Limnol. Oceanogr., 36(S), 1991, 1851-1864
0 199 1, by the American
Society
of Limnology
and Oceanography,
Inc.
Effects of iron, manganese, copper, and zinc enrichments on
productivity and biomass in the subarctic Pacific
Kenneth H. Coale
Moss Landing Marine Laboratories, Moss Landing, California 95039-0450
Abstract
Natural plankton populations from subarctic Pacific surface waters were incubated in 7-d experiments with added concentrations of Fe, Mn, Cu, and Zn. Small additions of metals (0.89 nM
Fe, 1.8 nM Mn, 3.9 nM Cu, and 0.75 nM Zn) were used to simulate natural perturbations in metal
concentrations potentially experienced by marine plankton. Trace metal concentrations, phytoplankton productivity, Chl a, and the species composition of phytoplankton and microzooplankton
were measured over the course of the experiment. Although the controls indicated little growth,
increases in phytoplankton productivity, Chl a, and cell densities were dramatic after the addition
of 0.89 nM Fe, indicating that it may limit the rates of algal production in these waters. Similar
increases were observed in experiments with 3.9 nM Cu added. The Cu effect is attributed to a
decrease in the grazing activities of the microzooplankton (ciliates) and increases in the rates of
production. Mn enrichment had its greatest effect on diatom biomass, whereas Zn enrichment had
its greatest effect on other autofluorescent organisms.
The extent of trace metal adsorption onto carboy walls was also evaluated. These results imply
that natural systems may be affected as follows: natural levels of Fe and Cu may influence phytoplankton productivity and trophic structure in open-obean, high-nutrient, low-biomass systems;
rates of net production are not limited by one micronutrient alone. Because of the effects of
adsorption and complexation, experiments must be carefully monitored for free vs. total metal
concentrations, and short-term incubations (1 d) may not be affected dramatically by small perturbations in trace metal micronutrients.
Metals are required micronutrients
in
many of the enzyme and electron transport
systems present in all living things. Due to
their concentrations,
redox behaviors, or
complexation,
trace metals are known to
limit plant growth in many terrestrial systems (Shkolnik 1984), yet their role in limiting phytoplankton
growth in the oceans
has remained largely speculative.
Over the last decade there has been a revolution in our understanding of trace metal
Acknowledgments
I express my appreciation and thanks to A. Michaels
and C. Michaels for shipboard assistance in processing
the productivity, Chl a, and autofluorescent samples;
to C. Michaels and B. Schreiber for counts of autofluorescent organisms; to M. Silver, A. Michaels, K. Buck,
and F. Chavez for discussions throughout the development and review of this manuscript; to M. Gordon,
S. Fitzwater, and J. Martin for the use of their sampling
van and bottles for the collection of these samples; and
to K. Bruland for support throughout the development
and execution of this project. Thanks are also extended
to the crew and officers of the RV Wecoma for their
assistance.
This work was supported by ONR contracts NO00 1483-0683 and NOOO14-87-0683to K. Bruland, N0001489- 10 10 to K. Johnson, and NSF grant OCE 89-23057
to K. Johnson and K. Coale.
concentrations
and distributions
in the
oceans. We now recognize that trace metals
exist at nanomolar and picomolar concentrations in seawater (Bruland 198 3) - orders
of magnitude
below the concentrations
found in terrestrial ecosystems. In surface
waters of the North Pacific total concentrations of Fe, Mn, Cu, and Zn are -0.1, 1, 1,
and 0.1 nM. Consequently, the hypothesis
that these trace metals may limit phytoplankton growth and biomass has become
popular in marine plankton ecology. Conversely, some trace metals have been shown
to be toxic to organisms at very low concentrations. Therefore, trace metals can have
either a positive or negative effect on phytoplankton productivity
and species distributions in the oceans.
Historically, Cu has been the most intensively studied trace metal with respect to
biological effects, not for its role as a micronutrient but as a toxicant to phytoplankton in marine systems. Recently upwelled
seawater may contain high levels of free Cu
and low levels of chelators, Fe, and Mn, the
combination of which is inhibitory to phytoplankton (Steemann Nielsen and Wium-
1851
c
1852
Coale
Andersen 1970; Sunda et al. 198 1). Barber
has reported experiments in which chelators
added to upwelled water have increased
phytoplankton
growth (Barber and Ryther
1969; Barber et al. 197 1). Although these
experiments were mechanistically inconclusive, the effect of added chelator has been
attributed to the binding of Cu and a reduction in its free ion activity.
Evidence from the culture experiments of
investigators using chemically defined media, in which the equilibrium concentration
of free ionic metal species can be controlled
by adding the trace metal chelators EDTA
or NTA, has suggested that cupric ion act.ivities at naturally occurring levels could
produce inhibitory
effects in some species
of phytoplankton (Sunda and Guillard 1976;
Anderson and Morel 1978; Brand et al.
1986). Due to the extremely small Cu requirements of phytoplankton
and the scientific preoccupation with pollution, there
have been few experiments that suggest that
Cu is a limiting micronutrient
in seawater.
Unlike Cu, Fe is potentially limiting to
phytoplankton
growth and is not known to
be toxic to organisms at concentrations
found in seawater. There have been numerous reports suggesting that Fe may limit
production in the sea. Menzel and Ryther
(196 1) demonstrated enhanced rates of 14C
uptake in phytoplankton samples from subtropical and tropical regions of the western
Atlantic upon the addition of Fe. However,
due to the high levels of Fe added in these
experiments and the potential for artifacts
in the productivity
methods used, it is difficult to speculate from these results whether
natural inputs of Fe could have any effect
on productivity
in these regions.
