Iron deficiency and phytoplankton growth in the equatorial Pacific

Drep-SeoRe~~arcl1II.Vol.43,No.4-6,pp.995-1015.1996

Pergamon

Copyright Q 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved

P11:S0967-0645(96)00033-1

0967+45/96Sl5.00+0.00

Iron deficiency and phytoplankton growth in the equatorial Pacific STEVE

E. FITZWATER,* KENNETH KENNETH S. JOHNSON*? (Received

H. COALE,* and MICHAEL

R. MICHAEL GORDON,* E. ONDRUSEKS

I February 1995; in revisedform 16 January 1996; accepted 2 February 1996)

Abstract-Several experiments were conducted in the equatorial Pacific at 14O”W during the Joint Global Ocean Flux Study, equatorial Pacific, 1992 Time-series I (TS-I, 23 March-9 April), Timeseries II (TS-II, 2-20 October) and FeLINE II cruises (10 March-14 April), to investigate the effects of added Fe on phytoplankton communities. Seven series of deckboard iron-enrichment experiments were performed, with levels of added Fe ranging from 0.13 to 1000 nM. Time-course measurements included nutrients, chlorophyll a and HPLC pigments. Results of these experiments showed that subnanomolar (sub-nM) additions of Fe increased net community specific growth rates, with a increases and nutrient decreases. Community growth rates followed resultant chlorophyll Michaelis-Menten type kinetics resulting in maximum rates of 0.99 doublings per day and a halfsaturation constant of 0.12nM iron. The dominant group responding to iron enrichment was diatoms. Copyright c> 1996 Elsevier Science Ltd

INTRODUCTION The “high-nitrate-low-chlorophyll” (HNLC) paradox exhibited in the equatorial Pacific region as well as the subarctic Pacific and Southern Ocean waters has intrigued investigators for decades. Over that time, several hypotheses have been put forward as possible explanations for these observations including; grazing control, ammonium inhibition of nitrate uptake, light limitation, physical processes and micronutrient limitation (Cullen, 1991; Chavez et al., 1991; Geider and La Roche, 1994). Of these, the “iron hypothesis” outlined by Martin and Fitzwater (1988) and Martin and Gordon (1988) has stimulated the most recent attention and controversy [Limnology and Oceanography, 36 (1991)]. Martin was not, however, the first to invoke the possibility of the importance of micronutrients such as Fe, on regulating phytoplankton growth in areas of high macronutrients (i.e. N03, PO4, Si04). During the 1920s and 193Os, marine scientists frequently mentioned the importance of iron as a factor controlling plant productivity in the sea (Gran, 193 1: Hart, 1934; Harvey, 1938). As early as 1934, Hart identified Fe as a possible factor contributing to low phytoplankton productivity rates observed in the Southern Ocean. However, with the exception of a few occasional studies, very little field research was performed on the effects of Fe (Menzel and Ryther, 1960; Barber and Ryther, 1969). Hampered by a lack of techniques necessary for trace-metal research and associated contamination during sample collection and handling, these early investigations led to ambiguous results.

* Moss Landing Marine Laboratories, P.O. Box 450, Moss Landing, CA 95039, U.S.A. t Monterey Bay Aquarium Research Institute, P.O. Box 628, Moss Landing, CA 95039, U.S.A z Department of Oceanography, SOEST, University of Hawaii, Honolulu, HI 96822, U.S.A. 995

996

S. E. Fitzwater

et al.

Knowledge about iron-phytoplankton relationships was gained primarily by those studying trace-element effects on phytoplankton in the laboratory. Laboratory studies involving metal ion buffers pointed to the possible role that deficiencies in one or more bioactive trace metals (Fe, Mn, Co, Ni, Cu and Zn) may have in controlling production in the oceans (Sunda et al., 1981; Anderson and Morel, 1982; Brand et al., 1983). Of the bioactive metals, iron appears to be the most important: it is an essential phytoplankton requirement for a multitude of physiological processes (e.g. synthesis of chlorophyll, cytochromes and nitrate reductase; Weinberg, 1989). The development of “clean techniques” in the 1980s made it possible to determine that open-ocean iron concentrations were so low that most of the phytoplankton’s requirements had to be met via fallout of atmospheric dust (Moore et al., 1984; Duce, 1986). This, in turn, led to the observation that open-ocean areas with unused major nutrients were located in regions with very low dust input; hence, the assumption by Martin et al. (1989, 1990, 1991) that a deficiency of this element might be limiting phytoplankton growth. We have since tested the “iron hypothesis” in subarctic, Antarctic and equatorial Pacific HNLC waters (Martin et al., 1989, 1990, 1991; Coale, 1988, 1991; Johnson et al., 1994). Water samples from these areas have all shown increased growth with iron additions. Several other independent field studies have also shown the positive effects of added iron in these areas (de Baar et al., 1990; Price et al., 1991; Helbling et al., 1991). To date, however, the nature of the iron enrichment response is not well understood, nor is the level at which added iron becomes effective in alleviating nutritional stress. Several experiments were performed during the FeLINE II (1992) and JGOFS EqPac time-series cruises (1992) in equatorial Pacific HNLC waters to further investigate the effects of Fe additions on resident phytoplankton population dynamics and the possible role of iron limitation in regulating new production in these waters.

METHODS Three enrichment experiments were carried out on each of the JGOFS EqPac surveys, Time-series I (TS-I, 23 March-9 April 1992) and Time-series II (TS-II, 2-20 October 1992), and one on the FeLINE II cruise (10 March-14 April 1992). Most of these experiments were conducted in the vicinity of O”, 14O”W (Table 1). Nitrate, nitrite, ammonia, phosphate and silicate concentrations on TS-I and TS-II were provided by P. Wheeler from Oregon State University. Nutrient concentrations on FeLINE II were determined at Moss Landing. HPLC pigment analysis on TS-I and TS-II were performed by the methods of Bidigare and Ondrusek (1996). Fluorometric chlorophyll a determinations were performed using standard methods. Because HPLC analysis was conducted on a limited number of samples, chlorophyll a numbers discussed here are fluorometer derived unless otherwise noted. Samples from the JGOFS EqPac experiments were provided for flow cytometry analysis (Zettler et al., 1996) and for microscopy identification (Fryxell and Kaczmarska, 1994) Experimental “Clean” sampling techniques for both trace-metal analysis and biological measurements have been described by us and others in previous studies (Patterson and Settle, 1976; Bruland et al., 1979; Fitzwater et al., 1982). Fe-enrichment studies incorporating these clean

Iron deficiency

and phytoplankton

997

growth

Table 1. Summary qf the iron-enrichment experiments conductedon the FeLINE IIand JGOFS cruises. Experiment Start dates, locations, depths of collection and iron concentrations are included. Initial Fe refers to homogeneous source waters measured by GFAAS and FIA-CD (see text). Ambient Fe refers to water-column values determined for the corresponding collection depth by GFAAS

Cruise

Experiment

FeLINE II JGOFS Time-series

I

JGOFS

II

Time-series

S-Fe- 1 S-Fe-2 S-Fe-3 F-Fe- 1 F-Fe-2 F-Fe-3

Depth

Added Fe

Initial Fe

Location

Date

(m)

(nM)

(nM)

Ambient (nM)

