Matthew P. Hurst a,, Kenneth W. Bruland. 1. Introduction

Transcription

1 Marine Chemistry 103 (2007) 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 Available online 23 August 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 ( μm), and particulate ( 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 57 Fe (+0.90 nm) and 68 Zn (+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 plasmamass spectrometry (HR-ICP-MS). More than 50% of the added 57 Fe partitioned to the colloidal fraction within 45 min of adding the tracer. Both the 57 Fe and 56 Fe colloidal fraction were removed from the dissolved phase at a faster rate than the soluble Fe fraction. In contrast, the colloidal 66 Zn and 68 Zn concentrations remained constant over the 5-day experiment, suggesting a unique removal mechanism for colloidal Fe. The net removal of dissolved 57 Fewasobservedtobe3to4timesmorerapidthandissolved 56 Fe, 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 68 Zn tracer resided in the soluble fraction and was assimilated by the diatom biomass (N10 μm size-fraction) at the same rate as 66 Zn. 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 Elsevier B.V. All rights reserved. Keywords: Iron; Zinc; Seawater; Inductively coupled plasma-mass spectrometry; Stable isotopes 1. Introduction Corresponding author. Present address. Department of Chemistry, Humboldt State University, 1 Harpst Street, Arcata, CA 95521, USA. Tel.: ; fax: address: mph3@humboldt.edu (M.P. Hurst). The development of sensitive analytical methods and trace metal clean techniques has allowed progress in defining the role of trace metals as an important factor influencing primary productivity in marine systems (Morel /$ - see front matter 2006 Elsevier B.V. All rights reserved. doi: /j.marchem

2 212 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) et al., 2003). These advancements have led to the understanding that bioactive trace metals, such as Fe and Zn, may approach concentrations in the marine environment where they can become the limiting nutrient (Martin and Fitzwater, 1988; Morel et al., 1994; Coale et al., 1996). Other studies have demonstrated that variations in Fe and Zn concentrations can cause shifts in the structure of phytoplankton communities in ocean surface waters (Hutchins et al., 1998; Crawford et al., 2003; Leblanc et al., 2005). Much of the research on the bioavailability of Fe and Zn has focused on the dissolved phase and several mechanisms for Fe and Zn sequestration by marine phytoplankton have been suggested (Hutchins et al., 1999; Ellwood and van den Berg, 2000; Barbeau et al., 2001; Shaked et al., 2005; Lohan et al., 2005). There is consensus that the dissolved speciation of Fe and Zn is dictated by the presence of biogenic ligands in ocean surface waters, and these ligands chelate N99.9% of dissolved Fe (Rue and Bruland, 1995; Boye and van den Berg, 2000) and 98% of dissolved Zn (Bruland, 1989; Ellwood and van den Berg, 2000). However, the dissolved phase is operationally defined as the fraction of sample that passes through a 0.2 or 0.4 μm filter, but in actuality, this fraction contains a continuum of colloidal and soluble species within which trace metals may partition. Data collected from laboratory incubation experiments (Nishioka and Takeda, 2000; Chen et al., 2003) and from field work in both open and coastal ocean regimes (Nishioka et al., 2001a,b; Wu et al., 2001) suggest that colloids play an unique role in Fe bioavailability, where the colloidal Fe fraction appears to be preferentially removed by phytoplankton. These investigations have demonstrated the need to distinguish between colloidal and soluble forms of Fe, and more research is needed to elucidate the mechanism(s) linking colloidal Fe and biological uptake, if any. Furthermore, very few studies have investigated the interactions between particulate trace metals and the dissolved phase, or the bioavailability of particulate trace metals with respect to phytoplankton. This is particularly important for Fe, which resides largely in the particulate pool within surface waters (Wells and Mayer, 1991; Sunda, 2001). Previous research has demonstrated that a portion of the particulate trace metals is bioavailable to marine phytoplankton in coastal shelf waters (Wells and Mayer, 1991; Wells et al., 2000), which suggests that understanding the regeneration of particulate trace metals is an absolute necessity if the total amount of bioavailable trace metals is to be estimated. Regeneration of biogenic Fe, Mn, and Zn amongst grazers and planktonic prey has been investigated with the use of radiotracers (Hutchins and Bruland, 1994; Hutchins and Bruland, 1995), but the technique was unable to study the cycling of nonradioactive trace metals within the experimental setup. Adequate methodology has yet to be developed to accurately assess the bioavailability of the particulate pool. To continue the progress towards a more complete understanding of the biogeochemical cycling of Fe and Zn, it is necessary to examine the exchange and partitioning of trace metals between the different physicochemical fractions within the dissolved and particulate phases. This investigation uses stable, low-abundance isotopes of Fe and Zn as tracers in an incubation experiment that simulated a vertical mixing event on the mid-continental shelf waters in the eastern Bering Sea, whereby the mixing of subsurface waters containing macronutrients and Fe with nutrient-depleted surface waters induced a diatom bloom. By adding the tracers to the dissolved phase, changes in the isotopic ratio within different physicochemical fractions over time were used to determine the net removal of dissolved trace metals by the phytoplankton biomass and the regeneration rate of particulate trace metals into the dissolved phase. Previous studies have used stable, low-abundance isotopes as tracers to examine adsorption/desorption processes of particulate trace metals (Cu, Zn, Ni) in the estuarine environment (Gee and Bruland, 2002) and bioaccumulation processes of Hg in lakes (Pickhardt et al., 2002). The present investigation focuses on the contributions of particulate, colloidal, and soluble trace metals to the dynamics of trace metal cycling in a shelf water system. The partitioning of the various isotopes in the different physico-chemical fractions reveals the importance of chemically labile and refractory particulate trace metals and colloidal trace metals in the overall cycling and recycling of Fe and Zn within such a system. 2. Material and methods 2.1. Sample collection and handling The surface sample was collected using a clean surface pump system that included an all PTFE Teflon diaphragm pump (Bruiser, Osmonics, Minnetonka, MN) and PFA Teflon tubing (Bruland et al., 2005). The sample inlet was mounted to a PVC fish system and lowered to 3 m below the sea surface. The speed of the ship was about 5 knots; thus, ensuring that the intake would not be influenced by the ship's wake. The unfiltered sample was delivered directly to an acid-cleaned and seawater-conditioned 55 L carboy in a Class 100 clean area; the carboy was filled to the halfway point. The 40 m subsurface water sample was collected using a Tefloncoated, 30 L GO-Flo bottle (General Oceanics, Miami, FL) lowered on a Kevlar hydroline (Bruland et al., 1979).

