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 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 1 sulfide (2–6 mM) and nighttime water column hypoxia (5–3 mg L ) on the tropical testudinum Banks ex Ko¨nig. WeP examined 0 34 stressor effects on growth, shoot survival, tissue sulfur (S , TS, d S) and leaf quantum efficiencies, as well as, porewater sulfides ( TSpw) and mesocosm water column O2 dynamics. SulfideP was injected into intact seagrass cores of T. testudinum exposing below-ground tissues to 2, 4, and 2 6mMS , 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 (Fv/Fm < 0.70).P The most depleted rhizome d S signatures were also observed at 65 psu, suggesting increased sulfide exposure. Hypoxia did notP influence TSpw and plant growth, but strengthened the hypersalinity response and decreased 0 S , indicating less efficient oxidation of TSpw. Following nighttime hypoxia treatments, ecosystem level metabolism responded to salinity 1 1 treatments. When O2 levels were reduced to 5 and 4 mg L , daytime O2 levels recovered to approximately 6 mg L ; however, this recovery was 1 1 more limited when O2 levels were lowered to 3 mg L . Subsequent to O2 reductions to 3 mg O2 L , 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; ; Florida Bay

1. Introduction ‘‘die-off’’ in Florida Bay, a shallow semi-enclosed estuary in South Florida (Robblee et al., 1991) and since this time has In subtropical/tropical estuaries and coastal lagoons, large been followed by smaller (<1km2) patchy episodes of contiguous seagrass meadows support a diversity of higher mortality on an annual basis (Zieman et al., 1999). It is consumers, promote sedimentation, assist in sediment stabi- hypothesized that exposure to one or a combination of lization and enhance nutrient retention. Therefore, it is environmental stressors such as high temperature, salinity, important to understand large-scale mortality events of meadow porewater sulfide and/or a biological agent (Labyrinthula sp.) forming seagrass (Robblee et al., 1991; Seddon et al., 2000; contribute to sudden mortality events of T. testudinum (Robblee Plus et al., 2003). In 1987 approximately 40 km2 of Thalassia et al., 1991; Carlson et al., 1994; Zieman et al., 1999; Koch and testudinum Banks ex Ko¨nig meadows experienced a major 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., * Corresponding author. Tel.: +1 561 297 3325; fax: +1 561 297 2749. E-mail addresses: [email protected] (M.S. Koch), [email protected] 1999). High temperatures that promote hypersaline conditions (M. Holmer), [email protected] (C.J. Madden). in the bay also stimulate microbial sulfate reduction rates,

0304-3770/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2007.03.004 M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110 105 important for organic matter decomposition in coastal marine four sulfide treatments (0, 2, 4, and 6 mM) and their sediments (Canfield, 1993; Holmer et al., 2003; Koch et al., interactions. After 60 days of salinity and 40 days of sulfide 2007b). Accelerated sulfate reduction rates increase exposure of treatments, a hypoxia treatment was initiated. Hypersalinity, seagrass roots and to porewater sulfide, a potent sulfide and hypoxia and their interactions were tested for their phytotoxin to aquatic macrophytes (Ingold and Havill, 1984; effects on plant growth, shoot density, leaf quantum efficiency, Koch and Mendelssohn, 1989; Koch et al., 1990; Goodman et al., tissue sulfur (TS, S0) and isotopic ratios (d34S), porewater 1995; Holmer and Bondagaard, 2001). Sulfide accumulates in sulfide levels, and water column O2 dynamics. sediments of Florida Bay (Carlson et al., 1994) and other tropical Salinity (Instant Inc.) was raised 1 psu day1 (11 regions because of high temperatures (Holmer and Kristensen, August 2003) to approximate in situ evaporative rates on 1996; Koch et al., 2007b) and the low capacity of carbonate shallow