2012 - Fluorometric Detection of Total Dissolved Zinc in the Southern Indian Ocean

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  Fluorometric detection of total dissolved zinc in the southern Indian Ocean Kathleen J. Gosnell  a,b, ⁎ , William M. Landing  b , Angela Milne  c a University of Connecticut, Department of Marine Sciences, 1080 Shennecossett Road, Groton, CT 06340-6048, USA b Florida State University, Department of Earth, Ocean and Atmospheric Science, Tallahassee, FL 32306-3048, USA c University of Plymouth, School of Geography, Earth and Environmental Sciences, Plymouth, PL4 8AA, England, United Kingdom a b s t r a c ta r t i c l e i n f o  Article history: Received 10 June 2011Received in revised form 4 November 2011Accepted 9 January 2012Available online 16 January 2012 Keywords: Dissolved zincSilicateFlow injection analysisSouthern Indian Ocean Zinc acts as a micronutrient in the ocean, capable of in 󿬂 uencing and potentially controlling phytoplanktonproductivity and community structure. Thus, it is important to quantify the distribution of dissolved Zn inthe oceans, in addition to understanding the biogeochemical behavior of this important element. Meetingthis objective hasbeenelusive sincedissolved Znconcentrationsintheupperwater column canbeextremelylow, and it is dif  󿬁 cult to eliminate contamination during sample collection and analysis. Our approach to thisproblem wastoutilizeaFlowInjection Analysis(FIA)method initiallydescribedbyNowickietal.(1994),andcollecting uncontaminated seawater using a trace-metal clean rosette system (Measures et al., 2008).Samples for total dissolved Zn analysis were during the 2009 CLIVAR I5 cruise across the southern IndianOcean (from Cape Town, South Africa to Fremantle, Australia). Dissolved Zn concentrations have not beenpreviously reported for this region. Extremely low dissolved Zn concentrations (0.02 nM) were observed insurface waters of the central Indian Ocean gyre, documenting the extreme biological depletion of Zn typicalof the open ocean. Concentrations of Zn and Si both increased with depth. The highest concentrations mea-suredfordissolved Zn(>3.5 nM) werecollected at1300 moff westernAustralian. Totaldissolved Znconcen-trations were observed to be oceanographically consistent, and well correlated with dissolved silicate acrossthetransect. Thelinearregression of totaldissolved Znvs.Siforallofthedatayielded aslope of0.059±0.003(nM Zn/  μ  M Si), which is consistent with the values reported for the north Paci 󿬁 c and thus support the pre-viously reported nutrient-type Zn – silicate relationship. Thezonal section ofthe dissolved Zn/Si ratiosalso ex-hibit broad maxima and minima, consistent with variable sources for Zn and different recycling rates for Znvs. Si.© 2012 Elsevier B.V. All rights reserved. 1. Introduction Trace metals operate as either potential toxicants or nutrients inaquatic systems. Several essential metals, such as iron (Fe), manga-nese (Mn) and zinc (Zn), are typically found in surface waters of the open ocean at concentrations that have been shown to be bio-limiting in laboratory cultures (Brand et al., 1983; Morel et al.,1994; Sunda and Huntsman, 1992).Zinc is essential for phytoplankton growth as a cofactor for nearly300 different enzyme systems, such as carbonic anhydrase, carboxy-peptidase, alkaline phosphatase, and alcohol dehydrogenase (Morelet al., 1994). Carbonic anhydrase catalyzes the reversible dehydrationof H 2 CO 3 , andas a result is utilizedfor inorganiccarbonacquisitionbyphytoplanktonduringphotosynthesis(BadgerandPrice,1994). Somestudiesreportthatlow-dissolvedZnconcentrationsintheopenoceancould possibly limit phytoplankton growth and carbon dioxide acqui-sition (Anderson et al., 1978; Morel et al., 1994; Ibrahim et al., 2008).However, limited data are available on the relationships betweendissolved Zn, biological activity, and inorganic carbon (Morel et al.,1994; Schulz et al., 2004).Based on the ionic composition of seawater, in combination