0”,14O”W 0”,14o”W 0”,140”w 0”,14O”W 5”S,14o”W 0”,14o”W 0”,14O”W

26 March 1992 26 March 1992 5 April 1992 9 April 1992 29 September 1992 4 October 1992 14 October 1992

40 40 25 25 25 25 25

10.0 2.5 2.5 0.25-1000 0.25, 2.5 2.0 2.0

< 0.03* 0.28 0.20 < 0.03 0.15 0.25 0.05t

10.03 10.03 < 0.03 <0.03 <0.03 <0.03 <0.03

Fe

*Estimated Fe concentration. tProbable contamination above this level

methodologies have also been described in detail elsewhere (Martin et al., 1989, 1990, 1991; Coale, 1988, 1991). Briefly, seawater, with its resident phytoplankton populations, was collected from the mixed layer using 30-l Go-F10 bottles (General Oceanics, Miami, FL) collected individually from depth, suspended on Kevlar& hydroline. Aliquots were placed in well rinsed, acid-cleaned, 2-l (TS-I and TS-II) or 20-l (FeLINE II) polycarbonate bottles. To homogenize samples, incubation bottles were filled sequentially, i.e. for an experiment requiring 120 1 (four Go-Flos) incubation bottles were filled in quarter amounts from each sampler. One homogenized aliquot was collected for Fe analysis from each of the six experiments done on cruises TS-I and TS-II, and returned to the laboratory for later analysis of the dissolved Fe concentration (Gordon et al., unpublished data). Incubation bottles were spiked with small-volume injections (- 200-250 ~1 l- ‘) of Fe in the form of FeC13 or FeS04 using trace-metal-clean procedures. Following Fe additions, the 2-l bottles were triple bagged in polyethylene zip-lock bags to avoid contamination from the incubator coolant water. Sample bottles were then placed in deck-top incubators and cooled with running surface seawater. All sample manipulations were carried out in a filtered air, class 100 clean van. Handling of the 20-l incubation carboys is described by Coale (1991). In spite of these precautions, sample manipulations resulted in the inadvertent contamination of some of the samples. Trace-metal analysis of experimental source waters (TS-I and TS-II) showed that some of the controls were contaminated with picomolar (PM) concentrations of Fe. Knowledge of this low-level Fe contamination provided the opportunity to study the effects of added iron at extremely low levels.

RESULTS Iron concentrations in the homogenized samples were found to be elevated relative to ambient seawater concentrations in four of the six experiments tested during TS-I and TS-II (Table 1). Concentrations in these contaminated controls, although in the pM range, were 5-9 times higher than ambient water-column levels collected from the same depth, 0.15-0.28 vs < 0.03 nM Fe (Gordon et al., in press). Experiment F-Fe-3 was found to have an initial source-water concentration of 0.05 nM Fe. However, replicate control bottles in this

998

S. E. Fitzwater

ef al.

experiment grew as well as samples with 2.0 nM Fe additions (see below). We suspect that this was a result of contamination of the control bottles after filling, which was not apparent in the separate aliquot taken for Fe analysis. Source water used for the experiment conducted on the FeLINE II cruise contained iron at concentrations ~0.3 nM, the detection limit observed in shipboard, flow injection-chemiluminescence Fe detection (FIA-CD) (Elrod et al., 1991; Johnson et al., 1994). Parameters measured in the control treatments were similar to those measured in the uncontaminated controls of experiment SFe-3 (see below). Therefore, the source-water Fe concentration for the FeLINE II experiment was estimated to be 50.03 nM. As in previous experiments, all Fe addition treatments resulted in enhanced chlorophyll a production (Fig. 1). Following a 2-3 day lag period, chlorophyll a values in the Fe-enriched

Time Series I and FeLine II

Time Series II

012345601234567

Days Fig, 1. Chlorophyll a concentrations as a function of time in the seven equatorial Pacific Feenrichment experiments. Open circles (0) arc control treatments; closed circles (0) are 2.0-2.5 nM Fe additions; and closed triangles (A) are 0.25 nM Fe additions. 100 (+) and 1000 nM Fe(W) additions were conducted during S-Fe-3. Note the low chlorophyll levels in the uncontaminated controls of FeLINE II and S-Fe-3, and that the 0.25 nM Fe treatments fall between controls and treatments 22 nM Fe.

Iron deficiency

and phytoplankton

999

growth

samples for the three experiments conducted on TS-I increased by 11.4,8.9 and 5.7 times the initial chlorophyll a concentrations (Table 2). Chlorophyll a values in the controls increased to maximal values 3.8,4.5 and 1.4 times initial concentrations, paralleling the concentration of Fe found in these samples (0.28,0.20 and 0.03 nM Fe, respectively). TS-II Fe-enrichment experiments also resulted in higher maximum values of chlorophyll a relative to controls with the exception of experiment F-Fe-3. Following a lag period, chlorophyll a values increased 16.9, 11.9 and 8.4 times the initial concentrations. Control values also increased to maximum levels of 5.6, 4.8 and 8.8 times initial concentrations for the three experiments, with the F-Fe-3 controls attaining slightly higher levels than the 2.05 nM Fe additions. Iron concentrations in the controls for F-Fe-l and F-Fe-2 were both slightly elevated (- 0.2 nM Fe) relative to the ambient level of < 0.03 nM Fe. Chlorophyll a levels in the FeLINE II experiment increased by 16.8 times initial concentrations, with the 10nM Fe addition increasing from 0.17 to 2.85 pg chlorophyll al-‘. Control values increased by only 3.3 times the initial chlorophyll a level. Concentrations of the other major nutrients were also reduced in the enrichment experiments, although not as severely as nitrate (see the Appendix). Phosphate and silicate concentrations were reduced by approximately 50% from initial levels in both controls and treatments by the end of the experiments. Initial ammonium and nitrite concentrations were co.50 PM. Generally, nitrite was reduced over the course of the experiments while ammonia remained relatively constant. In order to determine specific effects of iron additions on groups of phytoplankton, daily samples were taken for HPLC pigment analysis during four of the six experiments conducted on the JGOFS EqPac cruises. The dominant pigments detected and used as

Table 2.

Maximum chlorophyll a concentrations attained in controls and enrichments along with nitrate consumed

Cruise

FeLINE

II

Time-series S-Fe- 1

S-Fe-3

F-Fe-2 F-Fe-3

Nitrate

(pM) Consumed

Initial

t0.03t

10.00

0.17 0.17

0.56 2.94

3.3 17.3

3.8 3.8

2.8

0.28 2.78 0.20 2.70 0.00 2.53

0.23 0.23 0.22 0.22 0.26 0.26

0.87 2.63 0.99

3.8 11.4 4.5 8.9 1.4 5.7

3.7 3.7 3.3 3.3 2.6 2.6

2.5 3.3 2.5 2.8 0.4 2.3

0.15 2.65 0.25 2.75 0.05$ 2.55

0.14 0.14 0.29 0.29 0.35 0.35

0.78 2.36

5.6 16.9 4.8 11.9 8.8 8.4

3.6 3.6 6.3 6.3 4.8 4.8

1.9 3.3 3.4 5.1 4.2 4.6

Initial

1.4

I

S-Fe-2

Time-series F-Fe- 1

Chlorophyll a (pg I-‘) Maximum Increase

Fe*(nM)

1.96 0.37 1.48

II

*Corrected for initial contamination. tEstimated Fe concentration. IProbable contamination above this level.

1.40 3.46 3.07 2.95

1000

S. E. Fitzwater

et al.

diagnostic markers were chlorophyll a (phytoplankton biomass), fucoxanthin (diatoms), 19’-hexanoyloxyfucoxanthin (prymnesiophytes), zeaxanthin (cyanobacteria), peridinin (dinoflagellates) and chlorophyll b (prochlorophytes). As can be seen in Table 3, the pigments, other than chlorophyll a, that showed the most consistent and substantial increases in response to Fe additions were chlorophyll c, fucoxanthin, diadinoxanthin and beta-carotene, which are the main pigments in diatoms.