3 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) The subsurface sample was transferred through Teflon tubing to fill the carboy. The mixed sample was aliquoted to 2.5 L polycarbonate incubation bottles, which were acid-cleaned with 3 M HCl (trace metal grade, Fisher Scientific, Pittsburgh, PA) for 2 weeks at room temperature, filled with dilute 0.05 M HCl, and stored until use. Prior to the experiment, the incubation bottles were conditioned by rinsing with clean surface seawater aboard ship. All sample handling and preparation was conducted in a HEPA-filtered Class 100 laminar flow clean area Reagents All reagents were diluted with Milli-Q, except for quartz-distilled acids, which were diluted with quartzdistilled water (QH 2 O). The 0.5 M NH 4 C 2 H 3 O 2 buffer (ph=5.8) was made by diluting saturated solution (19 M) that was prepared by bubbling NH 3 through quartzdistilled HC 2 H 3 O 2 (QHAc). If necessary, the ph was adjusted using quartz-distilled HNO 3 (QHNO 3 )andnh 3. The 1 M NH 4 Cl buffer (ph=8.9) was prepared from aqueous NH 3 (Optima grade, Fisher Scientific) and trace metal grade HCl. Buffer rinse solutions were prepared by diluting the individual buffers 10-fold. Trace metal standards of 10.0 μg/g were prepared from 1000 μg/ml stock solutions (SPEX, Edison, NJ; Fisher Scientific), diluted with 1 M HNO 3 (trace metal grade, Fisher Scientific) and used to make working standards for both particulate and dissolved trace metal analyses. The 100 μm standards of 57 Fe (Isoflex USA, San Francisco, CA) and 68 Zn (Isotec, Matheson, Miamisburg, OH) were prepared from oxide salts by dissolving the isotopically-enriched material in 1 M HCl. The primary standards were analyzed by mass sector, high resolution-inductively coupled plasma-mass spectrometry (HR-ICP-MS) and were verified to have an isotopic abundance of 57 Fe or 68 Zn that were 97.6% and 98.7% enriched, respectively Incubations Two experimental groups (A and B) were used to study the net transfer of 57 Fe and 68 Zn tracers from the soluble fraction to other trace metal pools (colloids and particles) within the mixed water sample. Sample bottles in Experiment A were spiked with 57 Fe such that the concentration of 57 Fe increased by 0.90 nm. The incubation bottles in Experiment B were spiked similarly with 57 Fe, but 0.99 nm of 68 Zn was also added. Experiment A was used as the control for the added 68 Zn in Experiment B, while the control for the 57 Fe tracer was carried out by measuring the more abundant isotope 56 Fe. The addition of 57 Fe did not significantly change the amount of total Fe in the sample (2% of total). After adding the isotopes to the individual samples, the bottles were sealed by wrapping Parafilm and then electrical tape around the bottle opening. They were placed in a Plexiglas incubator located on the top deck of the ship, which was equipped with a flow-through seawater system that kept the incubator at surface water temperatures. Shade cloth was draped over the outside of the incubator to reduce the light level by 50%, which mimicked the sunlight irradiance as measured at depths of 3 4 m in the Bering Sea shelf waters. The size-fractionation procedure was initiated by randomly taking a sample bottle from the incubator, rinsing it thoroughly with clean seawater, and placing it in a Class 100 clean area. Bottles were processed on days 1.0, 2.7, and 4.8. Two bottles were spiked with 57 Fe and 68 Zn and filtered immediately to obtain a measurement at 0.01 days or 45 min. For each sampling day and experimental group, duplicate measurements were made with exception of a single data point for each experimental group on day 1.0. Each bottle was sampled only once to minimize potential contamination Sample filtration scheme Polycarbonate track-etched (PCTE) membrane filters (47 mm dia., Nuclepore, Whatman) were treated with 2 M HNO 3 for 3 days, 6 M HCl for 3 days, and finally with 1 M HNO 3 (trace metal grade, Fisher Scientific) for 3 days. The PCTE filters were kept in the same polyethylene container throughout the treatment, rinsed with Milli-Q water between treatments, and stored in 0.01 M HCl (trace metal grade, Fisher Scientific). Each sample was passed through consecutive 10 μm and 0.2 μm pore-size PCTE membrane filters mounted in Teflon filter sandwiches (Millipore, Bedford, MA) (Fig. 1). The unfiltered samples were also passed through an acid-cleaned 0.03 μm (200 kda) polyethylene hollow fiber flow-through filter (700 cm 2, Sterapore, Mitsubishi-Rayon, Tokyo, Japan) using a second outlet on the filtration apparatus. The large surface area within the Sterapore filter capsule allows for a high-volume flow and adequate flushing of the filter with sample prior to sample collection, similar to a 0.45 μm flow-through capsule, and protocol for maintaining the 0.03 μm filter has been previously described by Nishioka et al. (2001a). The soluble fraction was operationally-defined by the 0.03 μm nominal pore size; thus, the colloidal trace isotope concentrations were not measured directly, but instead estimated by taking the difference between measured values of the 0.2 μm and 0.03 μm filtrates. The

