Aquatic Botany 87 (2007) 104–110
www.elsevier.com/locate/aquabot
Thalassia testudinum response to the interactive stressors
hypersalinity, sulfide and hypoxia
Marguerite S. Koch a,*, Stephanie A. Schopmeyer a, Marianne Holmer b,
Chris J. Madden c, Claus Kyhn-Hansen c
a
Aquatic Plant Ecology Laboratory, Biological Sciences Department, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, USA
b
Institute of Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
c
South Florida Water Management District, Everglades Division, 3301 Gun Club Road, West Palm Beach, FL 33406, USA
Received 7 April 2006; received in revised form 9 February 2007; accepted 14 March 2007
Available online 18 March 2007
Abstract
A large-scale mesocosm (sixteen 500 L tanks) experiment was conducted to investigate the effects of hypersalinity (45–65 psu), porewater
sulfide (2–6 mM) and nighttime water column hypoxia (5–3 mg L1) on the tropical seagrass Thalassia testudinum Banks ex König. We
P examined
stressor effects on growth, shoot survival, tissue sulfur (S0, TS, d34S) and leaf quantum efficiencies, as well as, porewater sulfides ( TSpw) and
mesocosm water column O2 dynamics. Sulfide
was injected into intact seagrass cores of T. testudinum exposing below-ground tissues to 2, 4, and
P
6 mM S2, but rapid oxidation resulted in TSpw < 1.5 mM. Hypersalinity at 65 psu lowered sulfide oxidation and significantly affected plant
34
growth rates and quantum efficiencies (F v/F m < 0.70).
P The most depleted rhizome d S signatures were also observed at 65 psu, suggesting
increased sulfide exposure. Hypoxia did notPinfluence TSpw and plant growth, but strengthened the hypersalinity response and decreased rhizome
S0, indicating less efficient oxidation of TSpw. Following nighttime hypoxia treatments, ecosystem level metabolism responded to salinity
treatments. When O2 levels were reduced to 5 and 4 mg L1, daytime O2 levels recovered to approximately 6 mg L1; however, this recovery was
more limited when O2 levels were lowered to 3 mg L1. Subsequent to O2 reductions to 3 mg O2 L1, nighttime O2 levels rose in the 35 and 45 psu
tanks, stayed the same in the 55 psu tanks, and declined in the 65 psu tanks. Thus, hypersalinity at 65 psu affects T. testudinum’s oxidizing capacity
and places subtle demands on the positive O2 balance at an ecosystem level. This O2 demand may influence T. testudinum die-off events,
particularly after periods of high temperature and salinity. We hypothesize that the interaction between hypersalinity and sulfide toxicity in
T. testudinum is their synergistic effect on the critical O2 balance of the plant.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Salinity; Hypersalinity; Sulfide; Hypoxia; Anoxia; Seagrass; Thalassia testudinum; Florida Bay
1. Introduction
In subtropical/tropical estuaries and coastal lagoons, large
contiguous seagrass meadows support a diversity of higher
consumers, promote sedimentation, assist in sediment stabilization and enhance nutrient retention. Therefore, it is
important to understand large-scale mortality events of meadow
forming seagrass (Robblee et al., 1991; Seddon et al., 2000;
Plus et al., 2003). In 1987 approximately 40 km2 of Thalassia
testudinum Banks ex König meadows experienced a major
* Corresponding author. Tel.: +1 561 297 3325; fax: +1 561 297 2749.
E-mail addresses: [email protected] (M.S. Koch), [email protected]
(M. Holmer), [email protected] (C.J. Madden).
0304-3770/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquabot.2007.03.004
‘‘die-off’’ in Florida Bay, a shallow semi-enclosed estuary in
South Florida (Robblee et al., 1991) and since this time has
been followed by smaller (<1 km2) patchy episodes of
mortality on an annual basis (Zieman et al., 1999). It is
hypothesized that exposure to one or a combination of
environmental stressors such as high temperature, salinity,
porewater sulfide and/or a biological agent (Labyrinthula sp.)
contribute to sudden mortality events of T. testudinum (Robblee
et al., 1991; Carlson et al., 1994; Zieman et al., 1999; Koch and
Erskine, 2001; Borum et al., 2005; Koch et al., 2007a,b).
