan, T. F,; Brendel, P. J,; Theberge,S. M,; Nuzzio, D. B. J. Environ. Moni t. 2001, 3, 61-66. 15 Luther, III, G, W.; Rozan, T. F.; Taillefert, M,; Nuzzio, D. B; Di Meo, C.; Shank, T. M.; Lutz, R. A.; Cary, S, C, Nature 2001, 401, 813-816. 16 Di Meo, C. A,; Wakefield, J, R.; Cary, S. C, Deep-SeaRes. 1 1999, 46, 1279-1287. 17 Brendel, P,; Luther, III, G, W, Environ, Sci. Technol. 1995, 29, 751-761. 18 Theberge,S, M,; Luther, 111,G, W. Aq. Geochem,1997, 3, 191-21l. 19 Johnson, K. S.; Beehler, C. L.; Sakamoto-Arnold, C. M,; Childress, J, J, Sci ence 1986, 231, 1139- l 141. 20 Rozan, T. F.; Theberge, S. M.; Luther, III, G. W. Anal. Chim. Acta 2000, 415, 175-184. 21 Millero, F. J.; Hubinger, S., Fernandez, M.; Garnett, S. E. Environ. Sci. Technol., 1987, 21, 439-443, 22 Luther, III, G. W.; Ferdelinan, T. G.; Kostka, J. E.; Tsamakis, E. J.; Church, T. M. Biogeochem.1991, 14, 57-88,

Reprintedfrom ACS Symposium Senes gl I EnvironmentalElectrochemistry Analysesof TraceElement Biogeochemistry Martial Taillefert, I'im F. Rozan,Editors Pub!ished2002 by the AmericanChemical Society Universityof Delaware