4 214 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) Fig. 1. Fractionation and analysis scheme for the dissolved phase, which includes soluble (b0.03 μm) and colloidal ( μm) fractions, and particulate phase, which was size-fractionated ( μm, N10 μm) and further separated by chemical lability (acetic acid-leachable, refractory). All sample fractions were analyzed using HR-ICP-MS. headspace of the 2.5 L, unfiltered sample bottle was pressurized with approximately 5 psi of N 2 gas to promote passive filtration of each sample. Aliquots of the 0.2 μm and 0.03 μm filtrate (125 ml) were collected in acidcleaned low-density polyethylene (LDPE) bottles and acidified to ph = 1.7 (4 ml of sub-boiled, quartz-distilled 6 M HCl/L of seawater). All filtrate samples were stored for at least 3 months prior to being analyzed. Unfiltered samples were collected for the determination of chlorophyll a, particulate organic carbon (POC), particulate organic nitrogen (PON), and biogenic silica (BSi). These aliquots were filtered and analyzed as described by Leblanc et al. (2005). Filtered samples (b0.2 μm) were collected for dissolved macronutrients (nitrate, silicate, and phosphate) and measured onboard ship with a Lachat QuickChem 8000 Flow Injection Analysis system using standard methods (Parsons et al., 1984) Trace metal analyses Once the sample filtration was complete, the 47 mm diameter PCTE filters were removed from the filtering 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 QH 2 O 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 QHNO 3 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 HNO 3 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 HNO 3 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- MS with a PFA Teflon spray chamber and PFA-ST nebulizer (Elemental Scientific, Omaha, NE). Plasma and mass spectrometer parameters were optimized daily using the internal standard intensities of 103 Rh in low resolution and 69 Ga in medium resolution. A Pump-pro MPL peristaltic pump (Watson-Marlow Bredel, Wilmington, MA) was employed to remove waste from the spray chamber and to pump particulate bomb digested and leached solutions directly to the nebulizer. The sample introduction system, including autosampler (ASX-100, CETAC Technologies, Inc., Omaha, NE) and μ-sampler (Thermo- Electron), were enclosed and under positive pressure using a HEPA filter. Data were acquired in low resolution ( 111 Cd) and medium resolution ( 31 P, 56 Fe, 57 Fe, 59 Co, 63 Cu, 66 Zn, 68 Zn) modes. The intensities of these analytes, relative to the internal standard, were quantified using an external calibration curve. Dissolved trace metals (Fe, Zn, Cu, Co, Cd) were analyzed using chelating resin, column partitioning with HR-ICP-MS (CRCP-HR-ICP-MS) (Willie et al., 1998; Warnken et al., 2000; Willie et al., 2001; Ndungu et al., 2003). The acidified filtrate samples were UV-irradiated off-line prior to analysis to break down organic chelators present in the seawater (Ndungu et al., 2003). The CRCP- HR-ICP-MS methodology employed Toyopearl AF- Chelate 650 M (Tosohaas, Montgomeryville, PA) as the chelating iminodiacetate (IDA) resin, and the samples were buffered to a ph of 5.5 prior to being loaded onto the

