Phytoplankton pigments at the Weddell?Scotia confluence during
the 1993 austral spring
A.C.Sigleo, P.J.Neale1 and A.M.Spector1
US Environmental Protection Agency, Western Ecology Division, 2111 SE
Marine Science Drive, Newport, OR 97365-5260 and College of Oceanic and
Atmospheric Sciences, Oregon State University, Newport, OR 97365 and
1Smithsonian Environmental Research Center, PO Box 28, Edgewater, MD 20715,
USA
Abstract. During a 1993 austral spring cruise, a complex biomass was encountered near South Orkney
Island that ranged from a low-biomass, Chaetoceros tortissimus assemblage south of the front towards
the ice edge, to a high-biomass, Thalassiosira gravida-dominated assemblage at the northern edge.
The maximum levels of chlorophyll (Chl) a (up to 6 mg m?3) were higher than those observed in
previous high-performance liquid chromatography-based studies of pigments in the pelagic Southern
Ocean. The non-photosynthetic pigment chlorophyllide a comprised up to 75% of the chlorophyllous
pigments in the southern assemblage, but < 5% in the northern assemblage. Concentrations of the
xanthophylls diadinoxanthin (DD) and diatoxanthin (DT), used as indicators of mean irradiance,
indicated low-light-adapted populations. Low-light DD + DT/Chl a ratios in surface waters indicated
that vertical mixing limited phytoplankton residence time in the near-surface layer, and thus limited
exposure to maximum irradiance. Deck incubations of natural assemblages indicated that the dark
epoxidation reaction (i.e. the return of DT to DD) was a two-step reaction with the initial rate being
more rapid (t1/2 = 9.5 min) than the second (t1/2 = 55 min). Fucoxanthin, a major diatom pigment, was
more stable chemically in the water column than Chl a, and the vertical profiles of fucoxanthin
followed those of chlorophyllide a in some cases. The formation and apparent stability of chloro-
phyllide a and fucoxanthin are important considerations when estimating photosynthetically active
biomass over large regions of the ocean.
Introduction
Phytoplankton pigments are used to indicate phytoplankton biomass, composi-
tion by taxonomic group, and phytoplankton physiological condition or growth
stage (Jensen and Sakshaug, 1973; Jeffrey, 1974; Hallegraeff, 1981; Bidigare et al.,
1986, 1996; Everitt et al., 1990; Vernet et al., 1994). The following study focused
on the pigment compositions in an area of high phytoplankton biomass during
the austral spring and summer that characteristically occurs in the region between
Elephant Island and South Orkney Island, between the Scotia Sea to the north
and the Weddell Sea to the south (El-Sayed and Taguchi, 1981; Nelson et al.,
1989). In this region, an upper surface layer of cooler, fresher water from the
Weddell Sea flows north through the Scotia arc over the slightly warmer, denser
water of the Scotia Sea (Nelson et al., 1987, 1989; Bianchi et al., 1992). High-
altitude imagery for the region also indicates large amounts of phytoplankton
productivity in this hydrographic region (Sullivan et al., 1993).
Austral spring blooms related to increased solar irradiance and the ice retreat
reported for the Weddell Sea and Weddell?Scotia Confluence in the vicinity of
60?S are often dominated by diatoms, although the prymnesiophyte Phaeocystis
sp. can also provide a major portion of the biomass (El-Sayed and Taguchi, 1981).
Journal of Plankton Research Vol.22 no.10 pp.1989?2006, 2000
1989Published by Oxford University Press
Rich blooms of diatoms, indicated by chlorophyll (Chl) a concentrations up to
7 mg m?3, with exceptional bloom centers up to 25 mg m?3, were observed in
Gerlache and Bransfield Straits (Mandelli and Burkholder, 1966). Mandelli 
and Burkholder also observed a considerable range in biological vitality, with
?old? cells being characterized by low photosynthetic efficiency (Mandelli and
Burkholder, 1966).
Pigment studies using high-performance liquid chromatography (HPLC)
found that phytoplankton populations, represented by class-specific indicator
pigments, were variable throughout Antarctic waters (Bidigare et al., 1986, 1996;
Bidigare, 1989; Buma et al., 1990, 1992; Vernet et al., 1994). In the waters west of
the Antarctic Peninsula, the presence of the pigment 19-hexanoyloxyfucoxan-
thin during November 1991 indicated a prymnesiophyte-dominated population,
along with cryptomonads as indicated by alloxanthin (Vernet et al., 1994). 