DISCUSSION Iron contamination In most of our previous studies, addition of nanomolar amounts of Fe resulted in substantial increases in chlorophyll, with relatively small increases observed in control bottles (Martin et al., 1991; Coale, 1991; Johnson et al., 1994). In the experiments presented here, however, there was also considerable growth in many of the control bottles as well. This is the result of the iron contamination which occurred during collection of the samples. Replicates of the control treatments in most cases exhibited the same growth pattern, which suggests that iron contamination must have occurred during sample collection. The most likely source of the contamination was one or more of the four Go-Flos used in water collection. Two of the Go-Flos used to collect enrichment source water were also used for water-column trace-metal collections and found to give reliable, consistent values. The other two must have contaminated samples in the 0.3-0.4nM Fe range based on the observed contaminated control values. Because of extremely low levels and contamination problems, marine geochemists have found iron to be one of the most difficult elements to measure. Contamination of water samples at these low levels is not unusual even when samplers are meticulously acid-cleaned and stringent clean techniques are employed. For example, during both Time-series I and II, samples for trace-metal analysis were collected simultaneously from the MLML-TM Rosette and the “ultra-clean” trace-metal Go-Flos used to collect water for the Fe experiments. All eight Go-F10 samplers of the MLML-TM Rosette on TS-I had elevated levels of Fe (mean=0.38 nM Fe). Only two of the eight bottles tested during TS-II were contaminated above ambient levels, but one had an extremely high Fe value of 3.51 nM Fe (Hunter et al., 1994; Sanderson et al., 1995). Previous enrichment experiments that have also shown growth in the control treatments may have been due to contamination. During the 1990 FeLINE I equatorial Pacific cruise, Fe experiments were conducted along 14O”W at 9”N, 3”N, 0” and 3”s. Most of these experiments (Martin et al., 1991) showed very little growth in controls and enhanced growth in samples enriched with Fe additions of various types. The experiment at 3”S, however, was very difficult to interpret because elevated growth occurred in the controls as well as in the Fe additions. Johnson et al. (1994) conducted Fe experiments concurrent with ours where very little growth occurred in control bottles, indicating contamination of our controls (Fig. 2). Without Fe measurements of the homogenous source water, however, the experiment is not interpretable. The above findings illustrate the extreme difficulties encountered in the collection and manipulation of samples for enrichment studies. Not only is it necessary to use trace-metalclean techniques in Fe bottle experiments, but also, in light of these results, any Fe experiments must be accompanied by the analysis of source water for iron after collection.

HPLC-d~rivedpiglnerlf

Fe addition (nM) Day Pigment (ng I - ‘) Chlorophyll a Chlorophyll b Chlorophyll cl ,2 Chlorophyll c3 Chlorophyll c4 Fucoxanthin 19’-Butanoylocoxanthin 19’-Butanoylocoxanthin Diatoxanthin Diadinoxanthin Prasinoxanthin Chlorophyllide a Peridinin Zeaxanthin Alpha-carotene Beta-carotene

Tub/e 3.

0.28 4.96 997 22 170 53 20 546 142 116 47 103 0 16 53 24 3 22

Initial 0.00

225 34 16 4 5 12 12 25 I 11 0 0 8 51 14 0

S-Fe- 1

2788 0 369 62 72 2008 69 39 122 310 0 21 6 15 0 78

2.78 4.96 695 38 137 41 15 352 66 114 14 14 6 23 13 98 13 22

237

0

2

73

15

0

0

12

2

26

66

21

5

9

17

34

0.2 4.50

Initial 0.00

S-Fe-2

1408 29 256 II5 30 893 65 70 52 164 2 84 0 75 14 78

2.7 4.50 200 28 19 I 6 25 70 34 6 I3 0 3 4 57 I1 3

Initial 0.00 754 0 113 45 23 448 90 89 29 121 0 15 12 30 I2 0

0.25 3.15

F-Fe-2

IIcruises.

1632 IO 216 128 53 1298 143 100 138 30 0 II 0 113 8 41

2.25 2.75

concetlfrarions clefertniueditl Fe-enrichment rsperirnents conductedon the JGOFS Time-series land ,fionl tlw pruk dq qf’chloropll~~ll a. Iron additions and day qf’murimul chlorophll a we included

256 32 27 IO 9 35 80 37 3 16 3 4 4 62 12 5

Initial 0.00

1694 29 210 99 60 814 132 103 102 135 15 Ill 0 145 I8 42

0.05 4.19

F-Fe-3

Pigment cowerlrrafions

I790 0 205 91 76 878 13 64 87 199 I2 55 0 88 I2 42

2.05 3.79

we

1002

S. E. Fitzwater

et al

Johnson et al. Controls

0

1

2

3

4

5

6

Days Fig. 2. Control treatments (0 Fe additions) of Fe-enrichment experiments conducted concurrently during the FeLINE I equatorial Pacific cruise (Summer 1990) at 3”S, 14O”W. Note the increase in chlorophyll a concentrations (A, A) and subsequent decrease in NO3 (0, 0) in the Martin (unpublished data) controls (dotted lines, open symbols) compared to the Johnson et al. (1994) controls (solid lines, solid symbols).

Population

dynamics

Most previous Fe-enrichment experiments have primarily relied on bulk measurements of chlorophyll a over time as an indicator of limitation. That is, Fe limitation was invoked if more chlorophyll was produced in Fe-enriched samples than in controls. Non-quantitative SEM examination of initial and final samples of these experiments indicated that pennate diatoms were primarily responsible for the observed increase in chlorophyll (Martin et al., 1991). In order to determine the effects of iron addition on specific groups of phytoplankton during the equatorial experiments, HPLC-determined phytoplankton pigments were used to partition chlorophyll a biomass into various groups of phytoplankton using a taxonomic algorithm modified from Letelier et al. (1993) for equatorial communities. Letelier et al. (1993) however, restricted their estimates of chlorophyll a partitioning to the chlorophyll maximum layer where the cells are low-light adapted. Because the samples collected for the Fe experiments were from the upper-water column, some of the algorithm’s pigment ratios had to be adjusted for high-light adapted cells. Chlorophyll b to chlorophyll a ratios (a diagnostic marker for prochlorophytes) have been found to increase with depth (Goericke and Repeta, 1993). During the JGOFS EqPac cruises, the accessory pigments, lutien and prasinoxanthin, were below detection limits, indicating that most of the chlorophyll b could be attributed to prochlorophytes since they contain both monovinyl and divinyl chlorophyll b. Therefore the ratio of total chlorophyll b to divinyl chlorophyll a of 0.4, observed in the mixed layer during TT008 (Bidigare and Ondrusek, 1996) may be a better estimate for prochlorophyte partitioning in the Fe-

Iron deficiency

and phytoplankton

growth

1003

enrichment experiments than the value of 1.1 used by Letelier et al. (1993). Goericke and Repeta (1993) also measured a total chlorophyll b to divinyl chlorophyll a ratio of 0.4 in the mixed layer of the subtropical North Atlantic; this value was used in our algorithm to determine prochlorophyte partitioning. Various values of fucoxanthin to chlorophyll a ratios for diatoms have also been published, ranging from 0.3 to 1.23, depending on the species and location of collection (Schofield et al., 1990; Letelier et al., 1993; Bidigare et al., in press). The fucoxanthin to chlorophyll a ratio in diatoms from mixed layer natural samples has been estimated to be 0.69 (Everitt et al., 1990; Bidigare et al., in press) and this estimate fits our data well, particularly in the Fe-enriched samples where fucoxanthin is the dominant accessory pigment. Also, Geider et al. (1993) showed that under similar light conditions, the fucoxanthin to chlorophyll a ratio in the diatom Pheoductylum tricornutum remained constant when grown in iron-replete or -starved conditions. Therefore the ratio 0.69 was used for both controls and enriched samples. Finally, the diagnostic pigment for cyanobacteria, zeaxanthin, was adjusted for high-light regimes. Kana et al. (1988) demonstrated that the Synechococcus clone WH7803, when grown under various irradiance levels, varied the zeaxanthin to chlorophyll a ratio from 0.4 in low light to 2.0 in high light. Since our samples were from the upper-water column, the ratio of 2.0 was used. Even though the chlorophyll a partitioned amongst the various groups by the algorithm presented here does not exactly agree with the total measured chlorophyll LI(Table 4) until more work is done on pigment ratios of cultures grown under various light and iron conditions, this algorithm is an adequate estimate of the phytoplankton composition in Feenrichment experiments. The unaccounted chlorophyll a may be caused by concentrations changing at different rates than accessory pigments under these conditions. This may result in higher or lower ratios of chlorophyll a to accessory pigments than used in the algorithm and, therefore, an underestimation of some taxonomic groups. Since the only significant increases in accessory pigments with added Fe were those identified with diatoms (chlorophyll c, fucoxanthin, diadinoxanthin and beta-carotene) we believe most of the “unaccounted” chlorophyll a can be attributed to diatom growth. As can be seen in Table 4, the primary source of the observed increases in chlorophyll was due to diatom growth. Diatom chlorophyll a increased from levels initially 416% of the total chlorophyll biomass to 68-110% of the total at peak levels with additions greater than 2.0 nM. The contribution by all the other groups innumerated by the algorithm were variable in response to iron enrichment, either decreasing or remaining constant within a factor of 2. Samples provided for flow cytometry analysis of the EqPac experiments exhibited essentially the same pattern (Zettler et al., 1996). Although all size groups described by flow cytometry methods (small and large pennate diatoms, coccoliths, nanoplankton, ultraplankton, Prochlorococcus and Synechococcus) physiologically benefited from added Fe (e.g. increased fluorescence per cell, increased cell size) the greatest differences observed with Fe additions were exhibited by dramatic increases in the numbers of small (< 50 pm) and large ( > 50 pm) pennate diatoms. In experiment S-Fe-3, for example, small pennate cell numbers increased from initial numbers of 354-2610 cells ml-’ in the Fe additions by day 5 of the experiment. Microscopic examination of samples found the diatom populations in experiment S-Fe-l to be dominated by the pennate diatoms Cylindrotheca closterium and the previously undescribed pennate diatoms Nitzschia martinii and Nitzschia reimeri (Fryxell and Kaczmarska, 1994; Kaczmarska and Fryxell, 1994).