carbonate banks in the bay (0.5 psu day1; Koch et al., sediments to bind sulfide into solid-phase forms, particularly 2007a). After 29 days all tanks were at salinity treatment level pyrite. and sulfide treatments were initiated. Sulfide was injected via While porewater sulfide can cause stress in seagrass and syringe through two horizontal sippers (0.5 cm diameter other emergent marine macrophytes, our mesocosm and field tubes) with small holes alternately drilled along the tube which experimental work has shown that T. testudinum can grow and was previously inserted across the center of the core in opposite maintain high shoot densities in porewater with sulfide directions to distribute sulfide throughout the core. One end of concentrations in the millimolar range (2–10 mM; Erskine the tube was closed and the other fitted with a three-way valve and Koch, 2000; Koch et al., 2007b). We have also found in a extending into the water. Sippers were left in the cores short-term hydroponic experiment that T. testudinum can throughout the experiment. Tests of the sulfide injection exhibit a ‘‘die-back’’ response when exposed to high sulfide system were conducted using dye tracers in extra cores to (6 mM), high temperature (35 8C) and hypersalinity (55– ensure that sulfides were not readily advected to the overlying 60 psu) in combination (Koch and Erskine, 2001). The research water. Deoxygenated (N2) artificial seawater was adjusted to presented herein is a continuation of this work using large-scale porewater pH (7.0) with NaOH and sulfide added as mesocosms (detailed in Koch et al., 2007a) to examine the NaS7H2O. At the initiation of the sulfide injection treatments, synergistic effects of various levels of hypersalinity (45–65 psu 60 mL of 2 mM sulfide was added to each of the sulfide for 60 days) and porewater sulfide (2–6 mM for 40 days). These treatment cores and ambient artificial seawater injected into longer-term experiments accommodate the use of intact the controls. Sulfide concentrations in the injections were seagrass cores and a slow rate of salinity increase allowing raised 1 mMuntilinjection treatmentlevelswerereached.Due to osmotically adjust, simulating field conditions (Koch to a lack of sulfide accumulation in the cores after 4 days using et al., 2007a). In the present study, we also examined the 60 mL injections adding 0, 120, 240, and 360 mmol S2 day1, interaction of sulfide and salinity with nighttime water column the injection volume was raised to 120 mL day1 adding 0, hypoxia (5–3 mg L1) found to influence sulfide intrusion into 240, 480, and 720 mmol S2 day1. Based on minimum sulfate seagrass below-ground tissues (Pedersen et al., 2004; Borum reduction estimates from T. testudinum cores in our et al., 2005). mesocosms (33 mmol L1 day1, Koch et al., 2007b)and those measured in Florida Bay (154 mmol L1 day1,Jensen 2. Materials and methods and Koch, unpublished data) and 2 L of porewater, our sulfide amendments would have increased the total sulfide load 2to 2.1. Plant collection and mesocosm design 5times. During the last 9 days of the sulfide salinity experiment, Intact T. testudinum cores (15 cm diameter 20 cm nighttime hypoxia was simulated in 8 of the 16 tanks (two depth, 2840 cm3 sediment, 2 L porewater) were collected from each salinity treatment) and tank aeration tubes on the 25 July, 2003 from Florida Bay (25802047.000N, 80845011.400W). upper powersweeps removed. Nitrogen gas was bubbled in the Cores were transported to the FAU Marine lab (Boca Raton, tank water for 15–20 min at the initiation of the 12 h dark 1 FL) and placed into mesocosm tanks (1 m ht 1 m diameter cycle (17:00 h) until O2 wasloweredto5mgL (YSI 85). 1 with 1000 W metal halide lights) with coastal Atlantic seawater On days 2–4, O2 level was lowered to 4 mg L andondays 1 for 2 weeks (36 psu, 27 1 8C, 12:12 h light–dark cycle; PAR 5–9 O2 wasloweredto3mgL . The hypoxia treatments of 582 56 mmol m2 s1). Mesocosm tanks were equipped were run for 9 days along with sulfide additions. The with one powersweep for circulation at canopy height and one experiment was terminated after 60 days of exposure to for surface to bottom circulation with aeration. The mesocosms hypersalinity, 40 days of daily sulfide injections and 9 days of were run as a closed system with deionized water amended to hypoxia. compensate for evaporation and coastal seawater added weekly to maintain nutrient levels. 2.3. Plant response measurements

