Article in Press + Model

ARTICLE IN PRESS + MODEL

Marine Chemistry xx (2006) xxx–xxx www.elsevier.com/locate/marchem

An investigation into the exchange of iron and zinc between soluble, colloidal, and particulate size-fractions in shelf waters using low-abundance isotopes as tracers in shipboard incubation experiments ⁎ Matthew P. Hurst a, , Kenneth W. Bruland b

a Department of Chemistry and Biochemistry, University of California, Santa Cruz, 1156 High St., Santa Cruz, CA 95064, USA b Department of Ocean Sciences, University of California, Santa Cruz, 1156 High St., Santa Cruz, CA 95064, USA Received 6 September 2005; received in revised form 20 June 2006; accepted 10 July 2006

Abstract

A vertical mixing event was simulated in shipboard incubation experiments on the mid-continental shelf of the eastern Bering Sea to investigate Fe and Zn cycling between the soluble (b0.03 μm or 200 kDa), colloidal (0.03–0.2 μm), and particulate (0.2–10 μm, N10 μm) size-fractions. The particulate Fe and Zn were further separated into chemically labile (25% acetic acid-leachable) and refractory pools. The experiment employed 57Fe (+0.90 nM) and 68Zn (+0.99 nM) as stable, low-abundance isotope amendments to the soluble fraction, and the exchange of Fe and Zn between the different physico-chemical fractions was measured using high resolution-inductively coupled plasma- mass spectrometry (HR-ICP-MS). More than 50% of the added 57Fe partitioned to the colloidal fraction within 45 min of adding the tracer. Both the 57Fe and 56Fe colloidal fraction were removed from the dissolved phase at a faster rate than the soluble Fe fraction. In contrast, the colloidal 66Zn and 68Zn concentrations remained constant over the 5-day experiment, suggesting a unique removal mechanism for colloidal Fe. The net removal of dissolved 57Fewasobservedtobe3to4timesmorerapidthandissolved56Fe, which can be attributed to the regeneration of particulate Fe. Using a simple first-order model, it was determined that the net removal of 2.0 nM of dissolved Fe during the experiment was a consequence of dynamic cycling, whereby 2.9 nM of particulate Fe was regenerated and contributed to an overall removal of 4.9 nM of Fe from the dissolved phase. The amended 68Zn tracer resided in the soluble fraction and was assimilated by the diatom biomass (N10 μm size-fraction) at the same rate as 66Zn. This similarity in rates suggests that nearly all of the net removal of Zn was due to assimilation and that regeneration did not play a significant role in Zn cycling within the incubation experiment. This research demonstrates the advantage of using low-abundance isotopes as tracers and the importance of particulate and colloidal Fe in the overall biogeochemical cycling of Fe in ocean surface waters. © 2006 Elsevier B.V. All rights reserved.

Keywords: Iron; Zinc; Seawater; Inductively coupled plasma-mass spectrometry; Stable isotopes

1. Introduction

The development of sensitive analytical methods and ⁎ Corresponding author. Present address. Department of Chemistry, Humboldt State University, 1 Harpst Street, Arcata, CA 95521, USA. trace metal clean techniques has allowed progress in Tel.: +1 707 826 5720; fax: +1 707 826 3279. defining the role of trace metals as an important factor E-mail address: [email protected] (M.P. Hurst). influencing primary productivity in marine systems (Morel

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2 M.P. Hurst, K.W. Bruland / Marine Chemistry xx (2006) xxx–xxx et al., 2003). These advancements have led to the under- the experimental setup. Adequate methodology has yet to standing that bioactive trace metals, such as Fe and Zn, may be developed to accurately assess the bioavailability of the approach concentrations in the marine environment where particulate pool. To continue the progress towards a more they can become the limiting nutrient (Martin and Fitzwater, complete understanding of the biogeochemical cycling of 1988; Morel et al., 1994; Coale et al., 1996). Other studies Fe and Zn, it is necessary to examine the exchange and have demonstrated that variations in Fe and Zn concentra- partitioning of trace metals between the different physico- tions can cause shifts in the structure of phytoplankton chemical fractions within the dissolved and particulate communities in ocean surface waters (Hutchins et al., 1998; phases. Crawford et al., 2003; Leblanc et al., 2005). This investigation uses stable, low-abundance isotopes Much of the research on the bioavailability of Fe and of Fe and Zn as tracers in an incubation experiment that Zn has focused on the dissolved phase and several mech- simulated a vertical mixing event on the mid-continental anisms for Fe and Zn sequestration by marine phyto- shelf waters in the eastern Bering Sea, whereby the mixing plankton have been suggested (Hutchins et al., 1999; of subsurface waters containing macronutrients and Fe Ellwood and van den Berg, 2000; Barbeau et al., 2001; with nutrient-depleted surface waters induced a diatom Shaked et al., 2005; Lohan et al., 2005). There is consensus bloom. By adding the tracers to the dissolved phase, that the dissolved speciation of Fe and Zn is dictated by the changes in the isotopic ratio within different physico- presence of biogenic ligands in ocean surface waters, and chemical fractions over time were used to determine the these ligands chelate N99.9% of dissolved Fe (Rue and net removal of dissolved trace metals by the phytoplankton Bruland, 1995; Boye and van den Berg, 2000) and 98% of biomass and the regeneration rate of particulate trace dissolved Zn (Bruland, 1989; Ellwood and van den Berg, metals into the dissolved phase. Previous studies have used 2000). However, the dissolved phase is operationally stable, low-abundance isotopes as tracers to examine defined as the fraction of sample that passes through a 0.2 adsorption/desorption processes of particulate trace metals or 0.4 μm filter, but in actuality, this fraction contains a (Cu, Zn, Ni) in the estuarine environment (Gee and continuum of colloidal and soluble species within which Bruland, 2002) and bioaccumulation processes of Hg in trace metals may partition. Data collected from laboratory lakes (Pickhardt et al., 2002). The present investigation incubation experiments (Nishioka and Takeda, 2000; focuses on the contributions of particulate, colloidal, and Chen et al., 2003) and from field work in both open and soluble trace metals to the dynamics of trace metal cycling coastal ocean regimes (Nishioka et al., 2001a,b; Wu et al., in a shelf water system. The partitioning of the various 2001) suggest that colloids play an unique role in Fe isotopes in the different physico-chemical fractions reveals bioavailability, where the colloidal Fe fraction appears to the importance of chemically labile and refractory be preferentially removed by phytoplankton. These inves- particulate trace metals and colloidal trace metals in the tigations have demonstrated the need to distinguish bet- overall cycling and recycling of Fe and Zn within such a ween colloidal and soluble forms of Fe, and more research system. is needed to elucidate the mechanism(s) linking colloidal Fe and biological uptake, if any. 2. Material and methods Furthermore, very few studies have investigated the interactions between particulate trace metals and the dis- 2.1. Sample collection and handling solved phase, or the bioavailability of particulate trace metals with respect to phytoplankton. This is particularly The surface sample was collected using a clean surface important for Fe, which resides largely in the particulate pump system that included an all PTFE Teflon™ dia- pool within surface waters (Wells and Mayer, 1991; Sunda, phragm pump (Bruiser™, Osmonics, Minnetonka, MN) 2001). Previous research has demonstrated that a portion of and PFA Teflon™ tubing (Bruland et al., 2005). The the particulate trace metals is bioavailable to marine phy- sample inlet was mounted to a PVC fish system and toplankton in coastal shelf waters (Wells and Mayer, 1991; lowered to 3 m below the sea surface. The speed of the Wells et al., 2000), which suggests that understanding the ship was about 5 knots; thus, ensuring that the intake regeneration of particulate trace metals is an absolute would not be influenced by the ship's wake. The unfiltered necessity if the total amount of bioavailable trace metals is sample was delivered directly to an acid-cleaned and to be estimated. Regeneration of biogenic Fe, Mn, and Zn seawater-conditioned 55 L carboy in a Class 100 clean amongst grazers and planktonic prey has been investigated area; the carboy was filled to the halfway point. The 40 m with the use of radiotracers (Hutchins and Bruland, 1994; subsurface water sample was collected using a Teflon- Hutchins and Bruland, 1995), but the technique was unable coated, 30 L GO-Flo bottle (General Oceanics, Miami, to study the cycling of nonradioactive trace metals within FL) lowered on a Kevlar hydroline (Bruland et al., 1979). ARTICLE IN PRESS