Bidigare et al. found during October?November 1990 that the Phaeocystis-domi-
nated community within 50 km of the ice edge was replaced by a diatom-dominated
community in the open water 150 km from the ice edge (primarily Chaetoceros spp.
and Thalassiosira spp.) (Bidigare et al., 1996). In the Weddell?Scotia Confluence,
Buma et al. used cluster analysis of accessory pigment concentrations to define four
plankton communities that included a diatom-dominated community at the
Weddell?Scotia Confluence and three phytoflagellate communities slightly further
north and south of the Weddell?Scotia Confluence (Buma et al., 1990).
Chromophyte algae live in a dynamic, turbulent environment, frequently under
rapidly changing light conditions. To accommodate this variation in light inten-
sity, rapid (of the order of minutes) and reversible changes in the in vivo fluor-
escence per cell occur when diatom cells are transferred from low to high light
intensities (Sakshaug et al., 1987). The rapid change in the in vivo fluorescence
per cell is associated with rapid and reversible changes in the concentrations of
carotenoid pigments in the xanthophyll cycle (Sakshaug et al., 1987; Demers et
al., 1991; Brunet et al., 1993; Olaizola, 1993). 
The xanthophyll cycle consists of de-epoxidation/epoxidation reactions that
transform xanthophylls from the epoxide or oxygen-bound form to the de-
epoxide forms. In diatoms, the de-epoxidation reaction converts diadinoxanthin
(DD) to diatoxanthin (DT) under increasing irradiance or strong light, concur-
rent with changes in cell fluorescence (Demers et al., 1991). For this reason, some
investigations have considered using DT/DD ratios in the oceans as a potential
index of vertical mixing, and as an index of irradiance exposure (Olaizola et al.,
1992; Brunet et al., 1993; Olaizola, 1993). Brunet et al. found that measurements
of DD and DT provided an indication of the light environment of phyto-
plankton (Brunet et al., 1993). Their data demonstrated a direct relationship
between light intensity and DD + DT/Chl a ratios, indicating that phytoplankton
cells were adapting to local irradiances. We further examined DT/DD ratios in
the ocean during a day cycle, along with the rate of the reverse epoxidation reac-
tion.
In the present paper, primary and accessory phytoplankton pigments from a
series of stations near 60?S, 51?W (Table I) were used to estimate phytoplankton
composition, biomass and phytoplankton physiological condition. The pigments
A.C.Sigleo, P.J.Neale and A.M.Spector
1990
were measured across a zone of high biomass at the confluence of the Scotia and
Weddell Seas ~100 km north of the marginal ice zone as part of a larger investi-
gation on the effects of stratospheric ozone depletion and the concomitant
increase in UVB (280?320 nm) radiation on phytoplankton photosynthetic
processes (Neale and Spector, 1994; Sigleo and Neale, 1994; Ferreyra, 1995; Neale
et al., 1998). The standard method for determining Chl a and phaeopigment
concentrations using a fluorometer (Strickland and Parsons, 1972) also was used
as a rapid shipboard method for estimating plankton biomass. The physiological
status of phytoplankton assemblages was estimated from the relative proportions
of Chl a and chlorophyllide a (Jensen and Sakshaug, 1973; Gowen et al., 1983;
Ridout and Morris, 1985; Klein and Sournia, 1987). Chlorophyllide a is formed
by chlorophyllase activity that hydrolyzes the phytol from the Chl a molecule in
mature or senescent diatoms (Jeffrey, 1974; Simpson et al., 1976). Chlorophyll a
molecules that have degraded to chlorophyllide a are not photosynthetically
active (Simpson et al., 1976).
Method
Water samples were collected from the upper 200 m at the confluence of the
Weddell?Scotia water masses near 60?S, 51?W (Figure 1) between 18 October and
5 November 1993 from the R/V ?Nathaniel B. Palmer?. The vessel was equipped
with a General Oceanics 12 Niskin bottle rosette sampler surrounding a Sea Bird
Electronics Model SBE-9 conductivity, temperature and depth (CTD) profiler
designed for real-time operation. CTD data were logged on a Hewlett Packard
Vectra PC using standard Sea Bird software.
The formation of DT from the parent DD with variations in irradiance was
examined over a day cycle in samples collected by bucket over the port rail, and
immediately syringe filtered (100 ml) through 25 mm GF/F filters. The filters were
submerged in 100% acetone within 2 min from the time the bucket surfaced in
the water, as monitored by stopwatch, and analyzed as described below. The time
course of the reverse dark epoxidation reaction (DT ? DD) was estimated by
filling a brown glass bottle after a 15 min deck exposure to sunlight. The bottle
was capped, wrapped in black plastic and stored in the dark. Samples were with-
drawn from the bottle with a 100 ml syringe at ~15 min intervals.