2.2. Experimental design Plant growth as leaf elongation rates (Zieman, 1974) and net shoot numbers as percent survival were determined weekly. We determined the response of T. testudinum Banks ex Leaf quantum efficiency (Fv/Fm) was also measured weekly on Ko¨nig to four salinity (36 [ambient], 45, 55, and 65 psu) and 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 2.6. Statistical analysis (Fv/Fm ratio) has an optimal range in seagrass between 0.7 and 0.8 (Ralph, 1999; Durako et al., 2002). Therefore, we used In the pre-hypoxia phaseP of the experiment, salinity and an Fv/Fm ratio of 0.7 as a lower ‘‘stress’’ threshold in the sulfide treatment effects on TSpw and plant responses were interpretation of our results. analyzed using two-way analysis of variance and Tukey multiple mean comparison tests (Sigma Stat. 3.0). If normality 2.4. Sulfur analyses of plant tissue or equal variance assumptions were violated ranked data were used. Differences between hypoxia and non-hypoxia responses At the end of the experiment, plant tissue was separated into were examined using a t-test or Mann–Whitney Rank Sum test. leaf, root, and rhizome and freeze dried. Sulfur analyses (S0, If non-significant, data from hypoxia and control tanks were TS, d34S) were run on leaves and rhizomes from plants in the pooled. Tissue sulfur data in which hypoxia was significant was 35 and 65 psu treatments exposed to 6 mM sulfide and analyzed by three-way ANOVA. All statistical significance is at controls (0 mM sulfide). Leaf and rhizome d34S and TS analysis the p < 0.05 level unless otherwise stated. were made at Iso-Analytical Limited Inc. (Cheshire, UK). The sulfur isotope composition is expressed in the following 3. Results 34 standard d notation: d S=[(Rsample/Rstandard) 1] 1000, P 34 32 where R = S/ S. Precision was better than 0.4% based on 3.1. Porewater sulfide ( TSpw) internal standards. Rhizome and leaf elemental sulfur (S0) was determined according to Zopfi et al. (2001). Sulfide was injected into intact seagrass cores of T. testudinum exposing below-ground tissues to 2, 4, and 2 2.5. Physicochemical measurements 6mMS P, but rapid oxidation resulted in lower total porewater sulfide ( TSpw) throughout the experiment. Although 2 Every 5 days 10 mL of porewater was extracted and one approximately 10, 19, and 29 mmol S was injectedP into T. 5 mL subsample used to measure pore water sulfide concen- testudinum intact sediment cores over 40 days, the TSpw tration, salinity, and pH. The other 5 mL subsample was remained <1 mM in all sulfide treatment cores (Table 1 pre- immediately transferred into an alkaline buffer, converting all hypoxia). Assuming a 308 mmol day1 rate of sulfate reduction sulfides to S2 and immediately measured with a silver/sulfide in the mesocosm cores (see Section 2) equating to 12 mmol of ion electrodeP (Orion 420A). The resulting total porewater sulfide produced in the coresP over 40 days, and taking into sulfide pool ( TSpw) at pH 7 would have a composition of H2S account the sulfide added and TSpw measured (Table 1), the (pKa1 =7;pKa2 = 19) to HS of approximately 50:50. As oxidation rate of sulfide would have been approximately 71– porewater was sampled, 10 mL of deoxygenated seawater at 431 mmol L1 day1 or 92–99% of sulfide added and treatment salinity was added back to each core in order to produced. Root oxidation probably accounted for a large maintain