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The subsurface sample was transferred through Teflon™ of 57Fe did not significantly change the amount of total Fe tubing to fill the carboy. The mixed sample was aliquoted in the sample (2% of total). to 2.5 L polycarbonate incubation bottles, which were After adding the isotopes to the individual samples, acid-cleaned with 3 M HCl (trace metal grade, Fisher the bottles were sealed by wrapping Parafilm and then Scientific, Pittsburgh, PA) for 2 weeks at room temper- electrical tape around the bottle opening. They were ature, filled with dilute 0.05 M HCl, and stored until use. placed in a Plexiglas incubator located on the top deck Prior to the experiment, the incubation bottles were con- of the ship, which was equipped with a flow-through ditioned by rinsing with clean surface seawater aboard seawater system that kept the incubator at surface water ship. All sample handling and preparation was conducted temperatures. Shade cloth was draped over the outside in a HEPA-filtered Class 100 laminar flow clean area. of the incubator to reduce the light level by 50%, which mimicked the sunlight irradiance as measured at depths 2.2. Reagents of 3–4 m in the Bering Sea shelf waters. The size-fractionation procedure was initiated by All reagents were diluted with Milli-Q™,exceptfor randomly taking a sample bottle from the incubator, quartz-distilled acids, which were diluted with quartz- rinsing it thoroughly with clean seawater, and placing it in distilled water (QH2O). The 0.5 M NH4C2H3O2 buffer a Class 100 clean area. Bottles were processed on days 1.0, (pH=5.8) was made by diluting saturated solution (19 M) 2.7, and 4.8. Two bottles were spiked with 57Fe and 68Zn that was prepared by bubbling NH3 through quartz- and filtered immediately to obtain a measurement at 0.01 distilled HC2H3O2 (QHAc). If necessary, the pH was days or 45 min. For each sampling day and experimental adjusted using quartz-distilled HNO3 (QHNO3)andNH3. group, duplicate measurements were made with exception The 1 M NH4Cl buffer (pH=8.9) was prepared from of a single data point for each experimental group on day aqueous NH3 (Optima grade, Fisher Scientific) and trace 1.0. Each bottle was sampled only once to minimize po- metal grade HCl. Buffer rinse solutions were prepared by tential contamination. diluting the individual buffers 10-fold. Trace metal standards of 10.0 μg/g were prepared from 1000 μg/mL 2.4. Sample filtration scheme stock solutions (SPEX, Edison, NJ; Fisher Scientific), diluted with 1 M HNO3 (trace metal grade, Fisher Sci- Polycarbonate track-etched (PCTE) membrane filters entific) and used to make working standards for both (47 mm dia., Nuclepore™, Whatman) were treated with particulate and dissolved trace metal analyses. 2 M HNO3 for 3 days, 6 M HCl for 3 days, and finally 57 The 100 μM standards of Fe (Isoflex USA, San with 1 M HNO3 (trace metal grade, Fisher Scientific) for Francisco, CA) and 68Zn (Isotec, Matheson, Miamis- 3 days. The PCTE filters were kept in the same poly- burg, OH) were prepared from oxide salts by dissolving ethylene container throughout the treatment, rinsed with the isotopically-enriched material in 1 M HCl. The Milli-Q™ water between treatments, and stored in 0.01 M primary standards were analyzed by mass sector, high HCl (trace metal grade, Fisher Scientific). resolution-inductively coupled plasma-mass spectrome- Each sample was passed through consecutive 10 μm try (HR-ICP-MS) and were verified to have an isotopic and 0.2 μm pore-size PCTE membrane filters mounted in abundance of 57Fe or 68Zn that were 97.6% and 98.7% Teflon™ filter sandwiches (Millipore, Bedford, MA) enriched, respectively. (Fig. 1). The unfiltered samples were also passed through an acid-cleaned 0.03 μm (200 kDa) polyethylene hollow 2.3. Incubations fiber flow-through filter (700 cm2, Sterapore™, Mitsu- bishi-Rayon, Tokyo, Japan) using a second outlet on the Two experimental groups (A and B) were used to study filtration apparatus. The large surface area within the the net transfer of 57Fe and 68Zn tracers from the soluble Sterapore™ filter capsule allows for a high-volume flow fraction to other trace metal pools (colloids and particles) and adequate flushing of the filter with sample prior to within the mixed water sample. Sample bottles in Ex- sample collection, similar to a 0.45 μm flow-through periment A were spiked with 57Fe such that the con- capsule, and protocol for maintaining the 0.03 μm filter centration of 57Fe increased by 0.90 nM. The incubation has been previously described by Nishioka et al. (2001a). bottles in Experiment B were spiked similarly with 57Fe, The soluble fraction was operationally-defined by the but 0.99 nM of 68Zn was also added. Experiment A was 0.03 μm nominal pore size; thus, the colloidal trace iso- used as the control for the added 68Zn in Experiment B, tope concentrations were not measured directly, but while the control for the 57Fe tracer was carried out by instead estimated by taking the difference between mea- measuring the more abundant isotope 56Fe. The addition sured values of the 0.2 μm and 0.03 μm filtrates. The ARTICLE IN PRESS