withinorganic Zn complexes (Brand et al., 1983), low concentrations of Zn found in the open ocean could theoretically limit the growth of some phytoplankton species. While laboratory studies have investi-gated biological limitations by dissolved zinc (Sunda and Huntsman,1992; Ellwood and Hunter, 1999; Anderson et al., 1978; Schulz etal., 2004), understanding the impacts dissolved Zn might have on pri-mary production and phytoplankton community structure in theopen ocean has been hampered by a relative lack of reliable data fordissolved Zn concentrations. In addition, we are not aware of anypublished experimental results from an in-situ Zn fertilization exper-iment, in contrast to the many Fe fertilization experiments where theeffects from Fe addition on phytoplankton productivity have beenquanti 󿬁 ed. Crawford et al. (2003) reported slight chlorophyll enrich-ment from Zn additions during bottle incubation experiments in thesubarctic Paci 󿬁 c; on the other hand, Coale et al. (2003) did not  󿬁 ndthat incubations including Zn affected chlorophyll growth for theAntarctic Circumpolar Current (ACC) region. As Zn is not found at Marine Chemistry 132 – 133 (2012) 68 – 76 ⁎  Corresponding author. University of Connecticut, Department of Marine Sciences,1080 Shennecossett Road, Groton, CT 06340-6048, USA. E-mail address: (K.J. Gosnell).0304-4203/$  –  see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.marchem.2012.01.004 Contents lists available at SciVerse ScienceDirect Marine Chemistry  journal homepage:  limiting concentrations for the ACC (>2 nM), it is likely that theseship board incubation experiments were not conducted with Zn-starved phytoplankton. Hence, these studies only represent the startof what necessitates further investigation. There are still many mys-teries to unravel about the biogeochemical cycle of Zn in the oceans.Although trace metal sampling and handling techniques have ad-vanced immensely in the last 35 years, there are still many obstaclesto overcome in order to collect samples that are uncontaminated forZn and to make accurate analytical measurements. Zinc is a notori-ously pervasive contaminant, as it is used frequently on marine ves-sels and equipment. Additionally, Zn contamination can result fromunexpected sources such as Kimwipes and nitrile gloves. Accordingly,accurate measurements can only be made by using strict safeguardsduring sample collection and analysis (Bruland et al., 1979; Ellwoodand Van den Berg, 2000). These sampling and analytical techniquesare labor-intensive and require some level of expertise. As a result,there are only a few reliable pro 󿬁 les of dissolved zinc from the openocean. Currently only Morley et al. (1993) have reported dissolvedZn pro 󿬁 les for the southwestern Indian Ocean. In other regions,such as the northeast Paci 󿬁 c Ocean, data has primarily been acquiredatonlya fewstations(Bruland,1989;Lohanetal.,2002), yetthesere-sults have provided strong support for theories on biolimitation byZn, in addition to demonstrating the importance of trace metals inmarine ecosystems.Vertical and horizontal distributions of chemical tracers in theoceansarein 󿬂 uencedbyamyriadofchemical,biological,andphysicalprocesses, and the resulting distributions must be consistent with theeffects of those processes. The relationship between dissolved Zn andSihasbeenrecognizedsincethemid-1970swhencontamination-freesampling and analytical methods were publicized (Bruland et al.,1979). The srcinal work that demonstrated the strong correlationbetween dissolved Zn and silicate (Bruland et al., 1978) has beenexpanded to include a few pro 󿬁 les from the Atlantic (Bruland andFranks, 1983; Ellwood and Van den Berg, 2000), the Paci 󿬁 c (Bruland,1980, 1989; Lohan et al., 2002), and the southwestern Indian Ocean(Morley et al., 1993).Here we report on 42 pro 󿬁 les (0 – 1000 m) of total dissolved Znfrom the 2009 I5 CLIVAR Repeat Hydrography cruise in the southernIndian Ocean, which were measured using a  󿬂 ow-injection (FIA)scheme utilizing  󿬂 uorescence detection. The concentration range,the strong correlation with dissolved silicate, and overall oceano-graphic consistency of the data illustrate the suitability of the analyt-ical method (adapted from Nowicki et al., 1994) as well as theaccuracy and reliability of the data. By combining careful sample col-lectionwith a sensitiveanalytical method, our goal was to expandthedatabase for dissolved Zn in the oceans, and to help inform futurestudies of the biogeochemical cycling of Zn in the oceans. 