Phytoplankton pigments at Weddell?Scotia Conference
1991
Table I. Station locations and 1993 sample dates
Station Date Latitude (degrees:minutes) Longitude
A October 18 58:47.00 49:49.00
B October 18 59:40.00 50:00.00
C October 18 60:10.00 50:00.00
M, M October 20, 21 59:34.60 50:00.00
N October 24 60:03.55 52:09.41
P October 26 50:55.00 52:09.34
R, R October 29, November 5 59:36.99 52:09.92
For pigment analyses, 500 ml?2 l seawater samples were filtered through 47 mm
GF/F glass fiber filters. The filters were immediately submerged in 1.5 ml of 100%
acetone and extracted at ?20?C in the dark for 18?24 h. Extracted samples were
centrifuged for 5 min to remove cellular debris and analyzed at sea by reverse-
phase HPLC using a St Johns Associates liquid chromatograph with a Microsorb-
MV C18 column (4.6  100 mm, 3 ?m particle size; Rainin Instrument Corp.). The
pigments were separated using a three-step solvent gradient based on the solvent
compositions and ion-pairing solution of Mantoura and Llewellyn (Mantoura and
Llewellyn, 1983). After injection (Alcott Model 728 autosampler with a 20 ?l
loop), mobile phase A (80:15:5, methanol:water:ion-pairing solution) was
programmed for 2 min, phase B (90:8:2, methanol:water:ion-pairing solution) for
8 min and mobile phase C (100% methanol) for 32 min, all at a flow rate of
0.5 ml min?1. Pigments were detected sequentially with a Waters Model 420 fluor-
escence detector (excitation 400?460 nm, emission >600 nm) for chlorophylls and
phycoerythrin, and a Hewlett Packard 1050 variable wavelength UV?visible
A.C.Sigleo, P.J.Neale and A.M.Spector
1992
Antarctic
Peninsula
Weddell
Sea
A
M
B
C
R
14 5
2
P
N
South
America
60W 50W
50S
60S
70W
60
S
60W 50W 40W
Fig. 1. Location map for stations listed in Table I. Contour lines indicate total pigment concentra-
tions in mg m?3.
absorbance detector programmed at 440 nm for carotenoid pigments. Eluting
peaks were quantified with Hewlett Packard Model 3392A integrators.
The identity and relative retention time for each pigment were determined
after extraction from pure cultures of phytoplankton with well-established
pigment contents (Wright et al., 1991). The retention times and response factors
were confirmed with standards separated in the above manner by Welschmeyer
[Environmental Protection Agency (EPA) contract, EPA, Cincinnati]. The
chlorophyllides and phaeopigments for HPLC standards were derived chemically
from Chl a according to the procedures of Mantoura and Llewellyn (Mantoura
and Llewellyn, 1983). All pigment data are arithmetic means of duplicate injec-
tions. The analytical precision, expressed as the standard deviation for multiple
standard injections, was ?7% (n = 19) for the Antarctic cruise. Standards were
analyzed before each sample group, and after each 10 samples. Reagent blank
concentrations were three orders of magnitude below sample concentrations for
Chl a and undetectable for all other pigments.
Chlorophyll a and phaeopigment concentrations were also determined on a
Turner Designs Model 10 fluorometer calibrated with authentic Chl a (Sigma) in
90% acetone (Strickland and Parsons, 1972). The concentration of Chl a in the
standard was determined spectrophotometrically (Jeffrey and Humphrey, 1975).
The samples (34 ml) were prepared for analysis by filtration through Whatman
GF/F 25 mm filters and extracted in 10 ml of 90% acetone overnight at ?20?C in
the dark. Turner fluorometer measurements were taken before and after acidifi-
cation to determine both Chl a and total phaeopigments (Strickland and Parsons,
1972). Samples for phytoplankton counts were preserved with Lugol?s solution
and identified using the Uterm?hl method with an inverted Zeiss microscope
(Uterm?hl, 1958).
Results
During October?November 1993, surface phytoplankton abundance increased
sharply northward from 60?S, 50?W, ~100 km north of the ice edge (Figure 1; Table
I). At stations north of 60?S, there was a well-defined surface upper mixed layer
as illustrated by the sharp decline in pigment concentrations at depths of 60?100
m (Figure 2) that coincided with a pronounced pycnocline (Neale et al., 1998).
Experimental work was carried out primarily at Station P (26?28 October) and
Station R (29 October?8 November). During the latter period, westerly winds
continued at 10?15 m s?1, maintaining active and deep vertical mixing. Depression
of fluorescence near the surface was absent (Neale et al., 1998). Samples were
collected for pigment analyses in multiple parts of the biomass, processed identi-
cally, and analyzed at sea within 24 h of collection to eliminate problems of sample
degradation. Jeffrey and Hallegraeff (Jeffrey and Hallegraeff, 1987) suggested
that chlorophyllase may be released during filtration, particularly of mature cells,
and recommended pigment extractions in 100% acetone to inactivate the
chlorophyllase, a procedure followed in the present study. Also, we found that
2 min (or less) for filtration and submergence in acetone minimized the effects of
chlorophyllase activity. The differences in sample pigment concentrations, 
Phytoplankton pigments at Weddell?Scotia Conference
1993
therefore, were indicative of phytoplankton abundance and physiological
condition, and were not artifacts from differences in sample treatment, handling
or storage. Despite these precautions, there is still a possibility that some chloro-
phyllase was released during filtration.