*Probable

F-Fe-3

Time-series F-Fe-2

S-Fe-2

Time-series S-Fe- I

Initial 0.25 2.25 Initial 0.05* 2.05

Initial 0.28 2.78 Initial < 0.03 2.53

contamination

II

I

Experiment (nM Fe)

Treatment

12 80 9 23 19 0

Dinoflagellates

above this level.

26 607 1794 40 III6 1216

IO 738 2802 22 469 1235

Diatoms

85 IO1 168 98 I53 5

89 164 83 81 65 72

Prymnesiophytes

30 19 88 32 91 58

21 103 34 22 102 62

Chrysophytes

I ‘)

51 0 26 61 0 0

13 0 0 85 45 4

Prochlorophytes

(I (ng

to diutom growth

Chlorophyll

uttrihutedprinluril~~

23 3 56 25 43 34

23 0 0 34 26 21

Cyanophytes

221 745 2131 261 1338 1263

228 1074 2883 267 126 1394

Estimated chlorophyll

u

200 154 I632 256 1694 1790

225 991 2788 231 695 1408

Measured chlorophyll

u

I.11 0.99 I.31 1.02 0.79 0.71

1.01 I .08 I .03 I.13 1.04 0.99

Ratio (estimated: measured)

Tuhle 4. Purtitioning of’HPLC-derived chloroph,vN a,/tiw~d iti the mcjor ph~~toplunktortgroup.r in inifiul waters and UI musimum chloroph,vll levels in the Fe-enrichment esperinzents. Concentruiions of the curious groups were estimated using u tu\-onomic al~orirlw? n~orliJied,fiorn Let&r et al. ( 1993). Total chlorophyll a concentrations tieterntined,fron7 the algorithm (estimutrd) are compured with chlorophyll a measured hy HPLC techniqws (measured). The increase in chlorophyll a concentrutions cun he

Iron deficiency

and phytoplankton

growth

1005

Substantial increases in diatom growth in Fe-enrichment bottle experiments have been documented by several other investigations (Martin et al., 1989; Buma et al., 1991). Coale (1991) performing Fe-enrichment experiments in the subarctic Pacific, found that diatom growth (pennates spp., Nitzschia spp. and Pseudonitzschia) increased dramatically in the iron additions, comprising approximately 42% of the total biomass at the end of the experiments with the remainder comprised of cyanobacteria (Prokaryotes) and autofluorescent organisms (<25+m size class). Also, as in the experiments presented here, dinoflaggellate numbers decreased in the Fe-addition bottles. Preliminary findings from the recently completed IronEx I project (Martin et al., 1994), where inorganic Fe was added to equatorial Pacific waters in an unenclosed mesoscale experiment (64 km2), found qualitatively similar results in chlorophyll a concentrations to those observed in incubation experiments. Photosynthetic efficiency increased within 24 h for all phytoplankton groups (Kolber et al., 1994), and 3-fold increases in both chlorophyll and productivity (as measured by 14C) occurred in 334days. However, no single group became dominant and nitrate was not substantially reduced as is the case with bottle enrichments. These differences may be due primarily to the loss of iron from the system, which does not occur in bottle experiments. Criticism of past Fe-enrichment studies has centered on the observations that chlorophyll sometimes also increased in control bottles (Banse, 1990). Several authors have pointed out that this may indicate that grazing was artificially diminished in bottle experiments, which would then select for diatom growth (Dugdale and Wilkerson, 1990; Cullen, 1991). We do not believe that the above observed increases, particularly the substantial increases observed in Fe enrichments, can be attributed solely to diminished grazing pressure. Several studies have shown that grazing pressure by micro- and macrozooplankton actually increases in enrichment bottle experiments, either by the removal of large predators or in response to increased food sources (Buma et al., 1991; Chavez et al., 1991; Coale, 1991, 1995). Preliminary work using flow cytometry methods in these experiments also indicates that grazing increases in the Fe-addition bottles (Zettler, pers. communication). Grazing also increased in the unenclosed IronEx I “patch”, with the numbers of grazers increasing in response to phytoplankton growth (Martin et al., 1994). An alternate hypothesis, and one supported by these findings, is that possible contamination of control bottles with pM quantities of Fe, as well as the lack of a loss term from sinking or mixing (i.e. Fe regeneration and photo-reduction of dissolved and colloidal Fe species in deck-top incubations), may also be an important factor contributing to the observed growth in control bottles. Community

growth rates

As Banse (1991) reiterated, iron limitation is first of all a question of cell physiology, and, therefore, observing the specific growth rate during exponential phase (instead of final yields such as chlorophyll levels) is required to confirm Fe as a limiting parameter. Phytoplankton concentration at time t (N,) can be described by the following equation: N1 = NOexp[(u - m)t],

(1)

where NOis the initial concentration, u is the instantaneous phytoplankton growth rate, and m is the instantaneous death rate from grazing and enclosure effects. In enrichment experiments, we assume that mortality due to enclosure and grazing are initially the same for

1006

S. E. Fitzwater

et al.

both controls and Fe additions, and that differences exhibited over time are due to treatments only. Since we did not measure mortality directly, we define U’= u-m, where U’is the instantaneous net community growth rate. Equation (1) can then be expressed as: U’= ln(N,/No)/t.

(2)

Growth rates based on the cumulative increase of particulate organic nitrogen (PON) were calculated from nitrate removal during exponential growth using equation (2) where iVOis the initial PON concentration (an estimate of 0.50 PM PON was based on mixed-layer samples collected on FeLINE II) and N, is the concentration of PON after time. Specific growth rates, i.e. doublings per day (U), were estimated by linearly regressing the logtransformed cumulative PON data and dividing the slope, u’, by In 2 [Fig. 3(a)-(f)].

Time Series

I and FeLine II

Time Series 3

’ 4 b)

II

I e) F-Fe-1

S-Fe-1

01234560123456 Days Fig. 3. Linear regression analysis on the In-transformed cumulative PON data for the seven experiments. Dividing the slope of the line (u’. the instantaneous growth coefficient) by In 2 results in the specifiic growth rate, U, in doublings per day. Control treatments (0) and the 2.0-2.5 nM Fe additions (A) were conducted for each experiment. The 0.25 nM Fe additions (dashed lines) fall between the 0 additions and Fe additions 12.0nM [(d) and (e)]. 100 (V) and IOOOnM Fe (0) additions were conducted during S-Fe-3 (d) and follow the 2.5 nM Fe treatment closely.