In Florida Bay, hypersalinity (>50 psu), driven by high heat
loads and evaporation, is frequently found at the end of the dry
season, particularly during periods of drought (Boyer et al.,
1999). High temperatures that promote hypersaline conditions
in the bay also stimulate microbial sulfate reduction rates,
M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110
important for organic matter decomposition in coastal marine
sediments (Canfield, 1993; Holmer et al., 2003; Koch et al.,
2007b). Accelerated sulfate reduction rates increase exposure of
seagrass roots and rhizomes to porewater sulfide, a potent
phytotoxin to aquatic macrophytes (Ingold and Havill, 1984;
Koch and Mendelssohn, 1989; Koch et al., 1990; Goodman et al.,
1995; Holmer and Bondagaard, 2001). Sulfide accumulates in
sediments of Florida Bay (Carlson et al., 1994) and other tropical
regions because of high temperatures (Holmer and Kristensen,
1996; Koch et al., 2007b) and the low capacity of carbonate
sediments to bind sulfide into solid-phase forms, particularly
pyrite.
While porewater sulfide can cause stress in seagrass and
other emergent marine macrophytes, our mesocosm and field
experimental work has shown that T. testudinum can grow and
maintain high shoot densities in porewater with sulfide
concentrations in the millimolar range (2–10 mM; Erskine
and Koch, 2000; Koch et al., 2007b). We have also found in a
short-term hydroponic experiment that T. testudinum can
exhibit a ‘‘die-back’’ response when exposed to high sulfide
(6 mM), high temperature (35 8C) and hypersalinity (55–
60 psu) in combination (Koch and Erskine, 2001). The research
presented herein is a continuation of this work using large-scale
mesocosms (detailed in Koch et al., 2007a) to examine the
synergistic effects of various levels of hypersalinity (45–65 psu
for 60 days) and porewater sulfide (2–6 mM for 40 days). These
longer-term experiments accommodate the use of intact
seagrass cores and a slow rate of salinity increase allowing
plants to osmotically adjust, simulating field conditions (Koch
et al., 2007a). In the present study, we also examined the
interaction of sulfide and salinity with nighttime water column
hypoxia (5–3 mg L1) found to influence sulfide intrusion into
seagrass below-ground tissues (Pedersen et al., 2004; Borum
et al., 2005).
2. Materials and methods
2.1. Plant collection and mesocosm design
Intact T. testudinum cores (15 cm diameter 20 cm
depth, 2840 cm3 sediment, 2 L porewater) were collected
25 July, 2003 from Florida Bay (258020 47.000 N, 808450 11.400 W).
Cores were transported to the FAU Marine lab (Boca Raton,
FL) and placed into mesocosm tanks (1 m ht 1 m diameter
with 1000 W metal halide lights) with coastal Atlantic seawater
for 2 weeks (36 psu, 27 1 8C, 12:12 h light–dark cycle; PAR
of 582 56 mmol m2 s1). Mesocosm tanks were equipped
with one powersweep for circulation at canopy height and one
for surface to bottom circulation with aeration. The mesocosms
were run as a closed system with deionized water amended to
compensate for evaporation and coastal seawater added weekly
to maintain nutrient levels.
2.2. Experimental design
We determined the response of T. testudinum Banks ex
König to four salinity (36 [ambient], 45, 55, and 65 psu) and
105
four sulfide treatments (0, 2, 4, and 6 mM) and their
interactions. After 60 days of salinity and 40 days of sulfide
treatments, a hypoxia treatment was initiated. Hypersalinity,
sulfide and hypoxia and their interactions were tested for their
effects on plant growth, shoot density, leaf quantum efficiency,
tissue sulfur (TS, S0) and isotopic ratios (d34S), porewater
sulfide levels, and water column O2 dynamics.