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apparatus, folded in eighths, placed in acid-cleaned 7 mL high-density polyethylene (HDPE) vials, and kept frozen. The extraction of the labile trace element fraction was performed by aliquoting 2 mL of 25% QHAc into the 7 mL vial and allowing contact with the filter for 2 h at room temperature (Chester and Hughes, 1967). The leachate and QH2O rinses of the filter were removed, placed into acid-cleaned quartz beaker, and heated to dryness (Landing and Bruland, 1987). The resulting residue was redissolved in 1 M QHNO3 and individual aliquots of this solution were spiked with an internal standard solution to give a 10 ng/g Ga and 1 ng/ g Rh final concentration in solution. Each filter and the associated refractory material from the particulate sample was then microwave-bomb digested with 2 mL of concentrated HNO3 and 50 μL of concentrated HF (trace metal grade, Fisher Scientific) in PTFE Teflon™ bombs equipped with pressure relief valves (Savillex, Minnetonka, MN). The final digest solutions were diluted to a 1 M HNO3 solution and spiked with internal standards that yielded a final concentration of 10 ng/g Ga and 1 ng/g Rh. Isotope concentrations were measured using a Thermo-Electron Element 1 magnetic sector HR-ICP- Fig. 1. Fractionation and analysis scheme for the dissolved phase, MS with a PFA Teflon™ spray chamber and PFA-ST which includes soluble (b0.03 μm) and colloidal (0.03–0.2 μm) nebulizer (Elemental Scientific, Omaha, NE). Plasma and fractions, and particulate phase, which was size-fractionated (0.2– μ N μ mass spectrometer parameters were optimized daily using 10 m, 10 m) and further separated by chemical lability (acetic 103 acid-leachable, refractory). All sample fractions were analyzed using the internal standard intensities of Rh in low resolution 69 HR-ICP-MS. and Ga in medium resolution. A Pump-pro MPL peris- taltic pump (Watson-Marlow Bredel, Wilmington, MA) headspace of the 2.5 L, unfiltered sample bottle was was employed to remove waste from the spray chamber pressurized with approximately 5 psi of N2 gas to promote and to pump particulate bomb digested and leached passive filtration of each sample. Aliquots of the 0.2 μm solutions directly to the nebulizer. The sample introduc- and 0.03 μm filtrate (125 mL) were collected in acid- tion system, including autosampler (ASX-100, CETAC cleaned low-density polyethylene (LDPE) bottles and Technologies, Inc., Omaha, NE) and μ-Sampler (Thermo- acidified to pH=1.7 (4 mL of sub-boiled, quartz-distilled Electron), were enclosed and under positive pressure 6 M HCl/L of seawater). All filtrate samples were stored using a HEPA filter. Data were acquired in low resolution for at least 3 months prior to being analyzed. Unfiltered (111Cd) and medium resolution (31P, 56Fe, 57Fe, 59Co, samples were collected for the determination of chloro- 63Cu, 66Zn, 68Zn) modes. The intensities of these ana- phyll a, particulate organic carbon (POC), particulate or- lytes, relative to the internal standard, were quantified ganic nitrogen (PON), and biogenic silica (BSi). These using an external calibration curve. aliquots were filtered and analyzed as described by Dissolved trace metals (Fe, Zn, Cu, Co, Cd) were Leblanc et al. (2005). Filtered samples (b0.2 μm) were analyzed using chelating resin, column partitioning with collected for dissolved macronutrients (nitrate, silicate, HR-ICP-MS (CRCP-HR-ICP-MS) (Willie et al., 1998; and phosphate) and measured onboard ship with a Warnken et al., 2000; Willie et al., 2001; Ndungu et al., Lachat QuickChem 8000 Flow Injection Analysis 2003). The acidified filtrate samples were UV-irradiated system using standard methods (Parsons et al., 1984). off-line prior to analysis to break down organic chelators present in the seawater (Ndungu et al., 2003). The CRCP- 2.5. Trace metal analyses HR-ICP-MS methodology employed Toyopearl AF- Chelate 650 M (Tosohaas, Montgomeryville, PA) as the Once the sample filtration was complete, the 47 mm chelating iminodiacetate (IDA) resin, and the samples diameter PCTE filters were removed from the filtering were buffered to a pH of 5.5 prior to being loaded onto the ARTICLE IN PRESS

