Changes in iron concentrations and bio-availability during an open-ocean mesoscale iron enrichment in the western subarctic Pacific, SEEDS II

ARTICLE IN PRESS Deep-Sea Research II 56 (2009) 2796–2809

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Changes in iron concentrations and bio-availability during an open-ocean mesoscale iron enrichment in the western subarctic Pacific, SEEDS II Jun Nishioka a,b,, Shigenobu Takeda c, Yoshiko Kondo c, Hajime Obata d, Takashi Doi d, Daisuke Tsumune b, C.S. Wong e, W. Keith Johnson e, N. Sutherland e, Atsushi Tsuda d a

Pan-Okhotsk Research Center, Institute of Low Temperature Science, Hokkaido University, Sapporo, Hokkaido 060-0819, Japan Central Research Institute of Electric Power Industry, Abiko, Chiba 270-1194, Japan c Department of Aquatic Bioscience, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan d Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639, Japan e Climate Chemistry Laboratory, Institute of Ocean Sciences, Fisheries and Oceans Canada, PO Box 6000, Sidney, BC, Canada V8L 4B2 b

a r t i c l e in f o

a b s t r a c t

Topical issue on ‘‘SEEDS II: The Second Subarctic Pacific Iron Experiment for Ecosystem Dynamics Study.’’ The issue is compiled and guest-edited by the North Pacific Marine Science Organization (PICES) and International SOLAS. Available online 3 July 2009

A patch of water in the western subarctic gyre (low iron concentration, o0.02 nM) was fertilized twice with 322 and 159 kg of iron to induce a phytoplankton bloom. In order to understand the changes in iron distribution and bio-availability throughout the evolution and termination phase of the iron-induced bloom, iron concentrations were measured at stations inside and outside of the iron-fertilized patch, and shipboard culture experiments using iron and desferrioxamine B (DFB) inoculation to regulate iron availability were conducted 5 times with water collected from the center of the iron-fertilized patch on D2, D7, D11, D17 and D23. After the iron fertilization, we observed a significant increase in dissolved iron (1.38 nM at 5 m depth) at the center of the patch (D1). Dissolved iron concentrations subsequently decreased to an ambient level (0.08 nM) on D16–D17, despite the second iron fertilization made on D6. During the 4-day incubations of the shipboard culture experiments, excess DFB-inoculated treatment inhibited the phytoplankton growth compared to the controls for D2, D7 and D11 patch water. This indicated that available iron existed in the iron-fertilized patch at least until D11. Moreover, iron-inoculated treatments induced growth of large-sized phytoplankton with an accompanying silicate decrease for D7, D11 and D17 patch water, but not for D23 patch water. These results indicated that large diatoms, which can respond to additional iron inoculation, existed in the iron-fertilized patch in evolution and early termination phase of the iron-induced bloom (at least until D17); however, there was no significant amount of large diatoms, which could rapidly respond to iron, in late termination phase (D23) of the iron-induced phytoplankton bloom. & 2009 Elsevier Ltd. All rights reserved.

Keywords: Iron fertilization Iron dynamics Iron bio-availability Incubation experiment Diatom Western subarctic north Pacific

1. Introduction Several in-situ iron-enrichment experiments have been performed with the general goal to evaluate the question of whether iron availability controls phytoplankton production and uptake of carbon in high-nutrient and low-chlorophyll (HNLC) waters of the equatorial Pacific and Southern Ocean (Martin et al., 1994; Coale et al., 1996, 2004; Boyd et al., 2000; Gervais et al., 2002; Hoffmann et al., 2006). This ‘‘iron hypothesis (Martin, 1990)’’ was also investigated in the western subarctic Pacific (Subarctic Pacific Iron Experiment for Ecosystem Dynamics Study (SEEDS)) (Tsuda  Corresponding author at: Pan-Okhotsk Research Center, Institute of Low Temperature Science, Hokkaido University, Sapporo, Hokkaido 060-0819, Japan. Tel.: +8111706 7655; fax: +81117067142. E-mail address: [email protected] (J. Nishioka).

0967-0645/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr2.2009.06.006

et al., 2003) and eastern subarctic Pacific (Subarctic Ecosystem Response to Iron Enrichment Study (SERIES)) (Boyd et al., 2004) using mesoscale iron injections in order to enhance the biological and geochemical signals. All of these in-situ iron-enrichment experiments clearly revealed that iron limits phytoplankton growth in these HNLC regions. In SEEDS, which was conducted in 2001 summer, a single injection of iron caused a large increase in the phytoplankton standing stock and decreases in macronutrients and CO2 fugacity in the surface mixed layer. Chlorophyll-a concentration in the surface mixed layer in the iron patch increased by 20 times (from 0.7 to 20 mg m3) within 9 days from the iron injection. The large response was dependent on a species shift from openocean species to neritic diatoms dominated by chain-forming Chaetoceros debilis (Tsuda et al., 2003; Takeda and Tsuda, 2005). The SEEDS was characterized by the highest biogeochemical

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response (highest chlorophyll-a concentrations in the surface mixed layer) among the previous mesoscale iron-enrichment experiment (de Baar et al., 2005). A second mesoscale iron-enrichment study in the western subarctic Pacific (SEEDS II) was carried out in summer 2004. Since the short observation period (13 days) of SEEDS was not enough to examine the fate of the iron-induced phytoplankton bloom, the iron-fertilized patch was traced for 26 days, which included observations of the evolution and termination phase of the phytoplankton bloom in SEEDS II. SEEDS II was carried out at almost the same location, the same season and the same protocol to manipulate the iron concentration as SEEDS. However, the magnitude of the iron-induced phytoplankton bloom was quite different between SEEDS and SEEDS II, with a significantly lower response in SEEDS II (Tsuda et al., 2007). In SEEDS II, the chlorophyll-a concentration increased by only 3 times from the initial concentrations of 0.86 to 2.80 mg m3 (average in mixed layer), with a low drawdown of macronutrients (3.8 mM in nitrate decrease). In contrast to the large diatoms, which became the dominant species in SEEDS, pico-phytoplankton became the most dominant species throughout SEEDS II. Regarding the differences in response of phytoplankton to iron enrichment between SEEDS and SEEDS II, several explanations have been put forward. Tsuda et al. (2007) reported that the absence of a large phytoplankton standing stock due to mesozooplankton grazing was one of the major reasons for the weak biological response in SEEDS II. Additionally, bio-available iron retention time in the surface mixed layer after the iron injection is another possible important factor to determine the magnitude of the phytoplankton responses. Previous experiments indicated that the injected dissolved iron concentration decreased rapidly, and dramatic changes in the chemical/physical form of iron were observed during the experiments (Gordon et al., 1998; Bowie et al., 2001; Nishioka et al.,2003, 2005; Wells 2003; Croot et al., 2001, 2005; Boye et al., 2005). In SEEDS and SERIES, we observed that colloidal iron in the dissolved fraction was transformed to particulate iron. In SEEDS, SERIES and SOIREE, the particulate iron was retained in the surface mixed layer (Croot et al., 2001; Nishioka et al., 2003; Wong et al., 2006), and the transformation led to a reduction in the iron bio-availability (Nishioka et al., 2003). Wells (2003) also reported biological uptake was responsible for the loss of a part of the soluble iron in the IronEx II study, which was conducted in the equatorial Pacific Ocean. Rue and Bruland (1997), Boye et al. (2005) and Kondo et al. (2008) also indicated that the production of organic ligands, which behave as iron chelators, was induced by iron enrichment in the upper water column, and the organic ligands have strong influences on iron retention and transformation. Thus, chemical/physical iron transformation occurred during the iron-induced bloom evolution and termination phase, and iron transformation strongly influences iron bio-availability. Therefore, investigation of changing iron bio-availability during the experiment is important to evaluate the reason for the differences in the magnitude of the biogeochemical response between SEEDS and SEEDS II. Wells (1999) and Hutchins et al. (1999) reported that a bottle incubation experiment with fungal siderophore desferrioxamine B (DFB) is a useful approach to evaluate the amount of bio-available iron in seawater. They clearly demonstrated that addition of an excess concentration of DFB essentially eliminated iron uptake in natural phytoplankton populations, and DFB can be used to manipulate biologically accessible iron in iron-replete water. In this study, we measured dissolved and total dissolvable iron in the iron-fertilized patch (hereafter in-patch) and outside of the iron-fertilized patch (hereafter out-patch) during SEEDS II to examine the changes in iron distribution after the iron injection to