Salinity (Instant Ocean Inc.) was raised 1 psu day1 (11
August 2003) to approximate in situ evaporative rates on
shallow carbonate banks in the bay (0.5 psu day1; Koch et al.,
2007a). After 29 days all tanks were at salinity treatment level
and sulfide treatments were initiated. Sulfide was injected via
syringe through two horizontal sippers (0.5 cm diameter
tubes) with small holes alternately drilled along the tube which
was previously inserted across the center of the core in opposite
directions to distribute sulfide throughout the core. One end of
the tube was closed and the other fitted with a three-way valve
extending into the water. Sippers were left in the cores
throughout the experiment. Tests of the sulfide injection
system were conducted using dye tracers in extra cores to
ensure that sulfides were not readily advected to the overlying
water. Deoxygenated (N2) artificial seawater was adjusted to
porewater pH (7.0) with NaOH and sulfide added as
NaS7H2O. At the initiation of the sulfide injection treatments,
60 mL of 2 mM sulfide was added to each of the sulfide
treatment cores and ambient artificial seawater injected into
the controls. Sulfide concentrations in the injections were
raised 1 mM until injection treatment levels were reached. Due
to a lack of sulfide accumulation in the cores after 4 days using
60 mL injections adding 0, 120, 240, and 360 mmol S2 day1,
the injection volume was raised to 120 mL day1 adding 0,
240, 480, and 720 mmol S2 day1. Based on minimum sulfate
reduction estimates from T. testudinum cores in our
mesocosms (33 mmol L1 day1, Koch et al., 2007b) and
those measured in Florida Bay (154 mmol L1 day1, Jensen
and Koch, unpublished data) and 2 L of porewater, our sulfide
amendments would have increased the total sulfide load 2 to
5 times.
During the last 9 days of the sulfide salinity experiment,
nighttime hypoxia was simulated in 8 of the 16 tanks (two
from each salinity treatment) and tank aeration tubes on the
upper powersweeps removed. Nitrogen gas was bubbled in the
tank water for 15–20 min at the initiation of the 12 h dark
cycle (17:00 h) until O2 was lowered to 5 mg L1 (YSI 85).
On days 2–4, O2 level was lowered to 4 mg L1 and on days
5–9 O2 was lowered to 3 mg L1. The hypoxia treatments
were run for 9 days along with sulfide additions. The
experiment was terminated after 60 days of exposure to
hypersalinity, 40 days of daily sulfide injections and 9 days of
hypoxia.
2.3. Plant response measurements
Plant growth as leaf elongation rates (Zieman, 1974) and net
shoot numbers as percent survival were determined weekly.
Leaf quantum efficiency (F v/F m) was also measured weekly on
dark adapted (5 min) leaves using a diving PAM (Pulse
106
M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110
Amplitude Modulation; Walz, Germany). Quantum efficiency
(F v/F m ratio) has an optimal range in seagrass between 0.7
and 0.8 (Ralph, 1999; Durako et al., 2002). Therefore, we used
an F v/F m ratio of 0.7 as a lower ‘‘stress’’ threshold in the
interpretation of our results.
2.4. Sulfur analyses of plant tissue
At the end of the experiment, plant tissue was separated into
leaf, root, and rhizome and freeze dried. Sulfur analyses (S0,
TS, d34S) were run on leaves and rhizomes from plants in the
35 and 65 psu treatments exposed to 6 mM sulfide and
controls (0 mM sulfide). Leaf and rhizome d34S and TS analysis
were made at Iso-Analytical Limited Inc. (Cheshire, UK). The
sulfur isotope composition is expressed in the following
standard d notation: d34S = [(Rsample/Rstandard) 1] 1000,
where R = 34S/32S. Precision was better than 0.4% based on
internal standards. Rhizome and leaf elemental sulfur (S0) was
determined according to Zopfi et al. (2001).