M.P. Hurst, K.W. Bruland / Marine Chemistry xx (2006) xxx–xxx 5 resin column. To remove salts within the sample matrix, of 4 μg/L, and the diatom community was supported by the column was rinsed with an NH4C2H3O2 buffer the transport of macronutrients across the pycnocline solution prior to the elution of the analytes with 1 M from the subsurface waters. The subsurface water QHNO3. All trace metal analyses were performed in concentrations of nitrate, silicic acid, and phosphate medium resolution mode with an NH4C2H3O2 buffer, were 6 μM, 34 μM, and 1.5 μM, respectively. The except Cd, which was performed at low resolution with an mixed seawater sample used in the incubation ex- NH4Cl buffer. UV-oxidized seawater, which is relatively periment had measured concentrations for nitrate, silicic free of trace metals and complexing organic ligands acid, and phosphate of 3 μM, 18 μM, and 0.8 μM, (Donat and Bruland, 1988), was acidified with QHCl to respectively. the same extent as the samples and used for preparing The incubation experiment was designed to simulate a working standards. The average height of the time re- major storm-induced mixing event, a primary mechanism solved peak was quantified for each analyte using the for supplying nutrients to the surface waters in a shelf standard curve. water regime. By mixing the two water types in a 1:1 ratio, macronutrients and trace metal micronutrients originally 2.6. Certified values and blanks in the subsurface waters, in both dissolved and particulate forms, were introduced to the diatom community present The CRCP-HR-ICP-MS method determined total in the nitrate-limited surface waters. During the course of dissolved Fe and Zn in CASS-3 (Coastal Atlantic the experiment, there was an insufficient amount of nitrate Seawater Standard, National Research Council of Canada) to promote optimal growth within the diatom community − to be 21.3±0.9 nM (certified value=22.6±3.0 nM) for Fe by day 4.8. The limiting [NO3 ] is reflected in the Chl a and 18.4±1.5 nM (certified value=19.0±3.8 nM) for Zn. data, where there is a minimal increase between days 2.7 During the analysis of filtrate samples, the average method and 4.8 (Fig. 3). Particulate organic C/N ratios were blank values (n=6) over the course of the analytical run relatively stable at values between 7.2 and 7.4, except for for the individual isotopes were 0.031±0.008 nM (56Fe), day 4.8, where the ratio increased to 9.8. The BSi–Chl a 0.005±0.001 nM (57Fe), 0.028±0.008 nM (66Zn), and ratio remained steady at 30 g/g until day 4.8, where the 0.031±0.004 nM (68Zn). Although there is not a standard value then increased to 47 g/g. reference material for phytoplankton biomass, recoveries within the 95% confidence intervals were obtained for river sediment (SRM 1645, National Institute of Standards and Technology, USA) and marine sediment (BCSS-1, National Research Council of Canada) using the microwave-bomb digestion procedure.

3. Results and discussion

3.1. Hydrographic and nutrient data at shelf water sampling site

The sample site used in the incubation experiment was at 57°42.18N and 168°42.87W on the unusually broad mid-continental shelf in the eastern Bering Sea aboard the R/V Kilo Moana during August 20–25 in 2003. The water column profile revealed an abrupt vertical boundary between the surface mixed layer and subsurface bottom waters at a depth of 25 m (Fig. 2). The surface waters had a temperature of 11 °C and salinity of 32.0, while subsurface waters had a temperature of 4.5 °C and a salinity of 32.2. The concentrations for nitrate, silicic acid, and phosphate in the b μ b μ ∼ μ surface water were 0.1 M, 1 M, and 0.1 M, Fig. 2. Depth profile at the sample station on the mid-continental shelf respectively. The fluorescence maximum at 15 m depth in the eastern Bering Sea, with ⋄ representing temperature (C°) and ● corresponded to a chlorophyll a (Chl a) concentration representing chlorophyll a concentration (μg/L). ARTICLE IN PRESS

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The determination of isotopic ratios using HR-ICP- MS in medium resolution mode has been proven to be as accurate and precise as ratios obtained using thermal ionization mass spectrometry (TIMS), the standard technique for determining isotopic ratios (Roehl et al., 1995; Ingle et al., 2004). However, there still remain response biases in ICP-MS that can cause the measured isotopic ratio to deviate slightly from the natural abundance isotopic ratio, prompting the need to correct for these biases during each analytical run using solutions containing a natural abundance of the isotopes (Table 1). For Fe, the measured ratio was estimated at 0.0243 for particulate 57Fe/56Fe and 0.0241 for dissolved 57Fe/56Fe, yielding a small percent difference relative Fig. 3. The chlorophyll a concentration (μg/L) and nitrate concentra- to the reported natural abundance ratio of 0.0231. The 68 66 tion (μM) over the course of the incubation experiment. The circles measured values for the Zn/ Zn ratio in the par- − represent [Chl a] and the diamonds represent [NO3] (solid symbols ticulate and dissolved phase were 0.669 and 0.663, represent Experiment A, open symbols represent Experiment B). respectively, and compared well to the natural abundance ratio for 68Zn/66Zn of 0.671.

3.2. Isotope ratio method 3.3. Trace metals (all isotopes)

The isotope ratio method used in this investigation Ambient dissolved trace metal concentrations in the takes advantage of differences in the natural isotopic surface waters at the mid-shelf station were measured at abundance of 56Fe (91.75%) compared to 57Fe (2.12%) 0.22 nM for Fe and 0.25 nM for Zn, while the total and 66Zn (27.90%) compared to 68Zn (18.75%). Low- particulate Fe and Zn concentrations were 7.9 nM and abundance isotopes (97.6% purity 57Fe and 98.7% pu- 0.18 nM, respectively. The dissolved concentrations in the rity 68Zn) were initially added to the soluble fraction and subsurface waters were 4.4 nM for Fe and 1.6 nM for Zn, exchanged with Fe and Zn in the colloidal and par- and the total particulate Fe and Zn were 89 nM and ticulate pools, resulting in a shift of the isotopic ratio 0.81 nM, respectively. The mixed sample had an initial in each trace metal pool over time. To achieve a mea- dissolved Fe concentrations of 2.3 nM and a total par- surable shift in the isotopic ratio, it is important to have ticulate Fe concentration of 50 nM. For Zn, the dissolved enough trace metals in the particulate pool to exchange and particulate concentrations in the mixture were with the low-abundance isotope residing in the soluble 0.83 nM and 0.49 nM, respectively. and colloidal fractions. Also, it is optimal to use an The changes in dissolved and particulate Fe concen- isotope of low abundance as the tracer and compare it to trations over the course of the experiment are illustrated in the isotope of highest abundance so that the enrichment Fig. 4. These data include the total of all Fe isotopes and produces a maximum shift in the isotopic ratio. represent the average of replicates in Experiments A and