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the western subarctic Pacific water. Additionally, we conducted shipboard culture experiment using iron and DFB inoculation to regulate iron availability, with water that was collected from the center of the in-patch, in order to determine the changes in iron bio-availability throughout the evolution and termination phase of the iron-induced phytoplankton bloom. Then we discuss the reasons for the weak response of phytoplankton to the iron fertilization during SEEDS II.

2. Materials and methods 2.1. SEEDS II experiment A second iron-enrichment experiment in the western North Pacific (SEEDS II) was carried out by two research vessels: R.V. Hakuho-Maru and R.V. Kiro Moana from 13 July to 27 August 2004. A preliminary survey was performed by Hakuho-Maru from 13 July to 19 July 2004 with CTD observations, an underway survey system, and vertical bottle sampling to determine the biological, chemical and physical heterogeneity of the experimental area in order to select the experimental site. The first iron injection was conducted by Hakuho-Maru on 20 July (day 0, hereafter D0) and the second injection was conducted on 26 July (D6), and the in-patch water mass was traced for 25 days. The first two weeks of tracing the in-patch was conducted by HakuhoMaru with a combination of mapping using inert SF6 tracer and drogued GPS buoy, similar to those of previous experiments (Law et al., 1998; Tsumune et al., 2005). Then the observation was continued by Kilo-Moana until D22. Again, Hakuho-Maru took over the observation from D23 to D25. On D31, unfortunately, we failed to find the in-patch. Therefore, the observation periods for the in-patch were until D25 and D32 for the out-patch from the first iron injection (Tsuda et al., 2007). 2.2. Iron and SF6 tracer release The method of iron and SF6 tracer injection in SEEDS II was similar to that in SEEDS. Details of the method are described in Tsumune et al. (2005). Prior to the iron injection, two steel tanks containing a total of 4000-L in-situ subarctic seawater were saturated with SF6 bubbling over a 24-h period and sealed in order to reduce loss to the atmosphere. On the other hand, two 3000-L polyethylene tanks were filled with in-situ subarctic seawater and the water was acidified to pH 1.8 by adding concentrated hydrochloric acid to each tank. Heptahydrate iron sulfate (FeSO4  7H2O) was then added to the tanks, and the waters in each tank were mixed using a stirrer resulting in a solution of approximately 0.57 M iron. We replicated the procedure during the first iron injection. The first iron injection at 481100 N, 1661000 E began at 0:50 GMT on 20 July (D0). The injection was completed in 23 h with a speed of about 5 knots. Totally, 4000 L of SF6 saturated solution and 9700 L (assuming approximately 20% of the iron solution remained in the tanks) of iron solution (322 kg of iron) were injected at the depth of 5 m in the 30-m mixed layer over an area of 8  8 km2. The flow rate of the iron solution was 8 L min1 and that of the SF6 solution was 3 L min1. A second iron injection (159 kg of iron) was performed on day 6 without the SF6 tracer. During the second iron injection, the top of tubing for the iron injection was set on the water surface (0 m), in order to keep high ship speed (10 knots) for injecting iron to a wide area, and the iron solution was injected in the in-patch, which was traced using the SF6 signals. Times and quantities comprising the iron injection in the SEEDS II are presented in Table 1. Expected average concentrations of iron after the first and the second iron injection

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Table 1 Times and quantities comprising the iron infusion in the SEEDS II experiment. Number of iron infusion

First

Second

Date of release

Amount of FeSO4  7H2O (kg)

Release grid size (km2)

Depth of surface mixed layer (m)

Theoretical iron increase (nM)

Start (local time)

Finish (local time)

20 July (D0) 9:50

21 July (D1) 9:00

1600 (Fe: 322)

64

30

3.0

26 Jul (D6) 15:00

27 Jul (D7) 1:00

790 (Fe: 159)

200

30

0.5

were estimated to be about 3.0 and 0.5 nM in the in-patch, respectively. A Lagrangian reference frame was employed for the iron injection to minimize the effect of advection (Stanton, et al., 1998). A GPS buoy with WOCE-style drogue attached at about 20 m was used as a reference for the Lagrangian frame. A computer display onboard updated the relative position of the ship to the GPS buoy every 10 min during the iron and SF6 tracer release.

2.3. Sampling and analysis of iron during SEEDS II Continuous underway sampling of surface water was conducted to determine the area and position of the center of the inpatch (in-patch station) using the ship’s pumping system with an intake 6 m below the surface for measurements of SF6 and pCO2. The ship moved through the patch several times from various angles. The ship’s trajectory was navigated in the Lagrangian reference frame based on the reference GPS buoy. Vertical discrete sampling was conducted to characterize vertical profiles at in-patch station on D0, D1, D2, D4, D5, D7, D8, D10, D11, D12, D23, D25 and out-patch on D0, D2, D5, D8, D11, D25, D32 by Hakuho-Maru, and in-patch on D13, D14, D16, D17, D19 and D21 and out-patch on D9, D18, D24 by Kilo-Moana. These samples were collected from 5, 10, 20, 30, 50 and 75 m depth. On Hakuho-Maru, the seawater samples and hydrographic data were collected using a titanium cable and clean CTD-carousel multiple sampler system (CMS, SBE-911plus and SBE-32 water sampler, Sea Bird Electronics, Inc.), which housed 12 acid-cleaned Tefloncoated 12-L X-Niskin bottles, except D1. The samplers for trace metals were coated inside with Teflon, the drain cocks were replaced with all-Teflon stop cocks and acid-cleaned inside. On D1, surface samples were collected from 5 m depth using an acid-cleaned air-driven Teflon bellows pump (PFD-1 Asti Co. Ltd.) with Teflon tubing (12 mm i.d.), which was covered by PVC, with and without 0.22 mm Durapore filters (Opticap, Millipore). On Kilo-Moana, the samplers for trace metals were suspended by a Kevlar hydro-wire and tripped using Teflon messengers. For subsampling from the X-Niskin samplers, both on the Hakuho-Maru and on the Kilo-Moana, acid-cleaned 0.22-mm Durapore filters (Millipac 100, Millipore) in a polycarbonate housing were connected to the sampler spigot. Then the filtrate was collected under gravity pressure. Moreover, samples collected from the surface mixed layer (5, 10 m) for iron analysis were immediately size-fractionated by a clean in-line filtration system using a 200 kDa polyethylene hollow-fiber ultrafilter (Nishioka et al., 2001, 2005) in the clean-air laboratory, which was located onboard. At one of the out-patch stations (D32), a vertical profile from the surface to 3000 m depth was collected for sizefractionation study of iron in natural water.