There have been only a few experiments
with natural assemblages of phytoplankton
using realistic levels of Fe to suggest that it
limits growth in the oceans. Among these
few are a series of experiments by Brand
and coworkers (Brand et al. 1983) in which
a clear distinction between Fe requirements
of neritic vs. oceanic phytoplankton
was
demonstrated. Brand pointed out that the
similarity between trace metal requirements
and the pattern of phytoplankton
distributions suggests that Zn, Mn, and Fe may be
important selective, and therefore ecological, factors in controlling
phytoplankton
distributions.
This result has been supported by Sunda et al. (199 1) who investigated
the Fe growth requirements in oceanic and
coastal phytoplankton.
They measured
growth rates of both an estuarine and an
oceanic diatom (Thalassiosira pseudonana
and Thalassiosira oceanica) and found the
oceanic species to be growing at or near its
maximum growth rate at 25% the amount
needed for the coastal diatom. Recently
Martin and Fitzwater ( 1988) reported the
first convincing evidence that Fe may limit
productivity
in the subarctic Pacific. Subsequent experiments indicate that Fe may
stimulate phytoplankton
growth in North
Atlantic, equatorial Pacific, and Antarctic
waters (.I. H. Martin pers. comm.; Martin
et al. 1990, 199 1; de Baar et al. 1990).
The roles of Mn and Zn in controlling
phytoplankton
productivity
are less well
known. Both are required micronutrients;
Zn may elicit toxic responses in phytoplankton at high concentrations. Sunda and coworkers have reported experiments in which
ad’ditions of Mn have been shown to reverse
the effect of Cu and Zn toxicity in monoclonal and natural seawater cultures (Sunda
et al. 1981; Sunda and Huntsman 1983;
Sunda 1987). Brand et al. (1983) have shown
that some species may be limited at near
natural concentrations
of these metals;
however, few experiments with natural assemblages of plankton
have been perfo#rmed.
In this study, the effects of four trace metals (Fe, Mn, Cu, and Zn) on species composition, abundance, and productivity were
determined for natural plankton assemblages collected from the subarctic Pacific
(Ocean Station P). Small amounts of these
metals were added to simulate natural perturbations of these metals in this system.
Trace metal concentrations
and Cu complexation by natural organic ligands were
monitored over time to assess the effective
changes in trace metal availability over the
course of the experiment.
Methods
Seawater was collected, treated with met;als, and followed over time to assess effects
of added metals on productivity
and biomass. Water samples for this study were
collected in August 1987 from 20 m at Ocean
Trace metal efects in the subarctic
Station P (VERTEX 7, leg 2, 50°N, 145”W)
at dawn with Teflon-coated Go-Flo bottles
(General Oceanics) deployed on Kevlar hydrowire (Philadelphia Resins). Physical disruption and light shock of the phytoplankton were avoided by passing unpressurized
seawater through 5/8in. (1.6 cm) Bevaline
tubing into a darkened, filtered-air clean van
where six, modified, ZO-liter polycarbonate
incubation carboys were filled. The ZO-liter
carboys were used for the l-week incubations and Z-liter polycarbonate productivity
bottles were used for the 24-h incubations.
These relatively large carboys and productivity bottles were used to minimize containment
effects during the experiment
(Gieskes et al. 1979).
The carboys and bottles had been sequentially cleaned with ethanol, Micro, detergent, 3 N HCl (glass distilled), and finally,
pH 1 quartz-distilled
HCl (Q-HCl) in MilliQ water. Carboys and productivity
bottles
were allowed to retain the last weak Q-HCl
rinse for 3 weeks before being rinsed and
filled with clean seawater 1 d before the experiment. Prior to the experiment, this seawater was discarded and the carboys were
rinsed with sample. This treatment reduced
the risk of trace metal and organic contamination and ensured that minimal leachable/exchangeable sites remained on the interior container surfaces.
Upon filling with sample, 1 ml additions
of trace metal solutions in pH 1 Q-HCl (so
as not to significantly alter the pH or NO3
concentrations in the media) were added to
the ZO-liter carboys. Fe was added in a 1 : 1.5
Fe : EDTA solution in Milli-Q water to ensure initial Fe solubility yet not alter the
overall metal speciation in the incubation
media. Six carboys were used in this experiment: five treatments and one control. The
treatments were +0.89 nM Fe, +1.8 nM
Mn, +3.9 nM Cu, +0.75 nM Zn, +10.2
nM EDTA, and no additions (control). The
carboys were incubated on deck and in a
running surface seawater bath to maintain
surface seawater temperatures under PVC
screens. The screening provided spectrally
unmodified shading to 30% of the ambient
light level, estimated to correspond to the
light level at the depth of collection.
Four times during the course of the incubations, subsamples for trace metal anal-
1853
ysis, metal complexation,
Chl a, and productivity were taken. Samples for species
composition
and autofluorescence
were
taken initially and at the end of the experiment. These samples were drawn from the
carboys through a modified cap (Fig. 1) by
pressurizing the carboy (-0.0 12 bars) with
purified air filtered with a 0.2~pm Teflon
filter. Samples for total trace metal analysis,
Cu complexation, and 14Cproductivity were
collected in acid-cleaned, 0.5-liter polyethylene, FEP Teflon and Z-liter polycarbonate
bottles, respectively.
Trace metal samples were acidified at sea
and stored at pH 1.5 (with Q-HCl) for 2
weeks before analysis. Trace metal analysis
proceeded according to the methods of Bruland et al. (1979) as modified by Coale and
Bruland (1988).
Changes in Cu complexation
over time
were assessed by differential pulse anodic
stripping voltametry (DPASV) on board in
a clean-air analytical van. Due to limited
time and resources, only samples from the
control and Cu treatment were analyzed by
DPASV. The analytical procedures for the
DPASV analysis are reported elsewhere
(Coale and Bruland 1988).
Primary productivity
was measured on
samples drawn at or near dawn on days 1,
2,4, and 6. Productivity measurements were
made by adding 0.5 ml of a H14C03 standard to Z-liter polycarbonate bottles containing sample from the ZO-liter carboys.