5 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) resin column. To remove salts within the sample matrix, the column was rinsed with an NH 4 C 2 H 3 O 2 buffer solution prior to the elution of the analytes with 1 M QHNO 3. All trace metal analyses were performed in medium resolution mode with an NH 4 C 2 H 3 O 2 buffer, except Cd, which was performed at low resolution with an NH 4 Cl buffer. UV-oxidized seawater, which is relatively free of trace metals and complexing organic ligands (Donat and Bruland, 1988), was acidified with QHCl to the same extent as the samples and used for preparing working standards. The average height of the time resolved peak was quantified for each analyte using the standard curve Certified values and blanks The CRCP-HR-ICP-MS method determined total dissolved Fe and Zn in CASS-3 (Coastal Atlantic Seawater Standard, National Research Council of Canada) to be 21.3±0.9 nm (certified value=22.6±3.0 nm) for Fe and 18.4±1.5 nm (certified value=19.0±3.8 nm) for Zn. During the analysis of filtrate samples, the average method blank values (n=6) over the course of the analytical run for the individual isotopes were 0.031±0.008 nm ( 56 Fe), 0.005±0.001 nm ( 57 Fe), 0.028±0.008 nm ( 66 Zn), and 0.031±0.004 nm ( 68 Zn). Although there is not a standard 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. of 4 μg/l, and the diatom community was supported by the transport of macronutrients across the pycnocline from the subsurface waters. The subsurface water concentrations of nitrate, silicic acid, and phosphate were 6 μm, 34 μm, and 1.5 μm, respectively. The mixed seawater sample used in the incubation experiment had measured concentrations for nitrate, silicic acid, and phosphate of 3 μm, 18 μm, and 0.8 μm, respectively. The incubation experiment was designed to simulate a major storm-induced mixing event, a primary mechanism for supplying nutrients to the surface waters in a shelf water regime. By mixing the two water types in a 1:1 ratio, macronutrients and trace metal micronutrients originally in the subsurface waters, in both dissolved and particulate forms, were introduced to the diatom community present in the nitrate-limited surface waters. During the course of the experiment, there was an insufficient amount of nitrate to promote optimal growth within the diatom community by day 4.8. The limiting [NO 3 ] is reflected in the Chl a data, where there is a minimal increase between days 2.7 and 4.8 (Fig. 3). Particulate organic C/N ratios were relatively stable at values between 7.2 and 7.4, except for day 4.8, where the ratio increased to 9.8. The BSi Chl a ratio remained steady at 30 g/g until day 4.8, where the value then increased to 47 g/g. 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 N and W on the unusually broad mid-continental shelf in the eastern Bering Sea aboard the R/V Kilo Moana during August in 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 The concentrations for nitrate, silicic acid, and phosphate in the surface water were b0.1 μm, b1 μm, and 0.1 μm, respectively. The fluorescence maximum at 15 m depth corresponded to a chlorophyll a (Chl a) concentration Fig. 2. Depth profile at the sample station on the mid-continental shelf in the eastern Bering Sea, with representing temperature (C ) and representing chlorophyll a concentration (μg/l).

6 216 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) Fig. 3. The chlorophyll a concentration (μg/l) and nitrate concentration (μm) over the course of the incubation experiment. The circles represent [Chl a] and the diamonds represent [NO 3 ] (solid symbols represent Experiment A, open symbols represent Experiment B) Isotope ratio method The isotope ratio method used in this investigation takes advantage of differences in the natural isotopic abundance of 56 Fe (91.75%) compared to 57 Fe (2.12%) and 66 Zn (27.90%) compared to 68 Zn (18.75%). Lowabundance isotopes (97.6% purity 57 Fe and 98.7% purity 68 Zn) were initially added to the soluble fraction and exchanged with Fe and Zn in the colloidal and particulate pools, resulting in a shift of the isotopic ratio in each trace metal pool over time. To achieve a measurable shift in the isotopic ratio, it is important to have enough trace metals in the particulate pool to exchange with the low-abundance isotope residing in the soluble and colloidal fractions. Also, it is optimal to use an isotope of low abundance as the tracer and compare it to the isotope of highest abundance so that the enrichment produces a maximum shift in the isotopic ratio. 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 for particulate 57 Fe/ 56 Fe and for dissolved 57 Fe/ 56 Fe, yielding a small percent difference relative to the reported natural abundance ratio of The measured values for the 68 Zn/ 66 Zn ratio in the particulate and dissolved phase were and 0.663, respectively, and compared well to the natural abundance ratio for 68 Zn/ 66 Zn of Trace metals (all isotopes) Ambient dissolved trace metal concentrations in the surface waters at the mid-shelf station were measured at 0.22 nm for Fe and 0.25 nm for Zn, while the total particulate Fe and Zn concentrations were 7.9 nm and 0.18 nm, respectively. The dissolved concentrations in the subsurface waters were 4.4 nm for Fe and 1.6 nm for Zn, and the total particulate Fe and Zn were 89 nm and 0.81 nm, respectively. The mixed sample had an initial dissolved Fe concentrations of 2.3 nm and a total particulate Fe concentration of 50 nm. For Zn, the dissolved and particulate concentrations in the mixture were 0.83 nm and 0.49 nm, respectively. The changes in dissolved and particulate Fe concentrations over the course of the experiment are illustrated in Fig. 4. These data include the total of all Fe isotopes and 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 Particulate 1 M HNO 3 Fe (170 nm), Zn (75 nm) ±1.3% 0.668±1.4% n=9 Digested particulate sample Fe (1.3 μm), Zn (23 nm) ±0.5% 0.660±2.6% n=17 Leached particulate sample Fe (1.1 μm), Zn (50 nm) ±1.7% 0.679±2.0% n=15 Dissolved UVSW Fe (0.55 nm), Zn (not detected) ±7.2% ND n=13 UVSW standard (ph=1.7) Fe (1.6 nm), Zn (1.5 nm) ±5.8% 0.659±3.9% n=9 UVSW standard (ph=1.7) Fe (7.3 nm), Zn (6.1 nm) ±4.3% 0.667±4.0% n=11