2.5. Physicochemical measurements
Every 5 days 10 mL of porewater was extracted and one
5 mL subsample used to measure pore water sulfide concentration, salinity, and pH. The other 5 mL subsample was
immediately transferred into an alkaline buffer, converting all
sulfides to S2 and immediately measured with a silver/sulfide
ion electrodeP(Orion 420A). The resulting total porewater
sulfide pool ( TSpw) at pH 7 would have a composition of H2S
(pKa1 = 7; pKa2 = 19) to HS of approximately 50:50. As
porewater was sampled, 10 mL of deoxygenated seawater at
treatment salinity was added back to each core in order to
maintain porewater volume. Mesocosm tanks were monitored
daily for water column salinity and temperature and weekly for
light (Li-Cor spherical sensor) and O2 (YSI 85), with the
exception that during the hypoxia experiment O2 was measured
daily before and after lights were turned on and at the end of the
day before O2 levels were readjusted.
2.6. Statistical analysis
In the pre-hypoxia phasePof the experiment, salinity and
sulfide treatment effects on TSpw and plant responses were
analyzed using two-way analysis of variance and Tukey
multiple mean comparison tests (Sigma Stat. 3.0). If normality
or equal variance assumptions were violated ranked data were
used. Differences between hypoxia and non-hypoxia responses
were examined using a t-test or Mann–Whitney Rank Sum test.
If non-significant, data from hypoxia and control tanks were
pooled. Tissue sulfur data in which hypoxia was significant was
analyzed by three-way ANOVA. All statistical significance is at
the p < 0.05 level unless otherwise stated.
3. Results
3.1. Porewater sulfide (
P
TSpw)
Sulfide was injected into intact seagrass cores of T.
testudinum exposing below-ground tissues to 2, 4, and
6 mM S2P
, but rapid oxidation resulted in lower total porewater
sulfide ( TSpw) throughout the experiment. Although
approximately 10, 19, and 29 mmol S2 was injectedP
into T.
testudinum intact sediment cores over 40 days, the
TSpw
remained <1 mM in all sulfide treatment cores (Table 1 prehypoxia). Assuming a 308 mmol day1 rate of sulfate reduction
in the mesocosm cores (see Section 2) equating to 12 mmol of
sulfide produced in the coresP
over 40 days, and taking into
account the sulfide added and TSpw measured (Table 1), the
oxidation rate of sulfide would have been approximately 71–
431 mmol L1 day1 or 92–99% of sulfide added and
produced. Root oxidation probably accounted for a large
portion of the sulfide oxidized; however, oxidation from
overlying surface water cannot be discounted and therefore
plant oxidation rates were not calculated.
P
Even though sulfide oxidation rates were high, TSpw was
significantly higher at 65 psu in contrast to all other
hypersalinity treatments and the 35 psu control (Table 1).
Table 1
P
Total porewater sulfide concentration ( TSpw; pH avg. 7.5 0.13) measured in intact cores as a function of salinity and sulfide treatments pre-hypoxia (17, 23, 31
October and 7, 14 November 2003) and 8 days post-hypoxia 21 November 2003
P
TSpw (mM)
Salinity treatment (psu)
Sulfide injection treatment
35
45
55
65
Pre-hypoxia (mM)
0
2
4
6
0.11 0.04
0.19 0.05
0.27 0.02
0.75 0.29
0.26 0.09
0.17 0.05
0.33 0.12
0.33 0.08
0.13 0.06
0.21 0.02
0.15 0.02
0.40 0.19
0.52 0.17
0.58 0.23
0.46 0.05
0.74 0.11
Post-hypoxia (mM)
0
2
4
6
0.07 0.02
0.20 0.17
0.18 0.08
0.34 0.24
0.14 0.01
0.12 0.08
0.25 0.20
0.12 0.06
0.07 0.00
0.20 0.09
0.13 0.01
0.17 0.02
1.14 0.37
1.28 0.82
0.68 0.05
0.85 0.67
Means S.E. (n = 4 pre-hypoxia; n = 2 post-hypoxia).
M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110
107
Table 2
Two-way ANOVA results for Thalassia testudinum (a) leaf elongation rates and
(b) percent shoot survival across salinity and sulfide treatments based on
measurements taken post-hypoxia treatment 17–21 November 2003
Source of variation
d.f.