The H14C03 solution was made by diluting
a 5-mCi standard in UV-oxidized,
trace
metal-free seawater. Sample activities were
-0.02 PCi ml-l. Productivity
bottles were
incubated for 24 h before filtering (50 ml
for >0.45-pm fraction, 500 ml for the > 25pm fraction). Productivity
filters (25-mm
GFF and 25-pm Nitex) were placed in scintillation vials to which Aquasol was added.
All manipulations
of productivity
samples
took place in a darkened lab. Productivity
samples were counted by liquid scintillation
at UCSC (Univ. Calif., Santa Cruz) on a
Packard TM liquid scintillation counter using external standards ratio corrections. Chl
a determinations were made on 50-ml samples filtered onto GFF filters and extracted
with 90% ethanol for 24 h at 4°C. Analysis
was performed on board with a Turner III
fluorometer.
1854
Coale
Filtered air inport
m
Sample outport
Bevaline tubing
Fig. 1. Schematic diagram depicting incubation carboys and cap construction. These modifications to carboy
caps facilitate subsampling without contamination. High-purity filtered air (0.2 pm) was delivered through the
air inport (~0.2 atm) and sample was caught as it passed from the sample outport. The sample outport did not
touch the collection container and the entire cap was protected with clean plastic bags between sampling intervals.
Samples for species enumeration
were
collected and preserved with glutaraldehyde. Samples for counts of autofluorescent
organisms (25 ml) were preserved with glutaraldehyde, filtered on black-gridded filters
(0.45 pm, Sartorius), and frozen before
counting (Booth 1987). These samples were
counted for several size classes of autofluorescent algae at 400 x with standard techniques (Caron 1982).
In the lab, 100 ml from each treatment
were settled (3 d) before counting on an inverted microscope. Three size classes of ciliates and dinoflagellates were thus counted.
Cell densities of the abundant, linear, chainforming pennate diatoms were obtained
from photographs of settled material with
a digitizing pad, cursor, and video imageanalysis software (VIAS version 2.11).
C conversions for different taxonomic
groups were calculated with C: vol. relationships reported in the literature. Dinoflagellates and autofluorescent
organisms:
log,& (pg) = 0.758 x [log,,cell vol. (pm3)]
- 0.422 (Strathmann 1967); diatoms: 54%
of the value as calculated by Strathmann
(1967) (Sicko-Goad et al. 1984). These factors are similar to those calculated by Eppley et al. (1970) yet tend to estimate more
conservatively
the C in diatoms. Ciliates:
0.14 pg C pm-3 (Putt and Stoecker 1989);
cyanobacteria: 0.21 pg cell-’ (Waterbury et
al. 1987). Error estimates were based on errors propagated in the uncertainty of the
counts themselves.
Results
Trace metals-Results
from the trace
metal analysis show the effects of adsorption
over the time-course of the experiment (Ta-
1855
Trace metal efects- in’the subarctic
Table 1. Changes in total trace metal concentrations in the carboys over time. Percent adsorbed is calculated
as 100 x measured/(initial metal + addition).
Metal
lreatment
carboy
Fe
Mn?
Cu
Zn
Total initial
metal
0.36
1
1.3
0.3
Addition
(nMI
Midpoint
total
+0.89
+1.8
+3.9
+0.75
2.7
2
4.2
0.75
* Values represent increases in Fe as measured in the subsamples taken. This increase
the incubation
carboy, as described in the text.
t Due to poor extraction
efficiency, Mn values are only approximate
and must remain
ble 1). It appears that Fe increased in the
incubation carboy over time. However, adverse sea state, wind, and spray conditions
on deck could have easily introduced contaminant Fe into the subsamples taken for
trace metal analysis as a result of sample
manipulations
under exposed conditions.
Due to the design of the carboy caps (Fig.
l), it is unlikely that the incubations themselves were contaminated.
The caps remained closed and covered with plastic, and
the sample draw is a clean catch with no
backwash. Furthermore,
the large 20-liter
incubation volumes greatly reduce the effects of any small amount of contamination,
which is supported by the results of the experiments themselves (see below) and the
fact that no other carboy showed elevated
levels of Fe.
Had Fe contamination
been introduced
in the other carboys (to the level of 0.9 nM),
growth similar to the Fe treatments would
be expected. Analyses of Fe in incubation
containers showed no evidence for significant Fe adsorption. This result is inconclusive due to subsample contamination,
but
the contention that only a small amount of
added Fe adsorbed to the carboy walls and
contamination
of the carboy through the
sampling caps, as used, was insignificant is
supported by shipboard measurements of
Fe in similar experiments in the equatorial
Pacific (Elrod et al. unpubl.). In these experiments, identical incubation
carboys,
caps, and apparatus were used. Samples
withdrawn four times each day (under better sea conditions) indicated photochemical
cycling of Fe between oxidation states (II
and III) and -30% net removal of total Fe
from the dissolved phase over the first few
Midpoint
adsorbed
w
Final total
(nM)
3.1
1.
4.4
0.64
216*
30
19
29
is attributed
tentative
to Fe contamination
Final
adsorbed
(W
248*
60
15
39
in the subsamples
and not
(see text).
days of the experiment (Elrod et al. unpubl.).
The Mn results are not quantitative due
to the poor extraction efficiency of Mn by
this method. However, the estimates reported in Table 1 (based on 10% extraction
efficiency) indicate that a significant fraction
of the total Mn may be adsorbed to the
carboy walls. Similar results in the equatorial Pacific show -20% of the added Mn
becomes adsorbed. Cu on the other hand
showed an initial loss of 1 nM to the carboy
walls and little subsequent change thereafter. About half of the added Zn appeared to
be adsorbed initially
and changed little
thereafter.
These results are not surprising given the
inherent surface reactivity of these elements
in seawater. In any containment experiment
at natural pH, the levels of dissolved metals
would be expected to change due to adsorption, leaching of the container walls, or
photochemical
transformations
of metal
species during the course of the incubation.