7 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) 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 μm fraction (65 80%) (Fig. 4c) and in the refractory fraction ( 80%) (Fig. 4d). Fig. 4b illustrates the increase in dissolved Fe upon addition of the 57 Fe 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 disappearance 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 68 Zn 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 aceticacid 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 andf). The concentrations of Cu, Co, and Cd were also measured in the dissolved and soluble fractions (Fig. 6). There were no measurable differences between Experiments 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 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 Metal-to-carbon ratios Fig. 4. The Fe results from Experiment A+B (average of replicates) in the different fractions: (a) total Fe (particulate and dissolved), (b) dissolved Fe (soluble and colloidal), (c) total particulate Fe ( μm, N10 μm), and (d) total particulate Fe (acetic acid-leachable, refractory). The concentration in the initial sample mixture is labeled Fe mix.. By considering the Redfield formula (C 106 N 16 P 1 )for living plankton biomass and the removal of dissolved trace metals with 3 μm of nitrate by the diatom biomass over the 5-day experiment, the metal-to-carbon (Me/C) ratios were estimated for Fe (100 μmol/mol C) in Experiment (A+B), Zn (25 μmol/mol C) in Experiment A, Zn (50 μmol/mol C) in Experiment B, and Cd (10 μmol/mol C) in Experiment (A+B). The 100 μmol

8 218 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) 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 ( μm, N10 μm); and (d, h) total particulate Zn (acetic acid-leachable, refractory). The concentration in the initial sample mixture is labeled Zn mix..

9 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) the elevated Cd/C ratio, which was likely due to the interreplacement of Zn with Cd (Sunda and Huntsman, 2000) Zinc isotopes The 66 Zn results from Experiment A show a net transfer of 66 Zn from the dissolved to the particulate phase (Fig. 7). Initially, the 0.23 nm of dissolved 66 Zn was over 80% soluble. A decrease in the soluble 66 Zn 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 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 [Zn 2+ ] causes an increase in both the assimilation rate of Zn and the Zn/C ratio. Finally, the decrease in dissolved Zn to subnanomolar levels during the last 2 days in both Experiments A and B resulted in the observed drawdown of dissolved Cd and Fig. 7. The 66 Zn results from Experiment A in the different fractions: (a) dissolved 66 Zn (soluble and colloidal), (b) total particulate 66 Zn ( μm, N10 μm), and (c) total particulate 66 Zn (acetic acidleachable, refractory). The concentration in the initial sample mixture is labeled 66 Zn mix..