Iron deficiency

and phytoplankton

1007

growth

Enrichment experiments have consistently shown that Fe-deficient cells will preferentially synthesize chlorophyll, resulting in a decreased carbon:chlorophyll ratio, thereby overestimating growth (Coale, 1991). Therefore, nitrate uptake was used for growth estimates because nitrate is primarily assimilated by phytoplankton and subsequent grazing or cell death results in a release of ammonium or amino acids. Comparisons of U on an experiment during the FeLINE II cruise, where PON was directly measured, showed close agreement with rates derived by cumulative PON estimated from NO3 removal. Growth rates based on chlorophyll, however, were much higher, indicating enhanced chlorophyll production relative to biomass (Fig. 4). Increasing concentrations of Fe, added either deliberately or through contamination, resulted in increasing growth rates (Table 5). Morel et al. have demonstrated that uptake rates of Fe by phytoplankton follow Michaelis-Menten type kinetics (Morel et al., 1991; Price et al., 1994). Comparison of the growth rates determined here as a function of Fe concentration also follow Michaelis-Menten type kinetics where the specific community growth rate (U) is proportional to the substrate concentration (Fe) at low concentrations and reaches a maximum (U,,,) at substrate concentrations that exceed the half-saturation constant, Ku. The relationship is described as follows: Fe’ + LF~ ++ FeLr, C) Fein,

(3)

where Fe’ is the bioavailable, reactive Fe concentration, LFe the Fe binding ligand and Fein is the intracellular Fe. Growth rate, U, can then be expressed in the classical MichaelisMenten hyperbolic equation: U=

GdFe’l

(4)

KU + [Fe’] ’ 7

?? =PON

I

0123456789'

I

,

Y

I

1

Days Fig. 4. Particulate organic nitrogen (PON), chlorophyll a and nitrate concentrations for an experiment conducted on FeLINE II (1992). Growth rates calculated from PON measured directly and PON increases based on nitrate removal agree well. PON doublings per day=0.96, r2= 0.96; NO3 = 0.86 day-‘, r’= 0.99. Chlorophyll-derived growth rate was considerably higher, being 2.18day-‘, r’=0.92.

1008 Table

S. E. Fitzwater

5.

Growth

Experiment Spring 1992 FeLINE II Time-series S-Fe- 1

S-Fe-3

Days

<0.03* 10.03 0.2 2.78 0.20 2.70 <0.03 0.28 2.53 100 11000

consumed.

II’

n

r

1.44.2 2.4-4.2

0.169 0.609

4 3

0.561 0.979

0.24 0.88

81.9-5.9 1.94.9 2.0-5.0 2.04.5

0.518 0.667 0.576 0.643 0.136 0.382 0.666 0.571 0.513

0.987 0.999 0.964 0.979 0.894 0.849 0.965 0.991 0.891

0.75 0.96 0.83 0.93 0.20 0.55 0.96 0.82 0.74

0.369 0.509 0.673 0.306 0.727 0.574 0.809

0.963 0.998 1.ooo 0.973 0.993 0.961 0.984

0.53 0.73 0.97 0.44 1.05 0.83 1.17

0.197 0.693 0.720 0.196 0.513 0.669

0.974 0.947 0.854 0.963 0.995 1.000

0.28 0.92 1.04 0.28 0.74 0.97

0.15 0.40 2.65 0.25 2.25 0.05-I 2.05

F-Fe-3 Summer 1990 FeLINE I 0’.14O”W

*Probable tEstimated

Fe (nM)

nitrate

U (day-‘)

1.46.0 1.4-6.0 3.CK6.0 3.0-6.0 3.G6.0

II

F-Fe-2

3‘S,l4O’W

rates ,for the equatorial PaciJic Fe-enrichment studies calculated ,from E.~perimentsfiom the 1990 FeLINE I cruise are inciuded,for comparison

I

S-Fe-2

Fall 1992 Time-series F-Fe- 1

er al.

0.05* 1.05 5.05 0.05* 5.05 10.05

1.7-5.7

1.7-5.7 1.7-4.8 1.7-6.8 I .7-3.8 1.8-5.8 1.84.8

2.C5.0 2.Ck5.1 2.k5.2 1.94.9 1.94.9

1.94.9

contamination above this level from water-column data.

Least-squares estimates of the Michaelis-Menten equation by non-linear curve fit 0.99 doublings per day and a half-saturation constant (SYSTATTM) results in U,,,= (KU) = 0.12 nM Fe (Fig. 5). These growth rates are similar to those calculated by Zettler et al. (1996) based on cell numbers, for small and large pennate diatoms. They found a mean for the Fe addition samples and 0.23 doublings day-’ for the growth rate of 0.69day-’ “clean” control of S-Fe-3. A general statement on whether or not equatorial communities are iron-limited can be made based on the half-saturation constant of 0.12 nM Fe. As Morel et al. (1991) state, metal limitation (in cultures) is evident when ambient reactive metal levels (Fe’) are less than the half-saturation constant which, based on the above findings, could be invoked here. Mixedlayer dissolved Fe concentrations in Pacific equatorial waters typically range from 0.03 to 0.06 nM (Gordon et al., in press) which is one-half to one-quarter the measured Ku found in this study. Very little information is available on the effects of such sub-nM additions. Recent work by Takeda and Obata (1995) at O”, 159”W confirms the effects of iron additions

Iron deficiency

and phytoplankton

.-F 0.8

??

il

0

3

5

[Fe]*0.99 u=

0.6

[Fe]+0.12

,

L 0 ;

1009

growth

D

0.4

.. 0.0 0

* ’ E * I B * ’ * ‘,“/‘, 2 4 6 8 10 Iron

s 200

’ ’ ’ ’ * ’ . ’ 400 600 800 1000

(nM)

Fig. 5. Community growth rates (doublings per day) as a function of Fe concentration showing Michaelis-Menten type kinetics. A non-linear curve fit (SYSTATTM) results in U,,,,, of 0.97 day-’ and a half-saturation constant (KU) of 0.12 nM Fe. F-Fe-3 control treatments were not included in the calculation.

in the sub-nM range. They found that addition of 0.1 nM Fe almost doubled particulate organic carbon levels during a 5-day experiment. POC levels also increased proportionally with increased pM Fe additions. The proportionality of growth-rate response to increasing Fe additions in these studies is a positive indication of deficiency of the element. From the above reasoning, U,,,,, would be reached when Lw becomes saturated, i.e. the unbound ligand concentration, [LF& approaches zero. We then infer that the LF~ concentration is approximately 1 nM based on the total dissolved Fe added at U,,,. This, of course, is a generous estimate of LF~ because the ratio [Fe’]/[Fe& is presently unknown. However, the ratio may be high in bottle experiments due to the lack of loss terms for Fe and photo-reduction which is thought to render colloidal and dissolved Fe more bioavailable (Wells, 1989; Morel et al., 1991; Johnson et al., 1994). Therefore, 1 nM may be a very good estimate of the total iron-binding ligand available and is in the same range as that recently found for dissolved iron-binding ligands in Pacific waters determined by ligand competition-cathodic adsorption techniques (Rue and Bruland, 1995).

CONCLUSIONS Keeping in mind that “no single factor can be said to ‘control’ phytoplankton to the exclusion of others” (Chisolm and Morel, 1991), we still believe that phytoplankton community growth in major nutrient-rich waters such as the eastern equatorial Pacific is physiologically limited by iron deficiency. This is not to say, however, that phytoplankton do not grow or are not grazed in these environments. Obviously there are organisms that do very well with the small amounts of Fe available. High productivity rates in the equatorial regions (58-100 mmol C m-* day-‘) are regularly reported (Barber and Chavez, 1991); NH3 appears to be the preferred source of N (Price et al., 1991) and grazing appears to keep

S. E. Fitzwater et al.

1010

the population in semi-steady state (Miller et al., 1991; Frost, 1991; Price et al., 1994). Much of this growth can be attributed to the efficient recycling of iron within the euphotic zone (Hutchins et al., 1993) and new production stimulated by Fe upwelled with deep water Fe (Coale et al., 1996; Gordon et al., in press). However, the extremely low mixed-layer levels (co.03 nM) are not sufficient to enable phytoplankton communities to deplete ambient nutrients. The results presented here not only indicate that equatorial Pacific populations are Fedeficient, but also demonstrate that extremely low Fe concentrations increase growth rates as well as nitrate uptake and chlorophyll production. Although these Fe additions increased the physiological “health” of the entire community, as was found in the unenclosed IronEx project, the increased growth exhibited in bottle experiments was due primarily to diatoms. The results of this study indicate the difficulties involved in performing bottle experiments. Much has been said concerning the artifacts and biases of Fe-enrichment bottle experiments due to bottle effects, grazing, etc. (Geider and La Roche, 1994). However, very little has been said about the problem of Fe contamination. As previously mentioned, interpretation of the results of these enrichment studies was only possible because initial Fe concentrations were measured. This will have to be done in future experiments to verify levels of iron in the controls and treatments. We cannot stress enough the necessity of employing truly clean techniques in enclosed bioassay experiments. Ackno,~,/edger?tents-We

thank Craig Hunter and Sara Tanner for their substantial help with the field work and laboratory analyses. We also wish to thank the crews of the R.V. Wecoma and R.V. Thompson for their invaluable assistance. We dedicate this paper to the memory of John H. Martin, the primary “mover and shaker” of this program. This work was supported by grants from the ONR Chemistry Program (NO00 14-84-C-0619), NSF Marine Chemistry Program (OCE 8813565) and ONR Chemistry Program (NO01489-J-1010) to Drs Martin, Johnson and Coale. JGOFS contribution 234.