SS
(a) Leaf elongation rates
Salinity
3
Sulfide
3
Salinity sulfide
9
Residual
48
MS
8.11
0.79
0.92
8.03
2.70
0.26
0.10
0.17
17.85
0.28
Total
63
(b) Short shoot survival
Salinity
Sulfide
Salinity sulfide
Residual
rate
3
3
9
48
1,780
484
1,978
7,611
593
161
220
159
Total
63
11,852
188
F
p
16.17
1.58
0.61
<0.001
0.207
0.783
3.7
1.0
1.4
0.017
0.394
0.221
Hypoxia treatment was non-significant so data were pooled.
Interestingly,
even in the cores where no sulfide was injected,
P
TSpw accumulated in the 65 psu cores to levels significantly
higher than the 35 and 55 psu treatments (Table 1).
Fig. 1. Thalassia testudinum leaf quantum efficiency (Fv/Fm) in response to
salinity and sulfide treatments before and after hypoxia treatments
(mean S.D., n = 4).
3.2. Plant growth and ecophysiological response
P
In congruence with TSpw results, salinity was the only main
effect explaining the variation in T. testudinum growth and short
shoot survival (Table 2). At 65 psu, leaf growth rates dropped
below 2.0 mm day1 and were significantly lower than all other
salinity treatments (Table 3). Shoot survival rates were also
lowest at 65 psu and on average shoot survival declined as a
function of increasing salinity (85, 81, 78 and 70%, respectively).
Consistent with the growth and shoot mortality response, leaf
quantum efficiency responded to salinity treatments ( p < 0.01),
but not sulfide treatments (Fig. 1). There was a pattern of reduced
Table 3
T. testudinum growth rates (average 17 and 21 November) and shoot survival
(17 November) after exposure to salinity (60 days), sulfide (40 days) and
hypoxia (9 days) treatments
Treatments
Salinity (psu)
Sulfide (mM)
35
35
35
35
45
45
45
45
55
55
55
55
65
65
65
65
0
2
4
6
0
2
4
6
0
2
4
6
0
2
4
6
Leaf growth
(mm day1)
Shoot
survival (%)
2.68 0.15
2.26 0.23
2.16 0.15
2.35 0.05
2.60 0.28
2.74 0.22
2.69 0.20
2.67 0.04
2.75 0.30
2.86 0.23
2.28 0.06
2.60 0.35
1.93 0.05
1.88 0.25
1.69 0.08
1.60 0.15
87 5
78 3
93 5
80 5
74 8
81 5
81 4
86 5
77 4
81 4
79 4
75 8
68 6
85 8
69 11
59 9
Hypoxia treatments (9 days) were not significant so data were pooled (n = 4).
leaf fluorescence with increasing sulfide exposure, particularly at
ambient salinity; however, this trend was not consistent across
salinity treatments and leaf quantum efficiencies maintained
levels indicative of relatively ‘‘healthy’’ plants (>0.70). Only in
the 65 psu treatment did F v/F m ratios drop below 0.70.
3.3. Hypoxia experiment
While we predicted that the hypoxia treatments would
promote a higher O2 demand of the system, and thereby greater
porewater sulfide exposure in below-ground tissues, we
P only
found a significant plant response to salinity and
TSpw
remained relatively low (Table 1). Similar to the pre-hypoxia
response, sulfide levels were only raised in the 65 psu treatment
(Table 1) and sulfide treatments had no effect on plant growth,
but salinity was highly significant ( p < 0.01). Leaf fluorescence values were also affected by salinity and F v/F m ratios fell
even farther in the 65 psu tanks with hypoxia, while the F v/F m
levels were 0.70 in the other treatments (Fig. 1). These data
suggest that even under nightime hypoxia in the overlying
waters, T. testudinum plants by day were able to produce
enough O2 to oxidize porewater sulfide at 35 psu and moderate
hypersaline conditions, but this capacity was reduced at 65 psu.