porewater volume. Mesocosm tanks were monitored portion of the sulfide oxidized; however, oxidation from daily for water column salinity and temperature and weekly for overlying surface water cannot be discounted and therefore light (Li-Cor spherical sensor) and O2 (YSI 85), with the plant oxidation rates were not calculated. P exception that during the hypoxia experiment O2 was measured Even though sulfide oxidation rates were high, TSpw was daily before and after lights were turned on and at the end of the significantly higher at 65 psu in contrast to all other day before O2 levels were readjusted. 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 0.11 0.04 0.26 0.09 0.13 0.06 0.52 0.17 2 0.19 0.05 0.17 0.05 0.21 0.02 0.58 0.23 4 0.27 0.02 0.33 0.12 0.15 0.02 0.46 0.05 6 0.75 0.29 0.33 0.08 0.40 0.19 0.74 0.11 Post-hypoxia (mM) 0 0.07 0.02 0.14 0.01 0.07 0.00 1.14 0.37 2 0.20 0.17 0.12 0.08 0.20 0.09 1.28 0.82 4 0.18 0.08 0.25 0.20 0.13 0.01 0.68 0.05 6 0.34 0.24 0.12 0.06 0.17 0.02 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 MS Fp (a) Leaf elongation rates Salinity 3 8.11 2.70 16.17 <0.001 Sulfide 3 0.79 0.26 1.58 0.207 Salinity sulfide 9 0.92 0.10 0.61 0.783 Residual 48 8.03 0.17 Total 63 17.85 0.28 (b) Short shoot survival rate Salinity 3 1,780 593 3.7 0.017 Sulfide 3 484 161 1.0 0.394 Salinity sulfide 9 1,978 220 1.4 0.221 Residual 48 7,611 159 Total 63 11,852 188 Hypoxia treatment was non-significant so data were pooled.

PInterestingly, even in the cores where no sulfide was injected, Fig. 1. Thalassia testudinum leaf quantum efficiency (F /F ) in response to TSpw accumulated in the 65 psu cores to levels significantly v m higher than the 35 and 55 psu treatments (Table 1). salinity and sulfide treatments before and after hypoxia treatments (mean S.D., n = 4). 3.2. Plant growth and ecophysiological response P leaf fluorescence with increasing sulfide exposure, particularly at In congruence with TSpw results, salinity was the only main ambient salinity; however, this trend was not consistent across effect explaining the variation in T. testudinum growth and short salinity treatments and leaf quantum efficiencies maintained shoot survival (Table 2). At 65 psu, leaf growth rates dropped levels indicative of relatively ‘‘healthy’’ plants (>0.70). Only in 1 below 2.0 mm day and were significantly lower than all other the 65 psu treatment did Fv/Fm ratios drop below 0.70. salinity treatments (Table 3). Shoot survival rates were also lowest at 65 psu and on average shoot survival declined as a 3.3. Hypoxia experiment function of increasing salinity (85, 81, 78 and 70%, respectively). Consistent with the growth and shoot mortality response, leaf While we predicted that the hypoxia treatments would quantum efficiency responded to salinity treatments ( p < 0.01), promote a higher O2 demand of the system, and thereby greater but not sulfide treatments (Fig. 1). There was a pattern of reduced porewater sulfide exposure in below-ground tissues, weP only found a significant plant response to salinity and TSpw Table 3 remained relatively low (Table 1). Similar to the pre-hypoxia T. testudinum growth rates (average 17 and 21 November) and shoot survival response, sulfide levels were only raised in the 65 psu treatment (17 November) after exposure to salinity (60 days), sulfide (40 days) and hypoxia (9 days) treatments (Table 1) and sulfide treatments had no effect on plant growth, but salinity was highly significant ( p < 0.01). Leaf fluores- Treatments Leaf growth Shoot cence values were also affected by salinity and F /F ratios fell (mm day1) survival (%) v m Salinity (psu) Sulfide (mM) even farther in the 65 psu tanks with hypoxia, while the Fv/Fm 35 0 2.68 0.15 87 5 levels were 0.70 in the other treatments (Fig. 1). These data 35 2 2.26 0.23 78 3 suggest that even under nightime hypoxia in the overlying 35 4 2.16 0.15 93 5 waters, T. testudinum plants by day were able