2. Methods  2.1. Sample collection Seawater samples were collected and analyzed on board the  R/V Revelle  during the 2009 I5 CLIVAR Repeat Hydrography cruise(March 23, 2009 to May 14, 2009). The I5 cruise track from 2009 isdisplayed in Fig. 1. The I5 transect primarily followed approximately30° S latitude from Cape Town, South Africa to Fremantle, Australia,with an occasional deviation away from the 30° S track to obtainphysical and chemical data from prominent submarine ridges in theregion. The CLIVAR   ‘ Trace Metals ’  rosette was deployed at approxi-mately every other station, yielding 97 pro 󿬁 les for trace metals. Dis-solved zinc samples were collected about every fourth station,resultingin 42 pro 󿬁 les for dissolved Zn out of the 97 stations sampledfortracemetals.Duetothesternpositioningoftherosetteontheves-sel, station sampling frequency was dependent on weather and oceanconditions. We collect 12-depth pro 󿬁 les to 1000 m at each station fortwo reasons. In general, the majority of the cycling activity for bioac-tive trace elements occurs in the upper 1000 m. Furthermore, theTrace Metals component of CLIVAR is limited by ship time con-straints; we chose to collect more pro 󿬁 les from the upper 1000 mrather than to have collected fewer pro 󿬁 les extending deeper intothe water column. At two stations, samples were likewise collectedfrom 0 to 1300 m.Subsamples for total dissolved Zn were  󿬁 ltered through 0.4  μ  macid-washed 47 mm polycarbonate track-etched  󿬁 lters (GE-PoreticsK04CP04700) directly into 125 mL polyethylene sample bottles. Sub-sampling was conducted in a HEPA  󿬁 ltered air environment in a lab-oratory van (Measures et al., 2008).  2.2. Analytical preparation Allplasticlabwarewashandledandacidwashedusingtracemetalclean techniques. Bottles used for samples and analysis were made of low density polyethylene. Reagent and subsample bottles were dou-ble bagged and shipped in sealed coolers to minimize contamination.Reagents were used as received unless otherwise speci 󿬁 ed. All work-ing reagents were prepared using puri 󿬁 ed 18 Mohm cm 3 water (UHPwater) drawn from a Barnstead UHP deionization system in the cleanvan. The clean van (Measures et al., 2008) has been used successfullyonover 17 researchcruisessince 2003 withno signi 󿬁 cant contamina-tion issues, as was the case for the I5 cruise.Inside the main laboratory of the ship, a lab table was transformedinto a clean workspace by means of completely covering it with plas-tic sheeting. A MAC-10 HEPA blower (ENVIRCO, Inc.) was suspendedfrom the ceiling several feet above the table, then more plastic sheet-ingwasdrapedfromthebackandsidesoftheHEPAblowerandtight-ly sealed to the plastic covered table with duct tape, creating a cleanair environment. The  󿬂 ow-injection manifold was set up inside thiscleanairenvironment.The 󿬂 uorometerandsoftwarecontrolcomput-er were placed outside the  󿬂 ow hood and connected to the FIA pumpthrough the plastic.The critical reagent in this method is p-Tosyl-8-aminoquinoline(pTAQ: Chemica Inc. Gardena, CA) which forms a stable  󿬂 uorescentcomplex with dissolved Zn(II). The 0.05 M pTAQ stock was preparedby dissolving 0.291 g of pTAQ into 20 mL of nonionic surfactant baseTriton X-100 (poly(oxyethylene)isooctylphenol). Several individualbottles of this pTAQ stock were prepared and bagged separately, intheeventthatonewastoleakorspill.Thesestocksolutionswerepre-pared 1 – 2 months in advance, and shipped to the starting port (CapeTown, SA) so that