10 220 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) fraction was observed while the colloidal fraction displayed minimal change over time (Fig. 7a). The largest increase in 66 Zn occurred in the N10 μm leachable particulate fraction (Fig. 7c), and was a result of increasing biomass spurred by the introduction of nitrate and Fe (Fig. 7b). The 68 Zn in Experiment A demonstrated behavior similar to the 66 Zn (data not shown). This net transfer of Zn can be attributed to active uptake, although adsorption to the cell surfaces may also be a significant factor. Fig. 8. The 68 Zn results from Experiment B in the different fractions: (a) dissolved 68 Zn (soluble and colloidal), (b) total particulate 68 Zn ( μm, N10 μm), and (c) total particulate 68 Zn (acetic acidleachable, refractory). The concentration in the initial sample mixture is labeled 68 Zn mix.. In Experiment B, with the addition of dissolved 68 Zn, nearly all of the added 68 Zn remained in the soluble fraction after 45 min (0.01 days) (Fig. 8). After 1 day, 50% of the soluble 68 Zn had partitioned to the colloids or became associated with particulate matter. Drawdown of the dissolved 68 Zn concentration was primarily due to assimilation into the N 10 μm leachable particulate fraction, while the amount of refractory particulate 68 Zn was relatively unchanged over the course of the experiment (Fig. 8b c). By days 2.7 and 4.8, the particulate 68 Zn 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 68 Zn/ 66 Zn isotopic ratios for both the dissolved and particulate fractions in Experiment B were plotted with respect to time (Fig. 9). The new overall 68 Zn/ 66 Zn isotopic ratio in both the dissolved and particulate phases was predicted to shift to 3.13 upon addition of 68 Zn and is noted as the adjusted isotopic ratio in Fig. 9. After 45 min, the 68 Zn/ 66 Zn 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 68 Zn/ 66 Zn isotopic ratio in the dissolved phase remaining relatively constant is that regeneration of 66 Zn 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 70 Zn 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 68 Zn/ 66 Zn isotopic ratio in the particulate phase increased markedly 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 68 Zn 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 68 Zn, 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 66 Zn and 68 Zn and the low ionization efficiency in the plasma during analysis Iron isotopes Unlike Zn, where assimilation into the N10 μm sizefraction was the overwhelming factor that affected the partitioning of Zn, the Fe results indicate a more

11 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) Fig. 9. The 68 Zn/ 66 Zn isotopic ratio over time in Experiment B for (a) the dissolved phase and (b) the total particulate phase. The initial sample mixture prior to adding 68 Zn is labeled Zn mix.. The natural abundance ratio (solid line) and adjusted ratio (dotted line) are included. dynamic cycling between the various trace metal pools. Fig. 10a and d illustrate the concentrations of 56 Fe and 57 Fe in the dissolved phase over time, respectively, and are separated into both colloidal and soluble fractions. The colloidal 56 Fe decreased from 0.72 nm in the initial mixed sample to 0.08 nm by day 4.8. Similarly, the colloidal 57 Fe fraction decreased from 0.43 nm after the spike to 0.01 nm on day 4.8. The decrease in colloidal 56 Fe, and most notably 57 Fe, was concurrent with no appreciable change in colloidal Zn or Cu. This may be due to a separate aggregation process for Fe, possibly as Fe (hydr)oxides, relative to Zn and Cu; however, the decrease may also suggest that a dissolution mechanism involving photolysis or sequestration of colloidal Fe by strong Fe(III) chelators in the soluble phase is at work (Barbeau et al., 2001; Borer et al., 2005), with subsequent assimilation of this soluble Fe by the plankton community. The soluble species also showed a decrease in both 56 Fe and 57 Fe concentrations over time, yet the drawdown was less dramatic than that observed for the colloidal Fe. Due to the overwhelming concentration of particulate Fe relative to the dissolved Fe in the initial seawater mixture, a change in the total particulate 56 Fe concentration could not be quantified; however, changes in the physico-chemical fractions were observed. The net transfer of 1.2 nm dissolved 56 Fe into the particulate 56 Fe was within the standard error of ±2.1 nm for the particulate 56 Fe concentration (Fig. 10b). Over the initial 2.7 days, a decrease in the leachable particulate 56 Fe and an increase in refractory particulate 56 Fe were measured, where the increase in refractory 56 Fe occurred in the N10 μm size-fraction (Fig. 10c). This corresponds to the observed increase in biomass, and these data suggest that the chemically labile fraction (possibly adsorbed species on the surface of cells) was incorporated into the diatom biomass. The 25% acetic acid leach has been demonstrated to be a mild extraction of labile Fe associated with biomass (Landing and Bruland, 1987) and samples collected on the continental slope of the Bering Sea during a large diatom bloom contained only 6% leachable particulate Fe. Thus, the leachable Fe solubilized with the 25% acetic acid leach does not appear to solubilize the bulk of the biogenic particulate Fe associated with the larger diatoms. On day 4.8, there was an increase in chemically labile 56 Fe in the N 10 μm size-fraction, which corresponded to a decrease in refractory 56 Fe. This was a reversal of the overall trend for particulate 56 Fe discussed above, and may be associated with the nitrate-limiting conditions and the diatom community becoming senescent (Fig. 3). This nitrate limitation may have created conditions whereby the regeneration of refractory 56 Fe occurred faster than the formation of refractory 56 Fe in the biomass. As observed with the 56 Fe, the dissolved 57 Fe was incorporated into the N10 μm and refractory 57 Fe fraction (Fig. 10d f). The addition of 0.90 nm 57 Fe to the dissolved phase resulted in a minor increase ( 0.2 nm) in particulate 57 Fe after 45 min, an increase of 0.7 nm by 1 day, and 0.9 nm by 4.8 days. Due to the relatively small amount of 57 Fe in the particulate fraction at the start of the experiment, the rate of 57 Fe assimilation into the N10 μm and refractory fractions was much greater than the regeneration rate. This resulted in a continual increase in refractory 57 Fe over the course of the experiment. The