3.4. Mesocosm O2 dynamics
Following nighttime hypoxia treatments, ecosystem level
metabolism, influenced by seagrass and other biota, responded
differently to salinity treatments. If we examine O2 fluxes
(mg L1 and percent O2 saturation) over the 9-day hypoxia
experiment, several observations are noteworthy (Fig. 2): (1)
when O2 levels were reduced to 5 and 4 mg L1 at the end of the
light cycle (first 2 arrows Fig. 2), daytime O2 levels recovered to
108
M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110
3.5. Rhizome and leaf sulfur
Fig. 2. Tank O2 concentration and percent saturation at 08:00 h before lights
came on and at the end of the day before O2 was adjusted down with N2, and
after adjusting nighttime O2 to treatment levels of 5, 4, and 3 mg/L indicated by
arrows (mean S.D., n = 2).
approximately 6 mg L1 and approached or were greater than
100% saturation across salinity treatments. However, this
daytime O2 recovery was more limited when O2 levels were
lowered to 3 mg L1 (third arrow). (2) Subsequent to O2
reductions to 5 and 4 mg L1, O2 levels fell during the night in
all salinity treatments. However, when reduced to 3 mg O2 L1,
O2 levels rose in the 35 and 45 psu tanks, stayed the same in the
55 psu tanks, and declined in the 65 psu tanks during the night.
Thus, salinity had a direct influence on system level O2 demand
at night (Fig. 2).
Plant exposure to sulfide and hypoxia was reflected in
rhizome tissue sulfur data (Table 4). Rhizome S0 was 2.5 times
higher in the 6 mM sulfide treatment compared to the control
and these levels were significantly reduced by a factor of 2
under hypoxia. S0 was always greatest in the control versus
hypoxia treatment, suggesting that the hypoxic condition led to
less efficient reoxidation of H2S to S0. This transformation may
be significant because of the high component of the TS pool
accounted for by S0 (22–46%) in the 6 mM sulfide treatment
(Table 4). Although rhizome S0 content was not different
between salinity treatments when exposed to 6 mM sulfide, S0
was 44 and 60% lower at 65 psu compared to ambient salinity
under hypoxic versus control treatments, respectively. The most
depleted d34S signatures in the rhizomes were also observed in
the high salinity treatments which suggest that sulfide intrusion
may have been greater at 65 psu. Extrapolating S0 in Table 2 to
the total rhizome biomass (3.7 g core1) and using the total S2
load over the course of the experiment (28 mmol core1), we
calculated that up to 3% of the S2 added may have been
converted to S0 in the rhizome at 35 psu. In contrast, only 1.7%
was estimated for 65 psu with the same loading, indicating a
potential reduction in oxidizing efficiency at 65 psu.
S0 was not detected in the leaves of T. testudinum with the
exception of the 6 mM sulfide 35 psu treatment, the same
treatment combination that exhibited the highest levels of S0 in
the rhizomes (Table 4).
4. Discussion
Porewater sulfides in T. testudinum intact cores were readily
oxidized at ambient seawater salinity and moderate hypersaline
conditions. While we cannot assume that all of the oxidation was
plant mediated, a high capacity for T. testudinum to oxidize H2S
has been observed in situ in Florida Bay (Borum et al., 2005).
Also, the ability for seagrass as well as other submerged and
emergent aquatic macrophytes to oxidize their rhizosphere
(sediment microzone surrounding roots) and associated reduced
Table 4
Elemental sulfur (S0) with % of total sulfur (TS) in parentheses, total S (TS) and S isotopic ratios (d34S) of T. testudinum leaf and rhizome tissue in the control (0) and
6 mM sulfide treatments exposed to 35 and 65 psu salinity, and 9 days of hypoxia (H) treatment or aerated control (average S.D.; n = 2)
Treatment
S0 (mmol g1 dry wt)
TS (mmol g1 dry wt)
d34S
Hypoxia (%TS)
Control (%TS)
Hypoxia
Control
Hypoxia
Control
Rhizome
0 mM 35 psu
0 mM 65 psu
6 mM 35 psu
6 mM 65 psu
54 14
23 13
192 80
84 41
84 77 (13)
128 25 (26)
269 127 (46)
162 14 (28)
453 22
469 195
509 155
394 35
720 91
498 73
573 161
591 102
8.5 3.1
9.5 0.7
8.8 1.2
10.7 0.2
9.5 6.3
10.7 2.5
10.1 1.1
10.7 1.7
Leaf
0 mM
0 mM
6 mM
6 mM
nd
nd
nd
nd
nd
nd
6 9 (2)
nd
219 18
191 a
239 24
228 a
227 11
200 a
213 44
222 a
14.9 3.3
10.4 a
13.7 2.0
7.3 a
14.8 1.4
7.5 a
14.5 1.2
9.2 a
35 psu
65 psu
35 psu
65 psu
(12)
(5)
(42)
(22)
nd: not detectable.