to produce 35 6 2.35 0.05 80 5 enough O2 to oxidize porewater sulfide at 35 psu and moderate 45 0 2.60 0.28 74 8 hypersaline conditions, but this capacity was reduced at 65 psu. 45 2 2.74 0.22 81 5 45 4 2.69 0.20 81 4 45 6 2.67 0.04 86 5 3.4. Mesocosm O2 dynamics 55 0 2.75 0.30 77 4 55 2 2.86 0.23 81 4 Following nighttime hypoxia treatments, ecosystem level 55 4 2.28 0.06 79 4 metabolism, influenced by seagrass and other biota, responded 55 6 2.60 0.35 75 8 65 0 1.93 0.05 68 6 differently to salinity treatments. If we examine O2 fluxes 1 65 2 1.88 0.25 85 8 (mg L and percent O2 saturation) over the 9-day hypoxia 65 4 1.69 0.08 69 11 experiment, several observations are noteworthy (Fig. 2): (1) 65 6 1.60 0.15 59 9 1 when O2 levels were reduced to 5 and 4 mg L at the end of the Hypoxia treatments (9 days) were not significant so data were pooled (n = 4). 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

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 0 less efficient reoxidation of H2StoS . 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. 0 Fig. 2. Tank O2 concentration and percent saturation at 08:00 h before lights S was not detected in the leaves of T. testudinum with the came on and at the end of the day before O2 was adjusted down with N2, and exception of the 6 mM sulfide 35 psu treatment, the same 0 after adjusting nighttime O2 to treatment levels of 5, 4, and 3 mg/L indicated by treatment combination that exhibited the highest levels of S in arrows (mean S.D., n = 2). the rhizomes (Table 4). approximately 6 mg L1 and approached or were greater than 4. Discussion 100% saturation across salinity treatments. However, this daytime O2 recovery was more limited when O2 levels were Porewater sulfides in T. testudinum intact cores were readily 1 lowered to 3 mg L (third arrow). (2) Subsequent to O2 oxidized at ambient seawater salinity and moderate hypersaline 1 reductions to 5 and 4 mg L ,O2 levels fell during the night in conditions. While we cannot assume that all of the oxidation was 1 all salinity treatments. However, when reduced to 3 mg O2 L , plant mediated, a high capacity for T. testudinum to oxidize H2S O2 levels rose in the 35 and 45 psu tanks, stayed the same in the has been observed in situ in Florida Bay (Borum et al., 2005). 55 psu tanks, and declined in the 65 psu tanks during the night. Also, the ability for seagrass as well as other submerged and Thus, salinity had a direct influence on system level O2 demand emergent aquatic macrophytes to oxidize their rhizosphere at night (Fig. 2). (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 54 14 (12) 84 77 (13) 453 22 720 91 8.5 3.1 9.5 6.3 0 mM 65 psu 23 13 (5) 128 25 (26) 469 195 498 73 9.5 0.7 10.7 2.5 6 mM 35 psu 192 80 (42) 269 127 (46) 509 155 573 161 8.8 1.2 10.1 1.1 6 mM 65 psu 84 41 (22) 162 14 (28) 394 35 591 102 10.7 0.2 10.7 1.7 Leaf 0 mM 35 psu nd nd 219 18 227 11 14.9 3.3 14.8 1.4 0 mM 65 psu nd nd 191 a 200 a 10.4 a 7.5 a 6 mM 35 psu nd 6 9 (2) 239 24 213 44 13.7 2.0 14.5 1.2 6 mM 65 psu nd nd 228 a 222 a 7.3 a 9.2 a nd: not detectable. a No standard deviation calculated (n = 1). M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110 109 compounds, such as sulfide, is well established in the literature (June–July 2004) where we stimulated sulfate reductionP rates (Sand-Jensen et al., 1982; Smith et al., 1984; Lee and Dunton, five-fold with glucose amendments (28–29 8C) and TSpw 2000). Stable isotope data show that this reduced sulfur source is averaged 2.3 mM compared to <1mM in non-glucose incorporated into plant tissue (Raven and Scrimgeour, 1997), amended controls only moderate effects on T. testudinum also indicated in this study by depleted d34S signatures in the growth and no influence on shoot mortality were found (Koch rhizomes. et al., 2007b). There is growing evidence that excess H2S in seagrass can be Some of the discrepancies reported for sulfide toxicity in 0 oxidized