Total pigment concentrations in the euphotic zone ranged from 0.78 ?g l?1 (or
mg m?3) at Station C on 18 October to >14 ?g l?1 on 5 November at Station R
(Table II). Chlorophyll a concentrations ranged from 0.4 to 6.4 ?g l?1, and
comprised between 16 and 46% by weight (10?36 mol%) of the total pigments
(Table II). High concentrations of the accessory pigments fucoxanthin (up to
3.8 ?g l?1), Chl c (up to 1.5 ?g l?1) and the photoprotective carotenoids, DD and
DT were also present (Figure 3). Chlorophyll b concentrations were low (20 ng
l?1) or below the detection limit. Phycoerythrin, indicative of cyanobacteria, was
A.C.Sigleo, P.J.Neale and A.M.Spector
1994
Fig. 2. Vertical distributions of chlorophylls (left panels) and carotenoids (right panels) measured at
Stations B (18 October) and P (26 October). High concentrations of chlorophyllide a open squares
are evident. Fucoxanthin profiles (right panels) follow those of chlorophyllide a.
Phytoplankton pigments at Weddell?Scotia Conference
1995
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minor (~200 ng l?1) but persistent (<4%) at most stations. 19-Hexanoyloxyfuco-
xanthin, characteristic of prymnesiophytes such as Phaeocystis sp., was present at
Stations B, M and N in the range 20?150 ng l?1, or <5 mol% of the total. Peridinin
and violaxanthin, indicative of dinoflagellates and chlorophytes, respectively,
were detected (~20 ng l?1) only in a few samples (Table II).
Fucoxanthin was the most abundant carotenoid and generally co-varied with
Chl a, chlorophyllide a, or the sum of the two (Table III; Figure 2). The ratio of
chlorophyllide a:Chl a varied from 0.1 at Station R to 2.5 in the community at
Station P. The fucoxanthin:Chl a ratio also varied widely from ~0.6 to 2.4 
(Table III). The ratio of fucoxanthin to the arithmetic sum of Chl a + chloro-
phyllide a + methylchlorophyllide a, however, varied only between 0.5 and 0.6 at
Stations R and P, respectively (Table III). The concentration of chlorophyllide a
varied from 0.5 ?g l?1 at Station R to >3 ?g l?1 at Stations B and P (Figure 2). At
Stations B and P, the concentrations of chlorophyllide a increased when those of
Chl a decreased, as expected of a secondary product formed from a primary one.
Carotenoids, or accessory pigment to Chl a ratios, are used frequently as indi-
cators of phytoplankton taxa in natural waters (Everitt et al., 1990; Letelier et al.,
1993; Vernet et al., 1994; Bidigare et al., 1996). High concentrations of the indi-
cator pigment fucoxanthin (22?28% of the total pigments) suggest that diatoms
were the primary class of phytoplankton observed during this sampling. Using the
calculation factor of Everitt et al. (Everitt et al., 1990), the diatom contribution to
total Chl a was found by multiplying the diatom-derived fucoxanthin concen-
tration by 1.4 to give a value of up to 82% of the Chl a as being diatom derived.
Algal communities examined by inverted microscopy from the confluence of
the Scotia and Weddell Seas during the 1993 austral spring were dominated by
Chaetoceros tortissimus (45% of the total cell number) at Station P, whereas
Thalassiosira gravida was the most important species at Station R (96% of the
total cell number) (Irene Schloss, personal communication) (Table IV). Thalas-
siosira gravida occurred in large, particularly conspicuous colonies up to several
millimeters in length, consisting of single cells embedded in a gelatinous matrix.
A.C.Sigleo, P.J.Neale and A.M.Spector
1996
Table III. Ratios (w:w) of major and accessory pigments in the upper mixed layer (upper 40?80 m to
the surface)
Pigment ratios
Station Date C-ide a Fuco Fuco Chl c DD + DT
Chl a Chl a Chl a + C-ide a Chl a Chl a
+ Me-ide
A October 18 1.02 ? 0.34 2.34 ? 0.20 0.97 ? 0.20 0.57 ? 0.14 ?