In addition, sampling for cross-section profiling analysis through the patch was also conducted on days 3, 9 and 20. The locations of the cross-section lines were decided by underway surveys. Six to seven sampling stations were set on the line through the patch, and vertical discrete samplings were conducted by the same method as for the in-patch and out-patch stations. All unfiltered and filtrate samples were buffered at pH 3.2 with 10 M formic acid–2.4 M ammonium formate buffer solution in the laminar flow, clean-air hood in the clean-air laboratory. Concentrations of Fe (III) in the buffered samples were determined onboard with an automatic Fe (III) flow injection analytical system using chelating resin concentration and chemiluminescence detection (Obata et al., 1993, 1997). The determined iron is a chemically labile species, which strongly reacts with 8-hydroxyquinoline resin at pH 3.2. It should be noted that acidification to pH 3.2 is not sufficient to release all the iron from particulate forms (Obata et al., 1997). For this cruise, the samples were allowed to stand at pH 3.2 at room temperature to allow for a weak digestion of particulate matter resulting in analysis of what we defined as ‘‘total dissolvable iron’’. Therefore, ‘‘total dissolvable iron’’ in this study includes only the iron leached at pH 3.2. In this paper, observed iron concentrations are defined as total dissolvable iron (TD-Fe; unfiltered), dissolved iron (Diss-Fe; o0.22 mm), colloidal iron (Coll-Fe; 200 kDa–0.22 mm) and soluble iron (Sol-Fe; o200 kDa) (Nishioka et al., 2001).

2.4. Shipboard culture experiments Wells (1999) and Hutchins et al. (1999) reported an approach to regulate iron bio-availability in iron-replete systems. They used the fungal siderophore DFB to regulate iron availability and reported that addition of excess DFB essentially eliminated iron uptake by phytoplankton and heterotrophic bacteria. In SEEDS II experiment, shipboard culture experiments using iron and DFB inoculation to regulate iron bio-availability were conducted 5 times with water collected from the center of the iron-fertilized patch on D2, D7, D11, D17 and D23 (Table 2). Surface water was collected for the incubation experiment from 10 m depth at the inpatch station using the same clean sampling method as the samples for trace metals analysis. To conduct each culture experiment, two of the 12-L (or 10-L on D17 experiment on Kilo-Moana) X-Niskin samplers were used to collect the seawater, and the seawater was transferred to acid-cleaned 25-L polycarbonate (PC) carboy, which has a polypropylene spigot (Nalgen, Ltd.). Then iron-free nitrate, phosphate and silicate were added to the seawater in the 25-L PC carboy, except for the D2 and D17 experiment, and mixed well. In total 250-ml PC incubation bottles were filled with the mixed seawater after being passed

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Table 2 Initial condition and treatments of shipboard incubation experiment during SEEDS II experiment. Days since iron infusion

D2 D7 D11 D17 D23

Initial Chl-a concentration (mg/m3)

0.83 2.12 2.77 2.15 0.97

Initial nutrient concentration (mM) NO3+NO2

Si(OH)4

PO4

18.8 63.3 67.0 16.1 57.1

36.7 80.3 83.8 30.9 75.3

1.61 4.39 4.6 1.39 4.08

through a 220-mm acid-cleaned Teflon mesh to remove mesozooplankton from the water. All sample containers and Teflon mesh were thoroughly acid-washed with the trace metal cleaning procedure (Obata et al., 1993; Takeda and Obata, 1995) before use. We prepared triplicate bottles for (1) control, (2) iron inoculation treatment (hereafter Fe treatment) and (3) DFB inoculation treatment (hereafter DFB treatment) (Table 2). For the Fe treatment, FeCl3 was added to each 250-ml PC incubation bottle to a final concentration of 1 nM (except D2 experiment). The bottles for DFB treatments received an addition of 100 nM of DFB (Sigma). Control bottles received no addition of iron or DFB. Initial chlorophyll-a and nutrient concentrations in each treatment are shown in Table 2. The 250 ml PC incubation bottles were sealed to avoid contamination, and then incubated on deck in running surface seawater baths to maintain the surface seawater temperature during the 4-day incubation period. The incubation bottles were covered with neutral density screens during the 4day incubation period. The screening provided shading to ca. 30% of the ambient light level at the beginning of the experiment, estimated to correspond to the approx. light level at the depth of water collection. The triplicate control, DFB treatment and Fe treatment (except D2 experiment) were withdrawn after the 4-day incubation period. Initial samples (day 0) and samples after 4-day incubation (day 4) were used for measurement of the nutrients and sizefractionated chlorophyll-a concentrations. To examine the size fractionation of chlorophyll-a concentration, water samples from the incubation bottles were filtered onto 10-mm Nuclepore PC filters and Whatman GF/F glass fiber filter, subsequently, under gentle vacuum (o100 mmHg). The filtered samples were extracted in N,N-dimethylformamide for 24 h at 4 1C (Suzuki and Ishimaru, 1990), and fluorescence was measured with a Turner Designs fluorometer AU-10 with the non-acidification method (Welschmeyer, 1994). Nutrients samples were transferred from incubation bottles to 10 ml acrylic tubes and kept frozen until analysis. After return to the onshore laboratory, nutrient concentrations were determined using a nutrient analyzer AARCS (BRAN+LUEBBE).

3. Results and discussion 3.1. Hydrography and biological response during SEEDS II During the course of SEEDS II (20 July–21 August 2004), surface water temperatures increased from 8.4 to 11.9 1C. Vertical temperature profiles during the SEEDS II, SEEDS and SERIES experiments are shown in Fig. 1. Surface mixed layer depth (MLD) was highly variable throughout the course of SEEDS II. At the beginning of SEEDS II, MLD was 33 m until D8 and became shallower between D9 and D11 to 15 m. After that, MLD increased again to 30 m on D16, and became shallower again to 10 m at the end of the experiment. This change in temperature and MLD was