REFERENCES Anderson G. C. and F. M. M. Morel (1982) The influence of aqueous iron chemistry on the uptake of iron by the coastal diatom Thallasiosira weissflogii. Linmology and Oceanography, 27, 789-813. Banse K. (1990) Does iron really limit phytoplankton production in the offshore subarctic Pacific? Limnology and Oceanography,

35, 172-715.

Banse K. (1991) Rates of phytoplankton cell division and Oceanograplzy, 36, 18861898. Barber R. T. and F. P. Chavez (1991) Regulation Lintnology

and Oceanography,

in the field and in iron enrichment of primary

productivity

rate

experiments. in the equatorial

Limnolog~

Pacific.

36, 1803- 18 15.

Barber R. T. and J. H. Ryther (1969) Organic chelators: Factors affecting primary production in the Cromwell Current upwelling. Journal of E.yperinwntal Marine Biology and Ecology, 3, 191-199. Bidigare R. R. and M. E. Ondrusek (1996) Spatial and temporal variability of phytoplankton pigment distributions in the central equatorial Paciic Ocean. Deep-Sea Research II, 43, 809-833. Bidigare R. R., J. L. Itiarte, S. H. Kang, M. E. Ondrusek, D. Karentz and G. A. Fryxell (in press) Phytoplankton: Quantitative and qualitative assessments. In: Foundations fbr ecosystem research in the Western Antarctic Peninsula region. Antarctic Research Series, American Geophysical Union, Washington, DC. Brand L. E., W. G. Sunda and R. R. L. Guillard (1983) Limitation of marine phytoplankton reproduction rates by zinc, manganese and iron. Limnology and Oceanography, 28, 1182-l 198. Bruland K. W., R. P. Franks, G. A. Knauer and J. H. Martin (1979) Sampling and analytical methods for the determination of copper, cadmium, zinc and nickel in sea water at the nanogram per liter level. Ana&tica Chimica

Acta.

105. 233-245.

Buma A. G. J.. H. J. W. de Baar. R. F. Nolting and A. J. van Bennekom (1991) Metal enrichment the Weddell-Scotia Seas: Effects of iron and manganese on various plankton communities. Oceanography,. 36, 1865-l 878.

experiments Limnology

in and

Iron deficiency

and phytoplankton

growth

Chavez F. P., K. R. Buck, K. H. Coale, J. H. Martin, G. R. DiTullio, N. A. Welschmeyer, T. Barber (1991) Growth rates, grazing, sinking and iron limitation of equatorial

1011 A. C. Jacobson and R. Pacific phytoplankton.

Limnology and Oceanography, 36, 18 161833. Chisolm W. W. and F. M. M. Morel (1991) What controls phytoplankton production in nutrient-rich areas of the open ocean. Preface. Limnology and Oceanography, 36. Coale K. H. (1988) Copper complexation in the North Pacific Ocean. Ph.D. thesis, University of California, Santa Cruz. Coale K. H. (1991) Effects of iron, manganese, copper, and zinc enrichments on productivity and biomass in the subarctic Pacific. Limnology and Oceanography, 36, 1851-1864. Cullen J. J. (1991) Hypothesis to explain high-nutrient conditions in the open sea. Limnology and Oceanography, 36, 1578-1599. Cullen J. J. (1995) Status of the iron hypothesis after the Open-Ocean Enrichment Experiment. Limnology and Oceanography, 40, 1336-1343. de Baar H. J. W., A. G. J. Buma, R. F. Nolting, G. C. Cadee, G. Jaques and P. J. Treguer (1990) On iron limitation in the Southern Ocean: experimental observations in the Weddell and Scotian Sea. Marine Ecology Progress Series, 65, 105-122. Duce R. A. (1986) The impact of atmospheric nitrogen, phosphorus and iron species on marine biological productivity. In: The role of air-sea exchange in geochemical cycling, P. Baut-Menard, editor. Reidel, New York, pp. 497-529. Dugdale R. C. and F. P. Wilkerson (1990) Iron addition experiments in the Antarctic: A re-analysis. Global Biogeochemical Cycles, 4, 13-19. Elrod V. A., K. S. Johnson and K. H. Coale (1991) Determination of subnanomolar levels of iron (II) and total dissolved iron in seawater by flow injection analysis with chemiluminescence detection. Analytical Chemistry, 63, 883-888. Everitt D. A., S. W. Wright, J. K. Volkman, D. P. Thomas and E. J. Lindstrom (1990) Phytoplankton community compositions in the western equatorial Pacific determined from chlorophyll and carotenoid pigment Deep-Sea Research, 37, 975-997. distributions. Fitzwater S. E.. G. A. Knauer and J. H. Martin (1982) Metal contamination and primary production: Field and laboratory methods of control. Limnology and Oceanography, 27, 544-551. Frost B. W. (1991) The role of grazing in nutrient-rich areas of the open ocean. Limnology and Oceanography, 36, 1616-1631. Fryxell G. A. and I. Kaczmarska (1994) Specific variability in Fe-enriched cultures from the equatorial Pacific. Journal of Plankton Research, 16, 755-769. Geider R. J. and J. La Roche (1994) The role of iron in phytoplankton photosynthesis, and the potential for ironlimitation of primary productivity in the sea. Photosynthetic Research, 39, 275-301. Geider R. J., J. La Roche, R. M. Greene and M. Olaizola (1993) Response of the photosynthetic apparatus of Phaeodactylum tricornutum (Bacillariophyceae) to nitrate, phosphate, or iron starvation. Journal of Phycology, 29, 755-766. Goericke R. and D. J. Repeta (1993) Chlorophylls a and b and divinyl chlorophylls a and b in the open subtropical North Atlantic Ocean. Marine Ecology Progress Series, 101, 307-313. Gordon R. M., K. H. Coale and K. S. Johnson (in press) Iron distribution in the equatorial Pacific: Implications for new production. Limnology and Oceanography. Gran H. H. (1931) On the conditions for the production of plankton in the sea. Rapports et Proces-Verbaux des Reunions Conseil International pour I’E.xploration de la Mer, 75, 37-46. Hart T. J. (1934) On the phytoplankton of the south-west Atlantic and the Bellingshausen Sea, 1929-1931. Discovery Reports, VIII, l-268. Harvey H. W. (1938) The supply of iron to diatoms. Journal of the Marine Biological Association of the United Kingdom, 22, 205-2 19. Helbling E. W., V. Villafane and 0. Holm-Hansen (1991) Effect of Fe on productivity and size distribution of Antarctic phytoplankton. Limnology and Oceanography, 36, 1879-l 885. Hunter C. N., R. M. Gordon, S. E. Fitzwater and K. S. Johnson (1994) A rosette/winch system for the collection of trace metal clean, large volume seawater samples. Eos, Transactions of the American Geophysical Union, 75,150. Hutchins D. A., G. R. DiTullio and K. W. Bruland (1993) Iron and regenerated production: Evidence for biological iron recycling in two marine environments. Limnology and Oceanography, 38. 1242-1255. Johnson K. S., K. H. Coale, V. A. El&l and N. W. Tindale (1994) Iron photochemistry in seawater from the equatorial Pacific. Marine Chemistry, 46, 319-334.

1012

S. E. Fitzwater

et al.