B October 18 1.63 ? 0.39 1.93 ? 0.29 0.58 ? 0.05 0.56 ? 0.15 0.50 ? 0.09
C October 18 0.58 ? 0.29 0.15 ? 0.05 ? 0.33 + 0.11 ?
M October 20 1.05 ? 0.59 1.49 + 0.55 0.59 ? 0.05 0.64 ? 0.21 0.33 + 0.09
M October 21 0.62 ? 0.29 1.41 ? 0.77 0.67 ? 0.19 0.34 + 0.11 0.31 ? 0.04
N October 24 1.13 ? 0.43 1.29 ? 0.23 0.50 ? 0.01 0.56 ? 0.03 0.55 ? 0.32
P October 26 2.71 ? 1.2 2.43 ? 0.76 0.62 ? 0.09 0.91 ? 0.37 0.74 ? 0.31
R October 29 0.22 ? 0.08 0.77 ? 0.03 0.55 ? 0.02 0.24 ? 0.03 0.22 ? 0.02
R November 5 0.11 ? 0.01 0.59 ? 0.04 0.53 ? 0.03 0.23 ? 0.05 0.25 ? 0.01
Chl a, chlorophyll a; C-ide a, chlorophyllide a; Me-ide, methylchlorophyllide a; Chl c, chlorophyll c1&2; Fuco,
Fluorescence comparison
Samples analyzed by both HPLC with the fluorescence detector and the Turner
fluorometer showed higher Chl a by the conventional fluorometric method, with
the exception of one station (Figure 4). At that station, Station R, diatoms were
present as large aggregates that may have been more plentiful in the samples used
for HPLC, causing a bias in the data set. For the other stations in Figure 4 (B, M,
N, P and R, 29 October), the linear regression of fluorometer Chl a versus HPLC
Chl a has a slope of 2.6 and an intercept of 0.2, a value not significantly different
from zero (R2 = 0.75, n = 26). The 25 m samples from Station P and Station R
were outliers from this relationship and were omitted from the regression. The
regression of fluorometer Chl a versus total Chl a-derived pigments measured
with HPLC (Chl a + chlorophyllide a + methylchlorophyllide a) using the same
points had a slope of 1.06 when forced through the origin (R2 = 0.45).
Phytoplankton pigments at Weddell?Scotia Conference
1997
Fig. 3. Relative abundances in mole percent of major and accessory pigments, and chlorophyll a
degradation products at stations listed in Table I. Chl a, chlorophyll a; C-ide a, chlorophyllide a; 
Me-ide, methylchlorophyllide a; Chl c, chlorophyll c; Phyco, phycoerythrin; Fuco, fucoxanthin; DD,
diadinoxanthin.
Table IV. Phytoplankton species abundance at the Weddell?Scotia Confluence, 1993
Station and date Phytoplankton Cells ml?1
P October 26 Chaetoceros tortissimus 16,800
Thalassiosira gravida 6,740
Miscellaneous diatoms 3,400
Miscellaneous flagellates 10,900
R November 1 Thalassiosira gravida 735,000
Chaetoceros tortissimus 10,200
Miscellaneous diatoms 94,400
Phytoflagellates 35,700
Xanthophyll cycle
Surface water samples collected through the day at Station M showed DT/DD
ratios of 0.15?0.3, or ~13% DT (Figure 5). A CTD profile near noon at Station
M showed an upper mixed layer depth near 100 m, as illustrated by the pigment
profile (Figure 6). Samples from simultaneous deck incubations at Station M had
ratios of DT/DD of between 1 and 2 (i.e. >50?75% conversion to DT). The rate
of the dark epoxidation reaction (i.e. the return of DT to DD) was estimated from
natural seawater samples collected at Station R (6 November). The initial sample
had a DT/DD ratio of 0.8, or 44% DT. After 100 min, the remaining DT had
decreased to 7.6% (Figure 7).
Discussion
During the 1993 austral spring, the research vessel passed through a complex
biomass near South Orkney island that ranged from a Chaetoceros-dominated
assemblage south of the front towards the ice edge to a Thalassiosira-dominated
A.C.Sigleo, P.J.Neale and A.M.Spector
1998
Fig. 4. Comparison of Chl a concentrations determined by HPLC (filled circles) and Turner 
fluorometer (open circles, dashed lines) from stations at the Weddell?Scotia Confluence.
assemblage at the northwestern edge. In this region, the stable layer of less saline
water allowed the algal species that were growing most rapidly to accumulate and
form local biomass maxima (Smith and Nelson, 1985). Nelson et al. also noted
that increased biomass levels in the Weddell?Scotia water mass were enhanced
Phytoplankton pigments at Weddell?Scotia Conference
1999
Fig. 5. DT:DD ratios in surface waters at Station M during daylight hours on 22 October. Midday
DT:DD ratios (hatched circles) from deck incubations at Station M are included to illustrate the
effects of surface irradiance intensities.