SEEDS II bloom phase

Treatments for incubation

initial phase evolution evolution termination termination

Control, DFB addition Control, DFB addition, Fe addition Control, DFB addition, Fe addition Control, DFB addition, Fe addition Control, DFB addition, Fe addition

due to wind speed and increasing solar radiation and the warming of the water at the experiment site (Tsumune et al., 2009). The initial MLD in SEEDS II was approximately 3 times deeper than that of SEEDS experiment (8.5 m) and comparable to that in SERIES (Fig. 1). More details on the MLD during the experiments are described in Tsumune et al. (2009). Details on the horizontal diffusion of the iron-fertilized patch were also analyzed using horizontally observed SF6 concentration, and described in Tsumune et al. (2009). The initial concentrations of SF6 of SEEDS II were lower than that of SEEDS, and may be due to the deeper MLD for SEEDS II. The estimated diffusion coefficients in SEEDS and SEEDS II during the first 10 days are 9.0 and 4.9  101 m2 s1, respectively (Tsumune et al., 2009). Therefore, dilution rate during the first 10 days of SEEDS II was at least 5 times larger than that of SEEDS. Changes in chlorophyll-a concentration at 5 m depth in the inpatch and the out-patch stations during SEEDS II are shown in Fig. 2. Prior to the first iron injection in SEEDS II, surface chlorophyll-a concentration was in the range between 0.6 and 1.1 mg m3 (0.86 mg m3, average in MLD). The initial chlorophylla concentrations were comparable to that of SEEDS; however, the response to iron injection was markedly different. The SEEDS result was the largest increase of chlorophyll-a concentration in the surface mixed layer (10 m) among the mesoscale ironenrichment experiments (de Baar et al., 2005). The maximum observed chlorophyll-a concentration in the surface mixed layer in the in-patch was over 15 mg m3 in SEEDS. In contrast to SEEDS, the maximum chlorophyll-a concentration in the surface mixed layer was only 3.0 mg m3 in SEEDS II. These data suggest that the biological response to iron injection was significantly lower in SEEDS II than that in SEEDS. In SEEDS II, we spent a longer period for the experiment (26 days for in-patch observations); therefore, termination phase of the bloom, which was defined as a return to pre-bloom chlorophyll-a levels, was observed. The consumption of nutrients was also significantly different between SEEDS and SEEDS II. In SEEDS, the amount of decrease of the nutrient drawdown in the surface mixed layer was the largest among the previous iron-enrichment experiments. The maximum differences in nitrate and silicic acid concentration between the in-patch and out-patch were 13.4 and 24.8 mM, respectively. In SEEDS II, the nitrate concentration at the in-patch station significantly decreased at a higher rate than outside of the patch; however, the maximum difference between inside and outside of the patch at 5-m depth was only 3.8 mM. A significant difference in the silicic acid concentration was not observed. More details on the biological response of the experiment are described in Tsuda et al. (2007), Saito et al. (2009) and Yoshimura et al. (2009).

3.2. Iron concentrations in the patch during SEEDS II experiment Vertical profiles of natural iron concentrations in the experiment site area (in the out-patch on D32) are shown in Fig. 3, and

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Temperature (°C)

Temperature (°C) 0

Depth (m)

40

9

12

15

Day 0 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Day 15 Day 17 Day 19 Day 22 Day 24 Day 26

60

80

100

6

9

0

20

Temperature (°C) 12

15

SEEDS

0

20

0

3

6

9

12

15 30 -10 m

SERIES

6

30 -25 m

20

3

3

10 -15 m

0

0

SEEDS II

40

40

60

Day 0

Day-1

Day 2

Day 2

Day 4

Day 5 Day 7

Day 9

Day 12

Day 11

80

60

Day 7

Day13

Day 23

80

Day 25

100

100

Fig. 1. Vertical profiles of temperature inside of the iron-fertilized patch station during SERIES, SEEDS and SEEDS II. Red arrows indicate approximate surface mixed-layer depth.

0.0

0.5

Fe conc. (nM) 1.0

1.5

0.0

0.5

1.0

1.5

3

2.0

0

2.5

Depth (m)

Chlorophyll-a conc. µg/L

3.5

2 1.5

In Out

1

50 100 150 200 250

0.5

2.0

0

0 0

5

10 15 20 25 Days since beginning of experiment

30

35 500

Fig. 2. Temporal changes in total chlorophyll-a concentrations at 5 m depth in the iron-fertilized patch (in-patch) and outside of the patch.

all the data are shown in Table 3. In the experiment site of SEEDS II, Diss-Fe concentration at 5 m was 0.02 nM prior to the iron injection. The Diss-Fe level at 5 m outside of the patch was 0.02–0.12 nM throughout the experiment period. Vertical profiles of iron concentrations in the different size fractions at the experiment site area (Fig. 3), in the out-patch station on D32, indicated that Diss-Fe concentration (o0.22 mm) in the surface mixed layer in this area was 0.03–0.09 nM and had nutrient-type distributions, characterized by depletion in the surface mixed layer and increasing with depth (Fig. 3). Our vertical measurements of iron in the experiment area confirm previous observations that increased gradients in Diss-Fe concentrations with depth from subsurface to intermediate water were greater in the region relative to that of the eastern subarctic Pacific (Fujishima et al., 2001; Nishioka et al., 2003, 2007; Kinugasa et al., 2005; Brown et al., 2005). Vertical profiles of TD-Fe and Diss-Fe concentrations at different times in the SEEDS II in-patch are shown in Fig. 4. The changes in TD-Fe and Diss-Fe concentrations at 5 m in the surface mixed layer over the time in SEEDS II are shown in Fig. 5. All the data are shown in Table 3. At the first sampling point after the initial iron injection (D1; within 16 h from the first iron injection), we observed a significant increase of TD-Fe and Diss-Fe (1.73 and

Depth (m)

1000

Dfe Tfe Sfe

1500

2000

2500

3000

3500 Fig. 3. Vertical profiles of iron concentrations in the different size fractions in the experiment site area, in the out-patch station on D32.

1.38 nM, respectively) at 5 m depth (Figs. 4 and 5, Table 3), indicating that most of the added iron was in the dissolved form immediately after the iron injection. The vertical distributions of iron at the in-patch stations also indicate that iron concentration

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Table 3 Size-fractionated iron concentrations in the SEEDS II experiment. In-patch stations

Out-patch stations

Days since from first iron release

Depth (m)

Day 0

Sol-Fe (o200 kDa) (nM)

Diss-Fe (o0.22 mm) (nM)

TD-Fe (unfiltered) (nM)

Days since from first iron release

5 10 20 30 50 75

0.02 0.02 0.02 0.02 0.03 0.07

0.02 0.11 0.12 0.17 0.12 0.17

Day 0

Day 1

5 10 20 30 50 75

1.38 N.D. N.D. N.D. N.D. N.D.

1.73 N.D. N.D. N.D. N.D. N.D.

Day 2

5 10 20 30 50 75

0.22 0.12

0.23 0.16 0.11 0.19 0.18 0.16

Day 4

5 10 20 30 50 75

0.15 0.14

Day 5

5 10 20 30 50 75

Day 7

Diss-Fe (o0.22 mm) (nM)

TD-Fe (unfiltered) (nM)

5 10 20 30 50 75

0.02 0.02 0.02 0.02 0.03 0.07

0.02 0.11 0.12 0.17 0.12 0.17

Day 2

5 10 20 30 50 75

0.05 0.06 D.L. D.L. 0.02 0.15

0.22 0.20 0.18 0.20 0.20 0.20

0.40 0.30 0.13 0.37 0.13 0.12

Day 5

5 10 20 30 50 75

0.06 0.04 0.06 0.06 0.05 0.06

0.18 0.16 0.19 0.17 0.15 0.16

0.29 0.17 0.15 0.09 0.11 0.15

0.75 0.78 0.39 0.20 0.19 0.31

Day 8

5 10 20 30 50 75

0.07 0.06 0.08 0.07 0.08 0.10

0.28 0.26

0.32

0.10 0.12

0.15 0.16 0.17 0.13 0.07 0.11

0.72 0.81 0.85 0.71 0.20 0.21

Day 9

5 10 20 30 50 75

0.05 0.05 0.05 0.06 0.05 0.09

0.13 0.11 0.13 0.17 0.12 0.17

5 10 20 30 50 75

0.10 0.08

0.52 0.43 0.16 0.09 0.16 0.12

1.86 1.70 0.87 0.46 0.29 0.36

Day 11

5 10 20 30 50 75

0.12 0.10 0.11 0.12 D.L.