a
No standard deviation calculated (n = 1).
M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110
compounds, such as sulfide, is well established in the literature
(Sand-Jensen et al., 1982; Smith et al., 1984; Lee and Dunton,
2000). Stable isotope data show that this reduced sulfur source is
incorporated into plant tissue (Raven and Scrimgeour, 1997),
also indicated in this study by depleted d34S signatures in the
rhizomes.
There is growing evidence that excess H2S in seagrass can be
oxidized to S0 in root and rhizome tissue (Holmer et al., 2005;
Frederiksen et al., 2006), although chemical oxidation is a slow
process compared to microbial mediation (Pedersen et al.,
2004). In our 6 mM sulfide treatment at ambient salinity, we
found 42–46% of the TS pool in T. testudinum rhizome tissue
could be accounted for by S0. In the temperate meadow forming
seagrass, Zostera marina, a high S0 to TS ratio was found in
roots (68%) and rhizomes (30%) when sulfide production rates
were stimulated with glucose (Holmer et al., 2005). However,
S0 was only a small component (<4%) of the TS pool in situ
based on data from several sites in Denmark. T. testudinum
appears to possess a high ratio of S0 to TS and total amount of
S0 (23–128 mmol g1 dry wt) compared to Z. marina
(<20 mmol g1 dry wt; Holmer et al., 2005). Additionally,
accumulation of S0 in Z. marina is concentrated in the roots and
not the rhizome (Holmer et al., 2005; Frederiksen et al., 2006),
suggesting that sulfide may not be as readily transported to
rhizomes in Z. marina. Also supporting this idea is the fact that
Z. marina TS and d34S rhizome signatures closely track the leaf
rather than the roots in the field (Frederiksen et al., 2006). We
observed contrasting TS and d34S signatures between T.
testudinum leaf andPrhizome tissues in this study. At field sites
in the bay where TSpw was 4 mM in intact T. testudinum
beds, root and rhizome tissue TS were both high (920 34 and
642 148 mmol g1 dry wt, respectively) with depleted d34S
signatures (21 2 and 19 2), while leaves had 2–3 times
lower total sulfur (329 35 mmol g1 dry wt) and more
enriched d34S signatures (6 2) (Koch and Holmer,
unpublished data). Perhaps some of these differences between
the two species S0, TS, and d34S are accounted for by the very
high porosity of T. testudinum below-ground tissue (Tomlinson,
1969).
In seagrass, aerenchyma (air space tissue) oxidation and
the rate of O2 diffusion to the sediment has been correlated to
plant photosynthesis (Lee and Dunton, 2000; Pedersen et al.,
2004; Borum et al., 2005). At 65 psu, T. testudinum growth
and leaf quantum efficiencies were
P significantly reduced
concomitant with an increase in TS
Ppw. It was interesting
that at 65 psu, we found the highest TSpw not only in the
6 mM sulfide treatments but also the controls inPwhich
no sulfide was added. These data indicate that
TSpw
accumulation was the balance between sulfate reduction rates
and sulfide oxidation. T. testudinum’s capacity to oxidize
sulfide to S0 at 65 psu declined as evidenced by lower S0 in
rhizomes, possibly limiting a potential mechanism of sulfide
detoxification (Holmer et al., 2005). While our data show an
increased exposure to sulfide with 6 mM injections, we did
not find increased plant mortality or significantly slower
growth due to sulfide treatments alone or in response to
hypoxic treatments. In a subsequent mesocosm experiment
109
(June–July 2004) where we stimulated sulfate reduction
P rates
five-fold with glucose amendments (28–29 8C) and TSpw
averaged 2.3 mM compared to <1 mM in non-glucose
amended controls only moderate effects on T. testudinum
growth and no influence on shoot mortality were found (Koch
et al., 2007b).