Kaczmarska I. and G. A. Fryxell (1994) The genus Nitzschia: Three new species from the equatorial Pacific Ocean. Diatom Research, 9, 87-98. Kana T. M., P. M. Gilbert, R. Goericke and N. A. Welschmeyer (1988) Zeaxanthin and p-carotene in S~nechococcus WH7803 respond differently to irradiance. Limnology and Oceanography, 33, 1623-1626. Kolber 2. S. (1994) Iron limitation of phytoplankton photosynthesis in the equatorial Pacific Ocean. Nature, 371, 145-149. Letelier R. M., R. R. Bidigare, D. V. Hebel, M. Ondrusek. C. D. Winn and D. M. Karl (1993) Temporal variability of phytoplankton community structure based on pigment analysis. Limnology and Oceanography, 38, 1420-1437. Martin J. H. and S. E. Fitzwater (1988) Iron deficiency limits phytoplankton growth in the North-East Pacific subarctic. Nature, 331, 341-343. Martin J. H. and R. M. Gordon (1988) Northeast Pacific iron distributions in relation to phytoplankton Deep-Sea Research, 35. 177-196. producivity. Martin J. H.. R. M. Gordon, S. E. Fitzwater and W. W. Broenkow (1989) Vertex: phytoplankton/iron studies in the Gulf of Alaska. Deep-Sea Research, 36, 649-680. Martin J. H., S. E. Fitzwater and R. M. Gordon (1990) Iron deficiency limits phytoplankton growth in Antarctic Waters. Global Biogeochemical Cycles, 4, 5-12. Martin J. H., R. M. Gordon and S. E. Fitzwater (1991) The case for iron. Limnology and Oceanography, 36. 1793-I 802. Martin J. H. et al. (1994) Testing the iron hypothesis in ecosystems of the equatorial Pacific. Nature, 371, 123-129. Menzel D. W. and J. H. Ryther (1960) Nutrients limiting the production of phytoplankton in the Sargasso Sea, with special reference to iron. Deep-Sea Research, 35, 473490. Miller C. B., B. W. Frost, P. A. Wheeler, M. R. Landry, N. A. Welschmeyer and T. M. Powell (1991) Ecological dynamics in the subarctic Pacific, a possible iron-limited ecosystem. Limnology and Oceanography, 36. 16O(t 1615. Moore R. M., J. E. Milley and A. Chat (1984) The potential for biological mobilization of trace elements from aeolian dust in the ocean and its importance in the case of iron. Oceanologica Acta, 7, 221-228. Morel F. M., R. J. M. Hudson and N. M. Price (1991) Limitation of productivity by trace metals in the sea. Limnology and Oceanography, 36, 1742-l 755. Patterson C. C. and D. M. Settle (1976) The reduction of orders of magnitude errors in lead analyses of biological materials and natural waters by evaluating and controlling the extent and sources of industrial lead contamination introduced during sample collecting, handling and analysis. Publications of the National Bureau of Standards, 422, 321-35 1. Price N. M., L. F. Anderson and F. M. M. Morel (1991) Iron and nitrogen nurition of equatorial Pacific plankton. Deep-Sea Research, 38, 1361-1378. Price N. M., B. A. Ahner and F. M. M. Morel (1994) The equatorial Pacific Ocean: Grazer-controlled phytoplankton populations in an iron-limited ecosystem. Limnologq and Oceanography, 39, 520-534. Rue E. L. and K. W. Bruland (1995) Complexation of iron (III) by natural organic ligands in the central north Pacific as determined by competetive equilibration/adsorptive cathodic stripping voltametry. Marine Chemistr~~, 50, 1 17-138. Sanderson M. P.. C. N. Hunter. S. E. Fitzwater, R. M. Gordon and R. T. Barber (1995) Primary productivity and trace metal contamination measurements from a clean rosette system versus ultra clean Go-Flo bottles. Deep-Sea Research II. 42. 43 1440. Schofield O., R. R. Bidigare and B. B. Prezelin (1990) Spectral photosynthesis, quantum yield and blue-green light enhancement of productivity rates in the diatom Chaetoceros grucile and the prymnesiophyte Emilionia hu.ulej?. Marine Erology Progress Series. 84. 175-l 86. Sunda W. G.. R. T. Barber and S. A. Huntsman (1981) Trace metal control of phytoplankton growth in seawater: Importance of copper-manganese cellular interactions. Journal of Marine Research, 39, 567-586. Takeda S. and H. Obata (1995) Response of equatorial Pacific phytoplankton to subnanomolar Fe enrichment. Marine Chemistry, 50, 219-227. Weinberg E. D. (1989) Cellular regulation of iron assimilation. Quarterley Review, of Biology, 64, 261-290. Wells M. L. (1989) The availability of iron in seawater: A perspective. Biological Oceanography, 6, 463476. Zettler E. R.. R. J. Olson, B. J. Binder, S. W. Chisholm, S. E. Fitzwater and R. M. Gordon (1996) Ironenrichment bottle experiments in the equatorial Pacific: responses of individual phytoplankton cells. DeepSea Reseurch II, 43, 1017-1029.

Iron deficiency

and phytoplankton

1013

growth

APPENDIX Nutrient, chlorophyll and phaeophytin concentrations for the FeLINE II and JGOFS EqPac Fe-enrichment experiments. Experimental start dates and sampling day are included. Bold nitrate values were used to calculate cumulative PON growth rates (see text). Values in square brackets were considered outliers and initial values for nitrate substituted for growth calculations. Date

Days

Feline II 26 March 28 March

1992 1992

0.00 1.43

29 March

1992

2.42

30 March

1992

3.43

31 March

1992

4.18

S-Fe- 1 26 March 27 March

1992 1992

0.00 0.50 1.01

28 March

1992

1.92

29 March

1992

2.44 2.96

30 March

1992

3.96

31 March

1992

4.44 4.96

1 April 1992

5.88

S-Fe-2 5 April 1992 6 April 1992

0.00 1.00

7 April 1992

2.00

8 April 1992

3.04 3.50

9 April 1992

4.00 4.45

Fe

NH4

NO3

NO1

PO4

Si04

Chlorophyll

(nM)

(/lM)

(FM)

GM)

(FM)

(PM)

(pg 1-Q

Phaeophytin (pg 1-l)

Initial <0.03 10.00 <0.03 10.00 10.03 10.00 <0.03 10.00

NA NA NA NA NA NA NA NA NA

NA 3.4 3.4 3.1 3.2 3.4 2.6 2.4 1.0

NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA

NA 1.1 1.1 1.0 1.o 1.0 0.5 0.6 0.2

0.17 0.16 0.23 0.35 0.35 NA NA 0.56 2.94

0.07 0.05 0.06 0.08 0.09

Initial 0.28 2.78 0.28 2.78 0.28 2.78 0.28 2.78 0.28 2.78 0.28 2.78 0.28 2.78 0.28 2.78 0.28 2.78

NA 0.25 0.21 NA NA 0.24 0.40 0.13 0.10 0.18 0.19 0.16 0.21 NA NA 0.11 0.41 0.07 0.08

- 3.70 3.70 3.90 NA NA [2.69] [2.59] 5.14 4.98 4.01 4.05 2.47 2.35 NA NA 1.18 0.36 0.62 0.59

NA 0.31 0.30 NA NA 0.41 0.40 0.41 0.40 0.54 0.57 0.46 0.47 NA NA 0.28 0.17 0.15 0.13

NA 0.60 0.56 NA NA AP AP 0.68 0.69 0.76 0.76 0.55 0.55 NA NA 0.22 0.19 0.36 0.31

NA 2.87 2.86 NA NA 5.51 5.39 1.65 2.77 2.09 2.07 1.66 1.78 NA NA 1.50 0.88 1.07 1.09

-0.23 0.23 0.21 0.13 0.13 0.16 0.23 0.34 0.24 0.28 0.43 0.61 1.27 0.59 2.71 0.87 2.96 0.73 0.95

NA 0.14 0.10 0.04 0.06 0.05 0.06 0.09 0.06 0.06 0.07 0.08 0.13 0.19 0.45 0.21 NA 0.20 0.21

Initial 0.20 2.70 0.20 2.70 0.20 2.70 0.20 2.70 0.20 2.70 0.20 2.70

NA NA NA NA NA NA NA NA NA 0.10 0.12 0.18 0.13

3.29 3.72 3.48 3.10 3.20 2.82 2.51 1.71 1.39 1.64 2.03 0.75 0.49

0.43 0.47 0.54 0.51 0.49 0.37 0.31 0.26 0.26 0.28 0.32 0.12 0.16

0.68 0.67 0.64 0.61 0.57 0.60 0.59 0.47 0.43 0.65 0.67 0.37 0.27

2.37 2.35 2.62 2.89 2.79 2.34 2.14 2.14 2.26 AP AP 1.69 1.34

0.22 0.19 0.29 0.27 0.37 0.45 0.77 0.84 1.83 0.79 2.11 1.06 1.96

0.10 0.06 0.08 0.07 0.09 0.15 0.16 0.18 0.32 0.23 0.36 0.20 0.30

0.18 0.26

(conlimwd)

1014

S. E. Fitzwater

Date

Days

Fe @MI

et al

NH4

NO,

NO?