Table 1 Isotopic ratios measured±%RSD for Fe and Zn in different standards and samples Trace metal concentration and sample matrix Isotopic ratio Fe Zn Natural abundance ratio 0.0231 0.671 Particulate

1 M HNO3 Fe (170 nM), Zn (75 nM) 0.0242±1.3% 0.668±1.4% n=9 Digested particulate sample Fe (1.3 μM), Zn (23 nM) 0.0243±0.5% 0.660±2.6% n=17 Leached particulate sample Fe (1.1 μM), Zn (50 nM) 0.0243±1.7% 0.679±2.0% n=15 Dissolved UVSW Fe (0.55 nM), Zn (not detected) 0.0248±7.2% ND n=13 UVSW standard (pH=1.7) Fe (1.6 nM), Zn (1.5 nM) 0.0241±5.8% 0.659±3.9% n=9 UVSW standard (pH=1.7) Fe (7.3 nM), Zn (6.1 nM) 0.0241±4.3% 0.667±4.0% n=11 ARTICLE IN PRESS

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B. The Fe data were combined because there was no statistical difference in assimilation or regeneration of Fe due to the addition of Zn (Experiment B). Mass balance for total Fe was observed with a standard deviation of ±2.1 nM (Fig. 4a); however, adsorption of Fe to the walls of the container may have occurred at levels less than the standard error. The particulate Fe was primarily found in the N10 μmfraction(65–80%) (Fig. 4c) and in the refractory fraction (∼80%) (Fig. 4d). Fig. 4billustrates the increase in dissolved Fe upon addition of the 57Fe and the subsequent drawdown. Nearly 40% of the dissolved Fe was in the colloidal fraction initially, but the colloidal Fe was rapidly removed relative to the gradual disap- pearance of soluble Fe species. This removal of colloidal Fe has been reported previously in laboratory incubation experiments and in coastal ocean regimes under phytoplankton bloom conditions (Nishioka and Takeda, 2000; Nishioka et al., 2001b; Chen et al., 2003). The mass balance for Zn in Experiments A and B is plotted in Fig. 5a and e, respectively. On inspection, variations in the particulate Zn concentration and low levels of analyte could account for the fluctuations observed in the mass balance plots. The added 68Zn to the dissolved fraction of Experiment B contributed to an increase in the soluble Zn fraction (Fig. 5f). The soluble Zn in both Experiments A and B (Fig. 5b and f) decreased over time and was assimilated into the N10 μm and acetic- acid leachable particulate fractions (Fig. 5c–d, g–h). The colloidal Zn concentration was relatively constant in both Experiments A and B over time (Fig. 5b and f). The concentrations of Cu, Co, and Cd were also measured in the dissolved and soluble fractions (Fig. 6). There were no measurable differences between Experi- ments A and B for these trace metals and the mean values are presented. Dissolved Cu and Co concentrations were constant throughout the experiment, with values of 2.6– 2.7 nM and 0.32 nM, respectively. The Cd was removed from the dissolved phase and a decrease from 0.42 nM to 0.25 nM was observed. Approximately 50% of the Cu was in the colloidal fraction while essentially all the Co and Cd was in the form of soluble species.

3.4. Metal-to-carbon ratios

By considering the Redfield formula (C106N16P1)for living plankton biomass and the removal of dissolved trace metals with 3 μM of nitrate by the diatom biomass Fig. 4. The Fe results from Experiment A+B (average of replicates) in over the 5-day experiment, the metal-to-carbon (Me/C) the different fractions: (a) total Fe (particulate and dissolved), (b) ratios were estimated for Fe (100 μmol/mol C) in dissolved Fe (soluble and colloidal), (c) total particulate Fe (0.2– Experiment (A+B), Zn (25 μmol/mol C) in Experiment μ N μ 10 m, 10 m), and (d) total particulate Fe (acetic acid-leachable, A, Zn (50 μmol/mol C) in Experiment B, and Cd refractory). The concentration in the initial sample mixture is labeled (10 μmol/mol C) in Experiment (A+B). The 100 μmol Femix.. ARTICLE IN PRESS

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Fig. 5. The Zn results from Experiment A (a–d) and B (e–h) in the different fractions: (a, e) total Zn (particulate and dissolved); (b, f) dissolved Zn (soluble and colloidal); (c, g) total particulate Zn (0.2–10 μm, N10 μm); and (d, h) total particulate Zn (acetic acid-leachable, refractory). The concentration in the initial sample mixture is labeled Znmix.. ARTICLE IN PRESS

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the elevated Cd/C ratio, which was likely due to the interreplacement of Zn with Cd (Sunda and Huntsman, 2000).

3.5. Zinc isotopes

The 66Zn results from Experiment A show a net transfer of 66Zn from the dissolved to the particulate phase (Fig. 7). Initially, the 0.23 nM of dissolved 66Zn was over 80% soluble. A decrease in the soluble 66Zn

Fig. 6. Dissolved and soluble trace metal concentrations from A+B (average of replicates) for: (a) Cu, (b) Co, and (c) Cd.

