An assessment of hydrogen sulfide intrusion in the seagrass Halodule wrightii

Abstract

Hydrogen sulfide (H₂S, “sulfide”) is a naturally occurring component of the marine sediment. Eutrophication of coastal waters, however, can lead to an excess of sulfide production that can prove toxic to seagrasses. We used stable sulfur isotope ratio (δ³⁴S) measurements to assess sulfide intrusion in the seagrass Halodule wrightii, a semi-tropical species found throughout the Gulf of Mexico, Caribbean Sea, and both western and eastern Atlantic coasts. We found a gradient in δ³⁴S values (−5.58 ± 0.54‰+13.58 ± 0.30‰) from roots to leaves, in accordance with prior observations and those from other species. The results may also represent the first values reported for H. wrightii rhizome tissue. The presence of sulfide-derived sulfur in varying proportions (15–55%) among leaf, rhizome, and root tissues suggests H. wrightii is able to assimilate sedimentary H₂S into non-toxic forms that constitute a significant portion of the plant’s total sulfur content.

Introduction

Seagrasses are marine angiosperms that provide key ecological services to coastal ecosystems. Unfortunately, seagrasses are experiencing a global crisis in terms of habitat decline (Waycott et al., 2009). Excess nutrients in coastal waters can lead to an increase in sulfide levels in seagrass beds (Ruiz-Halpern et al., 2008). H₂S is a potent toxin that can easily cross cell membranes and enter the plant (intrusion), potentially inducing seagrass mortality (Koch & Erskine, 2001). Sulfide intrusion can be assessed using stable sulfur isotope (³²S, ³⁴S) measurements. Sulfate-reducing bacteria discriminate against the heavier ³⁴S isotope, preferring the lighter ³²S form. This yields sedimentary H₂S with a lower ³⁴S isotopic “signal” (Canfield, 2001). This signal can be quantified in plants, providing an estimate of the proportion of tissue sulfur derived from sedimentary sulfide (Frederiksen et al., 2006).

Thus far, the literature on sulfide intrusion in seagrasses shows relatively few measurements for species besides Zostera marina, Thalassia testudinum, and Posidonia oceanica (Holmer & Hasler-Sheetal, 2014). The objective of this study was to use stable isotope analysis to examine H₂S intrusion in the seagrass Halodule wrightii, a semi-tropical species found throughout the Gulf of Mexico, Caribbean, and parts of both the eastern and western Atlantic coasts (Green & Short, 2003). We also estimated the proportion of total sulfur derived from sedimentary sulfide in the root, rhizome, and leaf tissues. Our goal was to obtain a more complete picture of sulfide uptake and distribution in this species, including its influence on the sulfur content in major plant organs.

Methods

Forty-eight H. wrightii, 10 sediment, and five seawater samples were collected from Oso Bay, Corpus Christi, TX, near the campus of Texas A&M University-Corpus Christi (Figure 1). Leaf, rhizome, and root tissues were separated from each plant sample, oven-dried and ground to a fine powder using a ball mill. Sediment sulfide was extracted and precipitated as Ag₂S using a modified total reduced inorganic sulfur (TRIS) distillation method based on Backlund et al. (2005) and Fossing and Jørgensen (1989). A detailed protocol is available at https://doi.org/10.17504/protocols.io.b2b8qarw. Seawater sulfate was precipitated as barium sulfate (BaSO₄) under acidic conditions (Grasshoff et al., 1999). Tissue, Ag₂S, and BaSO₄ samples were sent to the Stable Isotopes for Biosphere Science (SIBS) Laboratory at Texas A&M University (College Station) for analysis of stable sulfur isotope ratios (δ³⁴S; per mil (‰) units) and total sulfur content (TS; % dw) using elemental analyzer combustion continuous flow isotope ratio mass spectroscopy. δ³⁴S represents the deviation in the ratio of ³⁴S/³²S from a particular sample relative to an international standard, and is defined as

(1) $$ {\unicode{x03B4}}^{34}{\mathrm{S}}_{\mathrm{sample}}\hskip0.35em =\hskip0.35em \left(\frac{(R)_{\mathrm{S}\mathrm{ample}}}{(R)_{\mathrm{VCDT}}}-1\right)\times \mathrm{1,000} $$

Figure 1. Sampling site in Oso Bay, Corpus Christi, TX.

where R represents the ³⁴S/³²S ratio and VCDT corresponds to the Vienna-Canyon Diablo Troilite international standard. δ³⁴S values can be negative or positive depending on whether a sample is depleted or enriched, respectively, for the S³⁴ isotope compared to the standard.