12 222 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) rapid removal of 57 Fe that occurred on the timescale of 1daycanbeattributedtobothbiologicaluptakeand adsorption processes. The formation of Fe(III) (hydr) oxides may have occurred upon addition of 57 Fe to the seawater samples, which would explain the initial loss of tracer from the dissolved phase Significance of the 57 Fe/ 56 Fe isotopic ratio Over the course of the experiment, it was observed that the dissolved 57 Fe decreased from 0.8 to 0.1 nm, a factor of 8 decrease, while the 56 Fe decreased from 2.2 to 1.0 nm, which decreased by only a factor of 2. These differences in net removal of 57 Fe and 56 Fe from the dissolved phase greatly contribute to the trends observed in the dissolved and particulate isotopic ratios plotted in Fig. 11. The addition of dissolved 57 Fe shifted the natural 57 Fe/ 56 Fe isotopic abundance ratio of to a new adjusted isotopic ratio of for the entire solution of dissolved and particulate Fe (Fig. 11a b). The 57 Fe/ 56 Fe isotopic ratio in the dissolved phase increased by over an order-of-magnitude and was at a value of 0.33 after Fig. 10. The 56 Fe (a c) and 57 Fe (d f ) results from A+B (average of replicates) in the different fractions: (a) dissolved 56 Fe (soluble and colloidal), (b) total particulate 56 Fe ( μm, N10 μm), and (c) total particulate 56 Fe (acetic acid-leachable, refractory), (d) dissolved 57 Fe (soluble and colloidal), (e) total particulate 57 Fe ( μm, N10 μm), and (f ) total particulate 57 Fe (acetic acid-leachable, refractory). The concentration in the initial sample mixture is labeled 56 Fe mix. and 57 Fe mix., respectively.

13 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) min (0.01 days) and then subsequently decreased to a value of 0.09 by day 4.8 (Fig. 11a). This isotopic ratio leveled off near an asymptote well above the adjusted 57 Fe/ 56 Fe isotopic ratio of for the entire system, a consequence of the high concentration of non-exchanged refractory Fe in the particulate phase. In order for the isotopic ratio in the dissolved phase to reach the adjusted value, all the particulate Fe would have to be regenerated. In contrast, the particulate phase did approach the adjusted 57 Fe/ 56 Fe isotopic ratio of This value is reached because the majority of 56 Fe and 57 Fe (98% of total Fe in solution) resides in the particulate phase at days 2.7 and 4.8 (Fig. 4a). Additionally, essentially all the initial 57 Fe spike became incorporated into the biomass (N 10 μm size-fraction) by the end of the experiment (Fig. 10d). Lastly, it was of interest to plot the 57 Fe/ 56 Fe ratio over time in terms of the leachable 56 Fe and 57 Fe particulate concentrations (Fig. 11c). Previous studies have suggested that the readily leachable forms of Fe are bioavailable to phytoplankton and that refractory Fe, as defined by the particulate Fe remaining after the 25% acetic acid leach, is assumingly not bioavailable (Bruland et al., 2001; Fitzwater et al., 2003). By only including the 57 Fe and 56 Fe associated with the dissolved and leachable particulate phases, an adjusted isotopic ratio of 0.10 was calculated. This is a reasonable way of plotting the changing isotopic ratios over time if refractory particulate Fe, as defined by the 25% acetic acid leach, was not becoming regenerated. However, Fig. 11c confirms that a substantial component of the refractory particulate Fe is regenerated and is an active component of Fe cycling in this system. Over the course of the first day, the leachable particulate Fe behaves similar to particulate Zn, with the 57 Fe/ 56 Fe isotopic ratio rapidly approaching the adjusted isotopic ratio. These results suggest that Fe in the dissolved phase and leachable particulate phase undergoes exchange on the timescale of approximately 1 day. However, the leachable particulate 57 Fe/ 56 Fe ratio does not continue to approach the adjusted 57 Fe/ 56 Fe ratio value, but instead decreased on days 2.7 and 4.8 (Fig.11c). This decrease can be attributed to the simultaneous occurrence of several processes involving the refractory particulate pool. These processes include: (1) the regeneration of operationally-defined refractory particulate 56 Fe that results in the concurrent formation of leachable particulate 56 Fe, (2) the regeneration of refractory particulate 56 Fe exceeding the assimilation of leachable particulate and dissolved 56 Fe, and (3) the conversion of leachable particulate 57 Fe to refractory particulate 57 Fe by the plankton community Net removal of trace metals The complex conditions found in natural waters make it difficult to isolate trace metal removal that is solely due to biological uptake. The processes that influence the assimilation of trace metals into the particulate pool include sorption processes, such as adsorption and precipitation, as well as the active uptake through biological processes (Sposito, 1986). Also, the rate of active uptake by phytoplankton has been shown to be Fig. 11. The 57 Fe/ 56 Fe isotopic ratio over time in Experiment A+B (average of replicates) for (a) the dissolved phase, (b) the total particulate phase, and (c) the leachable particulate phase. The initial sample mixture prior to adding 57 Fe is labeled Fe mix.. The natural abundance ratio (solid line) and adjusted ratio (dotted line) are included.