Fe/mol C value was in the range of previously reported Fe/C ratios of 50–300 μmol/mol C for coastal diatoms (Sunda and Huntsman, 1995b; Bruland et al., 2001), which were also measured in Fe-replete conditions and resulted in luxury uptake of Fe by diatoms. The higher Zn/ C ratio estimated for Experiment B (+Zn) relative to Experiment A was consistent with ratios reported for coastal diatoms by Sunda and Huntsman (1995a),and demonstrated that an increase in [Zn2+] causes an increase 66 in both the assimilation rate of Zn and the Zn/C ratio. Fig. 7. The Zn results from Experiment A in the different fractions: (a) dissolved 66Zn (soluble and colloidal), (b) total particulate 66Zn Finally, the decrease in dissolved Zn to subnanomolar (0.2–10 μm, N10 μm), and (c) total particulate 66Zn (acetic acid- levels during the last 2 days in both Experiments A and B leachable, refractory). The concentration in the initial sample mixture 66 resulted in the observed drawdown of dissolved Cd and is labeled Znmix.. ARTICLE IN PRESS

10 M.P. Hurst, K.W. Bruland / Marine Chemistry xx (2006) xxx–xxx fraction was observed while the colloidal fraction In Experiment B, with the addition of dissolved 68Zn, displayed minimal change over time (Fig. 7a). The nearly all of the added 68Zn remained in the soluble largest increase in 66Zn occurred in the N10 μm fraction after 45 min (0.01 days) (Fig. 8). After 1 day, leachable particulate fraction (Fig. 7c), and was a result ∼50% of the soluble 68Zn had partitioned to the colloids of increasing biomass spurred by the introduction of or became associated with particulate matter. Drawdown nitrate and Fe (Fig. 7b). The 68Zn in Experiment A of the dissolved 68Zn concentration was primarily due to demonstrated behavior similar to the 66Zn (data not assimilation into the N10 μm leachable particulate shown). This net transfer of Zn can be attributed to active fraction, while the amount of refractory particulate 68Zn uptake, although adsorption to the cell surfaces may also was relatively unchanged over the course of the be a significant factor. experiment (Fig. 8b–c). By days 2.7 and 4.8, the particulate 68Zn was 91% leachable, which agrees with the value of 96% that was measured in particulate samples collected within a diatom bloom located near the continental slope in the Bering Sea. To better understand Zn cycling in the experiment, the 68Zn/66Zn isotopic ratios for both the dissolved and particulate fractions in Experiment B were plotted with respect to time (Fig. 9). The new overall 68Zn/66Zn isotopic ratio in both the dissolved and particulate phases was predicted to shift to 3.13 upon addition of 68Zn and is noted as the adjusted isotopic ratio in Fig. 9. After 45 min, the 68Zn/66Zn isotopic ratio value in the dissolved phase was 3.8±0.6 and it slightly decreased over time to a value of 3.4±0.6 by 4.8 days (Fig. 9a). The implication of the 68Zn/66Zn isotopic ratio in the dissolved phase remaining relatively constant is that regeneration of 66Zn from the particulate phase did not occur in any significant amount, a consequence of the small particulate Zn pool within the sample. The use of 70Zn with a 0.6% natural abundance may have allowed for a better resolution of regenerated Zn, if any, by creating a larger perturbation of the natural abundance ratio in the dissolved phase. In contrast, the 68Zn/66Zn isotopic ratio in the particulate phase increased mark- edly from the natural abundance value of 0.67 to values of 2.7±0.4 on day 2.7 and 2.4±0.6 on day 4.8 (Fig. 9b). This change can be attributed to the assimilation of 68Zn into the N10 μm size-fraction and the low concentration of Zn originally in the particulate phase. Finally, the isotopic ratios in Experiment A, with no addition of 68Zn, fluctuated near the natural abundance ratio of 0.67, and the mean value for both the particulate and dissolved phases was estimated at 0.67±0.09. The large standard deviation can be attributed to the low levels of both 66Zn and 68Zn and the low ionization efficiency in the plasma during analysis.

3.6. Iron isotopes Fig. 8. The 68Zn results from Experiment B in the different fractions: 68 68 (a) dissolved Zn (soluble and colloidal), (b) total particulate Zn N μ (0.2–10 μm, N10 μm), and (c) total particulate 68Zn (acetic acid- Unlike Zn, where assimilation into the 10 m size- leachable, refractory). The concentration in the initial sample mixture fraction was the overwhelming factor that affected the 68 is labeled Znmix.. partitioning of Zn, the Fe results indicate a more ARTICLE IN PRESS