Fig. 6. Vertical distributions of chlorophylls (left panel) and carotenoids (right panel) measured at
Station M (21 October 21, 14:00 h GMT) illustrating the upper mixed layer as defined by phyto-
plankton pigments.
initially in early spring by a period of delay in zooplankton development, as some-
times occurs during upwelling events in other areas (Nelson et al., 1987). Sedi-
ment trap data indicate that a significant amount of surface-derived material sinks
as large aggregates after phytoplankton blooms (Alldredge et al., 1995). These
aggregates often contain relatively intact diatoms that reach the sea floor without
being ingested by zooplankton (Smetacek, 1985). Because zooplankton were not
found in tows at these stations, diatom senescence rather than zooplankton inges-
tion appears to have been the fate of these phytoplankton.
The absolute levels of Chl a (up to 6 ?g l?1 or 6 mg m?3) encountered in the
bloom were higher than those observed in many previous studies using HPLC in
the Southern Ocean (Bidigare et al., 1986; Buma et al., 1990, 1992), but are typical
of values obtained during blooms in productive coastal waters in temperate
regions (Klein and Sournia, 1987). In agreement with many previous Antarctic
observations (Mandelli and Burkholder, 1966; El-Sayed and Taguchi, 1981; 
Bidigare et al., 1996), diatom species formed a significant part (up to 96%) of the
austral spring biomass at the Weddell?Scotia Confluence during October?
November 1993, although small numbers of flagellates also were observed.
Fluorescence comparison
The standard method for determining Chl a and phaeopigment concentrations
using a fluorometer (Strickland and Parsons, 1972) can under- or overestimate
Chl a concentrations since this method does not separate the pigments into indi-
vidual compounds, as does chromatography (Jensen and Sakshaug, 1973; Gowen
et al., 1983; Buma et al., 1992; Jeffrey and Mantoura, 1997). Because absorption
A.C.Sigleo, P.J.Neale and A.M.Spector
2000
Fig. 7. DT:DD epoxidation reaction as a function of dark incubation time. Sample was taken at
Station R (6 November, 15:15 h local time) and exposed to 15 min of surface irradiance on deck before
beginning dark incubation.
and fluorescence bands of non-chlorophyllous accessory pigments and Chl degrad-
ation products overlap with those of Chl a, Trees et al. found an error range of
+53 to ?69% with an average underestimation of 39% (n = 300) in comparing the
two methods in ocean waters (Trees et al., 1985). Individual pigment measure-
ments indicated that chlorophyllide a alone produces a strong signal for Chl a
with the fluorometer (Trees et al., 1985). Shipboard measurements of Chl a
with a spectrofluorometric method also gave higher levels (18%) of Chl a as
compared to Chl a measured by HPLC (Buma et al., 1992). Neither the Turner
fluorometer nor the spectrofluorometer differentiates between the chlorophylls
and Chl degradation products, which can lead to errors in calculated pigment
concentrations. HPLC, on the other hand, separates and then accurately quanti-
fies all major pigments and their degradation products (Bidigare et al., 1985). Our
results support the conclusions of Gowen et al. (Gowen et al., 1983) and Trees et
al. (Trees et al., 1985), and indicate that HPLC separation of pigments is
recommended over bulk fluorometric methods for phytoplankton pigment
determinations.
Physiological status
The relative proportions of the porphyrin pigments vary with the physiological
state or growth stage of the algae (Jensen and Sakshaug, 1973). Plankton in early
growth stages contain up to 50% of porphyrins as Chl a (Jensen and Sakshaug,
1973). At maturity, chlorophyllide a (a degradation product) can increase to
40?50% of the total (Hallegraeff, 1981; Gowen et al., 1983; Ridout and Morris,
1985; Klein and Sournia, 1987). Post-bloom biomass can contain 40?60%
phaeophorbide a, a pigment formed by acidic conditions that can occur by
passage through zooplankton or by the last phase of in situ cellular senescence
(Hallegraeff, 1981; Gowen et al., 1983; Bidigare et al., 1996).
Data from closed-chamber experiments at Stations P and R during the Antarc-
tic cruise (Ferreyra, 1995) further support the differences in physiological status
of the phytoplankton at the two stations. In the experiment at Station P, the
concentrations of Chl a decreased from 1.2 ?g l?1 to 0.86 ? 0.5 ?g l?1 (n = 7) in 
6 days (26 October?1 November), indicating a rather mature assemblage. At
Station R (2?8 November), however, the results were quite different and Chl a
increased from 5.03 ?g l?1 to 7.4 ? 1.1 ?g l?1 (n = 9; A.C.Sigleo, unpublished data).