to S in root and rhizome tissue (Holmer et al., 2005; seagrass may be accounted for by specific O2 Frederiksen et al., 2006), although chemical oxidation is a slow production potential and plant porosity, an indicator of O2 process compared to microbial mediation (Pedersen et al., storage capacity (Tomlinson, 1969) and/or site specific 2004). In our 6 mM sulfide treatment at ambient salinity, we sediment O2 consumption rates (Borum et al., 2005; Koch found 42–46% of the TS pool in T. testudinum rhizome tissue et al., 2007b; Jensen and Koch, unpublished data). Stressors could be accounted for by S0. In the temperate meadow forming that effect photosynthesis, rhizosphere oxidation, and system- 0 seagrass, Zostera marina, a high S to TS ratio was found in wide O2 consumption rates will influence plant sulfide exposure roots (68%) and rhizomes (30%) when sulfide production rates and simply tissue anoxia which may directly or indirectly were stimulated with glucose (Holmer et al., 2005). However, cause seagrass mortality. We and others (Carlson et al., 1994) 0 S was only a small component (<4%) of the TS pool in situ have measured porewaterP sulfide concentrations in the based on data from several sites in Denmark. T. testudinum millimolar range (2–5 mM TSpw pH 6.5) in intact seagrass 0 appears to possess a high ratio of S to TS and total amount of meadows not experiencing ‘‘die-off’’ (Koch et al., in prep.)P and 0 1 S (23–128 mmol g dry wt) compared to Z. marina we have experimental results verifying that millimolar TSpw (<20 mmol g1 dry wt; Holmer et al., 2005). Additionally, does not solely cause mass mortality or ‘‘die-off’’ episodes of T. accumulation of S0 in Z. marina is concentrated in the roots and testudinum (Koch et al., 2007b). Thus, while sulfide in highly not the rhizome (Holmer et al., 2005; Frederiksen et al., 2006), reduced marine sediments can accumulate, sulfide exposure is suggesting that sulfide may not be as readily transported to ameliorated by efficient plant O2 production (Borum et al., rhizomes in Z. marina. Also supporting this idea is the fact that 2005), at least if other stressors, such as hypersalinity and Z. marina TS and d34S rhizome signatures closely track the leaf temperature (Koch and Erskine, 2001; Koch et al., 2007b; this rather than the roots in the field (Frederiksen et al., 2006). We study) do not limit plant oxidation potential. observed contrasting TS and d34S signatures between T. Stressors such a hypersalinity not only affect plant testudinum leaf andP rhizome tissues in this study. At field sites oxidation potential but also ‘‘system’’ O2 fluxinthewater in the bay where TSpw was 4 mM in intact T. testudinum column. In our mesocosms, nighttime water column O2 beds, root and rhizome tissue TS were both high (920 34 and significantly declined as a function of increasing salinity after 1 34 642 148 mmol g dry wt, respectively) with depleted d S hypoxia treatments. Accelerated O2 consumption is important signatures (21 2 and 19 2), while leaves had 2–3 times in ecosystems such as Florida Bay that are shallow and have 1 lower total sulfur (329 35 mmol g dry wt) and more low early morning O2 levels in the overlying water column 34 enriched d S signatures (6 2) (Koch and Holmer, (Borum et al., 2005). When seagrass exhaust their internal O2 unpublished data). Perhaps some of these differences between supply at night, they can passively diffuse O2 from the water the two species S0, TS, and d34S are accounted for by the very column (Pregnall et al., 1984; Greve et al., 2003; Pedersen high porosity of T. testudinum below-ground tissue (Tomlinson, et al., 2004; Borum et al., 2005). However, if O2 tension is low 1969). (30–35% of air saturation, Pedersen et al., 2004), diffusive flux In seagrass, aerenchyma (air space tissue) oxidation and rates will be slow, particularly when flow rates are low (Binzer therateofO2 diffusion to the sediment has been