For experiments such as these, it is important to determine how these concentrations
change. The magnitude of the changes observed here are still within what can be considered to be realistic limits for natural
changes in the subarctic Pacific.
Complexation -The results of the complexation experiments are consistent with
previous measurements of complexation in
this geographical area (Coale and Bruland
1988) (Table 2). The DPASV technique can
discriminate between inorganic forms of Cu
and those forms which are bound in organic
complexes. I have used this technique to
distinguish two organic ligands that show
strong affinity for Cu in the northeast Pa-
Coale
1856
Table 2. Change in concentrations of ligands showing affinity for Cu and pCu (-log,, of the Cu2bactivity)
in the control and Cu treatment. L, represents the concentration of the strong ligand (log Klcond= 11.55); L,
represents the concentration of the weaker ligand (log
R cond= 8.64). (Derivation of these parameters given
by Coale and Bruland 1988.)
O--0Fe
*-e&h
a--aCU
A-A
Zn
o-•oEDTA
. - l Control
CIarboy
Control
day 1
day 4
day 7
2.8
2.3
2.3
Cu added
day 1
day 4
day 7
0
0
0
7.0
4.4
2.3
13.5
13.4
13.4
4.1
8.4
10.1
10.6
11.1
10.9
lz 01’
0
o--oFe
*-*bin
a--nCU
A-A
Zn
q -•EDTA
n -m
Control
1
2
3
4
5
6
2
3
4
5
6
7
7
Day
Pig. 3. Primary production in the total fraction
(>O.45 pm) for each metal treatment and control as a
function of time (days). Incubation time for each point
was 24 h.
cific: a very strong ligand (L,) at low concentrations (2 nM) and present only in the
surface waters and a weaker ligand (LJ which
is ubiquitous throughout the water column
(5-l 0 nM). In general, little change in the
strong ligand L, was observed in the control
carboy, whereas the weaker ligand L2 systematically decreased. Free Cu2+ activity,
expressed as pCu (-log,, of the Cu2+ activity), remained nearly constant in the control
carboy at 13.4 throughout the experiment.
Addition of Cu to the Cu treatment carboy
completely saturated the strong ligand, and
an apparent increase in production of the
weaker ligand L2 was observed. pCu values
correspondingly increased from 10.6 to 11
during the experiment.
Chl a- Chl a was similar in all treatments
(0.23 + 0.05 pg liter- ‘) initially but diverged
dramatically between days 2 and 3 (Fig. 2).
Interestingly, the control increased by a fac-
0
1
7
Day
Fig. 2. Chl a for each metal treatment and control
as a function of time (days).
tor of two during the course of the experiment, likely due to decreased grazing in the
carboys. Final values expressed as percent
of the control for each treatment (0.47 pg
liter-l) were: Fe, 924%; Mn, 140%; Cu,
281%; Zn, 123%; EDTA, 104%.
Productivity- Production measurements
are shown in Figs. 3 and 4. Although Martin
and Fitzwater (1988) used NO3 uptake rates
to estimate production and did not determine production rates of the different size
fractions, the initial total productivity
measured here (18.4k1.2 pg C liter-l d-l) is
similar to that observed by Martin and coworkers at this site (21.9kO.7 pg C liter-’
d-l, Martin and Fitzwater 1988). Production results indicate 18.4+_ 1.2 pg C liter-’
fraction
and
d-l for the >0.45-pm
0.63kO.13 pg C liter-’ d-l for the >25-ym
fraction.
Final productivity
values, expressed as percent of the final control (29.04
pg C liter-l d-l) for the >0.7-pm size class
were: Fe, 360%; Mn, 168%; Cu, 155%; Zn,
134%; EDTA, 134%. For the > 25-pm size
class, final productivity values, expressed as
percent of the control (2.36 pg C liter-’ d-l)
were: Fe, 1,300%; Mn, 2 16%; Cu, 147%; Zn,
97%; EDTA,
112%. Biomass estimates
based on microscopic counts, average cell
volumes per size class, and published C : vol.
relationships are presented together with cell
densities and specific growth rates for each
size class: ciliates (Table 3), dinoflagellates
(Table 4), autofluorescent organisms (Table
5), diatoms and cyanobacteria (Table 6). The
errors in these tables represent the uncer-
Trace metal eflects in the subarctic
o--oFe
*-*Mn
a---aCU
A--A
Zn
o--oEDTA
a--=
Control
01
0
1
2
4
3
5
6
7
Day
Fig. 4. As Fig. 3, but in the large fraction (>25 pm).
tainty in the microscopic
samples (W).
counts of these
Discussion
Background-The
ecology of the subarctic Pacific is characterized by high plant nutrients (7-l 5 ,uM NO3 in surface waters) yet
relatively low, constant standing stocks of
Chl a averaging 0.3 pg liter-’ and rarely
reaching 1 (Anderson et al. 1977). Seasonal
variation in the rates of production with
little variation in Chl suggests that there are
concurrent variations in C flux or in grazing
that maintain the constant low phytoplankton biomass. In such a system, any perturbation of either production or grazing rates
may produce large changes in the system.
Despite high nutrient concentrations
and
relatively high primary production, nutrient
assimilation
rates measured on samples
from the subarctic suggest that phytoplankton from this area are nutrient stressed and
therefore not likely to be growing at their
maximum rate (Falkowski 1980). Although
there is wide belief that grazing is the dominant pressure controlling biomass in this
region (Frost 1991), it is conceivable that
trace metal toxicity or nutritional limitation
could contribute to this observed ecological
balance.
Present study-For
every case in this
study, biomass and productivity
increased
between initial and final control samples
(except for the dinoflagellates). As these ex-
Table 3. Cell densities, biomass estimates, and specific growth rates for each size class of ciliates.