0.31 0.26 0.29 0.29 0.40 0.31

Day 8

5 10 20 30 50 75

0.27 0.19

0.59 0.66 0.37 0.22 0.14 0.28

2.00 2.01 1.67 0.76 0.39 0.44

Day 18

5 10 20 30 50 75

0.03 0.04 0.03 0.05 0.07 0.09

0.09 0.10 0.12 0.11 0.13 0.16

Day 10

5 10 20 30 50 75

0.11 0.10

0.21 0.16 0.07 0.07 0.07 0.10

1.11 0.87 0.30

Day 24

5 10 20 30 50 75

0.10 0.08 0.08 0.08 0.14 0.12

0.20 0.16 0.13 0.15 0.22 0.29

Day 11

5 10 20 30 50 75

0.05 0.07

0.14 0.13 0.12 0.08 0.05 0.10

0.92 0.89 0.79 0.53 0.32 0.41

Day 25

5 10 20 30 50 75

0.06 0.06 0.06 0.06 0.06 0.10

0.04 0.05 0.06 0.06 0.06 0.11

Day 12

5 10 20 30 50 75

0.07 0.07

0.16 0.17 0.17 0.10 0.08 0.09

0.80 0.83 0.84 0.48 0.30 0.28

Day 32

Day 13

5 10 20 30

0.10 0.09 0.05 0.07

0.48 0.42 0.16 0.12

5 10 20 30 50 75 100 150 200 250 500

0.06 0.09 0.04 0.03 0.05 0.10 0.19 0.57 0.67 0.77 1.05

0.15 0.13 0.17 0.16 0.15 0.23 0.40 0.85 0.91 0.99 1.34

0.19 0.24

Depth (m)

Sol-Fe (o200 kDa) (nM)

0.12 0.30

0.37

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Table 3 (continued ) In-patch stations Days since from first iron release

Out-patch stations Depth (m)

Sol-Fe (o200 kDa) (nM)

Diss-Fe (o0.22 mm) (nM)

TD-Fe (unfiltered) (nM)

50 75

0.07 0.12

0.12 0.18

5 10 20 30 50 75

0.07 0.07 0.04 0.05 0.06 0.12

0.33 0.29 0.14 0.11 0.13 0.21

Day 16

5 10 20 30 50 75

0.08 0.10 0.08 0.08 0.09 0.14

0.37 0.30 0.19 0.15 0.16 0.24

Day 17

5 10 20 30 50 75

0.07 0.06 0.08 0.06 0.06 0.10

0.21 0.18 0.20 0.17 0.14 0.18

Day 19

5 10 20 30 50 75

0.05 0.05 0.07 0.05 0.06 0.16

0.18 0.17 0.13 0.11 0.11 0.17

Day 21

5 10 20 30 50 75

0.05 0.05 0.06 0.08 0.08 0.10

0.21 0.18 0.15 0.12 0.12 0.21

Day 23

5 10 20 30 50 75

0.01 0.01 0.02 0.02 0.05

0.08 0.09 0.04 0.03 0.05 0.09

0.24 0.16 0.14 0.14 0.13 0.20

Day 25

5 10 20 30 50 75

0.10 0.09

0.17 0.10 0.09 0.13 0.10 0.12

0.29 0.18 0.15 0.19 0.21 0.30

Day 14

Days since from first iron release

Depth (m)

750 1250 1500 2500 3000

Sol-Fe (o200 kDa) (nM)

Diss-Fe (o0.22 mm) (nM)

TD-Fe (unfiltered) (nM)

0.34

1.30 1.24 1.34 0.97 0.93

1.54 1.52 1.62 1.41 1.41

Sol-Fe: soluble iron, Diss-Fe: dissolved Fe, TD-Fe: total dissolvable Fe, D.L.: under detection limit.

was significantly enhanced in the surface mixed layer by iron injection (Fig. 4). Comparisons of the changes in TD-Fe and Diss-Fe concentrations at 5 m of the surface mixed layer between SEEDS and SEEDS II are shown in Fig. 6. The amount of initial injected iron (350 kg iron in SEEDS and 322 kg iron in SEEDS II) and initial injected area (80 km2 in SEEDS and 64 km2 in SEEDS II) were not significantly different between SEEDS and SEEDS II; however, the initial Diss-Fe concentration was lower in SEEDS II than that in SEEDS (Fig. 6), probably due to the deeper MLD during the first iron injection in SEEDS II. Theoretical calculations of the amount of injected iron, area and MLD indicate our first iron injection would cause a 3 nM iron increase (Table 1). Therefore, the 1.73 nM increase of TD-Fe indicates either some of the iron was lost from the surface mixed layer or not all the iron was measured. The second iron injection

on D6 also resulted in TD-Fe and Diss-Fe concentrations being higher (1.86 and 0.52 nM, respectively) on D7 (Fig. 5, Table 3). Vertical section profiles of TD-Fe and Diss-Fe concentrations in the in-patch on D3, 9 and 20 are also shown in Fig. 7. The vertical section profile on D2 also indicated that the first iron injection enhanced the iron concentration in the surface mixed layer. On the other hand, the increase in iron concentration on D9 was only at depths shallower than 5 m (Fig. 7) after the second iron injection, probably due to the method of the second iron injection (tubing for iron injection was set on the surface (0 m) of the water during the second injection). Then, both TD-Fe and Diss-Fe concentrations subsequently decreased to ambient level (0.18–0.29 and 0.05–0.17 nM, respectively) after D16–D17 (Fig. 5). On D20, we could not identify the in-patch even in the vertical section profile of TD-Fe (Fig. 7). During the experiment,

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0.5

1.0

Depth (m)

0.0

1.5

0

0

10

10

20

D01

0.5

1.0

D07

50

40

D12

40

D21 D23

D13

50 60

70

70

70

80

80

Depth (m)

2.0

0.0

2.5

0.5

1.0

1.5

2.0

0.0

2.5

0

0

0

10

10

10

20

D01 D02

30

40

D04

40

D08

2.5

D16 30

D10 D11

D05 D07

Total Dissolvable Fe conc. (nM) 0.5 1.0 1.5 2.0

20

20

30

50

D25

Total Dissolvable Fe conc. (nM)

Total Dissolvable Fe conc. (nM) 1.5

D17 D19

60

1.0

D16

30

60

0.5

1.5

D11

D14

0.0

1.0

30

50

80

0.5

20

D10

D05

0.0

10 D08

D04 40

1.5 0

20

D02

30

Dessolved Fe conc. (nM)

Dissolved Fe conc. (nM)

Dissolved Fe conc. (nM) 0.0

2803

D12

50

D13

40 50

60

60

70

70

70

80

80

80

D14

60

D17 D19 D21 D23 D25

Fig. 4. Vertical profiles of total dissolvable and dissolved iron concentrations in the iron-fertilized patch (in-patch) station during the experiment.