Some of the discrepancies reported for sulfide toxicity in
seagrass may be accounted for by species specific O2
production potential and plant porosity, an indicator of O2
storage capacity (Tomlinson, 1969) and/or site specific
sediment O2 consumption rates (Borum et al., 2005; Koch
et al., 2007b; Jensen and Koch, unpublished data). Stressors
that effect photosynthesis, rhizosphere oxidation, and systemwide O2 consumption rates will influence plant sulfide exposure
and simply tissue anoxia which may directly or indirectly
cause seagrass mortality. We and others (Carlson et al., 1994)
have measured porewaterPsulfide concentrations in the
millimolar range (2–5 mM TSpw pH 6.5) in intact seagrass
meadows not experiencing ‘‘die-off’’ (Koch et al., in prep.)
P and
we have experimental results verifying that millimolar TSpw
does not solely cause mass mortality or ‘‘die-off’’ episodes of T.
testudinum (Koch et al., 2007b). Thus, while sulfide in highly
reduced marine sediments can accumulate, sulfide exposure is
ameliorated by efficient plant O2 production (Borum et al.,
2005), at least if other stressors, such as hypersalinity and
temperature (Koch and Erskine, 2001; Koch et al., 2007b; this
study) do not limit plant oxidation potential.
Stressors such a hypersalinity not only affect plant
oxidation potential but also ‘‘system’’ O2 flux in the water
column. In our mesocosms, nighttime water column O2
significantly declined as a function of increasing salinity after
hypoxia treatments. Accelerated O2 consumption is important
in ecosystems such as Florida Bay that are shallow and have
low early morning O2 levels in the overlying water column
(Borum et al., 2005). When seagrass exhaust their internal O2
supply at night, they can passively diffuse O2 from the water
column (Pregnall et al., 1984; Greve et al., 2003; Pedersen
et al., 2004; Borum et al., 2005). However, if O2 tension is low
(30–35% of air saturation, Pedersen et al., 2004), diffusive flux
rates will be slow, particularly when flow rates are low (Binzer
et al., 2005) as they are in Florida Bay in internal basins and on
shallow mudbanks. In our hypoxia experiment, O2 levels
remained above 40% airP
saturation, perhaps accounting for the
continued oxidation of TSpw through passive diffusion and
no significant effect of short-term 6 mM sulfide exposure
through injections.
In summary, hypersalinity at 65 psu directly effects T.
testudinum’s oxidizing capacity (Koch et al., 2007a; this study)
and places subtle demands on the positive O2 balance at an
ecosystem level. This O2 demand, as well as that imposed by
Florida Bay sediments with high sulfate reduction rates (Jensen
and Koch, unpublished data), may account for frequent
observations of seagrass die-off events, particularly after
periods of high temperature and salinity. We hypothesize that
the interaction between hypersalinity and sulfide toxicity in T.
testudinum is their synergistic affect on the critical O2 balance
of the plant.
110
M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110
Acknowledgements
We thank the South Florida Water Management District for
funding this research and Everglades National Park for their
logistical support through the Interagency Science Center at Key
Largo, FL. The Florida Institute of Oceanography’s Keys Marine
Laboratory facility also provided field support. We thank the
graduate students, Tammy Orilio, Scott Hurley and Brent
Anderson, and post-doc, Dr. Ole Nielsen, for their assistance in
collecting seagrass cores and running the experiment. Neal
Tempel is recognized for his assistance in designing our
mesocosms. Sulfur analysis for this research was supported by
the Danish Natural Science Foundation 272-05-0408 and the
Thresholds EU project 003933-2. We thank the anonymous
reviewers who significantly improved the manuscript.
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