Size class &m)
<lo
Cell densities* (No. ml-l)
Initial
0.37kO. 13
+Fe
0.19+0.10
+Mn
0.97kO.20
+cu
0.67kO.02
+Zn
1.5Ot-0.26
+EDTA
0.71kO.18
Control
0.53kO.15
Cellular CT (pg liter-l)
Initial
0.02f0.01
+Fe
0.01 kO.01
+Mn
0.05-t0.01
+cu
0.03 kO.00
+Zn
0.08kO.01
+EDTA
0.04~0.01
Control
0.03-r-0.01
Specific growth rates+ (d-l)
+Fe
-0.19
+Mn
0.28
+cu
0.17
+Zn
0.40
+EDTA
0.19
Control
0.10
IO-30
>30
Total
3.oot-0.30
5.6OkO.50
10.2OkO.70
7.50&0.06
7.9740.59
8.3820.62
11.30+-0.70
0.05kO.05
0.58&O. 10
O.lOfO.10
0.13kO.08
0.46f0.13
0.49kO.13
0.18kO.09
3.67kO.48
6.29k1.18
11.87+- 1.00
8.23-t-0.21
10.06+- 1.31
9.71f1.29
12.53-t-1.03
2.39kO.24
4.4740.40
8.14k0.56
5.99kO.05
6.36kO.47
6.6940.49
9.02t-0.56
0.32-tO.32
3.69k0.64
0.64kO.64
0.83kO.5 1
2.92kO.83
3.11 k0.83
1.14kO.57
2.73kO.56
8.1741.04
8.83+ 1.20
6.85kO.56
9.36kl.31
9.84+ 1.33
10.19k1.14
0.18
0.35
0.26
0.28
0.29
0.38
0.70
0.20
0.27
0.63
0.65
0.37
0.15
0.34
0.23
0.29
0.28
0.35
* Calculated
from inverted microscope counts on preserved samples.
t Calculated
based on average cell volumes for each size class. These volumes were converted to C based on C: vol. ratios reported in the literature
(see text).
* Calculated
using cell numbers for each size class and correcting for an estimated 2.5-d lag period between initiation
of experiment
and rapid, log
phase growth, as seen in the productivity
and Chl time-course
(aficr Banse 1990).
1858
Coale
Table 4. As Table 3, but for dinoflagellates.
10-25
Cell densities* (No. ml-l)
Initial
105.00~5.00
-l-Fe
89.2Ok4.60
+Mn
48.3Ok3.10
+cu
53.00+6.00
+Zn
64.5Ok3.50
f EDTA
73.00+4.00
Control
161.00~6.00
Cellullar Ct (pg liter- I)
Initial
2.19kO.10
+Fe
1.86kO. 10
+Mn
1.O1kO.06
+cu
3.2OkO.13
+Zn
1.35-10.07
+ EDTA
1.53kO.08
Control
3.36k0.13
Specific growth rat& (d- I)
+Fe
-0.05
+Mn
-0.22
+cu
0.11
SZn
-0.14
+ EDTA
-0.10
Control
0.12
>25
Total
68.9Ok3.80
64.8Ot-3.90
7O.lO-t3.70
1lO.OOt-5.00
54.6Ok3.30
52.OOzk3.00
76.7Ok3.80
8.00+ 1.30
7.90+ 1.40
4.10+0.90
9.40+ 1.40
9.10+ 1.30
5.3Ok 1.00
1.10*0.20
181.90+10.10
161.90f9.90
122.5Ok7.70
272.40+ 12.40
128.20+8.10
130.3048.00
238.8Ok 10.00
9.3OkO.5 1
8.75kO.53
9.46-cO.50
14.85kO.68
7.37kO.45
7.02kO.41
10.3520.5 1
3.04kO.49
3.OOkO.53
1.56kO.34
3.57f0.53
3.46 kO.49
2.01 k0.38
0.42kO.08
14.54-t-1.11
13.61k1.15
12.03zkO.91
21.62k1.33
12.18+1.01
10.56ItO.87
14.14*0.7x
0.00
-0.19
0.05
0.04
-0.12
-0.57
-0.03
-0.11
0.12
-0.10
-0.10
0.08
-0.02
0.00
0.13
-0.07
-0.08
0.03
* Calculated
from inverted microscope counts on preserved samples.
flagellates.
t Calculated
based on average cell volumes for each size class. These
(see kw).
$ Calculated
using cell numbers for each sire class and correcting for
phase growth, as seen in the productivity
and Chl time-course
(after
No distinction
volumes
was made here between
were converted
an estimated 2.5-d lag period
Banse 1990).
periments were performed on deck in transit, there is no way to compare the control
to changes in biomass or productivity
in
situ. One likely explanation for the increase
in stocks in all incubations is that the samples were devoid of some grazers naturally
present in situ. Such grazers might be numerically rare but normally process relatively large volumes of water. Because the
samples were collected near dawn, the vertically migrating zooplankton, in particular,
should be under-represented in these samples. In all the settled samples counted, only
one copepod nauplius was observed, indicating that decreased grazing by metazoans
could have contributed to an increase in
productivity
and biomass in the control.
Whether the natural grazers were undersampled, I suggest that all treatments and
controls were similar in this respect and
therefore that the differences observed can
be attributed to the effects of the treatments.
The addition of metals seemed to produce
autotrophic
and heterotrophic
to C based on C : vol. ratios reported
between
initiation
of experiment
dino-
in the literature
and rapid,
log
dramatic effects in these incubations.