δ³⁴S values from seagrass tissues (root, rhizome, leaf), sediment sulfide, and seawater sulfate were used to calculate the F _sulfide parameter, an estimate of the percentage of the total sulfur content within a tissue that is derived from sedimentary sulfide (Frederiksen et al., 2006):

(2) $$ {F}_{\mathrm{sulfide}}\hskip0.35em =\hskip0.35em \frac{\unicode{x03B4}^{34}{\mathrm{S}}_{\mathrm{tissue}}-{\unicode{x03B4}}^{34}{\mathrm{S}}_{\mathrm{sulfate}}}{\unicode{x03B4}^{34}{\mathrm{S}}_{\mathrm{sulfide}}-{\unicode{x03B4}}^{34}{\mathrm{S}}_{\mathrm{sulfate}}}\times 100 $$

Results

The mean δ³⁴S value from 10 sediment samples was −27.38 ± 1.41‰, while that of the seawater sulfate samples was +21.11 ± 0.76‰ (Table 1). Mean δ³⁴S values for seagrass tissues ranged from −5.58 ± 3.73‰ for roots to +13.58 ± 2.04‰ for leaves (Table 2). F _sulfide values ranged from 15.51 ± 4.2% of the total sulfur content in leaves to 55.02 ± 7.68% in roots (Table 2).

Table 1. δ³⁴S values of sulfur sources (sediment TRIS or seawater sulfate) from H. wrightii bed in Oso Bay, Corpus Christi, TX

Note. Values are given as sample mean ± SD. N = number of observations.

Abbreviation: TRIS, total reduced inorganic sulfur.

Table 2. Total sulfur (TS) and δ³⁴S values for H. wrightii leaf, rhizome, and root samples from Oso Bay, Corpus Christi, TX

Note. Values are given as mean ± SD. Sample sizes for each tissue were N _leaf = 47, N _rhizome = 48, and N _root = 48.

While the proportion of sulfur derived from sediment sulfide varied, the total sulfur content (%TS) across tissues was similar (Table 2). Mean TS values ranged from 0.49 ± 0.18% in rhizomes to 0.55 ± 0.23% in roots. Variation in mean δ³⁴S, F _sulfide, and TS values among tissues was assessed with a one-way analysis of variance (ANOVA) test. Results showed significant differences for F _sulfide and δ³⁴S across seagrass tissues, but no statistically significant difference for TS (Figure 2).

Figure 2. Boxplots representing variation among F _sulfide (a), δ³⁴S (b), and total sulfur (c) values for H. wrightii tissues. p-values from one-way ANOVAs are included below each graph. Individual dots represent outliers, as defined by any number larger than 3rd Quantile (Q₃) + 1.5 interquartile range (IQR) or smaller than 1st Quantile (Q₁) – 1.5 IQR. Lines outside the box (whiskers) extend to the smallest and largest non-outliers.

Discussion

The mean δ³⁴S values for H. wrightii leaf and root tissues were higher than those previously reported for this species (+9.3‰ and −7.4‰, respectively, Holmer & Hasler-Sheetal, 2014) but the high level of variation for these measurements across studies suggests the differences may not be significant. Although we could find no previous report for H. wrightii rhizomes, the mean δ³⁴S value for this tissue was similar to one calculated across a number of seagrass species (+5.1‰, Holmer & Hasler-Sheetal, 2014).

We found a gradient in δ³⁴S values from roots to leaves, suggesting that H₂S enters the roots and then passes up to the rhizome and leaf tissue, either as sulfide itself or in an oxidized or metabolized form. This was quantified as F _sulfide, which estimated that approximately 55, 32, and 15% of the total sulfur content in roots, rhizomes, and leaves, respectively, came from sediment-derived H₂S. The range of values suggests a mixing of the sulfur pools (seawater sulfate and sedimentary sulfide) in the various tissues, similar to other species. A comparable gradient, however, was not observed for total sulfur content, which remained similar across tissue types. This could suggest that, while H₂S can intrude and become distributed throughout the plant, the level does not exceed H. wrightii’s normal metabolic requirements for sulfur.

Conclusion

Our findings suggest that significant sedimentary H₂S intrusion can occur in H. wrightii, entering through the roots and then becoming distributed throughout the plant. The results verified a trend previously observed for H. wrightii, and seagrasses in general. They also represent the first report we are aware of for rhizome tissue from this species. The relatively high proportions of total sulfur content derived from sedimentary sulfide in root, rhizome, and leaf tissue suggest H. wrightii is able to convert H₂S into non-toxic forms that can accumulate and mix with other sulfur-containing compounds derived from seawater sulfate, as demonstrated in Z. marina (Hasler-Sheetal & Holmer, 2015). Diverse levels of intrusion, however, did not translate into differences in total sulfur content among tissues, suggesting that H₂S-derived products may constitute a normal part of their sulfur budget.