correlated to et al., 2005) as they are in Florida Bay in internal basins and on plant photosynthesis (Lee and Dunton, 2000; Pedersen et al., shallow mudbanks. In our hypoxia experiment, O2 levels 2004; Borum et al., 2005). At 65 psu, T. testudinum growth remained above 40% airP saturation, perhaps accounting for the and leaf quantum efficiencies wereP significantly reduced continued oxidation of TSpw through passive diffusion and concomitant with an increase in TSPpw. It was interesting no significant effect of short-term 6 mM sulfide exposure that at 65 psu, we found the highest TSpw notonlyinthe through injections. 6 mM sulfide treatments but also the controls inP which In summary, hypersalinity at 65 psu directly effects T. no sulfide was added. These data indicate that TSpw testudinum’s oxidizing capacity (Koch et al., 2007a; this study) accumulation was the balance between sulfate reduction rates and places subtle demands on the positive O2 balance at an and sulfide oxidation. T. testudinum’s capacity to oxidize ecosystem level. This O2 demand, as well as that imposed by sulfide to S0 at 65 psu declined as evidenced by lower S0 in Florida Bay sediments with high sulfate reduction rates (Jensen rhizomes, possibly limiting a potential mechanism of sulfide and Koch, unpublished data), may account for frequent detoxification (Holmer et al., 2005). While our data show an observations of seagrass die-off events, particularly after increased exposure to sulfide with 6 mM injections, we did periods of high temperature and salinity. We hypothesize that not find increased plant mortality or significantly slower the interaction between hypersalinity and sulfide toxicity in T. growth due to sulfide treatments alone or in response to testudinum is their synergistic affect on the critical O2 balance hypoxic treatments. In a subsequent mesocosm experiment of the plant. 110 M.S. Koch et al. / Aquatic Botany 87 (2007) 104–110

Acknowledgements Holmer, M., Kristensen, E., 1996. Seasonality of sulfate reduction and pore water solutes in marine fish farm sediment: the importance of temperature We thank the South Florida Water Management District for and sedimentary organic matter. Biogeochemistry 32, 15–39. Ingold, A., Havill, D.C., 1984. The influence of sulphide on the distribution of funding this research and Everglades National Park for their higher plants in salt marshes. J. Ecol. 72, 1043–1054. logistical support through the Interagency Science Center at Key Koch, M.S., Erskine, J.M., 2001. Sulfide as a phytotoxin to the tropical seagrass Largo, FL. The Florida Institute of Oceanography’s Keys Marine Thalassia testudinum: interactions with light, salinity, and temperature. J. Laboratory facility also provided field support. We thank the Exp. Mar. Biol. Ecol. 266, 81–95. graduate students, Tammy Orilio, Scott Hurley and Brent Koch, M.S., Mendelssohn, I.A., 1989. Sulphide as a soil phytotoxin: differential responses in two marsh species. J. Ecol. 77, 565–578. Anderson, and post-doc, Dr. Ole Nielsen, for their assistance in Koch, M.S., Mendelssohn, I.A., McKee, K.L., 1990. Mechanism for the collecting seagrass cores and running the experiment. Neal hydrogen sulfide-induced growth limitation in wetland macrophytes. Lim- Tempel is recognized for his assistance in designing our nol. Oceanogr. 35, 399–408. mesocosms. Sulfur analysis for this research was supported by Koch, M.S., Schopmeyer, S.A., Kyhn-Hansen, C., Madden, C.J., Peters, J.S., the Danish Natural Science Foundation 272-05-0408 and the 2007a. Tropical seagrass species tolerance to hypersalinity stress. Aquat. Bot. 86, 14–24. Thresholds EU project 003933-2. We thank the anonymous Koch, M.S., Schopmeyer, S.A., Kyhn-Hansen, C., Madden, C.J., 2007b. reviewers who significantly improved the manuscript. Synergistic effects of high temperature and sulfide on tropical . J. Exp. Mar. Biol. 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