I
observed the most dramatic increases in Chl
a and rates of production in the Fe treatment (Figs. 2,3, and 4), similar to the results
of Martin and Fitzwater (1988). Martin and
coworkers attribute this effect primarily to
diatom limitation by Fe. The biomass and
size-fractioned productivity
data presented
here indicate that at least 40% of the biomass increase in the Fe treatment was due
to diatoms. The remainder of the production and biomass was in the < 25 -pm size
fraction (i.e. not diatoms). In my samples
however, autofluorescent
organisms, primarily flagellates (Chrysochromulina spp.,
Isochrysis spp., Chromulina spp., dinoflagellates, and other haptophytes), made up the
bulk of the biomass. The dominant diatoms
(Nitzschia spp. and Pseudonitzschia) did increase dramatically (Fig. 5) and contributed
-42% of the total biomass in the Fe treatment, which is an upper estimate because
it assumes that most of the dinoflagellates
1859
Trace metal efects in the subarctic
Table 5. As Table 3, but for autofluorescent organisms (excluding diatoms).
Size class &m)
2.5-10
Cell densities* (No. ml-l)
1,328*61
Initial
3,467f85
+Fe
l,462t-53
+Mn
1,978+32
+cu
2,244+62
+Zn
1,693f57
+EDTA
Control
1,254+40
Cellular Ct (jkg liter-‘)
22.7kl.l
Initial
+Fe
59.3+- 1.4
25.Ot-0.9
+Mn
+cu
33.8kO.5
+Zn
38.4+ 1.1
29.0+ 1.O
+EDTA
Control
21.420.7
Specific growth rates* (d-l)
+Fe
0.27
+Mn
0.03
+cu
0.11
+Zn
0.15
+EDTA
0.07
Control
-0.02
IO-20
120
19.847.3
27.4k0.4
11.5k4.7
222+ 11
34+-8
70+11
39+7
8.3k4.7
1.77kO.36
17.2k5.7
17.7k3.1
3.65k2.6
17.225.7
4.17k2.1
1.8k0.7
2.45 kO.04
1.02kO.42
19.8t- 1.0
3.07kO.70
6.2+- 1.0
3.440.7
4.0k2.3
0.86f0.18
8.3k2.8
8.6+- 1.5
1.8f 1.3
8.3k2.8
2.02 1.o
28.5f4.0
62.6+ 1.7
34.4k4.1
62.2t-3.0
43.2k3.0
43.5k4.8
26.9t-2.4
0.09
-0.16
0.69
0.16
0.36
0.19
-0.44
0.21
0.22
-0.24
0.21
-0.20
0.27
0.03
0.14
0.15
0.08
-0.01
Total
1,356+73
3,496,86
1,491+64
2,218_+46
2,282f72
1,78O-t74
1,297f50
* Calculated
from epifluorescence
microscopy
counts on samples filtered at sea.
t Calculated
based on average cell volumes for each size class. These volumes were converted to C based on C : vol. ratios reported
(see fexl).
$ Calculated
using cell numbers for each size class and correcting for an estimated 2.5-d lag period between initiation
of experiment
phase growth, as seen in the productivity
and Chl time-course
(after Banse 1990).
were autotrophic; they were, therefore, assumed to be represented in the counts of
autofluorescent organisms.
Ciliates were likely important grazers in
my incubations. Ciliate biomass was variable but lowest in the Cu treatment (Table
3). If low ciliate biomass is indicative of Cu
toxicity for grazers, then rates of ciliate grazing would also be adversely affected and their
prey would tend to increase (if the prey organisms were not similarly sensitive). Autofluorescent
organisms-dinoflagellates
and diatoms- which are potential prey items
for these ciliates, were more abundant in
the Cu treatment. Conversely, where ciliate
biomass was high (Mn treatment), other organisms, excluding diatoms, were lower in
biomass than in the Cu treatment. Therefore, the potential for species shifts due to
differential survival of the grazers is of great
importance in these experiments.
The effects of Cu on two planktonic ciliates have been studied in metal-ion buffer
in the literature
and rapid,
log
systems with EDTA and NTA (Stoecker et
al. 1986). Stoecker et al. found that Cu2+
activities as low as 1O- 12.*M decreased longterm growth rates in both species tested and
levels as low as 1O-lo M rapidly caused abnormal motility in these species. From the
results of Coale and Bruland (1988, 1990)
it appears that the surface waters of the subarctic Pacific are poised on the brink of Cu
toxicity for ciliates. Any increase in the Cu2+
activity would potentially
physiologically
impair these organisms. These observations
support the hypothesis that added Cu increases phytoplankton biomass via decreasing grazing pressure. These results indicate
that ciliates are among the more sensitive
forms of plankton to Cu toxicity.
Culture experiments of Brand and others
(e.g. Brand et al. 1986; Sunda and Guillard
1976) have determined Cu toxicity in a variety of phytoplankton
at Cu2+ activities of
10-l l M or greater. Although pCu in the
treatment was at this threshold (10.6-l 1.1
1860
Coale
Table 6. As Table 3, but for diatoms and cyanobacteria.
Diatoms
Cell densities* (No. ml-l)
Initial
117fll
+Fe
2,336*46
+Mn
1,724+41
+cu
1,069+32
+Zn
465+23
+EDTA
475+23
Control
440+21
Cellular Ct (pg liter-l)
Initial
2.54kO.24
+Fe
50.7 + 1.o
+Mn
37.4f0.9
+cu
23.2kO.7
+Zn
10.1 kO.5
+EDTA
10.3kO.5
Control
9.55kO.46
Specific growth rates* (d-l)
+Fe
0.86
+Mn
0.77
+cu
0.63
+zn
0.39
+ EDTA
0.40
Control
0.38
Cyanobacteria
327+34
1,143+57
1,137+57
8OOk40
1,002+50
I,71 I+82
637+32
0.07+0.01
0.24kO.01
0.24kO.01
0.17+0.01
0.21 kO.01
0.36kO.02
0.13+0.01
0.36
0.36
0.26
0.32
0.47
0.19
* Calculated
from photographs
of settled samples using an inverted microscope for diatoms. Cyanobacteria
were counted with epifluorcscencc
microscopy
on samples filtered at sea.
t Calculated
based on average cell volumes for the diatoms and 0.2 1 pg
cell-’ for the cyanobacteria.