they were ready to use at the onset of the cruise.The previous study by Nowicki et al. (1994) indicated that the pTAQ concentration in the mixed reagent could be varied from 40  μ  M to120  μ  M, depending on expected concentrations of Zn in the waterbeing analyzed. For the trace amounts of zinc expected in the south-ern Indian Ocean, 40  μ  M was chosen.The 1.0 M boric acid solution was made by dissolving approxi-mately 31 g of H 3 BO 3  (Mallinkrodt Chemicals) into 500 mL of micro-wave warmed (~90 °C) UHP water. The H 3 BO 3  was weighed out intoseveral individual vials for easier shipping and ship-board prepara-tion of the solution. The 2.0 M NaOH solution is commercially avail-able (VWR BDH3223-1).The 40  μ  M pTAQ   󿬂 uorometric mixed reagent was prepared byadding 25 mL of 2.0 M NaOH and 100 mL of 1.0 M boric acid to UHPwater, subsequently bringing the solution up to approximately1000 mL with UHP water, then  󿬁 nally adding 800  μ  L of the 0.05 MpTAQ stock to the buffered solution. Due to the viscous nature of the triton, the 0.05 M pTAQ/Triton solution was shaken vigorouslyto ensure thorough mixing.Quartz distilled 6 M HCl was used to prepare the eluent and acidrinse. The 0.08 M Q-HCl eluent carrier was made up by adding13.3 mL of 6 M Q-HCl to UHP water, and bringing the mixture to atotal volume of 1000 mL with UHP water. The 1.0 M Q-HCl acid 69 K.J. Gosnell et al. / Marine Chemistry 132 – 133 (2012) 68 – 76   rinse was prepared by pouring 83.3 mL 6 M Q-HCl into some UHPwater, then bringing the volume up to 500 mL with UHP water.Due to the minute quantities of Zn being measured, it was neces-sary to remove interfering cations, primarily calcium (Ca) and mag-nesium (Mg), from the column prior to the elution of Zn. This wasaccomplished by using diluted ammonium acetate buffer in the col-umn rinse step. The buffer rinse was made by mixing 40 mL of 2.0 M ammonium acetate buffer (pH 6.8) with 460 mL of UHPwater, yielding a solution of 0.16 M NH 4 Ac (pH 5.77).Primary and secondary zinc standards were made every week in125 mL polyethylene bottles. Initially a 40  μ  M zinc standard was pre-pared by adding 262  μ  L of 1000 ppm atomic absorption zinc standardsolution (Fisher Scienti 󿬁 c) into 100 mL of UHP water. The 40  μ  M Znstandard was further diluted to produce a 200 nM Zn standard. Allstandards and samples were acidi 󿬁 ed with 6 M Q-HCl to a concentra-tion of 0.024 M HCl to keep dissolved Zn(II) in solution.Working standards were made prior to each sample run in 30 mL polyethylene bottles. These working standards were prepared dailyusing  “ low-Zn ”  surface seawater to ensure that the standard matrixremained consistent with the sample matrix. Seawater was collectedfrom the  “ trace metal clean ”  rosette system (Measures et al., 2008), 󿬁 ltered through a 0.2  μ  m AcroPak (Pall) capsule  󿬁 lter into 1 L poly-ethylene bottles, and quickly acidi 󿬁 ed to 0.024 M with 6 M Q-HCl.This bulk seawater solution was stored acidi 󿬁 ed for a minimum of 24 h prior to being used in standard preparation. Working standardsof +0, +1, +2, and occasionally +4 nM Zn(II) were made by adding150, 300, and 600  μ  L of the 200 nM secondary standard into 30 mL of low-Zn surface seawater. Samples and standards were buffered priorto extraction to a pH of 5.05 (0.067 M NH 4 Ac) using 1.0 mL of 2 Mammonium acetate per 30 mL of sample or working standard.All solutions were driven through the FIA system by a RaininRabbit-Plus eight-channel peristaltic pump. Fisher 2-stop PVC tubing,measuring 1.52-mm i.d. (coded blue/yellow), was used for the buff-ered pTAQ reagent, and also for the sample and column rinse lines( 󿬂 ow rate=1.1 mL/min). Fisher 2-stop PVC pump tubing, diameter0.7-mm i.d. (coded white/white), was used for the eluent acid carrier( 󿬂 ow rate=0.9 mL/min). All remaining manifold lines were FEP Tef-lon tubing of 0.8-mm i.d.A cation exchange column of 8-hydroxyquinoline (8-HQ) resinwas used to extract and preconcentrate Zn from seawater (Landinget al., 1986). The