SEEDSII Fe (In 5 m)

Fe conc. (nM)

2.5

IN Dissolved Fe

2.0

IN Total dissolvable Fe

1.5 1.0 0.5 0.0 0

5

10

15

20

25

30

35

2.5 SEEDSII Fe (Out 5 m)

Fe conc. (nM)

2.0

OUT Dissolved Fe

1.5

Out Total dissolvable Fe

1.0 0.5 0.0 0

5

10 15 20 25 Days since beginning of experiment

30

35

Fig. 5. Temporal changes in total dissolvable and dissolved iron concentrations at 5 m depth in the iron-fertilized patch (in-patch) and outside of the patch (out-patch).

Roy et al. (2008) observed clear signal of fertilized iron in Fe (II) fraction with concentration of 4200 pM on D13. We should note that higher concentrations of iron were found on D3 than on D2, and on D8 than on D7, indicating that the horizontal distribution of iron in SEEDS II was heterogeneous and/or we have not collected the water samples at the exact center of the in-patch (Fig. 5). However, our data obviously indicate that there was clear difference of Diss-Fe concentration level at in-patch station between SEEDS and SEEDS II, during the first 6 days after initial iron injection. Surface Diss-Fe concentration immediately decreased to 0.15–0.17 nM on D5 in SEEDS II. In contrast to SEEDS II, a higher level of Diss-Fe was retained in the surface mixed layer (0.68 nM; Diss-Fe on D6) after the single iron injection at the start of SEEDS (Fig. 6). Initial patch dilution rate during SEEDS II was at least 5 times larger than that of SEEDS as described above. The difference, longer retention of iron in the in-patch in SEEDS, was probably due to the shallower MLD (8.5–15 m; Tsumune et al., 2005) and physical stability, such as small dilution of the in-patch, during SEEDS (Tsumune et al., 2005). Additionally, during SEEDS II, MLD was getting shallower between D9 and D11, due to surface warming and wind relax, and the shallowing of the MLD would have resulted in accelerating the loss of added iron by sinking of particulate iron, which was no longer retained in the surface mixed layer, and replacement of subsurface water due to surface in-patch advection, same as the processes observed in other iron-enrichment experiments (Croot et al., 2005; Croot et al., 2007).

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These results indicate that SEEDS would have had better iron conditions for diatoms to acquire iron than in SEEDS II, because SEEDS had a higher initial Diss-Fe concentration (due to the shallower MLD during injection) and longer retention of Diss-Fe in the in-patch due to the physical stability of the patch, such as small dilution and less dynamic MLD. Therefore, the differences in the bio-availability of iron are one possible explanation for the difference of biogeochemical response between SEEDS II and SEEDS.

Fe infusion

3

Fe conc. (nM)

Dissolved Fe (< 0.22 µm)

2

SEEDS

SEEDS II

1

0 5

0

10

15

20

25

30

3

Fe conc. (nM)

Total dissolvable Fe

2 SEEDS

SEEDS II

1

0 0

5

10 15 20 Days since biginning of experiment

25

30

Fig. 6. Comparison of dissolved iron (upper graph) and total dissolvable iron (lower graph) between SEEDS and SEEDS II.

Days 2

-30 -40 -50 TD-Fe

-60

Days 20

-10

-10

-20

-20

-30

-30

Depth (m)

0.3

Depth (m)

-20

-40 -50 TD-Fe

-60 -70

-70 -10 -8

-6

-4

-2 0 2 4 Distance (km)

6

8

-40 -50 -70

-14 -12-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14

10

-15

Days 2 -20

-20

-30

-30

-30

Depth (m)

-20 -40 -50 -60 -70 -2

0

2

Distance (km)

4

6

8

-40 -50 -60

Diss-Fe

-70 10

0 5 10 Distance (km)

15

20

Days 20 -10

-4

-5

Days 9 -10

-6

-10

Distance (km)

-10

-10 -8

TD-Fe

-60

Depth (m)

Depth (m)

Shipboard culture experiments using iron and DFB were conducted with in-patch water on D2 at the start of the experiment, on D7 and D11 during the bloom evolution period, and on D17 and D23 during the bloom termination period. In these shipboard culture experiments, we added enough macronutrients to the collected in-patch water (Table 2) and removed the meso-zooplankton before the incubation to evaluate the iron bio-availability. Although it has been reported that there is some utilization of iron that bound to DFB by phytoplankton (SoriaDengg and Horstmann, 1995) and bacteria (Yun et al., 2000), and also indirect photo-reduction can occur (Kunkely and Vogler, 2001), Wells (1999) and Hutchins et al. (1999) clearly demonstrate a useful approach to regulate iron bio-availability with the high conditional stability constant of DFB by onboard bottle incubation experiments. Croot and Johansson (2000) have estimated conditional stability constant of DFB as KFe0 L ¼ 1016.5. We assumed their KFe0 L value for our incubation study, although some other lower values were reported (Witter et al., 2000; Rose and Waite, 2003). Iwade et al. (2006) clearly indicated that addition of DFB-Fe (III) (10:1, Fe ¼ 100 nM) in seawater medium did not show iron dissociation from DFB-Fe (III). Therefore, KFe0 L of DFB should be strong enough as Croot and Johansson (2000) reported, and addition of the high (100 nM) concentration of DFB in our experiment specifically decreases the bio-available Fe (III)’ in the incubation bottles. Equilibrium calculations indicate that even with dissolved iron concentration up to 10 nM inorganic Fe (III) species would be limited to 1016 M, or several orders of magnitude below the levels needed to support phytoplankton growth (Wells, 1999; Hutchins et al., 1999; Iwade et al., 2006). These previous studies indicated that addition of DFB to the waters as iron and macro nutrient replete condition sharply inhibited iron uptake during short-term (0–6 h) experiments and kept iron-limited conditions for most phytoplankton communities, including eukaryotes and prokaryotes during the 5-day incubation period (Wells, 1999; Hutchins et al., 1999). The results of the shipboard culture experiments on D2, D7, D11, D17 and D23 are shown in Fig. 8 with the timing of the culture experiments during the SEEDS II phytoplankton bloom. Tsuda et al. (2007) reported that during SEEDS II, micro(410 mm), nano- (2–10 mm) and pico-sized (0.2–2 mm)

Days 9

-10

Depth (m)

3.3. Existence of bio-available iron and diatoms during the SEEDS II phytoplankton bloom

Diss-Fe -14-12-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14

-40 -50 -60

Diss-Fe

-70 -15

-10

-5

0 5 10 Distance (km)

15

Fig. 7. Vertical section profiles of total dissolvable and dissolved iron in the iron-fertilized patch (in-patch) on D3, D9 and D20 of SEEDS II.