PO4

Si04

W)

WI

Wf)

k-M

W)

Chlorophyll 0% 1-l)

Phaeophytin (M-‘)

S-Fe-2 10 April 1992

5.00

0.20 2.70

0.10 0.06

0.35 0.29

0.06 0.06

0.41 0.43

1.31 1.34

0.83 0.89

0.22 0.18

S-Fe-3 9 April 1992 10 April 1992

0.00 1.37

12 April 1992

3.00

13 April 1992

4.00

14 April 1992

5.00

15 April 1992

6.00

Initial <0.03 0.28 2.53 100.00 1000.00 <0.03 0.28 2.53 100.00 1000.00 < 0.03 0.28 2.53 100.00 1000.00 <0.03 0.28 2.53 100.00 1000.00 <0.03 0.28 2.53 100.00 1000.00

0.05 DL 0.05 DL 0.14 0.45 0.12 0.14 0.16 0.09 0.17 0.13 0.05 DL 0.02 0.05 NA NA NA NA NA 0.06 0.10 0.10 0.08 0.09

2.61 2.90 [2.95] [3.17] 2.92 3.31 2.65 2.75 2.68 2.58 2.59 2.89 2.69 2.16 2.08 1.27 2.35 1.61 0.63 NA NA 2.23 0.68 0.29 0.32 0.34

0.36 0.33 0.32 0.35 0.34 0.35 0.30 0.36 0.31 0.30 0.24 0.32 0.38 0.27 0.21 0.12 0.17 0.16 NA NA NA 0.28 0.14 0.09 0.10 0.11

0.76 0.79 0.77 0.77 0.76 0.73 0.55 0.56 0.53 0.56 0.43 0.63 0.60 0.54 0.58 0.55 NA NA NA NA NA 0.49 0.26 0.23 0.22 0.22

AP 2.40 2.88 2.54 2.45 2.29 2.45 2.36 2.24 2.14 2.26 NA NA NA NA NA 1.87 1.42 0.84 0.98 0.87 NA NA NA NA NA

0.26 0.15 0.11 0.15 0.12 0.13 0.14 0.16 0.27 0.27 0.39 0.14 0.25 0.60 0.71 1.10 0.26 0.38 1.41 2.08 1.87 0.37 0.78 1.48 1.10 1.06

NA 0.06 0.04 0.04 0.05 0.04 0.07 0.09 0.08 0.07 0.08 0.06 0.12 0.20 0.16 0.23 0.08 0.14 0.42 0.46 0.48 0.11 0.25 0.55 0.37 0.31

Initial 0.15 0.40 2.65 0.15 0.40 2.65 0.15 0.40 2.65 0.15 0.40 2.65 0.15 0.40 2.65 0.15 0.40 2.65

0.17 0.14 0.10 0.09 0.15 0.19 0.28 0.32 0.33 0.31 0.26 0.30 0.22 0.13 0.07 0.10 0.37 0.41 0.35

3.58 3.53 [3.71] [3.66] 3.71 3.95 3.79 3.26 2.79 2.08 2.94 1.65 0.27 1.68 0.34 0.21 0.80 0.00 0.00

0.29 0.21 0.23 0.23 0.19 0.22 0.23 0.21 0.22 0.23 0.15 0.14 0.12 0.12 0.11 0.10 0.19 0.14 0.12

0.66 0.78 0.65 0.68 0.66 0.67 0.66 0.75 0.72 0.68 0.81 0.63 0.56 0.70 0.54 0.52 0.68 0.52 0.49

2.48 2.18 2.53 2.57 2.60 3.11 2.51 1.97 1.62 1.41 2.45 0.87 0.52 2.62 1.22 1.31 1.77 1.66 1.55

0.14 0.28 0.30 0.28 0.44 0.55 0.64 0.53 0.74 1.36 0.49 0.93 2.36 0.71 1.36 1.41 0.78 1.14 1.00

0.04 0.07 0.07 0.06 0.08 0.11 0.12 0.13 0.16 0.20 0.16 0.23 0.31 0.13 0.30 0.21 0.13 0.18 0.13 (<“r,rr,~,ird)

F-Fe- 1 29 September 1992 1 October 1992

0.00 1.73

2 October

1992

2.75

3 October

1992

3.75

4 October

1992

4.76

5 October

1992

5.74

6 October

1992

6.77

Iron deficiency

Days

Date

F-Fe-2 4 October 6 October

1992 1992

0.00 1.73

7 October

1992

2.75

8 October

1992

3.75

9 October

1992

4.75

10 October

1992

5.75

11 October

1992

6.75

F-Fe-3 14 October 15 October

1992 1992

0.00 0.79

16 October

1992

1.79

17 October

1992

2.79

18 October

1992

3.79

19 October

1992

4.79

20 October

1992

5.79

AP, analytical problem; *Probable contamination

Fe

and phytoplankton

1015

growth

NH4

NO3

NO2

PO4

SiOl

(nW

WI

WW

WW

WV

Wf)

Initial 0.25 2.25 0.25 2.25 0.25 2.25 0.25 2.25 0.25 2.25 0.25 2.25

0.23 0.32 0.29 0.24 0.31 0.18 0.13 0.50 0.21 0.60 0.23 0.27 0.18

6.25 5.60 5.42 4.82 4.32 3.72 0.93 NA 1.19 2.87 0.07 0.39 0.20

0.01 0.34 0.35 0.27 0.25 0.20 0.11 0.13 0.14 0.24 0.12 0.16 0.11

0.78 0.90 0.91 0.80 0.79 0.79 0.68 0.59 0.53 0.60 0.40 0.42 0.35

Initial 0.05* 2.05 0.05* 2.05 0.05* 2.05 0.05* 2.05 0.05* 2.05 0.05* 2.05

0.36 0.31 0.36 0.33 0.39 0.15 0.13 0.39 0.34 0.10 0.17 0.00 0.00

4.76 5.55 5.78 [5.24] [5.11] 3.70 4.13 4.19 1.77 0.61 0.15 0.17 0.13

0.34 0.24 0.26 0.34 0.34 0.28 0.28 0.33 0.26 0.23 0.20 0.15 0.14

NA 0.78 0.78 0.73 0.70 0.66 0.67 0.67 0.54 0.53 0.40 0.38 0.35

DL, below detection above this level.

limit; NA, not available.

Chlorophyll

Phaeophytin

@g 1-V

@g I-‘)

3.48 3.60 3.40 4.32 4.18 1.80 1.56 1.31 1.23 NA NA 1.76 1.48

0.29 0.67 0.92 0.37 1.52 1.16 2.61 1.66 3.46 1.40 1.58 2.27 1.14

0.09 0.12 0.14 0.09 0.16 0.50 0.62 0.38 0.63 0.24 0.39 0.32 0.27

2.33

0.35 0.38 0.45 0.56 0.80 1.36 1.10 0.39 2.69 3.07 2.95 1.82 1.53

0.09 0.11 0.12 0.13 0.14 0.30 0.21 0.10 0.33 0.41 0.50 0.42 0.32

1.90 2.27 2.30 2.09 2.75 2.51 2.37 0.91 2.13 2.47 2.55 1.81