column consisted of 200  μ  L of 8-HQ slurry packedinto a 2 cm polyethylene column (Global FIA). The resin was securedin the column with porous polyethylene frits and attached as a  “ sam-ple loop ”  in the injection valve.TheFIAmanifolddiagramisdisplayedin Fig.2.Alldataacquisitionand valve positions were controlled with a Dell Latitude 131L laptop.Valve switching was controlled with port software. A ten-port multi-position valve (MP: Cheminert 04R-0251L, VICI Valco In-struments Co. Inc.), was used for selecting the sequence of solutions 󿬂 owing to the injection valve (IV: Cheminert 04Q-0014L, VICI ValcoInstruments Co. Inc.).The IV valve begins in the  “ Load ”  position with a strong acid rinse(1.0 M HCl) for 10s (~0.2mL), in order to wash all trace elementsfrom the manifold tubing and the resin column. This is followed by a4.0min sample loading period (~4.1 mL total), in which Zn is accumu-lated on the 8-HQ resin as the buffered sample (pH 5.05, 0.067 MNH 4 Ac)  󿬂 ows through the column. During the load period the 0.08 MHCl eluent bypasses the column, 󿬂 owing directly towards the detector,mixing withthe pTAQ reagentand establishingthesignal baseline. Fol-lowing sample loading, the column receives a 1.5 min rinse of thebuffered-UHP water (~1.6 mL) in order to elute calcium and magne-sium cations. Immediately after the columnrinse, theIVvalve switchesto the  “ Inject ”  position for a 1.0min elution period, and approximately0.9mL of the 0.08M Q-HCl eluent  󿬂 ows in the reverse directionthrough the column, releasing Zn into the eluent stream. Zinc cationsmix with the pTAQ reagent at a Te 󿬂 on mixing-T prior to  󿬂 owing to-wards theFIAlab PMT-FL  󿬂 uorometer. Oncecolumn elutionhas ceased,the IV valve switches back to the load position for a 10s column washwith 1.0 M Q-HCl after which the cycle starts over again. A completecycle takes approximately 6.8min. Valve timing and positioning forthis method is summarized in Table 1.Fluorometer wavelengths were controlled by internal wavelength 󿬁 lters inserted into the  󿬂 uorometer. Wavelength  󿬁 lters were cen-tered near the maximum excitation (377 nm) and emission(495 nm) wavelengths of the pTAQ-Zn(II)  󿬂 uorescent complex asreported by Nowicki et al. (1994). The excitation  󿬁 lter used was365 nm (narrow band-pass, 358 – 372 nm) and the emission  󿬁 lterused was 500 nm (broad band-pass, 465 – 535 nm).Fluorescence was monitored continuously during the load and in- ject cycles using FIAlab 5 Analysis software. Zinc concentrations were Fig. 1.  Station locations for the 2009 CLIVAR I5 cruise transect in the southern Indian Ocean. Stations began off the east coast of South Africa (Station 1) and ended off the west coastof Australia (Station 195).70  K.J. Gosnell et al. / Marine Chemistry 132 – 133 (2012) 68 – 76   assessed by measuring the peak height of the  󿬂 uorescence signal.Peak values (in units of relative  “ counts ” ) were recorded via FIAlabsoftware and extracted into Excel for further data processing. The 󿬂 uorescentresponsewaslinearfrom 0to atleast 4 nMtotaldissolvedZn. The standard deviation averaged 0.018 nM (n=5), and the detec-tion limit was 0.06 nM (3SD). Standard SAFe S1 (0.06 nM Zn; Johnsonet al., 2007) standards were repeatedly and routinely analyzed foreach station in order to assure that there was a consistent signalfrom the Zn-FIA method, and measured values resided within thereported range (0.05±0.02 nM Zn). The Zn-FIA accuracy was alsoveri 󿬁 ed during the inter-calibration GEOTRACES 2008 cruise. Bermu-da Area Time Series station (BATS) samples were measured at0.024 nM Zn for 10 m (GS), while 1000 m (GD) was measured as1.36 nM Zn; both results are comparable to other laboratory resultsduring the trials of GEOTRACES 2008.  