20

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D7 experiment 12

10

10

10

8

8

8

6 4

0 initial

control

DFB 100 nM

Chl.a ug/L

12

2

6

4

2

2 0

0

Fe 1 nM

initial

control

DFB 100 nM

Fe 1 nM

10

8

8

6

4

2

2

0

>10um <10um

3.5

6

4

0 control

DFB 100 nM

Fe 1 nM

DFB 100 nM

Fe 1 nM

D7, 11 Bloom evolution

Chlorophyll-a conc. µg/L

10

Chl.a ug/L

12

control

After 4 days incubation

D23 experiment

12

After 4 days incubation

initial

After 4 days incubation

D17 experiment

initial

6

4

After 4 days incubation

Chl.a ug/L

D11experiment

12

Chl.a ug/L

Chl.a ug/L

D2 experiment

2805

D17, 23 Bloom termination

3 2.5

D2 Initial

2 1.5 1

Chlorophyll-a concentration (In 5 m)

0.5 0

initial

control

DFB 100 nM

After 4 days incubation

Fe 1 nM

0

5

10

15

20

25

30

Days since beginning of experiment

Fig. 8. Size-fractionated initial and final chlorophyll-a concentrations (after 4 days of incubation) of shipboard culture experiment, and timing of the culture experiments during the SEEDS II phytoplankton blooming. The data are shown as an average value and error bar is shown as 71SD.

chlorophyll-a accounted for 27%, 43% and 30%, respectively, of the total chlorophyll-a concentration before the increase of phytoplankton. This ratio did not significantly change during the evolution phase of the iron-induced bloom, then the contribution of micro-plankton gradually decreased in bloom termination phase (Tsuda et al., 2007). In the D2 shipboard culture experiment (hereafter D2 experiment), micro-phytoplankton (410 mm) comprised 16.9% of the total chlorophyll-a concentration (0.83 mg m3) at the initial control. After 4-day incubation, total chlorophyll-a concentration in the control and the DFB-treatment bottles increased 2.8 and 2.1 times more than the initial value, respectively. In the D2 experiment, chlorophyll-a increases in the control bottles were relatively smaller than that of the D7 and the D11 experiment, even after the first iron injection to the in-patch. This is probably due to the low initial biomass of phytoplankton and the water for the D2 experiment had quite a low iron concentration water probably because of the heterogeneously uneven distribution in the in-patch and/or missing of center of the patch at our sampling (see Section 3.2). At the start of the D7 experiment, micro-phytoplankton comprised 18% of total chlorophyll-a concentration (2.13 mg m3). After 4 days of incubation, total chlorophyll-a concentration in the control bottles, in the DFB-treatment bottles and in the Fetreatment bottles increased by 4.5, 3.0 and 5.3 times than the initial value, respectively. At the start of the D11 experiment, micro-phytoplankton comprised 18% of total chlorophyll-a concentration (2.77 mg m3). After 4 days of incubation, total chlorophyll-a concentration in the control bottles, in the DFBtreatment bottles and in the Fe-treatment bottles increased by 3.2, 1.7 and 4.0 times than the initial value, respectively. The large increases in the control bottles compared to the initial bottles in the D7 and the D11 experiment indicate that bioavailable iron existed in the in-patch on D7 and D11. Moreover, the magnitude of the increase in the control bottles was significantly

lower (t-test, at the 95% confidence interval, P ¼ 0.05) than that of the Fe-treatment bottles of the D11 experiment, indicating that the concentration of bio-available iron in the in-patch D11 was not enough to induce the maximum potential of phytoplankton growth. Chlorophyll-a concentration in the DFB-treatment bottles, both in the D7 and in the D11 experiment, also increased from the initial value; however, it was less than that in the control bottles. The difference between the control and the DFB treatment probably indicates the amount of chlorophyll-a, which is increased by the bio-available iron that existed in the in-patch water and extracellular iron. Micro-phytoplankton (410 mm) comprised 64% and 70% of the total chlorophyll-a increase after 4 days of incubation in the DFB treatment in the D7 and in the D11 experiments, respectively. Wells (1999) suggested that larger cells had sufficient iron to sustain growth over several hours before depleting the intracellular iron reserves. Other previous studies also indicated that phytoplankton can often accumulate an excess of iron than that needed to support maximum growth, when accessible iron is abundant. Such luxury uptake at high iron concentrations was observed in a culture experiment of oceanic and coastal eukaryotic algae (Sunda and Huntsman, 1995, 1997; Sunda, 2001; Iwade et al., 2006; Yoshida et al., 2006). Therefore, the chlorophyll-a increase in the DFB treatment was probably caused by the utilization of the intracellular iron reserves by micro-phytoplankton. The difference in magnitude of chlorophylla increase in the DFB treatment between the D7 experiment and the D11 experiment may reflect the amount of intracellular iron reserves in the organism, and the amount may be reflected by the amount of accessible bio-available iron in the in-patch water when incubation samples were collected. Significant silicic acid decrease was observed in all treatments showing increases in chlorophyll-a in the D7 and the D11 experiment (Table 4). Therefore, increased large micro-phytoplanktons (410 mm) in these bottles were considered to be large diatoms.

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Table 4 Initial and final nutrient concentrations and its decrease in the shipboard incubation study.

D2 experiment Initial Control DFB treatment Fe treatment d Control d DFB treatment d Fe treatment D7 experiment Initial Control DFB treatment Fe treatment d Control d DFB treatment d Fe treatment D11 experiment Initial Control DFB treatment Fe treatment d Control d DFB treatment d Fe treatment D17 experiment Initial Control DFB treatment Fe treatment d Control d DFB treatment d Fe treatment D23 experiment Initial Control DFB treatment Fe treatment d Control d DFB treatment d Fe treatment

NO3+NO2 (mM)

Si(OH)4 (mM)

PO4 (mM)

18.8 16.8 17.6

36.7 36.4 36.6

1.61 1.5 1.5

2.0 1.2

0.3 0.1

63.3 58.2 59.5 57.9

80.3 74.6 77.4 75.9

4.39 4.07 4.16 4.04

5.0 3.8 5.4

5.6 2.8 4.4

0.32 0.23 0.34

67.0 60.8 63.0 59.4

83.8 78.1 80.9 78.6

4.60 4.24 4.38 4.16

6.2 4.1 7.6

5.7 2.8 5.2

0.36 0.22 0.44

16.1 13.8 14.3 10.2

30.9 29.5 30.0 27.3

1.39 1.20 1.26 0.97

2.3 1.8 5.9

1.4 0.9 3.6

0.19 0.13 0.42

57.1 56.5 57.0 55.3

75.3 74.0 75.5 74.9

4.08 4.01 4.07 4.00

0.6 0.1 1.8

1.4 0.2 0.5

0.08 0.01 0.08

0.1 0.1

Si:N ratioa

N:P ratioa

0.2 0.1

20 17

1.1 0.7 0.8

16 17 16

0.9 0.7 0.7

17 19 17

0.6 0.5 0.6

12 14 14

2.1

8 10 22

0.3

d control, d DFB treatment and d Fe treatment indicate decrease of nutrients concentration during 4 days of incubation. a

Si:N and N:P ratio indicate ratio of decrease of nutrient concentration during 4 days of incubation.