These volumes were converted to C based
on C : vol. ratios reported in the literature
(see &xl).
$ Calculated
using cell numbers for both organisms and corrected for an
estimated 2.5-d lag period between initiation
of experiment
and rapid,
log phase growth, as seen in the productivity
and Chl time-course (after
Banse 1990).
see Table 2), the organisms Brand and others used were isolated from temperate and
subtropical regimes where total Cu values
are lower and therefore possibly more sensitive to increased Cu2+ activities. Sunda
(1987) and others have proposed that phytoplankton exhibit differential sensitivity to
Cu toxicity depending on the regime to
which they are adapted. It could be that
phytoplankton from the subarctic are better
adapted to higher levels of Cu than their
temperate counterparts. Although Cu is not
thought to be nutritionally
limiting in seawater, Schenck (1984) has observed Cu limitation in dinoflagellates
at pCu values
> 12.5. pCu values in the control were - 13.4
(Table 2), possibly indicating that the increase in production and biomass in the Cu
treatment was in fact due to Cu limitation.
Fig. 5. Initial and final diatom biomass for each
metal treatment and control.
However, others have been unable to reproduce this result, so this notion must remain tentative.
Alternatively,
it has been suggested that
added Cu may have displaced nutritionally
limiting Fe from organic ligands, thereby
making it more available to phytoplankton.
Due to the high ranking of Cu on the IrvingWilliams series and its strong tendency to
form complexes with organic ligands, Cu
can effectively compete with such sites both
in solution and on surfaces, which would
make it difficult to distinguish the effects of
added metal. As Sunda and coworkers have
shown, Mn additions to cultured phytoplankton can alleviate Cu toxicity (Sunda
and Huntsman 1983; Sunda et al. 198 1).
Morel et al. (1991) have also shown that
there may be physiological
tradeoffs with
respect to phytoplankton
trace element requirements. They show that under certain
circumstances, Zn deficiency can be compensated by Cd. These synergistic interactions make it difficult to attribute community responses to any one perturbation.
The results of the complexation
measurements, however, can provide some
chemical limits to the availability
of metal
added. These experiments with Cu indicate
that although 4 nM Cu was added, it was
rapidly complexed by natural ligands, resulting in a free Cu2+ activity of - 10 pM.
Because the ligands showing affinity for Cu
also seem to be quite specific (Coale and
Bruland 1988), this increase of 4 nM would
be expected to displace about 10 pM of oth-
Trace metal efects in the subarctic
1861
v
3 100
v
3 75
8u
50
;
25
t-l
Fig. 6. Initial and final C : Chl a ratio for each metal
treatment and control. Photosynthetic C was calculated
to be the sum of the autofluorescent and diatom C as
described in the text.
Fig. 7. Initial and final photosynthetic assimilation
ratio for each metal treatment and control.
er metals. Such an amount is not thought
to be significant. Similar specificity by another class of organic ligands has been demonstrated for Zn (Bruland 1989). Although
we cannot be certain of which ligands are
responsible for this binding, it appears that
the ligands we observe showing high affinity
for Cu, Zn, and Cd are specific and do not
show high affinity for Fe (Coale and Bruland
1988; Bruland 1989).
The effect of the added chelator EDTA
apparently had little effect, except on the
cyanobacteria (Table 5). The apparent stimulation of the cyanobacterial biomass can
be attributed to decreases in Cu*+ activity;
however, the calculated impact of only 10
nM EDTA on trace metal speciation is relatively small (- 5%) and the increase in DOC
due to the EDTA addition is probably negligible (0.1%). If, in fact, cyanobacteria are
extremely sensitive to Cu*+ activity, as observed by Brand et al. (1986), then even a
small reduction in Cu activity, by EDTA
complexation
together with an adsorptive
loss of Cu to the carboy walls, could help
alleviate a toxic condition. Until this result
is reproduced, it will remain our tentative
interpretation.
Fe, Mn, Cu, and Zn treatments all showed
increases in biomass, or productivity,
or
both. Fe had the most dramatic effect on
productivity,
yet Cu had an similarly dramatic effect on biomass. Because Fe is not
thought to be toxic at such low concentrations, the effect of Fe is attributed to Fe
limitation (especially of diatoms and small
autofluorescent organisms) in these subarctic waters. Furthermore,
my estimates of
phytoplankton
biomass based on size and
density (determined by microscopy) suggest
a large change in the C : Chl a ratio over the
course of the incubation. (Photosynthetic C
was estimated from counts of autofluorescent cells plus diatoms; Chl a is known for
each treatment). The C : Chl a ratio in the
Fe treatment was -r/3 that of the final control value (Fig. 6).
The productivity
: Chl a ratios in these
treatments tell a similar story (Fig. 7). Curl
and Small (1965) suggested that a productivity assimilation ratio [pg C fixed (pg Chl
a)-’ d-l] between 0 and 72 indicates nutrient
depletion, whereas ratios between 72 and
120 indicate borderline nutrient deficiency.
I observed initial ratios of 74& 7 and final
ratios (except for the Fe and Cu treatments)
of 72+ 7. Dramatically
lower values were
obtained for the Fe and Cu treatments (24
and 34). The low values for Fe and Cu are
surprising, because I expected organisms
limited by metals to metabolize more efficiently in their presence. N03, P04, and Si04
were all relatively high initially (7.8, 0.79,
and 16.7 PM), yet even initial assimilation
ratios indicate borderline nutrient deficiency. If low internal ratios were due to a de-
1862
Coale
7 1.0
6 0.9
2 0.8
z 0.7
; 0.6
Ls 0.5
u
. . 0.4
pg 0.3
Y
-g 0.2
L 0.1
0.0
&g
+
+
+
+
2
+
-El
u”
Fig. 8. Initial and final productivity : C ratio for
each metal treatment and control.
ficiency in any of the metals added, one
would expect to see an increase in the photosynthetic assimilation ratio as the deficiency was corrected, followed by a decline
upon depletion of the next limiting nutrient.