2.3. Cadmium interference Since pTAQ forms a  󿬂 uorescent complex with Cd(II), dissolved Cdcan yield a positive interference. Based on laboratory tests using UHPwaterandlow-Znseawater,itappearsthatCd 󿬂 uorescenceisapprox-imately 30% that of Zn  󿬂 uorescence. The interference we observed islower than the 70% reported by Nowicki et al. (1994). Since total dis-solved Cd concentrations found in the ocean tend to be about 10% of the dissolved Zn concentrations (Bruland, 1980), any corrections forthe presence of Cd would be about − 3%. Calculated Cd interferencelevels were below the detection limit ( b 0.006 nM), as a result theseawater Zn concentrations we report were not corrected for Cdinterference. 3. Results and discussion  3.1. Zinc measurements A zonal section of total dissolved zinc concentrations from the2009 CLIVAR I5 cruise, prepared using Ocean Data View (Schlitzer,2011) is displayed in Fig. 3. All of the station pro 󿬁 les determinedfrom Zn-FIA appear to be oceanographically consistent, displayingthe expected nutrient-like pro 󿬁 le associated with zinc dynamics.Zinc pro 󿬁 les tend to be surface depleted, with concentrations around0.05 nM throughout the euphotic zone. Zinc remains depletedthroughout the upper 200 m of the southern Indian Ocean, beforethe deeper remineralization yields a steady increase in concentrationwith depth. Concentrations in the upper 200 m ranged from 0.02 nMto0.27 nMincludingthecoastalstations,whichbothexhibitedhigherZn concentrations in surface waters.Coastal water is typically enriched with trace metals compared tothe open ocean; therefore it is no surprise that Station 1 and Station195 both have elevated Zn in the upper 200 m. Station 1, collected just off the eastern South African coast, had 0.25 nM Zn in the surface,and remained below 0.5 nM Zn until the deepest sample at 285 m,where Zn was measured at 0.78 nM. Station 195 was sampled off the western coast of Australia, and displayed a similar trend to thatseen at Station 1, with surface zinc levels of 0.27 nM. Zinc values forstation195 did not exceed0.4 nM, which wasthe concentration mea-sured in the deepest sample at that station (183 m). Both coastal sta-tions were affected by minor scatter throughout the Zn pro 󿬁 le,possibly due to anthropogenic input (i.e. ship or beach runoff) orvia benthic  󿬂 ux enrichment from coastal sediments.Three typical Zn pro 󿬁 les are displayed in Fig. 4, representing sam-ples from the beginning, middle and end of the I5 transect. Station 9displayed higher surface concentrations than station 71, representingpossible coastal anthropogenic Zn input, while station 177 demon-strates the increased deep water concentrations typical of stationscollected closer to Australia. There is a notable increase in deepwater Zn levels sloping up towards both the South African and Aus-tralian coastlines, with the deeper Zn concentrations showing a mod-est increase east of South Africa at stations 1 through 17 (30° 35 ′  E to33° 76 ′  E), and west of Australia for stations 170 through 195 (104°82 ′  E to 114° 84 ′  E). We sampled to 1300 m at two stations. Thetwo deeper stations, 179 and 185, both displayed higher concentra-tions of Zn than those collected up to 1000 m (Fig. 5). The highest 10-portmulti - positionValve (MP) waste mixing-T(2) Buffered sample(3) Buffered UHP water rinse(1) 1.0 M HClcolumn wash FIAlab  PMTfluorometer0.08 M HClElution acid40 µM p-TAQ10-portinjectionValve (IV)8-HQ resin LoadElute Fig. 2.  Flow-injection manifold diagram for total dissolved zinc analysis. The ten-port injection valve (IV) rotates, sending solution (1), (2) or (3) through the column. The injectionvalve (IV) switches from  “ Load ”  to  “ Inject ”  after the sample has been loaded and the column has been rinsed (see Table 2 for the analytical cycle time steps).  Table 1 Valve timing and position for the Zn-FIA method. Thirty seconds is added to the actualrinsetime periodof1.0 mininordertoaccountfor 󿬂 ow timeoftherinse tothecolumn.Time (min) Inject valve (IV) Multi-position valve (MP)0:10 Load 1.0 M HCl4:10 Load Buffered sample5:40 Load Buffered wash6:40 Inject 0.08 M eluent (to waste)6:50 Load 1.0 M HCl71 K.J. Gosnell et al. / Marine Chemistry 132 – 133 (2012) 68 – 76 


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