M.P. Hurst, K.W. Bruland / Marine Chemistry xx (2006) xxx–xxx 11

Fig. 9. The 68Zn/66Zn isotopic ratio over time in Experiment B for (a) the dissolved phase and (b) the total particulate phase. The initial sample 68 mixture prior to adding Zn is labeled Znmix.. The natural abundance ratio (solid line) and adjusted ratio (dotted line) are included. dynamic cycling between the various trace metal pools. corresponds to the observed increase in biomass, and Fig. 10a and d illustrate the concentrations of 56Fe and these data suggest that the chemically labile fraction 57Fe in the dissolved phase over time, respectively, and (possibly adsorbed species on the surface of cells) was are separated into both colloidal and soluble fractions. incorporated into the diatom biomass. The 25% acetic The colloidal 56Fe decreased from 0.72 nM in the initial acid leach has been demonstrated to be a mild extraction mixed sample to 0.08 nM by day 4.8. Similarly, the of labile Fe associated with biomass (Landing and colloidal 57Fe fraction decreased from 0.43 nM after the Bruland, 1987) and samples collected on the continental spike to 0.01 nM on day 4.8. The decrease in colloidal slope of the Bering Sea during a large diatom bloom 56Fe, and most notably 57Fe, was concurrent with no contained only 6% leachable particulate Fe. Thus, the appreciable change in colloidal Zn or Cu. This may be leachable Fe solubilized with the 25% acetic acid leach due to a separate aggregation process for Fe, possibly as does not appear to solubilize the bulk of the biogenic Fe (hydr)oxides, relative to Zn and Cu; however, the particulate Fe associated with the larger diatoms. On day decrease may also suggest that a dissolution mechanism 4.8, there was an increase in chemically labile 56Fe in involving photolysis or sequestration of colloidal Fe by the N10 μm size-fraction, which corresponded to a strong Fe(III) chelators in the soluble phase is at work decrease in refractory 56Fe. This was a reversal of the (Barbeau et al., 2001; Borer et al., 2005), with overall trend for particulate 56Fe discussed above, and subsequent assimilation of this soluble Fe by the may be associated with the nitrate-limiting conditions plankton community. The soluble species also showed and the diatom community becoming senescent (Fig. 3). a decrease in both 56Fe and 57Fe concentrations over This nitrate limitation may have created conditions time, yet the drawdown was less dramatic than that whereby the regeneration of refractory 56Fe occurred observed for the colloidal Fe. faster than the formation of refractory 56Fe in the Due to the overwhelming concentration of particulate biomass. Fe relative to the dissolved Fe in the initial seawater As observed with the 56Fe, the dissolved 57Fe was mixture, a change in the total particulate 56Fe concen- incorporated into the N10 μm and refractory 57Fe fraction tration could not be quantified; however, changes in the (Fig. 10d–f). The addition of 0.90 nM 57Fe to the dis- physico-chemical fractions were observed. The net solved phase resulted in a minor increase (∼0.2 nM) in transfer of 1.2 nM dissolved 56Fe into the particulate particulate 57Fe after 45 min, an increase of 0.7 nM by 56Fe was within the standard error of ±2.1 nM for the 1 day, and 0.9 nM by 4.8 days. Due to the relatively small particulate 56Fe concentration (Fig. 10b). Over the amount of 57Fe in the particulate fraction at the start of the initial 2.7 days, a decrease in the leachable particulate experiment, the rate of 57Fe assimilation into the N10 μm 56Fe and an increase in refractory particulate 56Fe were and refractory fractions was much greater than the measured, where the increase in refractory 56Fe regeneration rate. This resulted in a continual increase in occurred in the N10 μm size-fraction (Fig. 10c). This refractory 57Fe over the course of the experiment. The ARTICLE IN PRESS

12 M.P. Hurst, K.W. Bruland / Marine Chemistry xx (2006) xxx–xxx rapid removal of 57Fe that occurred on the timescale of of 8 decrease, while the 56Fe decreased from 2.2 to 1daycanbeattributedtobothbiologicaluptakeand 1.0 nM, which decreased by only a factor of 2. These adsorption processes. The formation of Fe(III) (hydr) differences in net removal of 57Fe and 56Fe from the oxides may have occurred upon addition of 57Fe to the dissolved phase greatly contribute to the trends observed seawater samples, which would explain the initial loss of in the dissolved and particulate isotopic ratios plotted in tracer from the dissolved phase. Fig. 11. The addition of dissolved 57Fe shifted the natural 57Fe/56Fe isotopic abundance ratio of 0.023 to a new 3.7. Significance of the 57Fe/56Fe isotopic ratio adjusted isotopic ratio of 0.043 for the entire solution of dissolved and particulate Fe (Fig. 11a–b). The 57Fe/56Fe Over the course of the experiment, it was observed that isotopic ratio in the dissolved phase increased by over an the dissolved 57Fe decreased from 0.8 to 0.1 nM, a factor order-of-magnitude and was at a value of 0.33 after

Fig. 10. The 56Fe (a–c) and 57Fe (d–f) results from A+B (average of replicates) in the different fractions: (a) dissolved 56Fe (soluble and colloidal), (b) total particulate 56Fe (0.2–10 μm, N10 μm), and (c) total particulate 56Fe (acetic acid-leachable, refractory), (d) dissolved 57Fe (soluble and colloidal), (e) total particulate 57Fe (0.2–10 μm, N10 μm), and (f) total particulate 57Fe (acetic acid-leachable, refractory). The concentration in the initial sample 56 57 mixture is labeled Femix. and Femix., respectively. ARTICLE IN PRESS

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45 min (0.01 days) and then subsequently decreased to a active component of Fe cycling in this system. Over the value of 0.09 by day 4.8 (Fig. 11a). This isotopic ratio course of the first day, the leachable particulate Fe behaves leveled off near an asymptote well above the adjusted similar to particulate Zn, with the 57Fe/56Fe isotopic ratio 57Fe/56Fe isotopic ratio of 0.043 for the entire system, a rapidly approaching the adjusted isotopic ratio. These consequence of the high concentration of non-exchanged results suggest that Fe in the dissolved phase and leachable refractory Fe in the particulate phase. In order for the particulate phase undergoes exchange on the timescale of isotopic ratio in the dissolved phase to reach the adjusted approximately 1 day. However, the leachable particulate value, all the particulate Fe would have to be regenerated. 57Fe/56Fe ratio does not continue to approach the adjusted In contrast, the particulate phase did approach the adjusted 57Fe/56Fe ratio value, but instead decreased on days 2.7 57Fe/56Fe isotopic ratio of 0.043. This value is reached and 4.8 (Fig.11c). This decrease can be attributed to the because the majority of 56Fe and 57Fe (98% of total Fe in simultaneous occurrence of several processes involving solution) resides in the particulate phase at days 2.7 and the refractory particulate pool. These processes include: 4.8 (Fig. 4a). Additionally, essentially all the initial 57Fe (1) the regeneration of operationally-defined refractory spike became incorporated into the biomass (N10 μm particulate 56Fe that results in the concurrent formation of size-fraction) by the end of the experiment (Fig. 10d). leachable particulate 56Fe, (2) the regeneration of Lastly, it was of interest to plot the 57Fe/56Fe ratio over refractory particulate 56Fe exceeding the assimilation of time in terms of the leachable 56Fe and 57Fe particulate leachable particulate and dissolved 56Fe, and (3) the concentrations (Fig. 11c). Previous studies have suggested conversion of leachable particulate 57Fe to refractory that the readily leachable forms of Fe are bioavailable to particulate 57Fe by the plankton community. phytoplankton and that refractory Fe, as defined by the particulate Fe remaining after the 25% acetic acid leach, is 3.8. Net removal of trace metals assumingly not bioavailable (Bruland et al., 2001; Fitz- water et al., 2003). By only including the 57Fe and 56Fe The complex conditions found in natural waters make associated with the dissolved and leachable particulate it difficult to isolate trace metal removal that is solely due phases, an adjusted isotopic ratio of 0.10 was calculated. to biological uptake. The processes that influence the This is a reasonable way of plotting the changing isotopic assimilation of trace metals into the particulate pool ratios over time if refractory particulate Fe, as defined by include sorption processes, such as adsorption and the 25% acetic acid leach, was not becoming regenerated. precipitation, as well as the active uptake through However, Fig. 11c confirms that a substantial component biological processes (Sposito, 1986). Also, the rate of of the refractory particulate Fe is regenerated and is an active uptake by phytoplankton has been shown to be