At the start of the D17 experiment, micro-phytoplankton comprised 27% of total chlorophyll-a concentration (2.15 mg m3). After 4 days of incubation, a significant increase of chlorophyll-a concentration with silicic acid decrease was observed only in the Fe-treatment bottles; the increase was 2.7 times than initial value. Chlorophyll-a concentration in the control bottles did not increase (1.0 times) and decreased in the DFB-treatment bottles. Increase of micro-phytoplankton (410 mm) comprised 60% of total chlorophyll-a increase after 4 days of incubation in the Fe treatment in the D17 experiment. At the start of the D23 experiment, microphytoplankton comprised 35% of total chlorophyll-a concentration (0.97 mg m3). After 4 days of incubation, a significant increase of chlorophyll-a concentration was observed only in the Fe-treatment bottles, the increase being 3.3 times the initial value. Chlorophyll-a concentration in the control bottles increased only 1.4 times and decrease in the DFB-treatment bottles was observed

as in the D17 experiment. Silicic acid decreased only 0.5 mM in the Fe treatment. Increase of micro-phytoplankton (410 mm) comprised only 30% of the total chlorophyll-a increase, and major increased fraction was less than 10 mm, after 4 days of incubation in the Fe treatment in the D23 experiment. Small or no increase in the control bottles and the DFBtreatment bottles compared to the initial concentration in the D17 and D23 experiments indicate that bio-available iron did not exist in the in-patch on D17 and D23. Chlorophyll-a concentration increased in the Fe treatment in the micro-phytoplankton fraction, with silicic acid decrease in D17 experiment and without silicic acid decrease in D23 experiment. These results indicate that large diatoms, which can respond to additional iron inoculation, existed in the in-patch until D17, the early termination phase of the ironinduced bloom. However, weak response of micro-phytoplankton without small decrease of silicic acid in the Fe treatment in D23 experiment indicates that there was no significant amount of large diatoms that could rapidly respond to iron in the late termination phase (D23) of the iron-induced phytoplankton bloom. It is well known that Redfield stoichiometry changes systematically with changes in the phytoplankton community or physiology. Several reports showed that the Si:N uptake ratio of diatoms increased by a factor of 2–3 under iron-limited condition (e.g., Takeda, 1998; Hutchins and Bruland, 1998). In addition, the N:P uptake ratio of diatom declines by two-thirds under ironlimited condition (e.g., de Baar et al., 1997; Price, 2005). Regarding the nutrient drawdown ratio in our incubation study, the signal of iron stress is not clear in the Si:N uptake ratio, except in D23 control bottles, which may be because non-diatom phytoplanktons were the major part of the phytoplankton at the beginning of the incubation study (Tsuda et al., 2007) and they also increase during 4 days of incubation. On the other hand, the N:P uptake ratio is relatively high in D2, D7 and D11 experiments (16–20), and significant decrease of the ratio was observed in control and DFB treatment of the D23 experiment. The decrease of the N:P uptake ratio may indicate that physiology changes occurred to phytoplankton community in the D23 bottles, which was mainly dominated by non-diatom phytoplankton, as iron stress inhibited N uptake at the end of the iron-induced phytoplankton bloom.

3.4. Why the weak response in SEEDS II Fig. 9 summarizes the existence of bio-available iron and large diatoms at the in-patch station during SEEDS II based on the data from this study. In the shipboard culture experiment, phytoplankton increase in both of control and DFB treatment was observed for D2, D7 and D11 in-patch waters, although excess DFB-inoculated treatment inhibited the phytoplankton growth compared to controls. Moreover, addition of extra iron induced an increase in the phytoplankton growth compared to control for D11 in-patch water. These data indicate that bio-available iron existed in the in-patch water until at least D11, and also luxury iron uptake in the in-patch occurred until at least D11, which allowed cell division and biomass accumulation during the 4-day incubation period. However, the level of iron bio-availability was not enough to induce maximum potential of phytoplankton growth even in the evolution phase of the bloom in the in-patch on D11. This interpretation is supported by the report of Suzuki et al. (2009), who reported on phytoplankton physiological parameters during SEEDS II. They observed an increase in photochemical quantum efficiency (Fv/Fm) of the phytoplankton community at the inpatch station after the iron injection. The increase of Fv/Fm indicated that the photosynthetic physiological condition of the

ARTICLE IN PRESS J. Nishioka et al. / Deep-Sea Research II 56 (2009) 2796–2809

Bio-available Fe in the patch

Chlorophyll-a conc. µg/L

3.5

D7

D2

the diatoms to a very low biomass before D23 in the in-patch. Consequently we could not detect any large-size phytoplankton increase after the 4-day incubation period in Fe-treatment bottles in the D23 experiment. Therefore, our study also supports evidence that the weak response of phytoplankton to the iron injections during SEEDS II was caused by reasons that the levels of iron were insufficient to allow the initiation of full diatom bloom conditions as well as the high levels of meso-zooplankton grazing, which effectively grazed down the large diatoms biomass as suggested by Tsuda et al. (2007). That is, both the heavy grazing pressure and the rapid dilution of the injected iron did not allow the initially sparse diatom biomass to multiply sufficiently to form a bloom.

No bio-available Fe in the patch

Large diatom which can response to Fe

D11

No large diatom (low biomass)

D17

D23

3 2.5 2 1.5

In Out

1

2807

Acknowledgements

0.5 0 0

5

10 15 20 25 Days since beginning of experiment

30

35

Fig. 9. Existence of bio-available iron and diatoms, which were estimated from the shipboard incubation experiment, during SEEDS II.

phytoplankton assemblage was improved by the first and second iron injections. After D13, Fv/Fm in the in-patch decreased with time and was close to the initial level around D16, indicating that the growth of phytoplankton population in the in-patch was limited by iron bio-availability after D13, and the period was consistent with the Diss-Fe level of the in-patch decrease to that of the ambient level (D16; Fig. 5) and evaluated existence of bio-available iron (Fig. 9). Suzuki et al. (2009) also report noniron-containing flavoprotein flavodoxin in the phytoplankton assemblage at the in-patch station. Abundance of the flavodoxin could be a diagnostic marker for iron limitation for phytoplankton (La Roche et al., 1996; McKay et al., 1999). Their flavodoxin data also indicated that the bio-available iron was not sufficient to released diatoms from iron stress during SEEDS II. Therefore, increased iron level after first and second iron injections in SEEDS II led to an improvement in phytoplankton physiological parameters during the evolution phase; however, it was not enough for maximum diatom growth. Therefore, as we discussed in Section 3.1, differences of the bio-availability of iron, such as initial iron concentration and retention time, between SEEDS and SEEDS II are strongly related to the difference of biogeochemical response between SEEDS II and SEEDS. Moreover, Fe treatment in our shipboard culture experiment induced the growth of large-size phytoplankton with accompanying silicate decrease for D7, D11 and D17 in-patch water, but not for D23 in-patch water. From these data, it can be inferred that large diatoms that can respond to the additional iron inoculation existed in the in-patch in the evolution and the early termination phase of the iron-induced bloom (at least until D17). However, there was no significant amount of large diatoms that could rapidly respond to iron in the late termination phase (D23) of the in-patch station. Tsuda et al. (2007) suggested that one of the possible reasons why large diatoms did not bloom during SEEDS II is that the enhanced diatoms were grazed down due to the 3–5 times higher Neocalanus plumchrus biomass at the bloom evolution phase than SEEDS. In our shipboard culture experiment, we removed meso-zooplankton from the water for the incubation. Therefore, one possible explanation is that there were some diatoms when we collected the incubated seawater from the inpatch on D2, D7, D11, D17, but macro-zooplankton grazed down

We thank the captain and crew of R.V. Hakuho-Maru and KiloMoana, and our colleagues onboard during the cruise. This study was partly supported by the Central Research Institute of Electric Power Industry and Ministry of Education, Culture, Sports, Science and Technology, and by the Grant-in-Aid for Scientific Research in Priority Areas ‘‘Western Pacific Air-Sea Interaction Study (W-PASS)’’ under Grant no. 19030002. This paper resulted from the international collaborative field project developed under the umbrella of PICES, through its Advisory Panel on the/Iron Fertilization Experiment in the Subarctic Pacific Ocean /(IFEP).

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