These data indicate that the diatoms at Station R were in an active growth stage,
whereas the diatoms at Station P were in a more mature condition. The differ-
ence in physiological status at the two stations, as indicated by the Chl pigment
data, is sustained by Chl a-specific growth.
Since phaeophorbide a is formed by zooplankton grazing (Bidigare et al., 1986),
its absence in these samples suggests that zooplankton grazing was minimal at
that time, as also indicated by the lack of zooplankton in net tows. During this
sampling, the major fate of phytoplankton Chl a appears to have been cellular
maturity rather than predation. These results (October?November) differ from
those of Bidigare et al., who observed that the primary degraded porphyrin in
this region was phaeophorbide a, and concluded that summertime (January?
Phytoplankton pigments at Weddell?Scotia Conference
2001
February) phytoplankton concentrations were controlled in the Southern Ocean
by zooplankton grazing (Bidigare et al., 1986).
Because chlorophyllide a concentrations exceeded those of Chl a by factors of
2?3 (up to 90% of the porphyrins) in cells from Stations B and P, these results
from the Southern Ocean emphasize the need for complete chromatographic
pigment analyses to avoid errors in estimations of photosynthetically active
biomass using chlorophyllous pigments (Gowen et al., 1983; Bidigare et al., 1985;
Trees et al., 1985).
Fucoxanthin
Comparison of the vertical profiles for fucoxanthin and chlorophyllide a at
Stations B and P (Figure 2) indicates a strong covariance of the two pigments.
The profiles suggest that the carotenoid, fucoxanthin, is more stable than Chl a,
and as Chl a degrades to chlorophyllide a, fucoxanthin remains and can accumu-
late along with chlorophyllide a (Figure 2). Data from higher plant studies
support this conclusion, most notably during autumn pigment degradation prior
to leaf fall in which carotenoids decline at a visibly slower rate than Chl a, thus
producing autumn colors (Simpson et al., 1976). UVB exposure studies of Antarc-
tic phytoplankton (El Sayed et al., 1990) also found that Chl a decreased select-
ively relative to other pigments, whereas fucoxanthin remained constant over
long exposures. Poister et al. note from sediment trap studies that carotenoid
pigments in sedimenting algae were degraded to a lesser extent than Chl a
(Poister et al., 1999).
Xanthophyll cycle
The xanthophyll de-epoxidation/epoxidation reaction transforms DD to DT
under increasing irradiance or strong light (Demers et al., 1991). For the diatom
Chaetoceros muelleri, high-light-induced xanthophyll de-epoxidation occurred
with a first order rate constant of 1.6 min?1 (Olaizola, 1993). The conversion of
DD to DT appeared complete within 5 min (Olaizola, 1993). Under low light, DT
is converted back to DD by enzymatic epoxidation.
The DD/DT cycle in diatoms and other phytoplankton is reported to have a
photoprotective role by dissipating excess excitation energy in the antennae, thus
preventing damage to the photosynthetic system (Sakshaug et al., 1987; Demers
et al., 1991). In the work of Demers et al. (Demers et al., 1991), the sum of the two
pigments remained constant, indicating that no new DD synthesis occurred.
Latasa confirmed the photoprotective role by demonstrating that total DD
concentrations, i.e. the sum of DD + DT, were higher in diatom cells cultured
under high light irradiances, relative to diatoms cultured under lower irradiances
(Latasa, 1995). In other words, Latasa?s results indicated that additional DD was
synthesized in high-light-cultured diatoms, and then became available for
conversion to DT. In the present study, the xanthophylls DD and DT increased
during deck incubations at Station R, indicating new synthesis of DD. In the incu-
bation studies at Station R (2?6 November), the DD + DT sum increased from
A.C.Sigleo, P.J.Neale and A.M.Spector
2002
1.09 ?g l?1 in the water column (sample T0) to >4.4 ?g l?1 after 4 days (A.C.Sigleo,
unpublished data). The DD + DT/Chl a ratios were 0.22 initially, and increased
to 0.66 ? 0.08 (n = 9) after 4 days, indicating a 3-fold increase of these protective
pigments relative to Chl a. Thus, the deck-incubated samples at Station R were
transformed from a water column low-light-adapted community to a high-light-
adapted community. These results indicate that natural diatom assemblages in
the Southern Ocean, although generally low-light adapted for a low-light environ-
ment, can synthesize additional protective xanthophylls within days when light
conditions change.
Although DD + DT/Chl a ratios were slightly higher at Station P (Table III),
Olaizola et al. (Olaizola et al., 1992) and Brunet et al. (Brunet et al., 1993) have
shown that these ratios are valuable only during log growth stages and are unre-
liable when large quantities of chlorophyllides and phaeophorbides are present.