Fig. 11. The 57Fe/56Fe isotopic ratio over time in Experiment A+B (average of replicates) for (a) the dissolved phase, (b) the total particulate phase, 57 and (c) the leachable particulate phase. The initial sample mixture prior to adding Fe is labeled Femix.. The natural abundance ratio (solid line) and adjusted ratio (dotted line) are included. ARTICLE IN PRESS

14 M.P. Hurst, K.W. Bruland / Marine Chemistry xx (2006) xxx–xxx dependent upon the intercellular Me/C ratio and the a novel approach for quantifying the exchange of trace inorganic trace metal concentrations ([Fe′] and [Zn′]) in metals between different physico-chemical fractions the dissolved phase (Sunda and Huntsman, 1995a, found in marine systems. This study provided a better Sunda and Huntsman, 1995b). The presence of metal- understanding of Fe and Zn cycling in a shelf water binding ligands in the dissolved phase (Gee and Bruland, environment during a diatom bloom induced by vertical 2002) and the changing concentration of particles that mixing of the water column. occurs during a phytoplankton bloom (Jannasch et al., The regeneration of Zn from the particulate pool was 1988) also contribute to this complexity. not a significant part of Zn cycling in the mid-shelf Although multiple variables influence the net regime, largely due to the low concentrations of Zn in the removal of trace metals from the dissolved phase to particulate phase relative to the dissolved phase. The the particulate phase, the net and overall removal of Fe assimilation of soluble Zn by the N10 μm biota was the and Zn can be estimated using the low-abundance dominant process contributing to the net removal of Zn isotope approach. Using a pseudo-first order rate from the dissolved phase, while the colloidal Zn con- expression, the average rate constants for the net centration remained constant and did not appear to be removal of 66Zn and 68Zn were essentially the same in bioactive. both Experiment A (k=−0.21±0.01 day− 1) and Exper- Two main observations were deduced from the iment B (k=−0.22±0.04 day− 1), respectively, indicat- exchange of Fe between the soluble, colloidal, and ing that regeneration did not significantly contribute to particulate pools: (1) there was active regeneration of the net removal of Zn from the dissolved phase. If particulate Fe that affected the net removal of Fe from regeneration had played an important role in the removal the dissolved phase, and (2) there was rapid drawdown of Zn, the rate constant for 66Zn drawdown would have and apparent utilization of colloidal Fe by the diatom been substantially smaller in Experiment B. community (N10 μm size-fraction). The soluble and In contrast, the net removal rate constant for 57Fe leachable particulate fractions of Fe also decreased over from the dissolved phase (k=−0.57 day− 1) was 3.6 time and were converted to refractory particulate Fe, times that of 56Fe (k=−0.16 day− 1). Assuming there is but did so at a much slower rate than the colloidal about 20 nM of truly labile particulate 56Fe (roughly fraction. estimated as twice the 25% acetic acid leachable The experimental data demonstrate the importance of concentration), that the particulate 57Fe concentration particulate Fe in the water column and its role as a is negligible (reasonable assumption given the amount bioavailable source; however, further development of of 57Fe added to the dissolved phase), and the use of analytical methodology is needed to accurately distin- first-order rate kinetics to construct a simple model of guish between the bioavailable particulate Fe and the data (d[Fediss.]/dt=−[Fediss.]k1 +[Fepart.]k2), it was refractory Fe pools. Additionally, it is important to determined that ∼0.6 nM day− 1 of particulate Fe was distinguish between colloidal and soluble Fe within the − 1 regenerated back to the dissolved phase (k1 =0.57 day dissolved phase given that their behavior within Fe − 1 and k2 =0.030 day ). Thus, the net removal of 2.0 nM cycling was observed to be different. This includes more of dissolved Fe over the 4.8-day period was the result of research in determining the most appropriate partition 2.9 nM of regenerated particulate Fe and an overall cutoff for small particulate, colloidal, and soluble forms removal of 4.9 nM of dissolved Fe. The difference of Fe. Progress in this area could be advanced by better between the overall and net removal of Fe during the characterizing the colloids and determining whether experiment, with the overall removal being 2.4 times the they are ill-defined entities or biogenic macromolecules net removal, demonstrates the dynamic cycling of Fe providing a distinct function for the biology. between the dissolved and particulate phases and the importance of particulate Fe as a bioavailable source. Acknowledgements The contribution of particulate Fe to the dissolved phase within the shelf water system was substantial, especially The authors thank the National Science Foundation when the Fe requirements for primary productivity are for funding (grants OCE-0238347 and OCE-0137085). considered (Sunda and Huntsman, 1995b). We thank Bettina Sohst for the dissolved macronutrient data, Karine Leblanc and Clint Hare for POC, PON, 4. Conclusion BSi, and Chlorophyll data. We appreciate the advice and expertise of Rob Franks during the trace metal analyses The use of stable, low-abundance, trace metal using the ICP-MS. We also thank Geoffrey Smith for isotopes in shipboard incubation experiments presents assisting in sample collection. ARTICLE IN PRESS

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