On the basis of increases in total biomass,
N consumption was at most 14% of the original 7.8 PM NO3 -t- NO*, indicating that N
was not limiting.
Production per unit biomass of the autofluorescent organisms plus diatoms, however, tells a different story (Fig. 8). The initial ratio [pg C liter-’ d-l (pg C liter-l)-l]
was 0.59. The final ratio for all other treatments except Fe and Cu was 0.71 kO.03.
The final Fe treatment ratio was 0.9 3, significantly above all other treatments, whereas the final Cu treatment ratio was 0.53,
similar to the initial ratio.
Several physiological
scenarios can be
constructed to explain these results. Fe, and
possibly Cu are required micronutrients
in
chlorophyll,
coproporphyrin,
and protochlorophyll synthesis (Shkolnik 1984). It is
possible that phytoplankton
from the subarctic, enriched with Fe and Cu, will preferentially produce Chl over other compounds, at least initially. Cu and Fe (possibly
both) are also required for function of the
enzyme superoxide dismutase. This enzyme
is present in cells and serves to inhibit
breakdown of Chl by superoxide radicals.
Increased activity of this enzyme could also
lead to higher levels of Chl a in phytoplankton. The beneficial effect of Cu is not un-
ambiguous. Since Cu is known to produce
toxic effects at extremely low activities, the
increases in productivity,
Chl a, and biomass were unexpected. However, counts of
the ciliates present indicate that Cu may
have caused the decline of these grazers,
thereby allowing the biomass of phytoplankton to increase.
Whereas productivity
: Chl a ratios indicate either decreased photosynthetic
efficiency or preferential production of Chl,
production : biomass ratios indicate that
production occurs more efficiently in samples treated with Fe and that production on
a per unit C basis in samples treated with
Cu proceeds less efficiently. The increase in
biomass in the Cu treatment is therefore
attributed to decreases in grazing (no increase in plant efficiency) and increases in
biomass in other treatments, especially Fe,
to increases in photosynthetic
efficiency.
Because the subarctic is an area in which
the rates of production are thought to be
tightly coupled to the rates of grazing, one
would expect NH3 to be the principal form
of N cycled from consumer to producer. If
the supply of NH3 could not keep pace with
the phytoplankton
demand, either through
increased rates of production or decreased
grazer activity, phytoplankton would switch
from NH, and deplete NO,. In this respect
the effect of Fe and Cu may be to decouple
rates of production and grazing. This explanation has been suggested by A. Michales and supported by N. Price et al. for
high-nutrient,
low-biomass
areas of the
oceans (Morel et al. 199 1). Morel et al.
(199 1) showed no enhanced uptake rates of
NH3, but dramatic increases in NO3 uptake
upon the addition of Fe (Fe is also required
for NO3 reductase activity). They attributed
this result to the higher Fe requirement of
diatoms. Results presented here indicate that
other small phytoplankton,
predominantly
flagellates, may be Fe limited as well (Table
5). This dynamic, food-web-scale interaction may be the basis of limitation in these
waters.
Although Zn is severely depleted in the
surface waters and shows a concentration
depth profile similar to Si04, of all the metals added, Zn produced the least response.
Trace metal eflects in the subarctic
This result was initially puzzling until Bruland (1989) reported some of the first studies of Zn-binding ligands in the North Pacific. In that study, he found Zn-specific
ligands with conditional stability constants
on the order of 10’ l and concentrations of
1.2 nM in surface waters. In the present
study, however, only 0.75 nM of Zn was
added in the enrichment experiments. The
small effect of added Zn in these experiments can be attributed to the binding of
Zn by natural ligands present in excess of
this addition.
These results are in general agreement with
those of Martin and Fitzwater ( 1988), indicating Fe limitation of productivity
in the
subarctic. However, productivity
also appears to be limited by Mn (particularly for
the diatoms, Table 6) and possibly other
metals. If ciliates are growing at subtoxic
levels of Cu then phytoplankton
could be
limited, in an indirect way, by Cu as well.
Although the addition of Fe did exhibit the
most pronounced effect, there seems to be
more than one element that increases biomass and production. These results are contrary to the hypothesis that Fe is the only
limiting nutrient in this system. Therefore,
Liebig’s law of the minimum does not apply
for Fe as suggested by Martin and Fitzwater
(1988). Nonetheless, small additions of Fe
can dramatically increase the rates of production and stocks of Chl a in this region.
Because the photosynthetic assimilation ratio changes and varies dramatically
with
metal treatments, increases in Chl a alone
cannot be used to follow increases in biomass.
These results also have implications
for
measurement of productivity
by the 14C incubation method. It appears that very low
levels of trace metal contamination may not
significantly
affect production
measurements over the brief incubation periods typically used in productivity
measurements
(6-24 h). However, substantial effects could
be encountered in longer incubations.
It appears that there are taxonomic differences in the response of the system to
metal additions. Dinoflagellates respond differently than do diatoms, and both are different from cyanobacteria. Furthermore, the
1863
effect of metals on micrograzers in the incubation must also be considered, because
decreases in grazing pressure can give results similar to increases in production.
These changes in food-web dynamics are
also supported by the observations of Chavez et al. (199 1) in the equatorial Pacific.
Lastly, there are substantial decreases in
total metal concentrations due to adsorption onto the walls of our scrupulously
cleaned incubation containers. Conversely,
there are likely increases in total metal concentrations due to desorption from less rigorously cleaned containers. There also appear to be changes in trace metal
complexation over time. In order to attribute responses in the system to changes in
available metal, we must address such
changes in metal concentration
and complexation in further studies. Future incubation containers should probably be equilibrated with seawater for longer periods of
time.
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