The work of these authors suggests that DD does not degrade as readily as Chl
a, and, like fucoxanthin, may accumulate (see Figure 3, Station A).
DT/DD or DT + DD/Chl a ratios have been used as a potential index of verti-
cal mixing in oceanic environments (Olaizola et al., 1992; Brunet et al., 1993;
Olaizola, 1993). Samples during this cruise collected from surface waters during
the day indicated that surface-collected water samples contained low-light-
adapted phytoplankton with DT/DD ratios of 0.15?0.3, or ~13% DT (Figure 6).
Samples from simultaneous deck incubations at Station M had ratios of DT/DD
of between 1 and 2 (i.e. >50?75% conversion to DT), indicating that there was
sufficient light intensity above the surface of the water to form maximum ratios.
In temperate waters, Brunet et al. interpreted the lack of variation in DT/DD
ratios in vertical profiles with respect to surface waters as an indicator of recent
vertical transport (Brunet et al., 1993). Thus, we interpret the low-light adaptation
of surface water pigments as an indication of recent vertical transport of the
phytoplankton. We conclude that phytoplankton pigments from this experiment
indicate that the phytoplankton were acclimated to fluctuating, or low-light
conditions.
Epoxidation reaction
The dark epoxidation reaction (i.e. the return of DT to DD), estimated from
natural seawater samples collected at Station R (6 November), was found to be
a two-step reaction with a rapid initial phase (t1/2 = 9.5 min) and a slower second
phase (t1/2 = 55 min) (Figure 7). Olaizola also observed a probable two-step
process, but did not indicate a rate (Olaizola, 1993). Brunet et al. measured a time
of 3.5 h for the decrease or disappearance of DT after dark (Brunet et al., 1993).
The intermediate values in bucket-collected surface water samples suggest that
the profile samples were adapted to fluctuating low-light conditions, as found in
a vertically mixed water column (Demers et al., 1991). Vertical mixing may have
been a factor limiting phytoplankton residence time in the near-surface layer
since under high-light, surface-water conditions DT is formed from DD. Profiles
of the pigments indicated that they were homogeneously distributed within the
upper mixed layer at all stations (see Figure 6), except Station P (26 October),
Phytoplankton pigments at Weddell?Scotia Conference
2003
which showed a disintegrating mixed layer (Figure 2). These results are consist-
ent with the conclusions of Keller et al. that under natural conditions vertical
mixing may provide refuge from rapidly attenuated UVB in the water column
(Keller et al., 1997).
In summary, Chl a concentrations of up to 6.5 ?g l?1 within the biomass bloom
are comparable to those for major algal blooms in temperate waters, and indicate
that these are highly productive waters, capable of producing immense quantities
of biomass. The high Chl a concentrations reinforce concerns about the poten-
tially harmful effects of increased UVB during highly productive periods in
Antarctic waters. During this austral spring bloom, however, dynamic hydro-
graphic conditions, as indicated by low-light DD + DT/Chl a totals in surface
waters, suggest that turbulence and vertical mixing within the water column may
minimize phytoplankton residence time in surface waters, thereby decreasing
exposure to UVB radiation. To examine this hypothesis further, future work
needs to include UVB exposure biomarkers along with vertical mixing data.
Chlorophyllide a concentrations greatly exceeded those of Chl a towards the
termination of a phytoplankton bloom, introducing a potential bias of up to 75%
in estimates of photosynthetically active biomass at one station. Fucoxanthin, a
major diatom pigment, shows evidence of greater chemical stability in the water
column than Chl a. The formation and chemical stability of these two pigments are
important considerations when high-altitude or remote sensing surveys are used
to determine primary productivity over large regions of the ocean. The results
emphasize the need for field measurements to confirm actual plankton vitality.
Acknowledgements
We thank the other members of the UV-B/Ozone 93 team, the officers and crew
of the R/V ?Nathaniel B. Palmer?, and Antarctic Support Associates for logistic
support. We are grateful to R.Steele for providing phytoplankton cultures for
pigment extractions, I.Schloss for plankton identifications, S.F.Echols for deriv-
ing the chlorophyllides and phaeopigment standards, W.E.Frick for model
support, and to G.Hansen, D.Specht and G.Hannach for manuscript reviews. This
research was supported by National Science Foundation grant OPP-9220373 to
P.J.N. and J.J.Cullen, and by the US EPA. Pigment standards were isolated under
EPA contract 68-C9-0032 to Bionics Corporation. This document was subjected
to the agency?s peer and administrative review, and it was approved for publi-
cation as an EPA document. Mention of trade names or commercial products in
this paper does not constitute endorsement or recommendation for use.
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Received on October 6, 1999; accepted on May 15, 2000
A.C.Sigleo, P.J.Neale and A.M.Spector
2006