14 224 M.P. Hurst, K.W. Bruland / Marine Chemistry 103 (2007) dependent upon the intercellular Me/C ratio and the inorganic trace metal concentrations ([Fe ] and [Zn ]) in the dissolved phase (Sunda and Huntsman, 1995a, Sunda and Huntsman, 1995b). The presence of metalbinding ligands in the dissolved phase (Gee and Bruland, 2002) and the changing concentration of particles that occurs during a phytoplankton bloom (Jannasch et al., 1988) also contribute to this complexity. Although multiple variables influence the net removal of trace metals from the dissolved phase to the particulate phase, the net and overall removal of Fe and Zn can be estimated using the low-abundance isotope approach. Using a pseudo-first order rate expression, the average rate constants for the net removal of 66 Zn and 68 Zn were essentially the same in both Experiment A (k= 0.21±0.01 day 1 ) and Experiment B (k= 0.22±0.04 day 1 ), respectively, indicating that regeneration did not significantly contribute to the net removal of Zn from the dissolved phase. If regeneration had played an important role in the removal of Zn, the rate constant for 66 Zn drawdown would have been substantially smaller in Experiment B. In contrast, the net removal rate constant for 57 Fe from the dissolved phase (k= 0.57 day 1 ) was 3.6 times that of 56 Fe (k= 0.16 day 1 ). Assuming there is about 20 nm of truly labile particulate 56 Fe (roughly estimated as twice the 25% acetic acid leachable concentration), that the particulate 57 Fe concentration is negligible (reasonable assumption given the amount of 57 Fe added to the dissolved phase), and the use of first-order rate kinetics to construct a simple model of the data (d[fe diss. ]/dt= [Fe diss. ]k 1 +[Fe part. ]k 2 ), it was determined that 0.6 nm day 1 of particulate Fe was regenerated back to the dissolved phase (k 1 =0.57 day 1 and k 2 =0.030 day 1 ). Thus, the net removal of 2.0 nm of dissolved Fe over the 4.8-day period was the result of 2.9 nm of regenerated particulate Fe and an overall removal of 4.9 nm of dissolved Fe. The difference between the overall and net removal of Fe during the experiment, with the overall removal being 2.4 times the net removal, demonstrates the dynamic cycling of Fe between the dissolved and particulate phases and the importance of particulate Fe as a bioavailable source. The contribution of particulate Fe to the dissolved phase within the shelf water system was substantial, especially when the Fe requirements for primary productivity are considered (Sunda and Huntsman, 1995b). 4. Conclusion The use of stable, low-abundance, trace metal isotopes in shipboard incubation experiments presents a novel approach for quantifying the exchange of trace metals between different physico-chemical fractions found in marine systems. This study provided a better understanding of Fe and Zn cycling in a shelf water environment during a diatom bloom induced by vertical mixing of the water column. The regeneration of Zn from the particulate pool was not a significant part of Zn cycling in the mid-shelf regime, largely due to the low concentrations of Zn in the particulate phase relative to the dissolved phase. The assimilation of soluble Zn by the N10 μm biota was the dominant process contributing to the net removal of Zn from the dissolved phase, while the colloidal Zn concentration remained constant and did not appear to be bioactive. Two main observations were deduced from the exchange of Fe between the soluble, colloidal, and particulate pools: (1) there was active regeneration of particulate Fe that affected the net removal of Fe from the dissolved phase, and (2) there was rapid drawdown and apparent utilization of colloidal Fe by the diatom community (N 10 μm size-fraction). The soluble and leachable particulate fractions of Fe also decreased over time and were converted to refractory particulate Fe, but did so at a much slower rate than the colloidal fraction. The experimental data demonstrate the importance of particulate Fe in the water column and its role as a bioavailable source; however, further development of analytical methodology is needed to accurately distinguish between the bioavailable particulate Fe and refractory Fe pools. Additionally, it is important to distinguish between colloidal and soluble Fe within the dissolved phase given that their behavior within Fe cycling was observed to be different. This includes more research in determining the most appropriate partition cutoff for small particulate, colloidal, and soluble forms of Fe. Progress in this area could be advanced by better characterizing the colloids and determining whether they are ill-defined entities or biogenic macromolecules providing a distinct function for the biology. Acknowledgements The authors thank the National Science Foundation for funding (grants OCE and OCE ). We thank Bettina Sohst for the dissolved macronutrient data, Karine Leblanc and Clint Hare for POC, PON, BSi, and Chlorophyll data. We appreciate the advice and expertise of Rob Franks during the trace metal analyses using the ICP-MS. We also thank Geoffrey Smith for assisting in sample collection.

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